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A giant market and a powerful metabolism: L-lysine provided by Corynebacterium glutamicum

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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.

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... Since its discovery, C. glutamicum has started a new era in biotechnology, as a massive industrial producer of amino acids like L-glutamate, L-threonine, L-lysine, L-isoleucine, L-tryptophan, L-Phenylalanine, Ltyrosine, L-arginine, L-histidine, and so forth (Kinoshita et al., 1958;Becker and Wittmann, 2017;Wang, 2019;Zhang et al., 2018;Ikeda, 2006). Some of these amino acids can be further used as precursors for the production of other high-value-added chemicals like 1,5-diaminopentane (Mimitsuka et al., 2007;Eggeling and Bott, 2015), 1,4-diaminobutane, 5-aminolevulinic acid, glutaric acid, L-pipecolic acid, etc. (Tsuge and Matsuzawa, 2021). It is also known for the production of other industrially relevant products like organic acids, alcohols, polymers, among other things (Kind et al., 2014;Hadiati et al., 2014;Haas et al., 2019). ...
... Of these, L-lysine is one of the nine essential amino acids that cannot be synthesized in the organisms and must be supplied in the diet externally. It is the fastest-growing amino acid segment (Sanchez et al., 2018) and holds the second-largest share of the amino acid market (2 200 000 tons/year) (Joo-Young et al., 2016;Eggeling and Bott, 2015). Feedstuffs like corn, wheat or barley are deficient in L-lysine, when supplemented with the latter, converts the former into a balanced/nutritious food or feed for the consumption of animals like swine, poultry and other livestock. ...
... Year after year as the range of applications of L-lysine increases, its demand is also increasing at a similar rate (Xiao et al., 2020) by almost 20-fold in the past 20 or so years (Sanchez et al., 2018). L-lysine is produced almost exclusively with mutants of C. glutamicum (Eggeling and Bott, 2015). ...
Article
Ever since its discovery in 1957, Corynebacterium glutamicum has become a well-established industrial strain and is known for its massive capability of producing various amino acids (like L-lysine and L-glutamate) and other value-added chemicals. With the rising demand for these bio-based products, the revelation of the whole genome sequences of the wild type strains, and the astounding advancements made in the fields of metabolic engineering and systems biology, our perspective of C. glutamicum has been revolutionized and has expanded our understanding of its strain development. With these advancements, a new era for C. glutamicum supremacy in the field of industrial biotechnology began. This led to remarkable progress in the enhancement of tailor-made over-producing strains and further development of the substrate spectrum of the bacterium, to easily accessible, economical, and renewable resources. C. glutamicum has also been metabolically engineered and used in the degradation/assimilation of highly toxic and ubiquitous environmental contaminant, arsenic, present in water or soil. Here, we review the history, current knowledge, progress, achievements, and future trends relating to the versatile metabolic factory, C. glutamicum. This review paper is devoted to C. glutamicum which is one of the leading industrial microbes, and one of the most promising and versatile candidates to be developed. It can be used not only as a platform microorganism to produce different value-added chemicals and recombinant proteins, but also as a tool for bioremediation, allowing to enhance specific properties, for example in situ bioremediation.
... 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. ...
Article
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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
... Its annual world production was estimated at 2.2 million tons in 2015, with an increase in the market of 7% per year (Eggeling and Bott, 2015). ...
... L-lysine can be marketed as a crystalline preparation containing 98.5% L-lysine-HCl, an alkaline solution containing 50.7% L-lysine, or an L-lysine sulfate preparation containing 54.6% L-lysine (Eggeling and Bott, 2015). Of course, each L-lysine commercial form requires different downstream processing to achieve the degree of purity. ...
Chapter
This chapter presents the design, operation, and integral evaluation of a sugarcane molasses biorefinery, for the production of l-lysine, lactic acid, polyhydroxybutyrate, and other by-products (such as the recovery of residual biomass). A systematic approach, based on modeling and simulation tools, is presented with two main contributions: (a) the development of operation strategies for fermentation reactors, in order to increase the yield and productivity of the products and (b) the proposal of a technical-economic-environmental strategy, to evaluate the profitability and sustainability of a multi-product biorefinery. The proposed multicriteria evaluations are useful both to evaluate the performance of various process options from various points of view and to support decision-making at the initial stage of designing a biorefinery. The results show the operation regions of the fermentation reactors and the integral evaluation of a complete flowsheet to achieve a conceptual design of a biorefinery that is profitable with low environmental impact.
... 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). ...
Article
Full-text available
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. This article is protected by copyright. All rights reserved.
... Furthermore, the organism has been engineered to metabolize nonnative carbon sources such as xylose (Kawaguchi et al., 2006;Meiswinkel et al., 2013;Lange et al., 2017), N-acetylmuramic acid (Sgobba et al., 2018), mannitol (Laslo et al., 2012), starch (Seibold et al., 2006), or cellobiose (Adachi et al., 2013). Generally, C. glutamicum is well known for the large-scale industrial production of amino acids, mainly L-glutamate and L-lysine (Eggeling and Bott, 2015;Wendisch et al., 2016). During the past decade, however, the range of products has been expanded drastically and comprises nowadays not only other proteinogenic amino acids, such as L-valine (Blombach et al., 2007a;Schwentner et al., 2018), L-arginine (Park et al., 2014), L-tryptophan , or L-histidine (Schwentner et al., 2019), but also organic acids (Wieschalka et al., 2013), alcohols (Blombach et al., 2011), carotenoids (Henke and Wendisch, 2019), and proteins (Bakkes et al., 2020;Hemmerich et al., 2020). ...
Article
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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.
... Corynebacterium glutamicum, a GRAS strain accumulated considerable amounts of L-glutamate, was discovered by Japanese scientists in 1956, providing a feasible process for the fermentative production of amino acids [116] and other high-value chemicals [17,21,183]. For GFAAs, in addition to L-glutamate, C. glutamicum has been successfully engineered to produce L-arginine [129], L-proline [67,212], L-citrulline [29,46], L-ornithine [32], HYP, GABA, 5-ALA [37]. C. crenatum, a subspecies of C. glutamicum isolated from soil by Chinese scientists, is safe, robust, and possesses genetic tractability. ...
Article
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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.
... The market of amino acid, which is known to be a key sector of industrial biotechnology, has been rapidly growing recently (Vassilev et al., 2018;Xafenias et al., 2017). Specifically, such a high production rate for L-lysine, an amino acid (e.g., 2.2 million tons per year with 7% increase per year) has been reported due to the growing demand for meat because the L-lysine was extensively used as an additive in animal nutrition (Ajinomoto Co., 2013;Eggeling and Bott, 2015;Xafenias et al., 2017). Over the decades, various industrial biotechnologies, based on Grampositive soil bacteria (e.g., Corynebacterium glutamicum), have been utilized for the production of lysine due to their advantages (e.g., a safe production host) (Eggeling and Bott, 2005;Tatsumi and Inui, 2012;Vassilev et al., 2018;Wittmann and Becker, 2007). ...
Chapter
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.
... 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. ...
Article
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. ...
Article
Full-text available
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). ...
Article
Full-text available
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.
... 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)). ...
Article
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.
... 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). ...
Article
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.
... The Gram-positive bacterium C. gutamicum is used for large scale industrial production of amino acids (Eggeling and Bott, 2015) and has been engineered into a versatile platform organism for the production of fine and bulk chemicals from renewable feedstocks (Wolf et al., 2021). Determining the cellular fitness of C. glutamicum during glutamate production has limitations as glutamate secretion is triggered by conditions disturbing cell wall and/or membrane integrity (Eggeling et al., 2001;Radmacher et al., 2005). ...
Article
Corynebacterium glutamicum efficiently produces glutamate when growth is inhibited. Analyses of viability in this non-growing state requires time consuming plating and determination of colony forming units. We here establish impedance flow cytometry measurements to assess the viability of non-growing, glutamate producing C. glutamicum cultures within minutes.
... 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]. ...
Article
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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.
... 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]. ...
Article
Full-text available
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.
... 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. ...
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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. ...
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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 .
... 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). ...
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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, 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. ...
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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.
... 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]. ...
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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.
... The finding that pH adjustments with different acidic conditions had a significant influence on the growth rate, i.e., a lower growth was observed when the pH was adjusted with HCl rather than with H 3 PO 4 , may be explained by the fact that the salt concentration in the medium increased. Here, the internal osmolality was presumably increased by the accumulation of organic solutes to counteract dehydration (Eggeling and Bott, 2015). In addition, gene expression adapts to hyperosmotic conditions (Wood, 1999). ...
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Bacteria respond to pH changes in their environment and use pH homeostasis to keep the intracellular pH as constant as possible and within a small range. A change in intracellular pH influences enzyme activity, protein stability, trace element solubilities and proton motive force. Here, the species Corynebacterium glutamicum was chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 ± 0.5 in environments with external pH values ranging between 5.5 and 9. In recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell response to defined short-term pH perturbations/pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyze the physiological growth response of C. glutamicum to precise pH stress pulses at the single-cell level. Analysis by dMSCC of the growth behavior of colonies exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in viability with increasing stress duration w . Colony regrowth was possible for all tested pH values after increasing lag phases for which stress durations w were increased from 5 min to 9 h. Furthermore, single-cell analyses revealed heterogeneous regrowth of cells after pH stress, which can be categorized into three physiological states. Cells in the first physiological state continued to grow without interruption after pH stress pulse. Cells in the second physiological state rested for several hours after pH stress pulse before they started to grow again after this lag phase, and cells in the third physiological state did not divide after the pH stress pulse. This study provides the first insights into single-cell responses to acidic and alkaline pH stress by C. glutamicum .
... 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]. ...
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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.
... The strain Corynebacterium glutamicum ATCC 13032 is a Gram-positive, facultatively anaerobic soil bacterium, which produces L-glutamate under particular treatments or growth conditions (Kimura, 2005). The annual production of several tons of L-glutamate (Eggeling and Bott, 2005) as well as other metabolically engineered products, such as other amino acids (Eggeling and Bott, 2015;Wendisch et al., 2016), alcohols (Inui et al., 2004a;Niimi et al., 2011;Yamamoto et al., 2013;Jojima et al., 2015), biopolymers (Liu et al., 2007), organic acids (Hüser et al., 2005;Okino et al., 2008;Takeno et al., 2013), terpenoids (Heider et al., 2014;Kang et al., 2014) or diamines (Kind et al., 2010a,b;Schneider and Wendisch, 2010), have turned C. glutamicum into a versatile and enormously relevant biotechnological microorganism. Despite an ongoing biotechnological application of C. glutamicum and the resulting knowledge on this bacterium for more than 70 years (Vertes et al., 2013), its metabolic potential not yet exhausted. ...
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Corynebacterium glutamicum belongs to the microbes of enormous biotechnological relevance. In particular, its strain ATCC 13032 is a widely used producer of L-amino acids at an industrial scale. Its apparent robustness also turns it into a favorable platform host for a wide range of further compounds, mainly because of emerging bio-based economies. A deep understanding of the biochemical processes in C. glutamicum is essential for a sustainable enhancement of the microbe's productivity. Computational systems biology has the potential to provide a valuable basis for driving metabolic engineering and biotechnological advances, such as increased yields of healthy producer strains based on genome-scale metabolic models (GEMs). Advanced reconstruction pipelines are now available that facilitate the reconstruction of GEMs and support their manual curation. This article presents iCGB21FR, an updated and unified GEM of C. glutamicum ATCC 13032 with high quality regarding comprehensiveness and data standards, built with the latest modeling techniques and advanced reconstruction pipelines. It comprises 1042 metabolites, 1539 reactions, and 805 genes with detailed annotations and database cross-references. The model validation took place using different media and resulted in realistic growth rate predictions under aerobic and anaerobic conditions. The new GEM produces all canonical amino acids, and its phenotypic predictions are consistent with laboratory data. The in silico model proved fruitful in adding knowledge to the metabolism of C. glutamicum: iCGB21FR still produces L-glutamate with the knock-out of the enzyme pyruvate carboxylase, despite the common belief to be relevant for the amino acid's production. We conclude that integrating high standards into the reconstruction of GEMs facilitates replicating validated knowledge, closing knowledge gaps, and making it a useful basis for metabolic engineering. The model is freely available from BioModels Database under identifier MODEL2102050001.
... As a non-pathogenic Gram-positive bacterium, Corynebacterium glutamicum has been widely used in industrial biotechnology for the production of several million tons of amino acids annually, especially L-glutamate, L-lysine, and L-valine (Hasegawa et al., 2012;Eggeling and Bott, 2015;Wendisch, 2020). Besides amino acids, the current product spectrum that is accessible with C. glutamicum comprises organic acids, diamines, vitamins, aromates, and alcohols (Chen et al., 2016;Chung et al., 2017;Becker et al., 2018;Kogure and Inui, 2018;Sato et al., 2020). ...
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Corynebacterium glutamicum is one of the important industrial microorganisms for production of amino acids and other value-added compounds. Most expression vectors used in C. glutamicum are based on inducible promoter (P tac or P trc ) activated by isopropyl-β-D-thiogalactopyranoside (IPTG). However, these vectors seem unsuitable for large-scale industrial production due to the high cost and toxicity of IPTG. Myo-inositol is an ideal inducer because of its non-toxicity and lower price. In this study, a myo-inositol-inducible expression vector pMI-4, derived from the expression vector pXMJ19, was constructed. Besides the original chloramphenicol resistance gene cat , multiple cloning sites, and rrnB terminator, the pMI-4 (6,643 bp) contains the iolR q cassette and the myo-inositol-inducible promoter P iolT1 . The pMI-4 could stably replicate in the C. glutamicum host. Meanwhile, the non-myo-inositol degradation host strain C. glutamicum Δ iolG Δ oxiC Δ oxiD Δ oxiE for maintaining the pMI-4 was developed. Overexpression of hemA M and hemL using pMI-4 resulted in a significant accumulation of 5-aminolevulinic acid, indicating its potential application in metabolic engineering and industrial fermentation.
... This problem presumably occurs in any AVA producing C. glutamicum, independent of the biosynthetic route, and it would be interesting to study this effect in related biotransformations that produce AVA from L-lysine; (iii) from a process perspective, the pronounced accumulation of glutarate in later process phases could be due to two reasons. First, the cell factories exhibited reduced and finally no more growth during production, something which is typical for industrial fermentation processes, where producing strains are intentionally driven into growth limitation to support product build-up (Becker and Wittmann, 2012;Eggeling and Bott, 2015;Graf et al., 2018). The halted growth, however, made ArgD unbusy. ...
Article
5-aminovalerate (AVA) is a platform chemical of substantial commercial value to derive nylon-5 and five-carbon derivatives like δ-valerolactam, 1,5-pentanediol, glutarate, and 5-hydroxyvalerate. De-novo bio-production synthesis of AVA using metabolically engineered cell factories is regarded as exemplary route to provide this chemical in a sustainable way. So far, this route is limited by low titers, rates and yields and suffers from high levels of by-products. To overcome these limitations, we developed a novel family of AVA producing C. glutamicum cell factories. Stepwise optimization included (i) improved AVA biosynthesis by expression balancing of the heterologous davAB genes from P. putida, (ii) reduced formation of the by-product glutarate by disruption of the catabolic y-aminobutyrate pathway (iii), increased AVA export, and (iv) reduced AVA re-import via native and heterologous transporters to account for the accumulation of intracellular AVA up to 300 mM. Strain C. glutamicum AVA-5A, obtained after several optimization rounds, produced 48.3 g L⁻¹ AVA in a fed-batch process and achieved a high yield of 0.21 g g⁻¹. Surprisingly in later stages, the mutant suddenly accumulated glutarate to an extent equivalent to 30% of the amount of AVA formed, tenfold more than in the early process, displaying a severe drawback toward industrial production. Further exploration led to the discovery that ArgD, naturally aminating N-acetyl-l-ornithine during l-arginine biosynthesis, exhibits deaminating side activity on AVA toward glutarate formation. This promiscuity became relevant because of the high intracellular AVA level and the fact that ArgD became unoccupied with the gradually stronger switch-off of anabolism during production. Glutarate formation was favorably abolished in the advanced strains AVA-6A, AVA-6B, and AVA-7, all lacking argD. In a fed-batch process, C. glutamicum AVA-7 produced 46.5 g L⁻¹ AVA at a yield of 0.34 g g⁻¹ and a maximum productivity of 1.52 g L⁻¹ h⁻¹, outperforming all previously reported efforts and stetting a milestone toward industrial manufacturing of AVA. Notably, the novel cell factories are fully genome-based, offering high genetic stability and requiring no selection markers.
... Monosodium glutamate is used as food additive, as it elicits the umami taste (Hashimoto, 2017). L-Lysine is used as an additive for animal feed that is often limited in some essential amino acids (Eggeling and Bott, 2015). Industrial L-glutamate and L-lysine production are aerobic processes dependent on a functional respiratory chain and in particular on a functional CIII 2 CIV 2 supercomplex, which is the major energy-conserving complex in C. glutamicum with its absence causing a severe growth defect (Davoudi et al., 2019). ...
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Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII2 dimer flanked by a CIV on two sides. A menaquinone is bound in each of the QN and QP sites in each CIII and an additional menaquinone is positioned ∼14 Å from heme bL. A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV.
... Hence, recombinant production of nisin using a robust biotechnological workhorse may increase product yields and improve product purity by using defined media and well-established fermentation and down-stream processes. The Gram-positive bacterium Corynebacterium glutamicum is a well-established host for a wide range of compounds including high value active ingredients, therapeutic proteins and supplements for medial infusion solutions [38][39][40][41][42][43][44]. Recently, we successfully established C. glutamicum as a host to produce the bacteriocin pediocin PA-1, a class IIa bacteriocin that is not extensively modified [45]. ...
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Background The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products. Results We established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli . Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin. Conclusions We demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum . This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.
... Pip is a cyclic amino acid, a common lysine metabolite in plants and animals (Pérez-García et al., 2019). Pip is an important precursor of some drugs, such as bupivacaine, rapamycin (Eggeling and Bott, 2015), and sandramycin (He, 2006). Importantly, Pip was a compatible solute for Escherichia coli, Silicibacter pomeroyi, and Sinorhizobium meliloti (Gouesbet et al., 1994;Gouffi et al., 2000). ...
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N-hydroxy-pipecolic acid (NHP) is a hydroxylated product of pipecolic acid and an important systemic acquired resistance signal molecule. However, the biosynthesis of NHP does not have a natural metabolic pathway in microorganisms. Here, we designed and constructed a promising artificial pathway in Escherichia coli for the first time to produce NHP from biomass-derived lysine. This biosynthesis route expands the lysine catabolism pathway and employs six enzymes to sequentially convert lysine into NHP. This artificial route involves six functional enzymes coexpression: lysine α-oxidase from Scomber japonicus (RaiP), glucose dehydrogenase from Bacillus subtilis (GDH), Δ1-piperideine-2-carboxylase reductase from Pseudomonas putida (DpkA), lysine permease from Escherichia coli (LysP), flavin-dependent monooxygenase (FMO1) and catalase from Escherichia coli (KatE). Moreover, different FMO1s are used to evaluate the performance of produce NHP. A titer of 111.06 mg/L NHP was yielded in shake flasks with minimal medium containing 4 g/L lysine. By this approach, NHP has so far been produced at final titers reaching 326.42 mg/L by 48 h in a 5-L bioreactor. To the best of our knowledge, this is the first NHP process using Escherichia coli and the first process to directly synthesize NHP by microorganisms. This study lays the foundation for the development and utilization of renewable resources to produce NHP in microorganisms.
... 17−21 For biobased precursor supply, we used the Gram-positive, non-sporulating bacterium Corynebacterium glutamicum, which is widely applied in industrial biotechnology for the large-scale production of various amino acids. 22,23 C. glutamicum remains metabolically active under low-oxygen conditions, and several extensively engineered strains are known to allow the exclusive production of pyruvate or L-alanine from D-glucose in separate cultures. 24−28 In our case, however, both precursors are required to be provided in a reaction mixture for the synthesis of metaraminol ( Figure 1B). ...
... Biosensors have been widely used to develop high throughput screening methods and optimize pathway expression [40][41][42]. In our previous study, the serine-biosensor pDser, which was based on NCgl0581 (a transcription factor specifically responsive to L-serine in C. glutamicum), had been constructed in C. glutamicum for high-throughput screening of L-serine high-yield strains. ...
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l-Cysteine is an important sulfur-containing amino acid with numerous applications in the pharmaceutical and cosmetic industries. The microbial production of l-cysteine has received substantial attention, and the supply of the precursor l-serine is important in l-cysteine biosynthesis. In this study, to achieve l-cysteine overproduction, we first increased l-serine production by deleting genes involved in the pathway of l-serine degradation to glycine (serine hydroxymethyl transferase, SHMT, encoded by glyA genes) in strain 4W (with l-serine titer of 1.1 g/L), thus resulting in strain 4WG with l-serine titer of 2.01 g/L. Second, the serine-biosensor based on the transcriptional regulator NCgl0581 of C. glutamicum was constructed in E. coli, and the validity and sensitivity of the biosensor were demonstrated in E. coli. Then 4WG was further evolved through adaptive laboratory evolution (ALE) combined with serine-biosensor, thus yielding the strain 4WGX with 4.13 g/L l-serine production. Moreover, the whole genome of the evolved strain 4WGX was sequenced, and ten non-synonymous mutations were found in the genome of strain 4WGX compared with strain 4W. Finally, 4WGX was used as the starting strain, and deletion of the l-cysteine desulfhydrases (encoded by tnaA), overexpression of serine acetyltransferase (encoded by cysE) and the key enzyme of transport pathway (encoded by ydeD) were performed in strain 4WGX. The recombinant strain 4WGX-∆tnaA-cysE-ydeD can produce 313.4 mg/L of l-cysteine using glycerol as the carbon source. This work provides an efficient method for the biosynthesis of value-added commodity products associated with glycerol conversion.
... Vinasse is the most important industrial waste produced by sucroenergetic activities: after ethanol is distilled from fermented broth, all the remaining material is characterized as vinasse and the production ratio is about 10-15 L VINASSE L ETHANOL −1 . In general, the polluting potential is very high (COD between 21,000 and 34,000 mgO 2 L −1 ) and important concentrations of carbon compounds and mineral salts may be found in vinasses [6,21]. ...
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The Brazilian ethanol industry is very relevant in the global market; however, vinasse is generated in significant amounts and its management has become costly for distilleries. In this study, the aim was to evaluate vinasse as basal culture medium for P(3-hydroxybutyrate) (PHB) biosynthesis. Two bacterial strains were evaluated, a sucrose-consuming halophilic strain, Halomonas sp. HG03; and Ralstonia eutropha L359PCJ, which used glycerol from vinasse as carbon source. Firstly, shake flask bioprocesses analyzed cellular growth and PHB biosynthesis in vinasse-based media: in natura and concentrated vinasses were both evaluated in volumetric dilutions of 50% and 75% in mineral medium. Increasing vinasse concentration improved cellular growth rather than PHB accumulation for both bacteria. In vinasse-based treatments, Halomonas sp. HG03 had PHB content between 19.6 and 75.2% and R. eutropha L359PCJ, 48.4–68.5%. Further experiments in CSTR bioreactors used concentrated vinasse-based medium and R. eutropha L359PCJ had PHB content of 66.3%, concentration of residual cell dry weight (rCDW) = 9.4 g L⁻¹, PHB = 18.6 g L⁻¹, YX/S = 0.16 grCDW gGLYCEROL⁻¹, YP/S = 0.32 gPHB gGLYCEROL⁻¹, and 0.25 gPHB Lh⁻¹. Halomonas sp. HG03 had PHB content of 42.2%, rCDW = 10.2 g L⁻¹, PHB = 7.4 g L⁻¹ and YX/S = 0.22 grCDW gSUCROSE⁻¹, YP/S = 0.16 gPHB gSUCROSE⁻¹ and 0.14 gPHB Lh⁻¹. Finally, cost reductions by concentrated vinasse-based medium were evaluated. As glycerol source for PHB production by R. eutropha L359PCJ, vinasse reduced overall production costs by 22.2%. Unit production costs between US$ 2.8 and 5.4 kgPHB⁻¹ were determined for scenarios that combined vinasse-based medium with high cell density culture and improvements of productivity. The investment payback times ranged from 1.6 to 4.5 years. Graphical abstract
Article
L-lysine is one of the amino acids necessary for humans and animals and widely used in food processing, pharmaceutical preparations and feed additives. In recent years, rational design based on systems metabolic engineering and conventional optimization of fermentation parameters have contributed to the high production of L-lysine. As the demand for L-lysine in the world market is increasing year by year, intensive research has been devoted to efficient productivity and economic production costs. This review briefly explains the biosynthesis and regulation mechanism of L-lysine in Corynebacterium glutamicum, and then outlines the construction, scale-up culture, and product separation and purification strategies of L-lysine high-producing strains. In addition, emerging strategies for the breeding and fermentation of C. glutamicum for the production of L-lysine have been emphatically elucidated. In short, the commercialization of L-lysine production requires chassis strains with excellent production performance, efficient fermentation process, and the development of sustainable purification technologies.
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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.
Chapter
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.
Article
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.
Chapter
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.
Article
Natural products have greatly influenced the development of drugs to combat infectious diseases, cancer, and other disorders affecting human well-being. Only rarely, a natural product is used in an unmodified form for therapeutic purposes. More often, natural product derivatives are preferred due to improved activity or toxicity profiles. These compounds are usually produced using ‘hybrid’ processes that integrate organic synthesis and biosynthesis. Either a natural product is isolated from a biological source and then converted into the final drug by semisynthesis or a synthetically prepared precursor is introduced into the engineered biosynthesis of a living cell in a procedure called mutasynthesis. In this review, we will present recent developments in these two research areas, which take advantage of heterologous biosynthesis.
Preprint
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.
Chapter
We present a scarless recombineering-based method for introducing multiple point mutations into the genome of a temperate phage. The method uses the λ Red recombineering system to promote exogenous ssDNA oligos to anneal on the prophage lagging strand during host genome replication. DNA repair is suppressed by inducing the expression of a dominant-negative mutant protein of the methyl-directed mismatch repair system. Screening for recombinant cells without a selection marker is feasible due to its high recombination frequency, estimated as more than 40% after six cycles. The method enables scarless editing of the genome of a bacteriophage in 4-5 days.
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Feed amino acids have numerous applications, and the market demand for them is likely to grow. Microbial cell factories promise the sustainable production of feed amino acids; however, their performance is significantly affected by the availability of precursors, carbon metabolic flux, and transporter systems. To circumvent these potential roadblocks, high-performance microbial cell factories have been constructed by strengthening the supply of precursors, increasing metabolic pathway flux, and engineering transporters. In this review, limiting factors and recent technical advances affecting the production of feed amino acids in microbial cell factories are discussed. In addition, existing challenges and potential strategies for increasing the output of these amino acids are described.
Chapter
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.
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The Brazilian ethanol industry is one of the most important in the global market, however these important industrial activities have been generating significant amounts of vinasse and its management has become costly for distilleries. In this study, the aim was to evaluate concentrated and in natura vinasse as basal culture media for biotechnological processes. Different bacteria and processes were assessed: L-threonine production by E. coli THR14, with glucose as carbon source; PHB production by halophilic strain Halomonas sp. HG03, with sucrose as carbon source; and PHB biosynthesis by R. eutropha L359PCJ, which used glycerol from vinasse as carbon source. Strains were evaluated firstly in shake flasks cultivations using vinasse-based media. E. coli THR14 had no statistical difference for biomass and L-threonine concentrations among control and vinasse-based treatments (up to 50% v v ⁻¹ of in natura vinasse). Halomonas sp. HG03 and R. eutropha L359PCJ were cultivated in mineral media diluted by in natura (50% and 75% v v ⁻¹ ) and concentrated (50% and 75% v v ⁻¹ ) vinasses. Higher vinasse concentrations resulted in higher cellular growth rather than PHB accumulation for both bacteria. In vinasse-based treatments, Halomonas sp. HG03 had PHB content between 19.6 – 75.2% and R. eutropha L359PCJ, 48.4 – 68.5%. 50% (v v ⁻¹ ) of concentrated vinasse was the most attractive condition for PHB production by both bacteria. Further experiments in CSTR bioreactors used this nutritional condition and R. eutropha L359PCJ had PHB content of 66.3%, concentrations of residual cell dry weight (rCDW) = 9.4 g L ⁻¹ and PHB = 18.6 g L ⁻¹ , with Y X/S = 0.16 g g GLYCEROL ⁻¹ , Y P/S = 0.32 g g GLYCEROL ⁻¹ and 0.25 g PHB Lh ⁻¹ . Halomonas sp. HG03 had PHB content of 45.7%, rCDW = 9.8 g L ⁻¹ , PHB = 8.3 g L ⁻¹ and Y X/S = 0.18 g g SUCROSE ⁻¹ , Y P/S = 0.16 g g SUCROSE ⁻¹ and 0.12 g PHB Lh ⁻¹ . Finally, cost reductions of PHB production by R. eutropha L359PCJ with concentrated vinasse-based medium were evaluated in silico by using SuperPro Designer. As a partial source of glycerol and other nutrients for PHB production by R. eutropha L359PCJ, vinasse reduced overall production costs by 13%. Simulated processes that used concentrated vinasse-based media combined with improvements of PHB productivity and higher cellular densities had production costs between US$ 3.9 – 7.5/kg PHB and 2.6 – 7.3 years of payback time.
Chapter
This chapter describes two related recombineering-based techniques: "Duplication Insertion" (Dup-In) and "Direct- and Inverted Repeat stimulated excision" (DIRex). Dup-In is used for transferring existing mutations between strains, and DIRex for generating almost any type of mutation. Both techniques use intermediate insertions with counter-selectable cassettes, flanked by directly repeated sequences that enable exact and spontaneous excision of the cassettes. These constructs can be transferred to other strains using generalized transductions, and the final intended mutation is obtained following selection for spontaneous loss of the counter-selectable cassette, which leaves only the intended mutation behind in the final strain. The techniques have been used in several strains of Escherichia coli and Salmonella enterica, and should be readily adaptable to other organisms where λ Red recombineering or similar methods are available.
Article
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.
Chapter
Corynebacterium glutamicum, as an important microbial chassis, has great potential in industrial application. However, complicated genetic modification is severely slowed by lack of efficient genome editing tools. The Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been verified as a very powerful tool for mediating genome alteration in many microorganisms but cannot work well in C. glutamicum. We recently developed two Francisella novicida (Fn) CRISPR-Cpf1 assisted systems for genome editing via homologous recombination in C. glutamicum. Here, we describe the protocols and demonstrated that N iterative rounds of genome editing can be achieved in 3 N + 4 or 3 N + 2 days, respectively.
Article
Terpenoids represent the largest group of secondary metabolites with variable structures and functions. Terpenoids are well known for their beneficial application in human life, such as pharmaceutical products, vitamins, hormones, anticancer drugs, cosmetics, flavors and fragrances, foods, agriculture, and biofuels. Recently, engineering microbial cells have been provided with a sustainable approach to produce terpenoids with high yields. Noticeably, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has emerged as one of the most efficient genome-editing technologies to engineer microorganisms for improving terpenoid production. In this review, we summarize the application of the CRISPR-Cas system for the production of terpenoids in microbial hosts such as Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Pseudomonas putida. CRISPR-Cas9 deactivated Cas9 (dCas9)-based CRISPR (CRISPRi), and the dCas9-based activator (CRISPRa) have been used in either individual or combinatorial systems to control the metabolic flux for enhancing the production of terpenoids. Finally, the prospects of using the CRISPR-Cas system in terpenoid production are also discussed.
Article
The gene whcE of Corynebacterium glutamicum plays a positive role in oxidative stress responses and the WhcE protein interacts with SpiE. By utilizing 2D-PAGE analysis, we identified the otsB gene to be under the control of whcE . The transcription of otsB , encoding trehalose 6-phosphatase, was stimulated by oxidative stress, and whcE and spiE were involved in diamide-mediated transcriptional stimulation. The Δ otsB strain was created and found to be sensitive to the thiol-specific oxidant diamide, suggesting a role of the gene in stress responses. Genes located upstream of otsB , such as NCgl2534 and otsA , formed an operon and purified WhcE was able to bind to the promoter region of the operon (P NCgl2534 ), but the binding was only possible in the presence of the oxidant diamide. In addition, the transcriptional activation of P NCgl2534 by WhcE was demonstrated in in vivo assays and the transcription was stimulated in cells exposed to the oxidant diamide. These findings indicate that WhcE is a transcriptional activator, and otsB , which is involved in trehalose biosynthesis, has a role in oxidative stress responses in C. glutamicum .
Article
L-lysine is a crucial nutrient for both humans and animals, and its main commercial use is as a supplement in animal feed to promote chicken and other animal growth. Fluorescence biosensors based on the transcriptional regulator have been developed for high-throughput screening of L-lysine producers. However, due to its inability to specifically detect lysine, this fluorescent biosensor cannot be employed to screen high-yielding strains. Here, we present a novel technique for observing L-lysine concentrations within individual Corynebacterium glutamicum cells. The transcriptional regulator LysG and its binding site, as well as the phytoene desaturase that catalyzes the synthesis of the red pigment, make up the functional core of the biosensor. The lysine-sensitive mutant LysG(E123Y, E125A), which improved the sensitivity of biosensors, was generated by site-directed saturation mutagenesis. In addition, we increased the lysine-induced chromogenic biosensor response to 320 mM by optimizing the L-lysine export mechanism and the pathway for the synthesis of lycopene precursors. The direct identification of producers with elevated L-lysine accumulation is thus made straightforward by colorimetric screening. Lys-8, a lysine producer with a maximum lysine titer of 316.2 mM, was sorted out based on the biosensor. The enzymatic colorimetric biosensor constructed here is a simple tool with great potential for the development of high-level lysine-producing C. glutamicum.
Article
myo -, scyllo -, and d - chiro -inositol are C 6 cyclic sugar alcohols with various biological functions, which also serve as carbon sources for microbes. Inositol catabolism starts with an oxidation to keto-inositols catalyzed by inositol dehydrogenases (IDHs).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Book
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.
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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.
Chapter
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
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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.
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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.
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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.