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A giant market and a powerful metabolism: L-lysine provided by Corynebacterium glutamicum
Abstract
L-lysine is made in an exceptional large quantity of currently 2,200,000 tons/year and belongs therefore to one of the leading biotechnological products. Production is done almost exclusively with mutants of Corynebacterium glutamicum. The increasing L-lysine market forces companies to improve the production process fostering also a deeper understanding of the microbial physiology of C. glutamicum. Current major challenges are the identification of ancillary mutations not intuitively related with product increase. This review gives insights on how cellular characteristics enable to push the carbon flux in metabolism towards its theoretical maximum, and this example may also serve as a guide to achieve and increase the formation of other products of interest in microbial biotechnology.
... This limitation is enforced by reducing µ (with Excel Solver or Goal Seek) such that the process reaction meets this limiting OUR. 154 As shown in Figure A.2a, the fed batch has an 154 See Note 123. Further note that the specific stoichiometric coefficients in Reaction A.6 depend on the growth rate, so these are recomputed at each time interval. ...
... This limitation is enforced by reducing µ (with Excel Solver or Goal Seek) such that the process reaction meets this limiting OUR. 154 As shown in Figure A.2a, the fed batch has an 154 See Note 123. Further note that the specific stoichiometric coefficients in Reaction A.6 depend on the growth rate, so these are recomputed at each time interval. ...
... For reference, lysine fermentation is carried out at 500 m 3[154]. designs of such large bioreactors, their associated piping and valves, and their heat removal equipment (internal coils or external loop through a heat exchanger) are not practicably cleaned and sterilized to a level suitable for ...
“Cultured meat” technologies aim to replace conventional meat with analogous or alternative bioproducts from animal cell culture. Developers of these technologies claim their products, also known as “cell-based” or “cultivated” meat, will be safer and more environmentally friendly than conventional meat while offering improved farm-animal welfare. To these ends, Open Philanthropy commissioned this assessment of cultured meat’s potential to measurably displace the consumption of conventional meat.
Recognizing that the scalability of any cultured-meat products must in turn depend on the scale and process intensity of animal cell production, this study draws on techno-economic analysis and due-diligence perspectives in industrial fermentation and upstream biopharmaceuticals to assess the extent to which animal cell culture could be scaled like a fermentation process.
The analysis identifies a number of significant barriers to the scale-up of animal cell culture. Bioreactor design principles indicate a variety of issues associated with bulk cell growth in culture: Low growth rate, metabolic inefficiency, catabolite and CO2 inhibition, and bubble-induced cell damage will all limit practical bioreactor volume and attainable cell density. With existing bioreactor designs and animal cell lines, a significant engineering effort would be required to address even one of these issues.
Economic challenges are further examined. Equipment and facilities with adequate microbial contamination safeguards are expected to have high capital costs. Suitable formulations of amino acids and protein growth factors are not currently produced at scales consistent with food production, and their projected costs at scale are likewise high. The replacement of amino-acid media with plant protein hydrolysates is discussed and requires further study.
Capital- and operating-cost analyses of conceptual cell-mass production facilities indicate production economics that would likely preclude the affordability of their products as food. The analysis concludes that metabolic efficiency enhancements and the development of low-cost media from plant hydrolysates are both necessary but insufficient conditions for the measurable displacement of conventional meat by cultured meat.
... C. glutamicum is a non-pathogenic, aerobic, Gram-positive, biotin-auxotrophic soil bacterium that was initially described to be a natural producer of L-glutamate (Kinoshita et al., 1957). Since its discovery, the most appreciated application for this organism is L-glutamate and L-lysine production (Eggeling and Bott, 2015), however, the organism has also been employed for the production of other industrially relevant amino acids such as L-methionine, L-threonine, L-valine, L-tryptophan, phenylalanine, and isoleucine (Becker and Whittmann, 2015;Eggeling and Bott, 2015;Wendisch et al., 2016). The ease of cultivation of C. glutamicum combined with the generally regarded as safe (GRAS) status and robustness towards environmental stress contributed to the success of this bacterium in biotechnology. ...
... C. glutamicum is a non-pathogenic, aerobic, Gram-positive, biotin-auxotrophic soil bacterium that was initially described to be a natural producer of L-glutamate (Kinoshita et al., 1957). Since its discovery, the most appreciated application for this organism is L-glutamate and L-lysine production (Eggeling and Bott, 2015), however, the organism has also been employed for the production of other industrially relevant amino acids such as L-methionine, L-threonine, L-valine, L-tryptophan, phenylalanine, and isoleucine (Becker and Whittmann, 2015;Eggeling and Bott, 2015;Wendisch et al., 2016). The ease of cultivation of C. glutamicum combined with the generally regarded as safe (GRAS) status and robustness towards environmental stress contributed to the success of this bacterium in biotechnology. ...
Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is an acetylated amino sugar nucleotide that naturally serves as precursor in bacterial cell wall synthesis and is involved in prokaryotic and eukaryotic glycosylation reactions. UDP-GlcNAc finds application in various fields including the production of oligosaccharides and glycoproteins with therapeutic benefits. At present, nucleotide sugars are produced either chemically or in vitro by enzyme cascades. However, chemical synthesis is complex and non-economical, and in vitro synthesis requires costly substrates and often purified enzymes. A promising alternative is the microbial production of nucleotide sugars from cheap substrates. In this study, we aimed to engineer the non-pathogenic, Gram-positive soil bacterium Corynebacterium glutamicum as a host for UDP-GlcNAc production. The native glmS, glmU, and glmM genes and glmM of Escherichia coli, encoding the enzymes for UDP-GlcNAc synthesis from fructose-6-phosphate, were over-expressed in different combinations and from different plasmids in C. glutamicum GRS43, which lacks the glucosamine-6-phosphate deaminase gene (nagB) for glucosamine degradation. Over-expression of glmS, glmU and glmM, encoding glucosamine-6-phosphate synthase, the bifunctional glucosamine-1-phosphate acetyltransferase/N-acetyl glucosamine-1-phosphate uridyltransferase and phosphoglucosamine mutase, respectively, was confirmed using activity assays or immunoblot analysis. While the reference strain C. glutamicum GlcNCg1 with an empty plasmid in the exponential growth phase contained intracellularly only about 0.25 mM UDP-GlcNAc, the best engineered strain GlcNCg4 accumulated about 14 mM UDP-GlcNAc. The extracellular UDP-GlcNAc concentrations in the exponential growth phase did not exceed 2 mg/L. In the stationary phase, about 60 mg UDP-GlcNAc/L was observed extracellularly with strain GlcNCg4, indicating the potential of C. glutamicum to produce and to release the activated sugar into the culture medium. To our knowledge, the observed UDP-GlcNAc levels are the highest obtained with microbial
... In general, NADPH plays an important role in the production of lysine by corynebacteria [86]. Phosphoglucose isomerase mutant bacteria had a strong positive effect on lysine accumulation. ...
... For the improvement of the biotechnological properties of C. glutamicum, CRISPR genome editing technology is also used [86]. This system has been successfully used in the context of metabolic engineering for the suppression of single genes and to enhance lysine production [92]. ...
The review is devoted to the analysis of the current achievements of Corynebacterium glutamicummetabolic engineering for the production of lysine. Key genes of lysine biosynthesis in C. glutamicum andways of creating new genetically modified strains are considered. The role of different plasmids, vector cas-settes, and promoter types for the regulation of gene expression in C. glutamicum is described. Information isprovided on the use of carbon-containing substrates (hexose, pentose, lactic acid, mannitol) for the produc-tion of lysine. Possibilities of using CRISPR technology in genetic engineering of C. glutamicum are consid-ered. Genetic changes in C. glutamicum allowed the use of alternative substrates and contributed to theincrease of lysine accumulation in the culture f luid. The data that may be used for the creation of new lysineoverproduction strains are summarized.
... To explore the potential of CRISPR-dCpf1 system for pathway engineering via endogenous gene regulation, four genes (gltA, pck, pgi, and hom) were selected as targets for enhancing lysine production in C. glutamicum (Figure 4A). Repression of gltA (encoding citrate synthase) and pck (encoding phosphoenolpyruvate carboxykinase) is expected to increase availability of oxaloacetate, which is the precursor for lysine biosynthesis (van Ooyen et al., 2012;Eggeling and Bott, 2015;Zhou and Zeng, 2015;Park et al., 2018). The disruption of pgi (encoding glucose-6-phosphate isomerase) would benefit lysine production by improving NADPH supply via enhancing pentose-phosphate pathway flux (Marx et al., 2003). ...
... homoserine dehydrogenase (encoded by hom) activity would also result in lysine accumulation due to an enhanced flux to lysine synthesis (Eggeling and Bott, 2015). ...
Corynebacterium glutamicum is an important workhorse for industrial production of diversiform bioproducts. Multiplex control of metabolic pathway genes is crucial for maximizing biosynthesis of desired products. However, few tools for simultaneously regulating multiple genes in C. glutamicum have been reported. Here, a CRISPR-dCpf1-based multiplex gene repression system was developed for C. glutamicum. This system successfully repressed two fluorescent reporter genes simultaneously by expressing a dCpf1 (E1006A, D917A) and a designed single crRNA array. To demonstrate applications of this CRISPR-dCpf1 system in metabolic engineering, we applied this system to repress four genes involved in lysine biosynthesis (gltA, pck, pgi, and hom) with a single array, which increased the lysine titer and yield for over 4.0-fold. Quantitative PCR demonstrated that transcription of all the four endogenous target genes were repressed by over 90%. Thus, the CRISPR-dCpf1 system is a simple and effective technique for multiplex gene repression in C. glutamicum and holds promise for metabolic engineering of C. glutamicum to produce valuable chemicals and fuels.
... The Gram-positive soil bacterium Corynebacterium glutamicum is widely used as anindustrial workhorse primarily for the production of L-glutamate and L-lysine [1], and has been genetically engineered as a broad platform for production of several important industrial products [2,3]. Currently, C. glutamiucm is mostly used for aerobic production processes, but its facultative anaerobic metabolism allows to design efficient two-stage processes for the production of reduced chemicals including an aerobic growth phase and an anaerobic production phase [4,5]. ...
... Prior to inoculation of the main-culture, cells of an overnight culture were washed twice with 100 mM potassium phosphate buffer (pH 7.0). C. glutamicum main cultures were grown in CGXII minimal medium [1] supplemented with 10 g L −1 or 20 g L −1 glucose as a carbon source for growth experiments in shake flasks and bioreactors, respectively. ...
In aerobic environments, bacteria are exposed to reactive oxygen species (ROS). To avoid an excess of ROS, microorganisms are equipped with powerful enzymatic and non-enzymatic antioxidants. Corynebacterium glutamicum, a widely used industrial platform organism, uses mycothiol (MSH) as major low molecular weight (LMW) thiol and non-enzymatic antioxidant. In aerobic bioreactor cultivations, C. glutamicum becomes exposed to oxygen concentrations surpassing the air saturation, which are supposed to constitute a challenge for the intracellular MSH redox balance. In this study, the role of MSH was investigated at different oxygen levels (pO 2) in bioreactor cultivations in C. glutamicum. Despite the presence of other highly efficient antioxidant systems, such as catalase, the MSH deficient ∆mshC mutant was impaired in growth in bioreactor experiments performed at pO 2 values of 30%. At a pO 2 level of 20%, this growth defect was abolished, indicating a high susceptibility of the MSH-deficient mutant towards elevated oxygen concentrations. Bioreactor experiments with C. glutamicum expressing the Mrx1-roGFP2 redox biosensor revealed a strong oxidative shift in the MSH redox potential (E MSH) at pO 2 values above 20%. This indicates that the LMW thiol MSH is an essential antioxidant to maintain the robustness and industrial performance of C. glutamicum during aerobic fermentation processes.
... They are widely used for the production of bioproducts. Especially, C. glutamicium whose ability to produce amino acids from sugar and ammonia has been utilized for industrial-scale production of several amino acids such as glutamate, lysine, isoleucine, tryptophan and threonine [95,96]. C. glutamicium has also been engineered to produce a wide variety of biochemicals, such as polymer subunits and biofuels [97,98]. ...
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) has been extensively exploited as a genetic tool for genome editing. The RNA guided Cas nucleases generate DNA double-strand break (DSB), triggering cellular repair systems mainly Non-homologous end-joining (NHEJ, imprecise repair) or Homology-directed repair (HDR, precise repair). However, DSB typically leads to unexpected DNA changes and lethality in some organisms. The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools. Hence, in this review, we focus on the non-DSB and template-free genome editing, i.e., base editing (BE) and prime editing (PE) in bacteria and plants. We first highlight the development of base and prime editors and summarize their studies in bacteria and plants. We then discuss current and future applications of BE/PE in synthetic biology, crop improvement, evolutionary engineering, and metabolic engineering. Lastly, we critically consider the challenges and prospects of BE/PE in PAM specificity, editing efficiency, off-targeting, sequence specification, and editing window.
... It contains a mycolyl-AG-PGN complex (compare with Figure 1) in addition to lipo(arabino)mannan in its cell wall. Furthermore, C. glutamicum is one of the main species used in the biotechnological industry, especially for the production of amino acids [163]. ...
The cell walls of Gram-positive bacteria contain a variety of glycopolymers (CWGPs), a significant proportion of which are covalently linked to the peptidoglycan (PGN) scaffolding structure. Prominent CWGPs include wall teichoic acids of Staphylococcus aureus, streptococcal capsules, mycobacterial arabinogalactan, and rhamnose-containing polysaccharides of lactic acid bacteria. CWGPs serve important roles in bacterial cellular functions, morphology, and virulence. Despite evident differences in composition, structure and underlaying biosynthesis pathways, the final ligation step of CWGPs to the PGN backbone involves a conserved class of enzymes—the LytR-CpsA-Psr (LCP) transferases. Typically, the enzymes are present in multiple copies displaying partly functional redundancy and/or preference for a distinct CWGP type. LCP enzymes require a lipid-phosphate-linked glycan precursor substrate and catalyse, with a certain degree of promiscuity, CWGP transfer to PGN of different maturation stages, according to in vitro evidence. The prototype attachment mode is that to the C6-OH of N-acetylmuramic acid residues via installation of a phosphodiester bond. In some cases, attachment proceeds to N-acetylglucosamine residues of PGN—in the case of the Streptococcus agalactiae capsule, even without involvement of a phosphate bond. A novel aspect of LCP enzymes concerns a predicted role in protein glycosylation in Actinomyces oris. Available crystal structures provide further insight into the catalytic mechanism of this biologically important class of enzymes, which are gaining attention as new targets for antibacterial drug discovery to counteract the emergence of multidrug resistant bacteria.
... L-Lysine is usually recognized as the primary limiting amino acids in various grains and is produced mainly by submerged fermentation. It represents around 80% of world market, and in 2015, the world market for L-lysine was around 2.2 million tons per year [11]. The major costs involved in L-lysine production are due to raw materials [12]. ...
Screening of amino acid-producing microorganisms isolated from natural sources
... In this study, a completely novel microbial conversion process is explored to obtain δ-valerolactam from lysine, which just needs one-step purification before polymerization. Since lysine is fermented from glucose by Corynebacterium glutamicum or engineered E. coli (Eggeling and Bott 2015;Ying et al. 2014), a completely bio-based δ-valerolactam production chain could be established from biomass to promote the development of the bioplastic nylon industry. ...
Nylon 5 and nylon 6,5 are recently explored as new commercial polyamides, of which the monomer includes δ-valerolactam. In this study, a novel catalytic activity of lysine 2-monooxygenase (DavB) was explored to produce δ-valerolactam from l-pipecolic acid (L-PA), functioning as oxidative decarboxylase on a cyclic compound. Recombinant Escherichia coli BS01 strain expressing DavB from Pseudomonas putida could synthesize δ-valerolactam from l-pipecolic acid with a concentration of 90.3 mg/L. Through the co-expression of recombinant apoptosis-inducing protein (rAIP) from Scomber japonicus, glucose dehydrogenase (GDH) from Bacillus subtilis, Δ¹-piperideine-2-carboxylae reductase (DpkA) from P. putida and lysine permease (LysP) from E. coli with DavB, δ-valerolactam was produced with the highest concentration of 242 mg/L. α-Dioxygenases (αDox) from Oryza sativa could act as a similar catalyst on l-pipecolic acid. A novel δ-valerolactam synthesis pathway was constructed entirely via microbial conversion from feedstock lysine in this study. Our system has great potential in the development of a bio-nylon production process.
Key points
• DavB performs as an oxidative decarboxylase on L-PA with substrate promiscuity.
• Strain with rAIP, GDH, DpkA, LysP, and DavB coexpression could produce δ-valerolactam.
• This is the first time to obtain valerolactam entirely via biosynthesis from lysine.
... Extracted protein from food wastes or biomass is an essential source of amino acids used for bulk platform chemical production. Globally, the production of amino acids is nearly five million tons within the few decades (Eggeling and Bott 2015). l-Lysine and l-glutamic acid can be hydrolysed from biomass, potentially serving as useful feedstock chemicals for a variety of commodity chemicals like N-methylpyrrolidone and N-vinylpyrrolidone which can be produced from glutamic acid. ...
Supercritical fluid extraction (SFE) with CO2 is a valuable alternative technique in which organic solvents are used in a series of laboratories and different industrial processes. In early research, water was used as the common solvent for the extraction process, but recently CO2 has received much attention as a supercritical fluid at different industrial levels. The industry zones, especially the rubber industries, prefer to use SFE with CO2 because this combination offers many advantages such as sample recovery, maintenance of purity factor, high selectivity in products, and a very short processing time, around 10–60 min. SFE with CO2 is very effective for reducing product contamination and improving environmental safety. CO2 as a solvent when used widely in various industrial processes and with SFE does not produce any emissions harmful to the environment. SFE technologies are used in different industrial applications that have shown substantial development in recent years. In this chapter, the role of SFE in rubber industries, and the importance of the rubber industry in Malaysia, with potential SFE applications, are summarized as possible future directions in research, especially for new investigators working in this area.
... Extracted protein from food wastes or biomass is an essential source of amino acids used for bulk platform chemical production. Globally, the production of amino acids is nearly five million tons within the few decades (Eggeling and Bott 2015). l-Lysine and l-glutamic acid can be hydrolysed from biomass, potentially serving as useful feedstock chemicals for a variety of commodity chemicals like N-methylpyrrolidone and N-vinylpyrrolidone which can be produced from glutamic acid. ...
The Amazon is an ecological system that comprises a considerable part of South America and that presents a great biodiversity of plants and fruits. Most of them have bioactive compounds, such as polyphenols and carotenoids, that are associated with the reduced risk of development of various chronic and degenerative diseases. To obtain such substances, the supercritical fluid extraction stands out among other extraction methods due to environmental concerns, since this technology uses green solvents such as carbon dioxide (CO2), water (H2O), and ethanol (EtOH). In this way, the objective of this work was to present the main solvents used in pressurized solvent extraction, as well as the main bioactive compounds obtained in such processes, and their consequent biological applications.
... Corynebacterium glutamicum, which has a long history in the production of amino acids, especially glutamate and lysine, is another microbial chassis commonly used [60][61][62]. The feedback inhibition resistant aspartokinase has been developed to provide lysine production with plenty of ASA precursor [63], which is also an important precursor compound for the biosynthesis of ectoine. ...
Ectoine and hydroxyectoine as typical representatives of compatible solutes are not only essential for extremophiles to survive in extreme environments, but also widely used in cosmetic and medical industries. Ectoine was traditionally produced by Halomonas elongata through a “bacterial milking” process, of which the marked feature is using a high-salt medium to stimulate ectoine biosynthesis and then excreting ectoine into a low-salt medium by osmotic shock. The optimal hydroxyectoine production was achieved by optimizing the fermentation process of Halomonas salina . However, high-salinity broth exacerbates the corrosion to fermenters, and more importantly, brings a big challenge to the subsequent wastewater treatment. Therefore, increasing attention has been paid to reducing the salinity of the fermentation broth but without a sacrifice of ectoine/hydroxyectoine production. With the fast development of functional genomics and synthetic biology, quite a lot of progress on the bioproduction of ectoine/hydroxyectoine has been achieved in recent years. The importation and expression of an ectoine producing pathway in a non-halophilic chassis has so far achieved the highest titer of ectoine (~ 65 g/L), while rational flux-tuning of halophilic chassis represents a promising strategy for the next-generation of ectoine industrial production. However, efficient conversion of ectoine to hydroxyectoine, which could benefit from a clearer understanding of the ectoine hydroxylase, is still a challenge to date.
... Extracted protein from food wastes or biomass is an essential source of amino acids used for bulk platform chemical production. Globally, the production of amino acids is nearly five million tons within the few decades (Eggeling and Bott 2015). l-Lysine and l-glutamic acid can be hydrolysed from biomass, potentially serving as useful feedstock chemicals for a variety of commodity chemicals like N-methylpyrrolidone and N-vinylpyrrolidone which can be produced from glutamic acid. ...
Phenolic compounds are a group of compounds varying from complex polymerized compounds to simple phenols. They mainly contain hydroxybenzoic acids and hydroxycinnamic acids based on their type of structure. These compounds have been stated to show miscellaneous health benefits including anti-allergic, anti-cancer, anti-inflammatory, and anti-microbial activity. These health-related benefits make it important to develop techniques for their efficient extraction and quantification minimizing reduction in potency and retaining their functional value. Supercritical fluid extraction (SFE) is one of such processes offering advantages with respect to extraction time, solvent consumption, extraction yields, and reproducibility. This chapter provides the insight about the phenolic compounds and their extraction from various plant by-products by SFE.
... Even though these pathways are not de novo, exogenous feeding of amino acids like L-lysine and glutamate (for valerolactam and butyrolactam production, respectively) is possible while still maintaining a completely biobased process, as these molecules are readily biosynthesized and available. Indeed, they are the most-produced amino acids worldwide, primarily through microbial fermentation, with production estimates exceeding 6 million tons (Eggeling and Bott 2015;Lee and Wendisch 2017;Wang et al. 2016;Wendisch 2020) (L-lysine production specifically projected to exceed 2.5 million tons by 2020 (Vassilev et al. 2018)). ...
Lactams, cyclic carboxamide acids, are important building blocks as monomers for the manufacture of polyamides (nylons), with a market of millions of tons per year. Likewise, their non-natural building blocks, straight chain ω-amino acids, also have a wide range of applications as pharmaceuticals, therapeutic agents, and precursors to other platform chemicals. Current industrial lactam production requires petrochemically-derived routes that involve the use of harsh chemicals and reaction conditions. Microbial production provides a more sustainable method for production, from cost effective renewable resources. This review provides an extensive overview of progress toward the microbial production of lactams, particularly 4C butyrolactam, 5C valerolactam and 6C caprolactam, and their ω-amino acid precursors. Additionally, recent advances in the field as well as proposed microbial production pathways will be discussed, as well as future perspectives for the production of these important bulk chemicals.
... While the fermentative production of canonical amino acids such as L-glutamate and L-lysine is operated at a million-ton scale with Corynebacterium glutamicum [108] , the fermentative synthesis of halogenated amino acids has been reported the first time by the group of Wendisch. [109] Metabolic engineering of C. glutamicum resulted in a recombinant strain suitable for the production of L-7-Cl-Trp from sugars, ammonium and chloride salts. ...
Halogenating enzymes are able to introduce halogen substituents under ambient conditions using non‐hazardous reagents with intriguing selectivity, which is highly desired in green chemistry. Although C–H functionalization such as halogenation is a well‐known transformation in synthetic chemistry, the selective incorporation of halogens using conventional chemical approaches often remains challenging. Therefore, enzyme‐based strategies have been emerging as valuable alternatives in recent years. Inspired by manifold developments of enzymatic halogenation, this review focuses on advances of halogenating enzymes and their application with particular emphasis on FAD‐dependent halogenases (FDHs). Catalytic strategies, application scope and engineering of FDHs are outlined pointing to the increasing utility of halogenases as promising biocatalysts. Current limitations as well as potential future developments of their synthetic utility are being discussed.
... Microbes have catabolic pathways for the breakdown of these monomers, which can be naturally and metabolically engineered towards seaweed-based call factories. [59,68], but catabolic pathways could be successfully reconstituted in the bacterium to use mannitol [8 ], glucuronate [69], galactose [70] and xylose [71,72]. Likewise, S. cerevisiae naturally utilizes only a small selection of the seaweed sugars [66], but has meanwhile been engineered to degrade xylose [73], alginate and its hexuronic acid constituents [74][75][76], and its natural mannitol pathway was activated [75]. ...
Sustainable production from seaweed has grown into an area of intense research and development. Meanwhile, more than 30 million tonnes of seaweed are produced, of which 70% are used as food and 30% have other applications such as feed, fertilizer, chemicals, and energy. Towards biorefining seaweed in an environmentally friendly and economically viable manner, we need efficient approaches that convert its biomass and residuals into added value products. Smart cell factories and fermentation strategies which can be integrated into future seaweed biorefineries are at the heart of the development and therefore receive increasing attention. Here, we review advances in the field including novel fermentation routes from seaweed to chemicals, materials, pharmaceuticals, fuels and energy, and discuss challenges and opportunities.
... The Gram positive, non-sporulating bacterium Corynebacterium glutamicum is widely used in industrial biotechnology for the large scale production of various amino acids such as -lysine (> 1.4 million t/a) and -glutamate (> 2 million t/a) [12]. Furthermore, the production of biobased organic acids such as pyruvate, lactate, and succinate has been reported using genetically engineered C. glutamicum [13]. ...
3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of interest in the chemical industry as a building block for novel polymers and has wide potential for pharmaceutical applications due to its antioxidant, anti-inflammatory, and antiviral properties. In the present study, we designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of D-xylose for microbial production of PCA. The engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L ⁻¹ ) from D-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-mol PCA C-mol XYL ⁻¹ , which corresponds to 38 % of the maximum theoretical yield and is 14-fold higher compared to the parental producer strain on D-glucose. By establishing a one-pot bioreactor cultivation process followed by subsequent process optimization, the same maximum titer and a total amount of 16.5 ± 1.1 g was reached. Downstream processing of PCA from this fermentation broth was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. Since PCA turned out to be electrochemically unstable in combination with several anode materials, a three-chamber electrolysis setup was established to crystallize PCA and to avoid direct anode contact. This resulted in a maximum final purity of 95.4 %. In summary, the established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.
... Corynebacterium glutamicum is a Gram-positive, non-pathogenic soil bacterium that was isolated in a screen for microorganisms which excrete the flavor enhancer L-glutamate (Kinoshita et al., 1957). It is used in industry for the biotechnological production of amino acids and proteins (Eggeling and Bott, 2015;Freudl, 2017). Furthermore, strains for efficient production of many other metabolites have been constructed, such as organic acids (Wieschalka et al., 2013), diamines (Wendisch et al., 2018), and various other compounds (Becker and Wittmann, 2012). ...
The oxidation of NADH with the concomitant reduction of a quinone is a crucial step in the metabolism of respiring cells. In this study, we analyzed the relevance of three different NADH oxidation systems in the actinobacterial model organism Corynebacterium glutamicum by characterizing defined mutants lacking the non-proton-pumping NADH dehydrogenase Ndh (Δ ndh ) and/or one of the alternative NADH-oxidizing enzymes, L -lactate dehydrogenase LdhA (Δ ldhA ) and malate dehydrogenase Mdh (Δ mdh ). Together with the menaquinone-dependent L -lactate dehydrogenase LldD and malate:quinone oxidoreductase Mqo, the LdhA-LldD and Mdh-Mqo couples can functionally replace Ndh activity. In glucose minimal medium the Δ ndh mutant, but not the Δ ldhA and Δ mdh strains, showed reduced growth and a lowered NAD ⁺ /NADH ratio, in line with Ndh being the major enzyme for NADH oxidation. Growth of the double mutants Δ ndh Δ mdh and Δ ndh Δ ldhA , but not of strain Δ mdh Δ ldhA , in glucose medium was stronger impaired than that of the Δ ndh mutant, supporting an active role of the alternative Mdh-Mqo and LdhA-LldD systems in NADH oxidation and menaquinone reduction. In L -lactate minimal medium the Δ ndh mutant grew better than the wild type, probably due to a higher activity of the menaquinone-dependent L -lactate dehydrogenase LldD. The Δ ndh Δ mdh mutant failed to grow in L -lactate medium and acetate medium. Growth with L -lactate could be restored by additional deletion of sugR , suggesting that ldhA repression by the transcriptional regulator SugR prevented growth on L -lactate medium. Attempts to construct a Δ ndh Δ mdh Δ ldhA triple mutant were not successful, suggesting that Ndh, Mdh and LdhA cannot be replaced by other NADH-oxidizing enzymes in C. glutamicum .
... opposed to CoQ biosynthesis, the aromatic precursor is almost fully modified before being prenylated in the second to last step of MK biosynthesis (Meganathan and Kwon, 2009). C. glutamicum is used in the food and feed industry for the million-ton-scale amino acid production (Eggeling and Bott, 2015;Wendisch, 2020). C. glutamicum shows stable growth to high cell densities (Riesenberg and Guthke, 1999;Pfeifer et al., 2017) and has been engineered for production of, e.g., N-functionalized amino acids (Mindt et al., 2020), diamines (Wendisch et al., 2018;Chae et al., 2020), alcohols (Jojima et al., 2015;Siebert and Wendisch, 2015), and organic acids (Wieschalka et al., 2013;Purwanto et al., 2018). ...
Coenzyme Q 10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para -hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli . CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.
... Corynebacterium glutamicum is a Gram-positive, non-spore-forming facultative anaerobic bacterium with a moderate to high GC content belonging to the phylum of actinobacteria [1]. The microbe is traditionally used to manufacture amino acids through fermentation [2], including the premium products l-glutamate [3,4], l-lysine [5][6][7], l-arginine [8], and l-tryptophan [9]. Remarkable efforts in metabolic engineering have widened the product portfolio of C. glutamicum to over 70 different compounds [10], including bulk biofuels [11,12], and bulk chemicals such as lactate [13,14], succinate [13,15,16], cis,cis-muconate [17], cadaverine (diaminopentane) [18][19][20][21], aminovalerate [22], glutarate [22][23][24][25], and 3-amino-4-hydroxybenzoate [26]. ...
The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity.
... An interesting candidate for biotechnological production of bacteriocins is C. glutamicum, one of the most extensively used platform organisms for biotechnological production of more than 70 compounds including high value active ingredients and therapeutic proteins Eggeling and Bott, 2015;Wolf et al., 2021;Yim et al, 2014Yim et al, , 2016. This platform organism bears the advantage that well-defined, low-cost, minimal media as well as appropriate industry-scale production processes are available. ...
Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms.
Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACDCgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.
... It is worth noting that the overexpression of ddh gene in strain XQ-5-6 increased the L-lysine production (300 mM) from 29.3 ± 3.49 g/L (strain XQ-5-6) to 41.9 ± 4.57 g/L (strain XQ-5-8). Interestingly, although the L-lysine yield of strain XQ-5-8 (i.e., 41.9 ± 4.57 g/L) was lower than that of strain XQ-5-3 (i.e., 53.8 ± 3.98 g/L), the q Lys,max. of strain XQ-5-8 (i.e., 0.30 ± 0.04 g/(g·h)) was 20% higher than that of strain XQ-5-3 (i.e., 0.25 ± 0.03 g/(g·h)) (Figure 5e), possibly due to the fact that the L-lysine precursor (i.e., meso-DAP) was biosynthesized in one step rather than four steps [35] since the dehydrogenase pathway is the only pathway for L-lysine production of strain XQ-5-8. These results also showed that the dehydrogenase pathway has potential to increase L-lysine production. ...
The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.
... Other amino acids, such as l-threonine and l-tryptophan, are also industrially produced via microbial fermentation by C. glutamicum. The worldwide production of amino acids has doubled to nearly 5,000,000 tons in the past decade (Eggeling and Bott 2015). ...
Corynebacterium glutamicum, a gram-positive and facultative anaerobic bacterium, is widely used for the industrial production of amino acids, such as l-glutamate and l-lysine. C. glutamicum grows and produces amino acids under aerobic conditions. When restricted under anaerobic conditions, it produces organic acids, such as l-lactate and succinate, through metabolic shift. With the increasing threat of global warming, these organic acids have drawn considerable attention as bio-based plastic monomers. In addition to the organic acids, the anaerobic bioprocess is also used to produce other value-added compounds, including isobutanol, ethanol, 3-methyl-1-butanol, 2,3-butanediol, l-alanine, and l-valine. Therefore, C. glutamicum is now a versatile cell factory for producing a wide variety of useful chemicals under both aerobic and anaerobic conditions. The growth and metabolism of the bacterium depend on the oxygen levels, which modulate the rearrangement of the carbon flux by reprogramming gene expression patterns and intracellular redox states. Anaerobic cell growth and l-lysine production as well as aerobic succinate production have been demonstrated by engineering the metabolic pathways or supplying a terminal electron acceptor instead of oxygen. In this review, we discuss the physiological and metabolic changes in C. glutamicum associated with its application as a cell factory under different oxygen states. Physiological switching in bacteria is initiated with the sensing of oxygen availability. While such a sensor has not been identified in C. glutamicum yet, the molecular mechanism for oxygen sensing in related bacteria is also discussed.
Key Points
• C. glutamicum produces a wide variety of useful compounds under anaerobic conditions.
• C. glutamicum is a versatile cell factory under both aerobic and anaerobic conditions.
• Metabolic fate can be overcome by engineering metabolic pathways.
... The Gram-positive, nonsporulating bacterium Corynebacterium glutamicum is widely used in industrial biotechnology for the large scale production of various amino acids such as L-lysine (>1.4 million t/a) and L-glutamate (>2 million t/a) (Eggeling & Bott, 2015). Furthermore, the production of biobased organic acids such as pyruvate, lactate, and succinate has been reported using genetically engineered C. glutamicum (Wieschalka et al., 2013). ...
3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of high interest in the chemical industry and has wide potential for pharmaceutical applications. We designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of d-xylose for microbial production of PCA. Shake flask cultivation of the engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L⁻¹) from d-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-mol PCA per C-mol d-xylose, which corresponds to 38 % of the maximum theoretical yield. Under growth-decoupled bioreactor conditions, a comparable PCA titer and a total amount of 16.5 ± 1.1 g PCA could be achieved when d-glucose and d-xylose were combined as orthogonal carbon substrates for biocatalyst provision and product synthesis, respectively. Downstream processing of PCA was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. This resulted in a maximum final purity of 95.4 %. The established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.
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... This essential amino acid is the world's leading feed supplement and finds further application in the polymer, cosmetic, and pharmaceutical industries (Koffas and Stephanopoulos, 2005). The global L-lysine market grows by 6-7% annually, and the production volume is expected to reach 4 million tons in 2023 (Cheng et al., 2018;Eggeling and Bott, 2015). Currently, the L-lysine industry is based on fermentation using cane and beet molasses and starch hydrolysates from corn, cassava and wheat (Ikeda, 2003;Wittmann and Becker, 2007). ...
Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L⁻¹ l-lysine from mannitol at a yield of 0.26 mol mol⁻¹ and a maximum productivity of 2.1 g L⁻¹ h⁻¹. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol⁻¹ using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol⁻¹. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.
... Initial studies have proved that strains are mainly engineered from the conventional l-lysine producers Corynebacterium glutamicum (C. glutamicum) [10] and Escherichia coli (E. coli) [3], which can produce DAP from renewable carbon resources and achieve potentially high level yields [6,11]. ...
Nylon is a polyamide material with excellent performance used widely in the aviation and automobile industries, and other fields. Nylon monomers such as hexamethylene diamine and other monomers are in huge demand. Therefore, in order to expand the methods of nylon production, we tried to develop alternative bio-manufacturing processes which would make a positive contribution to the nylon industry. In this study, the engineered E. coli-overexpressing Lysine decarboxylases (LDCs) were used for the bioconversion of l-lysine to cadaverine. An integrated fermentation and microfiltration (MF) process for high-level cadaverine production by E. coli was established. Concentration was increased from 87 to 263.6 g/L cadaverine after six batch coupling with a productivity of 3.65 g/L-h. The cadaverine concentration was also increased significantly from 0.43 g cadaverine/g l-lysine to 0.88 g cadaverine/g l-lysine by repeated batch fermentation. These experimental results indicate that coupling the fermentation and membrane separation process could benefit the continuous production of cadaverine at high levels.
... Even though these pathways are not de novo, exogenous feeding of amino acids like L-lysine and glutamate (for valerolactam and butyrolactam production, respectively) is possible while still maintaining a completely biobased process, as these molecules are readily biosynthesized and available. Indeed, they are the most-produced amino acids worldwide, primarily through microbial fermentation, with production estimates exceeding 6 million tons (Eggeling and Bott 2015;Lee and Wendisch 2017;Wang et al. 2016;Wendisch 2020) (L-lysine production specifically projected to exceed 2.5 million tons by 2020 (Vassilev et al. 2018)). ...
Lactams, cyclic carboxamide acids, are important building blocks as monomers for the manufacture of polyamides (nylons), with a market of millions of tons per year. Likewise, their non-natural building blocks, straight chain ω-amino acids, also have a wide range of applications as pharmaceuticals, therapeutic agents, and precursors to other platform chemicals. Current industrial lactam production requires petrochemically-derived routes that involve the use of harsh chemicals and reaction conditions. Microbial production provides a more sustainable method for production, from cost effective renewable resources. This review provides an extensive overview of progress toward the microbial production of lactams, particularly 4C butyrolactam, 5C valerolactam and 6C caprolactam, and their ω-amino acid precursors. Additionally, recent advances in the field as well as proposed microbial production pathways will be discussed, as well as future perspectives for the production of these important bulk chemicals.
... It is worth noting that the overexpression of ddh gene in strain XQ-5-6 increased the L-lysine production (300 mM) from 29.3 ± 3.49 g/L (strain XQ-5-6) to 41.9 ± 4.57 g/L (strain XQ-5-8). Interestingly, although the L-lysine yield of strain XQ-5-8 (i.e., 41.9 ± 4.57 g/L) was lower than that of strain XQ-5-3 (i.e., 53.8 ± 3.98 g/L), the q Lys,max . of strain XQ-5-8 (i.e., 0.30 ± 0.04 g/(g·h)) was 20% higher than that of strain XQ-5-3 (i.e., 0.25 ± 0.03 g/(g·h)) ( Fig. 5e), possibly due to the fact that the L-lysine precursor (i.e., meso-DAP) was biosynthesized in one step rather than four steps [35] since the dehydrogenase pathway is the only pathway for L-lysine production in strain XQ-5-8. These results also showed that the dehydrogenase pathway has potential to increase L-lysine production. ...
Background: The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and great potentials to increase the production of L-lysine.
Results: The aim of this work is to enhance the carbon flux in dehydrogenase pathway to promote L-lysine production. Firstly, the effect of ammonium (NH4⁺) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in high NH4⁺ environment. In order to reduce the requirement of NH4⁺, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3±4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189±8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35±0.05 g/(g·h) in a 5-L jar fermenter.
Conclusions: The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.
... The natural LysG-P lysE biosensor was applied in several studies to monitor the cellular production of Lys, Arg, and His and to screen their better producer (Binder et al. 2012;Schendzielorz et al. 2014;Eggeling and Bott 2015). In our designed programming evolution system here, the natural LysG-P lysE was used as the Lys biosensor. ...
4-Hydroxyisoleucine (4-HIL) is a promising drug for treating diabetes. In our previous study, 4-HIL was synthesized from self-produced L-isoleucine (Ile) in Corynebacterium glutamicum by expressing an Ile dioxygenase gene. Although the 4-HIL production of recombinant strain SZ06 increased significantly, a by-product, L-lysine (Lys) was accumulated because of the share of the first several enzymes in Ile and Lys biosynthetic pathways. In this study, programming adaptive laboratory evolution (ALE) was designed and conducted in SZ06 to promote 4-HIL biosynthesis. At first, a programming evolutionary system pMK was constructed, which contains a Lys biosensor LysG-P lysE and an evolutionary actuator composed of a mutagenesis gene and a fluorescent protein gene. The evolutionary strain SZ06/pMK was then let to be evolved programmatically and spontaneously by sensing Lys concentration. After successive rounds of evolution, nine mutant strains K1 − K9 with significantly increased 4-HIL production and growth performance were obtained. The maximum 4-HIL titer was 152.19 ± 14.60 mM, 28.4% higher than that in SZ06. This titer was higher than those of all the metabolic engineered C. glutamicum strains ever constructed. The whole genome sequencing of the nine evolved strains revealed approximately 30 genetic mutations in each strain. Only one mutation was directly related to the Lys biosynthetic pathway. Therefore, programming ALE driven by Lys biosensor can be used as an effective strategy to increase 4-HIL production in C. glutamicum .
... It also could be used for synthesis of 5-hydroxyvalerate 5 , 1,5-pentanediol and other valuable chemicals. 5AVA could be manufactured from L-lysine (L-lys), which could be produced 2.2 million tons per year 3,6 . In view of the important applications of 5AVA in the field of synthesis, it is necessary to put forward relatively higher requirement for the development of the biotechnological 5AVA production. ...
Microorganisms can utilize biomass to produce valuable chemicals, showing sustainable, renewable and economic advantages compared with traditional chemical synthesis. As a potential five-carbon platform polymer monomer, 5-aminovalerate has been widely used in industrial fields such as clothes and disposable goods. Here we establish an efficient whole-cell catalysis for 5-aminovalerate production with ethanol pretreatment. In this study, the metabolic pathway from L-lysine to 5-aminovalerate was constructed at the cellular level by introducing L-lysine α-oxidase. The newly produced H2O2 and added ethanol both are toxic to the cells, obviously inhibiting their growth. Here, a promising strategy of whole-cell catalysis with ethanol pretreatment is proposed, which greatly improves the yield of 5-aminovalerate. Subsequently, the effects of ethanol pretreatment, substrate concentration, reaction temperature, pH value, metal ion additions and hydrogen peroxide addition on the whole-cell biocatalytic efficiency were investigated. Using 100 g/L of L-lysine hydrochloride as raw material, 50.62 g/L of 5-aminovalerate could be excellently produced via fed-batch bioconversion with the yield of 0.84 mol/mol. The results show that a fast, environmentally friendly and efficient production of 5-aminovalerate was established after introducing the engineered whole-cell biocatalysts. This strategy, combined with ethanol pretreatment, can not only greatly enhance the yield of 5-aminovalerate but also be applied to the biosynthesis of other valuable chemicals.
Corynebacterium glutamicum is a major workhorse in industrial biotechnology for 60 years. As the world's flagship for amino acids, the microbe produces l‐glutamate and l‐lysine at a scale of seven million tons per year. In addition, it has been upgraded into a most versatile cell factory and meanwhile provides more than 80 different natural and non‐natural compounds for chemical, energy, food and feed, cosmetic, pharmaceutical, and medical applications. In this chapter, we introduce this well‐performing bacterium and highlight pioneering discoveries as well as milestones in technology and application. Moreover, we summarize recent advances to streamline C. glutamicum for novel types of products and valorization of newly arising sustainable feedstocks.
The supply and usage of energetic cofactors in metabolism is a central concern for systems metabolic engineering, particularly in case of energy intensive products. One of the most important parameters for systems wide balancing of energetic cofactors is the ATP requirement for biomass formation YATP/Biomass. Despite its fundamental importance, YATP/Biomass values for non-fermentative organisms are still rough estimates deduced from theoretical considerations. For the first time, we present an approach for the experimental determination of YATP/Biomass using comparative ¹³C metabolic flux analysis (¹³C MFA) of a wild type strain and an ATP synthase knockout mutant. We show that the energetic profile of a cell can then be deduced from a genome wide stoichiometric model and experimental maintenance data. Particularly, the contributions of substrate level phosphorylation (SLP) and electron transport phosphorylation (ETP) to ATP generation become available which enables the overall energetic efficiency of a cell to be characterized. As a model organism, the industrial platform organism Corynebacterium glutamicum is used. C. glutamicum uses a respiratory type of energy metabolism, implying that ATP can be synthesized either by SLP or by ETP with the membrane-bound F1FO-ATP synthase using the proton motive force (pmf) as driving force. The presence of two terminal oxidases, which differ in their proton translocation efficiency by a factor of three, further complicates energy balancing for this organism. By integration of experimental data and network models, we show that in the wild type SLP and ETP contribute equally to ATP generation. Thus, the role of ETP in respiring bacteria may have been overrated in the past. Remarkably, in the genome wide setting 65% of the pmf is actually not used for ATP synthesis. However, it turns out that, compared to other organisms C. glutamicum still uses its energy budget rather efficiently.
Thepsefdh_D221Q gene coding for a mutant formate dehydrogenase (PseFDG_D221Q) from Pseudomonas, which catalyzes the formate oxidation with the simultaneous formation of NADPH, has been expressed in the cells of lysine-producing Corynebacterium glutamicum strains. The psefdh_D221Q gene was introduced into С. glutamicum strains as part of an autonomous plasmid or was integrated into the chromosome with simultaneous inactivation of host formate dehydrogenase genes. It was shown that the С. glutamicum strains with NADP+ -dependent formate dehydrogenase have an increased level of L-lysine synthesis in the presence of formate, if their own formate dehydrogenase is inactivated. L-lysine, formate dehydrogenase, NADPH, Corynebacterium glutamicum The work was carried out using the equipment of the Multipurpose Scientific This work was carried out on the equipment of the Multipurpose Scientific Installation of «All-Russian Collection of Industrial Microorganisms», National Bio-Resource Center, NRC «Kurchatov Institute»- GosNIIgenetika. This work was financially supported by the Ministry of Education and Science of Russia (Unique Project Identifier - RFMEFI61017X0011).
In Corynebacterium glutamicum, cyclic adenosine monophosphate (cAMP) serves as an effector of the global transcriptional regulator GlxR. Synthesis of cAMP is catalyzed by the membrane-bound adenylate cyclase CyaB. In this study, we investigated the consequences of decreased intracellular cAMP levels in a ΔcyaB mutant. While no growth defect of the ΔcyaB strain was observed on glucose, fructose, sucrose, or gluconate alone, the addition of acetate to these growth media resulted in a severe growth inhibition, which could be reversed by plasmid-based cyaB expression or by supplementation of the medium with cAMP. The effect was concentration- and pH-dependent, suggesting a link to the uncoupling activity of acetate. In agreement, the ΔcyaB mutant had an increased sensitivity to the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The increased uncoupler sensitivity correlated with a lowered membrane potential of acetate-grown ΔcyaB cells compared to wild-type cells. A reduced membrane potential affects major cellular processes, such as ATP synthesis by F1F
O
-ATP synthase and numerous transport processes. The impaired membrane potential of the ΔcyaB mutant could be due to a decreased expression of the cytochrome bc1-aa3 supercomplex, which is the major contributor of proton-motive force in C. glutamicum. Expression of the supercomplex genes was previously reported to be activated by GlxR-cAMP. A suppressor mutant of the ΔcyaB strain with improved growth on acetate was isolated, which carried a single mutation in the genome leading to an Ala131Thr exchange in GlxR. Introduction of this point mutation into the original ΔcyaB mutant restored the growth defect on acetate. This supported the importance of GlxR for the phenotype of the ΔcyaB mutant and, more generally, of the cAMP-GlxR system for the control of energy metabolism in C. glutamicum.
Biotechnology is the basis for numerous processes for the production of food and feed, pharmaceuticals, chemical products and energy sources. It is also the technology that prepares raw biological materials and systems (cells and their components) for use in such processes. Although humans have been using microorganisms, animals, plants and enzymes for millennia, it is only modern biotechnology that has made it possible to optimise them specifically for defined processes.
l-glutamate family amino acids (GFAAs), consisting of l-glutamate, l-arginine, l-citrulline, l-ornithine, l-proline, l-hydroxyproline, γ-aminobutyric acid, and 5-aminolevulinic acid, are widely applied in the food, pharmaceutical, cosmetic, and animal feed industries, accounting for billions of dollars of market activity. These GFAAs have many functions, including being protein constituents, maintaining the urea cycle, and providing precursors for the biosynthesis of pharmaceuticals. Currently, the production of GFAAs mainly depends on microbial fermentation using Corynebacterium glutamicum (including its related subspecies Corynebacterium crenatum), which is substantially engineered through multistep metabolic engineering strategies. This review systematically summarizes recent advances in the metabolic pathways, regulatory mechanisms, and metabolic engineering strategies for GFAA accumulation in C. glutamicum and C. crenatum, which provides insights into the recent progress in l-glutamate-derived chemical production.
Many countries have set up policies to decrease fossil fuel dependency and petro‐based synthesis of commodity chemicals. Fermentative biofuels and bioresource recovery processes are expected to assist considerably in this context of sustainable transition to a bio‐based economy. The utilization of renewable resources such as waste biomass is often considered an attractive feature of fermentative bioprocesses. However, limitations regarding the robustness of process and selectivity of target products are often considered bottlenecks to their sustainable commercialization. Particularly, in conventional fermentation processes, microorganisms produce undesired by‐products to attain intracellular redox balance, which leads to a low yield of target products. Recently, electro‐fermentation has emerged as an innovative approach for changing metabolic pathways of fermentative microorganisms towards target products with higher yields and productivities by changing intracellular redox potential. Lab‐scale EF studies have successfully demonstrated superior performance over conventional fermentation to produce a wide variety of biofuels and commodity chemicals. This book chapter provides an overview of fundamental and applied aspects of various value‐added products synthesis with the EF process and identifies research gaps for future development.
The annual world production of amino acids is currently estimated at more than seven million tons and is expected to reach ten million tons by 2022. This giant market has been underpinned largely by amino acid fermentation technologies in which Corynebacterium glutamicum has played a leading role. Various genetic engineering tools and global analysis techniques for this bacterium have been developed and successfully applied with a great impact on the amino acid industry. In particular, systems biology for this bacterium is almost fully capable of predicting targets to be engineered and metabolic states that will yield maximum production, thus allowing “systems metabolic engineering” and development of industrially competitive production strains. Additionally, whole genomes of classically derived industrial producers have been analyzed by “reverse engineering” to identify important genetic traits, enabling the establishment of new industrial processes and the creation of genetically defined producers from scratch. This “genome breeding” strategy was first developed using C. glutamicum as a model and currently yields producers that are more efficient than classical ones. These advances in strain development technology have almost achieved the optimization of entire cellular systems as cell factories for amino acid production, as demonstrated by their ability to produce glutamate and lysine at concentrations now exceeding 150 g/L with estimated production yields towards sugar at almost 70%. This chapter describes advances in the production of amino acids by C. glutamicum and presents the latest details of the technology and strategies used for molecular strain improvement.
The Gram-positive soil bacterium Corynebacterium glutamicum is a leading workhorse for the biotechnology industry. Worldwide, engineered strains of the microbe synthetize approximately 6 million tons of the amino acids l-glutamate and l-lysine. In addition to these well-known traditional fermentation products, the portfolio of C. glutamicum has impressively expanded over the past years and meanwhile comprises more than 70 natural and non-natural chemicals, materials, fuels, and health-care products. A major challenge to create superior strains for industrial production has always been and still is the design part, i.e. the identification of the most appropriate targets for genomic optimization into a desired phenotype. Metabolic flux analysis is about deciphering the activity of biochemical reactions and pathways of central carbon metabolism. It probably provides the most understandable insight into the core machinery of a living cell. This core part of metabolism displays the controlling centrepiece for synthetizing all the different molecules of recognized industrial value. One can therefore easily understand that metabolic flux analysis had a huge impact on the industrial career of C. glutamicum since its pioneering days in the 1950s, when the microbe was discovered. Initial approaches used simple isotopic tracer studies and mathematical balancing of fermentation data to assess metabolic fluxes in C. glutamicum for the first time. These techniques have been systematically upgraded into comprehensive technologies and enabled a number of seminal studies of fluxes in C. glutamicum, which provided fascinating and unexpected insights into its lifestyle and laid the foundation for metabolically engineered strains, used today in industry. Today, C. glutamicum probably displays the best studied microorganism on the level of metabolic fluxes. New developments promise an even brighter future.
Corynebacterium glutamicum (C. glutamicum) has been used for decades as the major producer of amino acids in industrial biotechnology. In the past years, substantial progress has been achieved of understanding fundamental knowledge about its gene expression and regulation, protein secretion, metabolism, and physiology, which enables engineering this Gram-positive bacterium to become a flexible and efficient platform for production of recombinant proteins and small molecules of important applications. In this chapter, we briefly summarize the protein secretion system, protein expression system, gene editing tools, small molecule production by rational design, and directed evolution of this bacterium. As it can be predicted in future, C. glutamicum holds the potential to be upgraded into one of the most successful industrial microorganisms in the world when the modern disciplines and technologies (synthetic biology, systems biology, CRISPR genomic editing, etc.) are applied in a combinatorial and advanced fashion.
Translating ribosomes require elongation factor P (EF-P) to incorporate consecutive prolines (XPPX) into nascent peptide chains. The proteome of Corynebacterium glutamicum ATCC 13032 contains a total of 1,468 XPPX motifs, many of which are found in proteins involved in primary and secondary metabolism. We show here that synthesis of EIIGlc , the glucose-specific permease of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) encoded by ptsG, is strongly dependent on EF-P, as an efp deletion mutant grows poorly on glucose as sole carbon source. The amount of EIIGlc is strongly reduced in this mutant, which consequently results in a lower rate of glucose uptake. Strikingly, the XPPX motif is essential for the activity of EIIGlc , and substitution of the prolines leads to inactivation of the protein. Finally, translation of GntR2, a transcriptional activator of ptsG, is also dependent on EF-P. However, its reduced amount in the efp mutant can be compensated for by other regulators. These results reveal for the first time a translational bottleneck involving production of the major glucose transporter EIIGlc , which has implications for future strain engineering strategies.
The growth rate μ of bacteria depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for a few bacterial species, in particular Escherichia coli , but are missing for most bacterial phyla. In this study, we systematically analysed the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum , a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower μ max . Ribosomes were quantified via single-molecule localization microscopy (SMLM) using fluorescently tagged ribosomal proteins and via RNA/protein ratio. Both methods revealed a non-linear relationship with little change in ribosome abundance below μ = 0.4 h ⁻¹ and a steep increase at higher μ. Unlike E. coli , C. glutamicum keeps a large pool of active ribosomes at low μ, but the translation elongation rate declines from ~9 amino acids s ⁻¹ at μ max to <2 aa s ⁻¹ at μ < 0.1 h ⁻¹ . A model-based approach shows that depletion of translation precursors at low growth rates can explain the observed decrease in translation elongation rate. Nutrient up-shift experiments support the hypothesis that maintenance of excess ribosomes during poor nutrient conditions enables C. glutamicum to quickly restart growth when conditions improve.
In nature and in technical systems, microbial cells are often exposed to rapidly fluctuating environmental conditions. These conditions can vary in quality, e.g., the existence of a starvation zone, and quantity, e.g., the average residence time in this zone. For strain development and process design, cellular response to such fluctuations needs to be systematically analysed. However, the existing methods for physically imitating rapidly changing environmental conditions are limited in spatio-temporal resolution. Hence, we present a novel microfluidic system for cultivation of single cells and small cell clusters under dynamic environmental conditions (dynamic microfluidic single-cell cultivation (dMSCC)). This system enables the control of nutrient availability and composition between two media with second to minute resolution. We validate our technology using the industrially relevant model organism Corynebacterium glutamicum. The organism was exposed to different oscillation frequencies between nutrient excess (feasts) and scarcity (famine). The resulting changes in cellular physiology, such as the colony growth rate and cell morphology, were analysed and revealed significant differences in the growth rate and cell length between the different conditions. dMSCC also allows the application of defined but randomly changing nutrient conditions, which is important for reproducing more complex conditions from natural habitats and large-scale bioreactors. The presented system lays the foundation for the cultivation of cells under complex changing environmental conditions.
Scyllo-inositol has been identified as a potential drug for the treatment of Alzheimer's disease. Therefore, cost-efficient processes for the production of this compound are desirable. In this study, we analyzed and engineered Corynebacterium glutamicum with the aim to develop competitive scyllo-inositol producer strains. Initial studies revealed that C. glutamicum naturally produces scyllo-inositol when cultured with myo-inositol as carbon source. The conversion involves NAD+-dependent oxidation of myo-inositol to 2-keto-myo-inositol followed by NADPH-dependent reduction to scyllo-inositol. Use of myo-inositol for biomass formation was prevented by deletion of a cluster of 16 genes involved in myo-inositol catabolism (strain MB001(DE3)Δiol1). Deletion of second cluster of four genes (oxiC-cg3390-oxiD-oxiE) related to inositol metabolism prevented conversion of 2-keto-myo-inositol to undesired products causing brown coloration (strain MB001(DE3)Δiol1Δiol2). The two chassis strains were used for plasmid-based overproduction of myo-inositol dehydrogenase (IolG) and scyllo-inositol dehydrogenase (IolW). In BHI medium containing glucose and myo-inositol, a complete conversion of the consumed myo-inositol into scyllo-inositol was achieved with the Δiol1Δiol2 strain. To enable scyllo-inositol production from cheap carbon sources, myo-inositol 1-phosphate synthase (Ino1) and myo-inositol 1-phosphatase (ImpA), which convert glucose 6-phosphate into myo-inositol, were overproduced in addition to IolG and IolW using plasmid pSI. Strain MB001(DE3)Δiol1Δiol2 (pSI) produced 1.8 g/L scyllo-inositol from 20 g/L glucose and even 4.4 g/L scyllo-inositol from 20 g/L sucrose within 72 h. Our results demonstrate that C. glutamicum is an attractive host for biotechnological production of scyllo-inositol and potentially further myo-inositol-derived products.
Biomass is a realistic alternative renewable resource to produce fuels and chemicals, currently derived from non-renewable sources. The needs to replace non-renewable resources have fostered numerous studies on biomass-based platform chemicals. Supercritical fluid exhibits a wide range of chemical and physical properties, varying from gas-like to liquid-like behaviour due to the variations in the ionic product and dielectric constant. The diversity in its properties is beneficial especially in biomass conversion. Extensive researches are needed to produce platform chemicals from biomass using supercritical fluid technology with the aim to improve its economic feasibility.
l-lysine is an essential amino acid that contains various functional groups including α-amino, ω-amino, and α-carboxyl groups, exhibiting high reaction potential. The derivatization of these functional groups produces a series of value-added chemicals, such as cadaverine, glutarate, and d-lysine, that are widely applied in the chemical synthesis, cosmetics, food, and pharmaceutical industries. Here, we review recent advances in the biotechnological production of l-lysine and its derivatives and expatiate key technological strategies. Furthermore, we also discuss the existing challenges and potential strategies for more efficient production of these chemicals.
The compatible solute mannosylglycerate (MG) has exceptional properties in terms of protein stabilization and protection under salt, heat, and freeze-drying stresses as well as against protein aggregation. Due to these characteristics, MG possesses large potential for clinical and biotechnological applications. To achieve efficient MG production, Corynebacterium glutamicum was equipped with a bifunctional MG synthase (encoded by mgsD and catalyzing the condensation of 3-phosphoglycerate and GDP-mannose to MG) from Dehalococcoides mccartyi . The resulting strain C. glutamicum (pEKEx3 mgsD ) intracellularly accumulated about 111 mM MG (60 ± 9 mg g CDW ⁻¹ ) with 2% glucose as a carbon source. To enable efficient mannose metabolization, the native manA gene, encoding mannose 6-phosphate isomerase, was overexpressed. Combined overexpression of manA and mgsD from two plasmids in C. glutamicum resulted in intracellular MG accumulation of up to ca. 329 mM [corresponding to 177 mg g cell dry weight (CDW) ⁻¹ ] with glucose, 314 mM (168 mg g CDW ⁻¹ ) with glucose plus mannose, and 328 mM (176 mg g CDW ⁻¹ ) with mannose as carbon source(s), respectively. The product was successfully extracted from cells by using a cold water shock, resulting in up to 5.5 mM MG (1.48 g L ⁻¹ ) in supernatants. The two-plasmid system was improved by integrating the mgsD gene into the manA -bearing plasmid and the resulting strain showed comparable production but faster growth. Repeated cycles of growth/production and extraction of MG in a bacterial milking-like experiment showed that cells could be recycled, which led to a cumulative MG production of 19.9 mM (5.34 g L ⁻¹ ). The results show that the newly constructed C. glutamicum strain produces MG from glucose and mannose and that a cold water shock enables extraction of MG from the cytosol into the medium.
Cadaverine, a diamine monomer, broadly exists in living organisms participating in metabolism processes. As a promising substitute of 1,6-diaminohexane, cadaverine can polymerize with dicarboxylic acids and yield biobased polyamides, polyamide (PA) 5×, showing ecofriendly and excellent mechanical properties in the fields of electronics, automobile, material, storage, and others. Due to the increasing attentions on environment problems, biopolyamide is expected to be an ideal and green material to substitute conventional chemistry polyamide. This review summarizes the properties, potential applications, and production strategies about cadaverine and mainly focuses on the recent developments by cellular engineering Escherichia coli and Corynebacterium glutamicum as main workhorses. In addition, the key enzyme lysine decarboxylase decorated by means of immobilization and mutation to efficiently catalyze lysine and its catalysis mechanisms are also discussed. In order to achieve industrial applications, the indispensable steps of separation and purification are described as well and perspectives for cadaverine manufacturing from renewable resources are further provided.
Green chemical production by microbial processes is critical for the development of a sustainable society in the twenty-first century. Among the important industrial microorganisms, the gram-positive bacterium Corynebacterium glutamicum has been utilized for amino acid fermentation, which is one of the largest microbial-based industries. To date, several amino acids, including l-glutamic acid, l-lysine, and l-threonine, have been produced by C. glutamicum. The capability to produce substantial amounts of amino acids has gained immense attention because the amino acids can be used as a precursor to produce other high-value-added chemicals. Recent developments in metabolic engineering and synthetic biology technologies have enabled the extension of metabolic pathways from amino acids. The present review provides an overview of the recent progress in the microbial production of amino acid-derived bio-based monomers such as 1,4-diaminobutane, 1,5-diaminopentane, glutaric acid, 5-aminolevulinic acid, l-pipecolic acid, 4-amino-1-butanol, and 5-aminolevulinic acid, as well as building blocks for healthcare products and pharmaceuticals such as ectoine, l-theanine, and gamma-aminobutyric acid by metabolically engineered C. glutamicum.
Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2g•L(-1)•h(-1), and a final titer of 88g•L(-1). The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production.
Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways.
In this study, the feedback inhibition of Corynebacterium glutamicum PEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein
design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate
inhibition. Introducing one of the point mutations (N917G) into the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ∼37% improved lysine production. In vitro enzyme assays and 13C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively,
in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which
increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation
on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived
products.
Corynebacterium glutamicum is an important organism for industrial biotechnology; particularly, in amino acid production (e.g. l-lysine). Production scales often reach reactor working volumes of several hundred cubic meters, which triggers inhomogeneous distribution of substrates and dissolved gasses due to increasing mixing times. Individual cells which follow the flow profile through the reactor are experiencing oscillating microenvironments. Oscillations can have an influence on the process performance, which is a subject of scale-down experiments. In this work, l-lysine-producing C. glutamicum DM1933 was assessed for its robustness against continuous dissolved oxygen and substrate supply oscillation in two-compartment scale-down bioreactors. Aerobic, substrate-limited stirred tank and non-aerated, substrate-excess plug flow compartments were applied for oscillation. Inhomogeneity of substrate and oxygen supply was observed to cause rapid side product turnover, redistribution of oxygen uptake from oxygen limited into fully aerobic zones, and intermediate medium acidification. However, process inhomogeneity did not impair productivity or growth at plug flow residence times of several minutes. In a focused analysis of proteome, metabolome, transcriptome, and other physiological parameters, no changes were identified in response to process inhomogeneity. In conclusion, fed-batch processes with C. glutamicum DM1933 possess remarkable robustness against oxygen and substrate supply oscillation, which is a unique property in the field of published scale-down studies. Microbial physiology of C.
glutamicum appears to be ideally adapted to both homogeneous and inhomogeneous conditions. This ensures exceptional suitability for cultivation at increased mixing times, which is suggested to constitute an important basis for the long-lasting success in large scale bioprocess application.
Methanol is considered an interesting carbon source in “bio-based” microbial production processes. Since Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies of the response
of this organism to methanol. The C. glutamicum wild type was able to convert 13C-labeled methanol to 13CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be upregulated,
indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol
dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde.
Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde
dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The Δald ΔadhE and Δald ΔmshC deletion mutants were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF, recently identified by us. Similar to the case with ethanol, methanol catabolism
is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA,
which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogenous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway.
DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase, led to the isolation of four transcriptional regulators, i.e., RamA, GntR1,
GntR2, and IolR. Determination of the phosphoenolpyruvate carboxykinase activity of the ΔramA, ΔgntR1 ΔgntR2, and ΔiolR deletion mutants indicated that RamA represses pck during growth on glucose about 2-fold, whereas GntR1, GntR2, and IolR activate pck expression about 2-fold irrespective of whether glucose or acetate served as the carbon source. The DNA binding sites of
the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The
iolR gene is located upstream and in a divergent orientation with respect to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the ΔiolR mutant and the parental wild-type strain revealed strongly (>100-fold) elevated mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT1, and iolR. IolR therefore is presumably subject to negative autoregulation. A consensus DNA binding motif (5′-KGWCHTRACA-3′) which
corresponds well to those of other GntR-type regulators of the HutC family was identified. Taken together, our results disclose
a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism.
Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations
at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that
C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.
Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable
for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance.
Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection
and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this
new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries
to directly isolate l-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause l-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid
exchanges in the targeted murE codon, which caused different l-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single
step, thus establishing a new general concept in synthetic biology.
Bacterial cell size and morphology are enormously diverse. The molecular
factors of morphogenesis are well understood in certain bacterial models and fairly
conserved throughout a broad spectrum of bacterial species, as follows. In most
bacteria, the tubulin-like FtsZ protein polymerizes at the mid cell , thereby generating
the scaffold of the bacterial cell division septum. Actin-like MreB homologues are
required for cell elongation at the lateral walls of Escherichia coli or Bacillus subtilis.
Whereas FtsZ is conserved in Corynebacterium glutamicum, mreB homologues are
absent in the corynebacterial genomes sequenced to date. Furthermore, in these
bacteria, cell elongation occurs at the polar ends in a mycelial fashion. This process
is structurally maintained from the inside of the cell by oligomers created throughself-interaction of DivIVA, a coiled-coil-rich cytoskeletal protein that interacts with
the molecular machinery of the polar cell wall synthesis. In addition, the molecular
factors involved in the spatio-temporal regulation of bacterial cell division are
also missing in C. glutamicum. However, certain serine/threonine kinases have
been reported recently in this organism that could be implicated in a tight regulation
of cytokinesis through protein phosphorylation. Since numerous antibiotics target
bacterial cell division or cell elongation genes, a detailed understanding of these
processes could enable the development of novel antibiotics for treating bacterial
infections caused by pathogenic Corynebacteria or by the closely related
Mycobacteria, Nocardia, or Rhodococcus.
Because of their abundance in hemicellulosic wastes arabinose and xylose are an interesting source of carbon for biotechnological production processes. Previous studies have engineered several Corynebacterium glutamicum strains for the utilization of arabinose and xylose, however, with inefficient xylose utilization capabilities. To improve xylose utilization, different xylose isomerase genes were tested in C. glutamicum. The gene originating from Xanthomonas campestris was shown to have the highest effect, resulting in growth rates of 0.14 h−1, followed by genes from Bacillus subtilis, Mycobacterium smegmatis and Escherichia coli. To further increase xylose utilization different xylulokinase genes were expressed combined with X. campestris xylose isomerase gene. All combinations further increased growth rates of the recombinant strains up to 0.20 h−1 and moreover increased biomass yields. The gene combination of X. campestris xylose isomerase and C. glutamicum xylulokinase was the fastest growing on xylose and compared with the previously described strain solely expressing E. coli xylose isomerase gene delivered a doubled growth rate. Productivity of the amino acids glutamate, lysine and ornithine, as well as the diamine putrescine was increased as well as final titres except for lysine where titres remained unchanged. Also productivity in medium containing rice straw hydrolysate as carbon source was increased.
Funding Information No funding information provided.
We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes.
The phylum Actinobacteria harbors many important human pathogens and also provides one of the richest sources of natural products, including numerous antibiotics and other compounds of biotechnological interest. Thus, a reliable phylogeny of this large phylum and the means to accurately identify its different constituent groups are of much interest. Detailed phylogenetic and comparative analyses of >150 actinobacterial genomes reported here form the basis for achieving these objectives. In phylogenetic trees based upon 35 conserved proteins, most of the main groups of Actinobacteria as well as a number of their superageneric clades are resolved. We also describe large numbers of molecular markers consisting of conserved signature indels in protein sequences and whole proteins that are specific for either all Actinobacteria or their different clades (viz., orders, families, genera, and subgenera) at various taxonomic levels. These signatures independently support the existence of different phylogenetic clades, and based upon them, it is now possible to delimit the phylum Actinobacteria (excluding Coriobacteriia) and most of its major groups in clear molecular terms. The species distribution patterns of these markers also provide important information regarding the interrelationships among different main orders of Actinobacteria. The identified molecular markers, in addition to enabling the development of a stable and reliable phylogenetic framework for this phylum, also provide novel and powerful means for the identification of different groups of Actinobacteria in diverse environments. Genetic and biochemical studies on these Actinobacteria-specific markers should lead to the discovery of novel biochemical and/or other properties that are unique to different groups of Actinobacteria.
l-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by 13C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP).
Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed
in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand
of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an l-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity
offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover,
as demonstrated for l-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation
via the PPP.
Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial
strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate
a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made
considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited
number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study,
we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify
the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected
residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including
most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another
14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays.
The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter l-lysine within 30 h of fed-batch fermentation in a bioreactor.
Approximately one third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. This bacterium has an unusual lipid-rich cell wall containing a vast repertoire of antigens, providing a hydrophobic impermeable barrier against chemical drugs, thus representing an attractive target for vaccine and drug development. Apart from the mycolyl-arabinogalactan-peptidoglycan complex, mycobacteria possess several immunomodulatory constituents, notably lipomannan and lipoarabinomannan. The availability of whole-genome sequences of M. tuberculosis and related bacilli over the past decade has led to the identification and functional characterization of various enzymes and the potential drug targets involved in the biosynthesis of these glycoconjugates. Both lipomannan and lipoarabinomannan possess highly variable chemical structures, which interact with different receptors of the immune system during host-pathogen interactions, such as Toll-like receptors-2 and C-type lectins. Recently, the availability of mutants defective in the synthesis of these glycoconjugates in mycobacteria and the closely related bacterium, Corynebacterium glutamicum, has paved the way for host-pathogen interaction studies, as well as, providing attenuated strains of mycobacteria for the development of new vaccine candidates. This review provides a comprehensive account of the structure, biosynthesis and immunomodulatory properties of these important glycoconjugates.
Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose
uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general
PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization
genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent
glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2,
and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence
of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with Ks values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via
IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways
for l-lysine production were tested. The l-lysine yield by C. glutamicum DM1729, a rationally engineered l-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δhpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729Δhpr enabled l-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher l-lysine yield.
Oxoglutarate dehydrogenase (ODH) and pyruvate dehydrogenase (PDH) complexes catalyze key reactions in central metabolism,
and in Corynebacterium glutamicum there is indication of an unusual supercomplex consisting of AceE (E1), AceF (E2), and Lpd (E3) together with OdhA. OdhA
is a fusion protein of additional E1 and E2 domains, and odhA orthologs are present in all Corynebacterineae, including, for instance, Mycobacterium tuberculosis. Here we show that deletion of any of the individual domains of OdhA in C. glutamicum resulted in loss of ODH activity, whereas PDH was still functional. On the other hand, deletion of AceF disabled both PDH
activity and ODH activity as well, although isolated AceF protein had solely transacetylase activity and no transsuccinylase
activity. Surprisingly, the isolated OdhA protein was inactive with 2-oxoglutarate as the substrate, but it gained transsuccinylase
activity upon addition of dihydrolipoamide. Further enzymatic analysis of mutant proteins and mutant cells revealed that OdhA
specifically catalyzes the E1 and E2 reaction to convert 2-oxoglutarate to succinyl-coenzyme A (CoA) but fully relies on the
lipoyl residues provided by AceF involved in the reactions to convert pyruvate to acetyl-CoA. It therefore appears that in
the putative supercomplex in C. glutamicum, in addition to dihydrolipoyl dehydrogenase E3, lipoyl domains are also shared, thus confirming the unique evolutionary position
of bacteria such as C. glutamicum and M. tuberculosis.
In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase,
achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated
to a flux shift from the TCA cycle toward anaplerotic carboxylation.
Background
Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step.
Methodology/ Principal Findings
By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene.
Conclusions/ Significance
Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.
The influence of acetohydroxy acid synthase (AHAS) on l-lysine production by Corynebacterium glutamicum was investigated. An AHAS with a deleted C-terminal domain in the regulatory subunit IlvN was engineered by truncating the
ilvN gene. Compared to the wild-type AHAS, the newly constructed enzyme showed altered kinetic properties, i.e., (i) an about
twofold-lower Km for the substrate pyruvate and an about fourfold-lower Vmax; (ii) a slightly increased Km for the substrate α-ketobutyrate with an about twofold-lower Vmax; and (iii) insensitivity against the inhibitors l-valine, l-isoleucine, and l-leucine (10 mM each). Introduction of the modified AHAS into the l-lysine producers C. glutamicum DM1729 and DM1933 increased l-lysine formation by 43% (30 mM versus 21 mM) and 36% (51 mM versus 37 mM), respectively, suggesting that decreased AHAS activity
is linked to increased l-lysine formation. Complete inactivation of the AHAS in C. glutamicum DM1729 and DM1933 by deletion of the ilvB gene, encoding the catalytic subunit of AHAS, led to l-valine, l-isoleucine, and l-leucine auxotrophy and to further-improved l-lysine production. In batch fermentations, C. glutamicum DM1729 ΔilvB produced about 85% more l-lysine (70 mM versus 38 mM) and showed an 85%-higher substrate-specific product yield (0.180 versus 0.098 mol C/mol C) than
C. glutamicum DM1729. Comparative transcriptome analysis of C. glutamicum DM1729 and C. glutamicum DM1729 ΔilvB indicated transcriptional differences for about 50 genes, although not for those encoding enzymes involved in the l-lysine biosynthetic pathway.
The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter
of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain
C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone
was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward
the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%,
the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfbr. The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased
supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase
seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds
as well as for products directly stemming from the PPP.
One of the most important organisms in biotechnology, Corynebacterium glutamicum is currently used to produce 2 million tons of amino acids per year for a rapidly expanding market. Until now, research and information have been scattered among individual papers which are often difficult to locate in a timely manner. As the first complete compilation of major findings, Handbook of Corynebacterium glutamicum is a comprehensive source of scientific and technical information required for the understanding and manipulation of C. glutamicum. The book summarizes the current knowledge in the field ofC. glutamicum research from its discovery in 1957 through the most recent studies at the genomic and systemic level, and provides a basis for future work. Written by experts from industry and academia, chapters cover all major aspects of C. glutamicum, including physiology, biochemistry, genetics, and industrial applications. Just as C. glutamicum has proven its profitability in industry and research, this book will demonstrate its value to the scientists striving to understand and develop even more efficient producer strains of this promising microorganism.
A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.
Microbial metabolite export is an essential issue in industrial biotechnology. The reason is simple: otherwise, the development of advanced engineered strains might fail. Export belongs to the entire reaction sequence of substrate uptake and conversion to the valuable product finally accumulating in the medium. The relevance of export is probably best studied in the case of amino acid production with Escherichia coli or Corynebacterium glutamicum where specific exporters, when deleted, prevent amino acid production, or when overexpressed, increase amino acid production. However, antibiotic production also relies on exporters as the final step in cellular synthesis of these important metabolites. Another area where the importance of exporters is assessed is the use of whole cells for bioconversions occurring in organic solvents. This review covers these areas, gives a tabular overview on exporters recognized in biotechnology and describes also the varied methodology of exporter identification.
Keywords:
export;
l-cysteine;
exporter isolation;
l-glutamate;
antibiotica;
efflux;
toxic compounds
Abstract Cyanophycin, inclusions in cyanobacteria discovered by the Italian scientist Borzi in 1887, were characterized as a polyamide consisting of aspartic acid and arginine. Its synthesis in cyanobacteria was analyzed regarding growth conditions, responsible gene product, requirements, polymer structure and properties. Heterologous expression of diverse cyanophycin synthetases (CphA) in Escherichia coli enabled further enzyme characterization. Cyanophycin is a polyamide with variable composition and physiochemical properties dependent on host and cultivation conditions in contrast to the extracellular polyamides poly-γ-glutamic acid and poly-ϵ-l-lysine. Furthermore, recombinant prokaryotes and transgenic eukaryotes, including plants expressing different cphA genes, were characterized as suitable for production of insoluble cyanophycin regarding higher yields and modified composition for other requirements and applications. In addition, cyanophycin was characterized as a source for the synthesis of polyaspartic acid or N-containing bulk chemicals and dipeptides upon chemical treatment or degradation by cyanophycinases, respectively. Moreover, water-soluble cyanophycin derivatives with altered amino acid composition were isolated from transgenic plants, yeasts and recombinant bacteria. Thereby, the range of dipeptides could be extended by biological processes and by chemical modification, thus increasing the range of applications for cyanophycin and its dipeptides, including agriculture, food supplementations, medical and cosmetic purposes, synthesis of the polyacrylate substitute poly(aspartic acid) and other applications.
The exploration of scale-down models to imitate the influence of large scale bioreactor inhomogeneities on cellular metabolism is a topic with increasing relevance. While gradients of substrates, pH, or dissolved oxygen are often investigated, oscillating CO2/HCO3 (-) levels, a typical scenario in large industrial bioreactors, is rarely addressed. Hereby, we investigate the metabolic and transcriptional response in Corynebacterium glutamicum wild type as well as the impact on L-lysine production in a model strain exposed to pCO2 gradients of (75-315) mbar. A three-compartment cascade bioreactor system was developed and characterized that offers high flexibility for installing gradients and residence times to mimic industrial-relevant conditions and provides the potential of accurate carbon balancing. The phenomenological analysis of cascade fermentations imposed to the pCO2 gradients at industry-relevant residence times of about 3.6 min did not significantly impair the process performance, with growth and product formation being similar to control conditions. However, transcriptional analysis disclosed up to 66 differentially expressed genes already after 3.6 min under stimulus exposure, with the overall change in gene expression directly correlateable to the pCO2 gradient intensity and the residence time of the cells.
Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products.
Enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. Here we present an approach to rapidly generate sets of enzymes overriding this control. It is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. The sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened the library in a strain carrying the sensor pSenLys-Spc, which detects L-lysine, L-arginine and L-histidine. Six of the resulting N-acetyl-L-glutamate kinase proteins were further developed and characterized and found to be at least 20-fold less sensitive towards L-arginine inhibition than the wild-type enzyme. Overexpression of the mutein ArgB-K47H-V65A in C. glutamicumΔargR led to the accumulation of 34 mM L-arginine in the culture medium. We also screened mutant libraries of lysC-encoded aspartate kinase and hisG-encoded ATP phosphoribosyltransferase. We isolated 11 LysC muteins, enabling up to 45 mM L-lysine accumulation, and 13 HisG muteins, enabling up to 17 mM L-histidine accumulation. These results demonstrate that in vivo screening of enzyme libraries by using metabolite sensors is extremely well suited to identify high-performance muteins required for overproduction.
An l-glutamine overproducing mutant of an Escherichia coli K-12 derived strain was selected from randomly mutagenized cells in the course of l-alanyl-l-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism for l-glutamine overproduction in this mutant. Three mutations were identified that are related to the l-glutamine overproduction phenotype; namely, an intergenic mutation in the 5' -flanking region of yeiG and two non-synonymous mutations in gyrA (Gly821Ser and Asp830Asn). Expression of yeiG, which encodes a putative esterase, was enhanced by the intergenic mutation. The non-synonymous mutations in gyrA, a gene that encodes the DNA gyrase α subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (topA) and topoisomerase IV (parC-parE), an increase in l-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase in l-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed an l-glutamine hyper-producing strain by attenuating cellular l-glutamine degradation activity. Although the yeiG together with gyrA reconstituted mutant produced 200 mM l-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mM l-glutamine.
Bio-based production promises a sustainable route to myriads of chemicals, materials and fuels. With regard to eco-efficiency, its future success strongly depends on a next level of bio-processes using raw materials beyond glucose. Such renewables, i.e., polymers, complex substrate mixtures and diluted waste streams, often cannot be metabolized naturally by the producing organisms. This particularly holds for well-known microorganisms from the traditional sugar-based biotechnology, including Escherichia coli, Corynebacterium glutamicum and Saccharomyces cerevisiae which have been engineered successfully to produce a broad range of products from glucose. In order to make full use of their production potential within the bio-refinery value chain, they have to be adapted to various feed-stocks of interest. This review focuses on the strategies to be applied for this purpose which combine rational and evolutive approaches. Hereby, the three industrial platform microorganisms, E. coli, C. glutamicum and S. cerevisiae are highlighted due to their particular importance.
Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysC(fbr)dld(Psod)pyc(Psod)malE(Psod)fbp(Psod)gapX(Psod) was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of d-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35h(-1) and lysine carbon yields of approximately 90C-mmol (C-mol)(-1). Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine.
In this study, we demonstrate increased lysine production by flux coupling using the industrial work horse bacterium Corynebacterium glutamicum, which was mediated by the targeted interruption of the tricarboxylic acid (TCA) cycle at the level of succinyl-CoA synthetase. The succinylase branch of the lysine production pathway functions as the bridging reaction to convert succinyl-CoA to succinate in this aerobic bacterium. The mutant C. glutamicum ΔsucCD showed a 60% increase in the yield of lysine when compared to the advanced lysine producer which was used as parent strain. This mutant was highly vital and exhibited only a slightly reduced specific growth rate. Metabolic flux analysis with (13)C isotope studies confirmed that the increase in lysine production was mediated by pathway coupling. The novel strain exhibited an exceptional flux profile, which was closer to the optimum performance predicted by in silico pathway analysis than to the large set of lysine-producing strains analyzed thus far. Fluxomics and transcriptomics were applied as further targets for next-level strain engineering to identify the back-up mechanisms that were activated upon deletion of the enzyme in the mutant strain. It seemed likely that the cells partly recruited the glyoxylate shunt as a by-pass route. Additionally, the α-ketoglutarate decarboxylase pathway emerged as the potential compensation mechanism. This novel strategy appears equally promising for Escherichia coli, which is used in the industrial production of lysine, wherein this bacterium synthesizes lysine exclusively by succinyl-CoA activation of pathway intermediates. The channeling of a high flux pathway into a production pathway by pathway coupling is an interesting metabolic engineering strategy that can be explored to optimize bio-production in the future.
Amino acids have been produced with the aid of microorganisms for nearly 40 years now. The economic importance of these cellular
building blocks is enormous. Demand for them is rising continuously and currently more than 106 tonnes/year are required. Continual efforts to increase production performance are directed towards the microorganisms themselves,
as well as towards technical improvements of the respective processes. A special position within the amino-acid-producing
microorganisms is traditionally occupied by Corynebacterium glutamicum. Molecular research in conjunction with NMR studies of flux has revealed fascinating new properties of this particular organism,
including the existence of a new type of exporter and reverse fluxes within the anaplerosis. The knowledge gained will enable
the further improvement of production strains and furthermore extend fundamental insights into metabolite flux management
within bacteria in general.
The detection and quantification of specific metabolites in single bacterial cells is a major goal for industrial biotechnology. We have developed a biosensor based on the transcriptional regulator Lrp that detects intracellular l-methionine and branched-chain amino acids in Corynebacterium glutamicum. In assays, fluorescence output showed a linear relationship with cytoplasmic concentrations of the effector amino acids. In increasing order, the affinity of Lrp for the amino acids is l-valine, l-isoleucine, l-leucine and l-methionine. The sensor was applied for online monitoring and analysis of cell-to-cell variability of l-valine production by the pyruvate dehydrogenase-deficient C. glutamicum strain ΔaceE. Finally, the sensor system was successfully used in a high-throughput (HT) FACS screen for the isolation of amino acid-producing mutants after random mutagenesis of a non-producing wild type strain. These applications illustrate how one of nature's sensor devices - transcriptional regulators - can be used for the analysis, directed evolution and HT screening for microbial strain development.
We here developed a series of Corynebacterium glutamicum strains with gradual decreased specific citrate synthase (CS) activity and quantified in a multifaceted approach the consequences of residual activity on the transcriptome, metabolome, and fluxome level as well as on L-lysine formation and growth. We achieved an intended gradual L-lysine yield increase and recognized and overcame further new limitations in the L-lysine biosynthesis pathway to result in a strain with the highest yield reported so far when assayed under comparable conditions. As a non-intended outcome, a detailed flux analysis revealed an almost constant flux through CS at 10% remaining CS activity, whereas the metabolome data revealed an increase in the oxaloacetate and acetyl-CoA concentrations. Hence reduced CS activity is apparently efficiently buffered by increased concentrations of CS substrates, implying a certain robustness of the central metabolism in response of the imposed gene expressions.
Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to uptake and phosphorylate glucose; no other route has yet been identified. Disruption of the ptsH gene in wild-type C. glutamicum resulted, as expected, in a phenotype exhibiting little growth on any of the PTS sugars: glucose, fructose, and sucrose. However, a suppressor mutant that grew on glucose but not on the other two sugars was spontaneously isolated from the PTS-negative strain WTΔptsH. The suppressor strain SPH2, unlike the wild-type strain, exhibited a phenotype of resistance to 2-deoxyglucose which is known to be a toxic substrate for the glucose-PTS of this microbe, suggesting that strain SPH2 utilizes glucose via a different system involving a permease and native glucokinases. Analysis of the C. glutamicum genome sequence using Escherichia coli galactose permease, which can transport glucose, led to the identification of two candidate genes, iolT1 and iolT2, both of which have been reported as myo-inositol transporters. When cultured on glucose medium supplemented with myo-inositol, strain WTΔptsH was able to consume glucose, suggesting that glucose uptake was mediated by one or more myo-inositol-induced transporters. Overexpression of iolT1 alone and that of iolT2 alone under the gapA promoter in strain WTΔptsH rendered the strain capable of growing on glucose, proving that each transporter played a role in glucose uptake. Disruption of iolT1 in strain SPH2 abolished growth on glucose, whereas disruption of iolT2 did not, revealing that iolT1 was responsible for glucose uptake in strain SPH2. Sequence analysis of the iol gene cluster and its surrounding region identified a single-base deletion in the putative transcriptional regulator gene Cgl0157 of strain SPH2. Introduction of the frameshift mutation allowed strain WTΔptsH to grow on glucose, and further deletion of iolT1 abolished the growth again, indicating that inactivation of Cgl0157 under a PTS-negative background can be a means by which to express the iolT1-specified glucose uptake bypass instead of the native PTS. When this strategy was applied to a defined lysine producer, the engineered strain displayed increased lysine production from glucose.
Here, we describe the development of a genetically defined strain of l-lysine hyperproducing Corynebacterium glutamicum by systems metabolic engineering of the wild type. Implementation of only 12 defined genome-based changes in genes encoding central metabolic enzymes redirected major carbon fluxes as desired towards the optimal pathway usage predicted by in silico modeling. The final engineered C. glutamicum strain was able to produce lysine with a high yield of 0.55 g per gram of glucose, a titer of 120 g L(-1) lysine and a productivity of 4.0 g L(-1) h(-1) in fed-batch culture. The specific glucose uptake rate of the wild type could be completely maintained during the engineering process, providing a highly viable producer. For these key criteria, the genetically defined strain created in this study lies at the maximum limit of classically derived producers developed over the last fifty years. This is the first report of a rationally derived lysine production strain that may be competitive with industrial applications. The design-based strategy for metabolic engineering reported here could serve as general concept for the rational development of microorganisms as efficient cellular factories for bio-production.
A sufficient supply of NADPH is a critical factor in l-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP+ to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain
on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated
from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence
of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant
was engineered into an l-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l-lysine producer was evaluated. The amounts of l-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement
of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l-lysine production was attributed to GapN. These results demonstrate effective l-lysine production by C. glutamicum with an additional source of NADPH during glycolysis.
Poly-epsilon-lysine (epsilon-PL) is a non-toxic biopolymer with antimicrobial properties. The production of epsilon-PL by Streptomyces noursei NRRL 5126 shake-flask culture was optimized by identifying the most significant medium components which affect epsilon-PL production (glycerol, proteose peptone and ammonium sulphate) by Placket-Burman design and by application of an evolutionary operation (EVOP) to determine the optimal concentrations of these components. The epsilon-PL yield increased from 41.81 g/l in basal medium to 98.07 g/l in the EVOP-optimized medium containing 3% glycerol, 1% proteose peptone and 0.8% ammonium sulphate. Further improvements in media composition and culture conditions will be required to obtain yields comparable to those obtained with current commercial strains such as Streptomyces albulus.
The numerous physiological functions of the nonessential amino acid L-aspartate, the semi-essential amino acid L-arginine, and the essential amino acid L-lysine, made them attractive for a wide range of nutritional and/or therapeutic applications. Furthermore, the administration of these amino acids as mixtures or as dipeptides for higher bioavailability is scientifically approved, and various commercial products of these forms are already available on the market. Although the industrial production of dipeptides is, with few exceptions, in an early stage, several strategies have been established and are compared in this review. Additionally, the recent developments in the technical production of aspartate-arginine and aspartate-lysine dipeptides from the biopolymer cyanophycin produced in microorganisms are discussed. Cyanophycin-derived dipeptides are produced exclusively by biotechnological procedures, probably possess higher bioavailability and may be used as better alternatives to the widely applied amino acid mixtures. Thus, the pivotal advantages and the potential applications of these dipeptides as well as of their constituting amino acids in nutrition and therapy are also discussed. Special emphasis is dedicated to arginine due to its numerous physiological roles in many cardiovascular, genitourinary, gastrointestinal, and immune disorders.
Toward the creation of a robust and efficient producer of l-arginine and l-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92up, and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (ΔargR), the production was further increased. The best mutation set, ΔargR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome
analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical l-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of
the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation
in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased
production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus allowing significantly higher productivity of arginine/citrulline
even at the suboptimal 38°C.
The amino acid L-lysine is produced on a large scale using mutants of Corynebacterium glutamicum. However, as yet recombinant DNA techniques have not succeed in improving strains selected for decades by classic mutagenesis for high productivity. We here report that seven biosynthetic enzymes were assayed and oversynthesis of the dihydrodipicolinate synthase resulted in an increase of lysine accumulation from 220 mM to 270 mM. The synthase, encoded by dapA, is located at the branch point of metabolite distribution to either lysine or threonine and competes with homoserine dehydrogenase for the common substrate aspartate semialdehyde. When graded dapA expression was used, as well as quantification of enzyme activities, intracellular metabolite concentrations and flux rates, a global response of the carbon metabolism to the synthase activity became apparent: the increased flux towards lysine was accompanied by a decreased flux towards threonine. This resulted in a decreased growth rate, but increased intracellular levels of pyruvate-derived valine and alanine. Therefore, modulating the flux at the branch point results in an intrinsically introduced growth limitation with increased intracellular precursor supply for lysine synthesis. This does not only achieve an increase in lysine yield but this example of an intracellularly introduced growth limitation is proposed as a new general means of increasing flux for industrial metabolite over-production.
Corynebacterium glutamicum possesses high in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase (PEPCk) during growth on glucose, resulting together with anaplerotic carboxylation reactions in a PEP/pyruvate/oxaloacetate substrate cycle. The present study investigated the changes in intracellular fluxes and metabolite concentrations that are caused by altered PEPCk activity in L-lysine-producing C. glutamicum MH20-22B, applying a recently developed (13)C labeling-based strategy for anaplerotic flux resolution and quantification. Abolition of PEPCk activity by deletion of the respective pck gene resulted in increased intracellular concentrations of oxaloacetate L-aspartate, alpha-ketoglutarate, pyruvate, and L-lysine and in a 60% enhanced flux toward L-lysine biosynthesis, whereas increasing the PEPCk activity by pck overexpression had opposite effects. The results of the combined measurements of enzyme activities, in vivo fluxes, and metabolite concentrations were exploited to elucidate the in vivo regulation of anaplerotic reactions in C. glutamicum, and implications for the metabolic engineering of amino-acid-producing strains are discussed.
Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance. In the post-genomic era, a novel methodology which avoids this drawback presents itself. This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background. Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum. Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wild-type and production strains identified a Val-59-->Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311-->Ile mutation in the aspartokinase gene (lysC), and a Pro-458-->Ser mutation in the pyruvate carboxylase gene (pyc). Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production. The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l). Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h. This high-speed fermentation achieved the highest productivity (3.0 g l(-1) h(-1)) so far reported for microbes producing L-lysine in fed-batch fermentation.
A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.
Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.
In the biotechnological production of L-lysine and L-glutamate by Corynebacterium glutamicum media based on glucose, fructose or sucrose are typically used. Glutamate production by C. glutamicum was very similar on glucose, fructose, glucose plus fructose and sucrose. In contrast, lysine production of genetically defined C. glutamicum strains was significantly higher on glucose than on the other carbon sources. To test whether malic enzyme or fructose-1,6-bisphosphatase might limit growth and lysine on fructose, glucose plus fructose or sucrose, strains overexpressing either malE which encodes the NADPH-dependent malic enzyme or the fructose-1,6-bisphosphatase gene fbp were generated. Overexpression of malE did not improve lysine production on any of the tested carbon sources. Upon overexpression of fbp lysine yields on glucose and/or fructose were unchanged, but the lysine yield on sucrose increased twofold. Thus, fructose-1,6-bisphosphatase was identified as a limiting factor for lysine production by C. glutamicum with sucrose as the carbon source.
Corynebacterium glutamicum, well known for the industrial production of amino acids, grows aerobically on a variety of mono- and disaccharides and on alcohols and organic acids as single or combined sources of carbon and energy. Members of the genera Corynebacterium and Brevibacterium were here tested for their ability to use the homopolysaccharide starch as a substrate for growth. None of the 24 type strains tested showed growth on or degradation of this substrate, indicating that none of the strains synthesized and secreted starch-degrading enzymes. Introducing the Streptomyces griseus amy gene on an expression vector into the lysine-producer C. glutamicum DM1730, we constructed a C. glutamicum strain synthesizing and secreting alpha-amylase into the culture broth. Although some high-molecular-weight degradation products remained in the culture broth, this recombinant strain effectively used soluble starch as carbon and energy substrate for growth and also for lysine production. Thus, employment of our construct allows avoidance of the cost-intensive enzymatic hydrolysis of the starch, which commercially is used as a substrate in industrial amino acid fermentations.
Toward the elucidation of advanced mechanisms of L-lysine production by Corynebacterium glutamicum, a highly developed industrial strain B-6 was analyzed from the viewpoint of gene expression. Northern blot analysis showed that the lysC gene encoding aspartokinase, the key enzyme of L-lysine biosynthesis, was up-regulated by several folds in strain B-6, while no repression mechanism exists in L-lysine biosynthesis of this bacterium. To analyze the underlying mechanisms of the up-regulation, we compared the transcriptome between strain B-6 and its parental wild-type, finding that not only lysC but also many other amino acid-biosynthetic genes were up-regulated in the producer. These results suggest that a certain global regulatory mechanism is involved in the industrial levels of L-lysine production.
Based on the progress in genomics, we have developed a novel approach that employs genomic information to generate an efficient amino acid producer. A comparative genomic analysis of an industrial L-lysine producer with its natural ancestor identified a variety of mutations in genes associated with L-lysine biosynthesis. Among these mutations, we identified two mutations in the relevant terminal pathways as key mutations for L-lysine production, and three mutations in central metabolism that resulted in increased titers. These five mutations when assembled in the wild-type genome led to a significant increase in both the rate of production and final L-lysine titer. Further investigations incorporated with transcriptome analysis suggested that other as yet unidentified mutations are necessary to support the L-lysine titers observed by the original production strain. Here we describe the essence of our approach for strain reconstruction, and also discuss mechanisms of L-lysine hyperproduction unraveled by combining genomics with classical strain improvement.