[Show abstract][Hide abstract] ABSTRACT: Photosynthetic energy conversion using natural systems is increasingly being investigated in the recent years. Photosynthetic microorganisms such as cyanobacteria exhibit light dependent electrogenic characteristics in photo bio-electrochemical cells (PBEC) that generate substantial photocurrents, yet the current densities are lower than their photovoltaic counterparts. Recently we demonstrated that a cyanobacterium named Nostoc sp. employed in PBEC could generate up to 35 mW m(-2) even in a non-engineered PBEC. With the insights obtained from our previous research, a novel and successful attempt has been made in the current study to genetically engineer the cyanobacteria to further enhance its extracellular electron transfer. The cyanobacterium Synechococcus elongatus PCC 7942 was genetically engineered to express a non-native redox protein called outer membrane cytochrome S (OmcS). OmcS is predominantly responsible for metal reducing abilities of exoelectrogens such as Geobacter sp. The engineered S. elongatus exhibited higher extracellular electron transfer ability resulting in ∼ 9 fold higher photocurrent generation on the anode of a PBEC than the corresponding wild-type cyanobacterium. This work highlights the scope for enhancing photocurrent generation in cyanobacteria thereby benefiting faster advancement of the photosynthetic microbial fuel cell technology. This article is protected by copyright. All rights reserved.
Full-text · Article · Sep 2015 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6'-hydroxylase (F6'H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6'H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C2'H) from sweet potato. By comparing these two structures, we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.
Full-text · Article · May 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Establishment of novel metabolic pathways for biosynthesis of chemicals, fuels and pharmaceuticals has been demonstrated in Escherichia coli due to its ease of genetic manipulation and adaptability to varying oxygen levels. E. coli growing under microaerobic condition is known to exhibit features of both aerobic and anaerobic metabolism. In this work, we attempt to engineer this metabolism for production of 1,2-propanediol. We first redirect the carbon flux by disrupting carbon-competing pathways to increase the production of 1,2-propanediol microaerobically from 0.25 to 0.85 g/L. We then disrupt the first committed step of E. coli's ubiquinone biosynthesis pathway (ubiC) to prevent the oxidation of NADH in microaerobic conditions. Coupling this strategy with carbon flux redirection leads to enhanced production of 1,2-propanediol at 1.2 g/L. This work demonstrates the production of non-native reduced chemicals in E. coli by engineering its microaerobic metabolism.
No preview · Article · May 2015 · Journal of Industrial Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Metabolic engineering is a powerful tool for the sustainable production of chemicals. Over the years, the exploration of microbial, animal and plant metabolism has generated a wealth of valuable genetic information. The prudent application of this knowledge on cellular metabolism and biochemistry has enabled the construction of novel metabolic pathways that do not exist in nature or enhance existing ones. The hand in hand development of computational technology, protein science and genetic manipulation tools has formed the basis of powerful emerging technologies that make the production of green chemicals and fuels a reality. Microbial production of chemicals is more feasible compared to plant and animal systems, due to simpler genetic make-up and amenable growth rates. Here, we summarize the recent progress in the synthesis of biofuels, value added chemicals, pharmaceuticals and nutraceuticals via metabolic engineering of microbes.
Full-text · Article · May 2015 · Chemical Society Reviews
[Show abstract][Hide abstract] ABSTRACT: Tyrosine is a proteinogenic aromatic amino acid that is often used as a supplement of food and animal feed, as well as a (bio-)synthetic precursor to various pharmaceutically or industrially important molecules. Extensive metabolic engineering efforts have been made towards the efficient and cost-effective microbial production of tyrosine. Conventional strategies usually focus on eliminating intrinsic feedback inhibition and redirecting carbon flux into the shikimate pathway. In this study, we found that continuous conversion of phenylalanine into tyrosine by the action of tetrahydromonapterin (MH4)-utilizing phenylalanine 4-hydroxylase (P4H) can bypass the feedback inhibition in Escherichia coli, leading to tyrosine accumulation in the cultures. First, expression of the P4H from Xanthomonas campestris in combination with an MH4 recycling system in wild-type E. coli allowed the strain to accumulate tyrosine at 262 mg/L. On this basis, enhanced expression of the key enzymes associated with the shikimate pathway and the MH4 biosynthetic pathway resulted in the elevation of tyrosine production up to 401 mg/L in shake flasks. This work demonstrated a novel approach to tyrosine production and verified the possibility to alleviate feedback inhibition by creating a phenylalanine sink.
No preview · Article · Feb 2015 · Journal of Industrial Microbiology and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: The biological production of high value commodity 1,2-propanediol has been established by engineering the glycolysis pathway. However, the simultaneous achievement of high titer and high yield has not been reported yet, as all efforts in increasing the titer have resulted in low yields. In this work, we overcome this limitation by employing an optimal minimal set of enzymes, channeling the carbon flux into the 1,2-propanediol pathway, increasing NADH availability, and improving the anaerobic growth of the engineered Escherichia coli strain by developing a cell adaptation method. These efforts lead to 1,2-propanediol production at a titer of 5.13 g/L with a yield of 0.48 g/g glucose in 20 mL shake flask studies. On this basis, we pursue the enhancement of 1-propanol production from the 1,2-propanediol platform. By constructing a fusion diol dehydratase and developing a dual strain process, we achieve a 1-propanol titer of 2.91 g/L in 20 mL shake flask studies. To summarize, we report the production of 1,2-propanediol at enhanced titer and enhanced yield simultaneously in E. coli for the first time. Furthermore, we establish an efficient system for the production of biofuel 1-propanol biologically.
No preview · Article · Dec 2014 · ACS Synthetic Biology
[Show abstract][Hide abstract] ABSTRACT: Depression is a mental disorder affecting 350 million people over the world. According to the data released by World Health Organization (WHO), less than 50% of the patients on the globe (less than 10% in some regions) have received medical treatment. Deficiency of the neurotransmitter serotonin (5-hydroxytryptamine) in the central nervous system is thought to be a related physiological factor for bad mood. 5-Hydroxytryptophan (5-HTP) is the direct biosynthetic precursor to serotonin in human and animals. It has been proved to be clinically effective in treating depression, as well as insomnia, fibromyalgia, obesity, etc. Currently, commercial production of 5-HTP is merely achieved by the extraction from the seeds of an African plant Griffonia simplicifolia due to lack of (bio-)synthetic methods. Here we demonstrate a novel biotechnological production process via combined protein and metabolic engineering approaches. Instead of using the unstable animal tryptophan 5-hydroxylases, we reconstituted, screened, and modified bacterial phenylalanine 4-hydroxylase (P4H) activity in the microbial host Escherichia coli. Sequence and structure-based protein engineering dramatically reversed its substrate preference from phenylalanine to tryptophan, leading to high catalytic activity in converting tryptophan to 5-HTP. Most strikingly, the E. coli endogenous tetrahydromonapterin (MH4) can be utilized as an efficient coenzyme when a heterologous MH4 regeneration system is reconstituted. Whole cell bioconversion enabled the high-level production of 5-HTP from tryptophan in shake flasks. Furthermore, metabolic engineering efforts were made to achieve the total biosynthesis of 5-HTP from glucose by grafting the 5-hydroxlation reaction into the tryptophan overproducing strains. This approach does not require the addition of precursors or expensive pterin coenzymes into the medium but only utilizes abundant renewable carbon sources. This microbial platform holds great potential for scale-up production of 5-HTP.
[Show abstract][Hide abstract] ABSTRACT: Combinatorial biosynthesis has enabled the development of novel or artificial biosynthetic pathways for the generation of pharmaceuticals, chemicals and biofuels. The establishment of these pathways usually requires novel or engineered enzymes with desired functions. The coenzyme B12-dependent diol dehydratase from Klebsiella oxytoca was previously demonstrated to dehydrate its natural substrate 1,2-propanediol for 1-propanol production. In this work, we engineer the enzyme to catalyze a longer chain C4 triol (1,2,4-butanetriol) for the production of 1,4-butanediol via structure-based redesign. To achieve this, a systematic study of the active site is performed using its natural substrate 1,2-propanediol. Analysis of the enzyme in substrate-free and substrate-bound forms leads to the identification of key amino acids involved in substrate binding and orientation. A rational design strategy is then developed to increase the enzyme selectivity and activity towards 1,2,4-butanetriol. Following in silico screening, the mutants with the highest potential to interact with 1,2,4-butanetriol are selected for enzyme kinetics study. This approach results in mutants with increased activity towards 1,2,4-butanetriol as compared to the wild type enzyme. Whole cell conversion studies result in more efficient dehydration of 1,2,4-butanetriol into 4-hydroxybutyraldehyde and its subsequent native reduction into 1,4-butanediol in Escherichia coli.
[Show abstract][Hide abstract] ABSTRACT: Due to the concerns on oil crisis and environment pollution, increasing attention has been paid to developing sustainable alternatives for the production of fuels and chemicals. Metabolic engineering has been proven to be a promising way to manufacture these molecules, which is generally considered renewable and environmental friendly. Muconic acid (MA) and salicylic acid (SA) are important organic acids having wide commercial applications. MA is a potential platform chemical for the production of nylon and plastics, while SA is mainly used for producing pharmaceuticals, such as aspirin, lamivudine and skincare products. At present, MA and SA are mainly produced by chemical synthesis from petro-derived aromatic chemicals, such as benzene, which is toxic and nonrenewable. Here, we report the design and optimization of a novel pathway for microbial production of MA and its precursor SA. First, a well-developed phenylalanine producing Escherichia coli strain was engineered into a SA overproducer by introducing isochorismate synthase and isochorismate pyruvate lyase. High-titer SA production was achieved using this recombinant stain from simple carbon sources. SA was further converted into MA by introducing another two enzymes salicylate 1-monoxygenase and catechol 1,2-dioxygenase. Finally, a de novo MA biosynthetic pathway was assembled. Modular optimization enabled the production of 1.5 g/L MA within 48 h in shake flask experiments, a result showing scale-up potential. This study not only established an efficient microbial platform for the production of MA and SA, but also provided a useful pathway design strategy for the biosynthesis of other important catabolic metabolites.
[Show abstract][Hide abstract] ABSTRACT: A novel biosynthetic pathway was designed and verified reversely leading to the production of 5-hydroxytryptophan (5-HTP) from glucose. This pathway takes advantage of the relaxed substrate selectivities of relevant enzymes without employing the unstable tryptophan 5-hydroxylase. First, high-titer of 5-HTP was produced from 5-hydroxyanthranilate (5-HI) by the catalysis of E.coli TrpDCBA. Then, a novel salicylate 5-hydroxylase was used to convert the non-natural substrate anthranilate to 5-HI. After that, the production of 5-HI from glucose was achieved and optimized with modular optimization. In the end, we combined the full pathway and adopted a two-stage strategy to realize the de novo production of 5-HTP. This work demonstrated the application of enzyme promiscuity in non-natural pathway design.
No preview · Article · Oct 2014 · ACS Synthetic Biology
[Show abstract][Hide abstract] ABSTRACT: Non-oxidative decarboxylases belong to a unique enzyme family that does not require any cofactors. Here we report the characterization of a 2,3-dihydroxybenzoic acid (2,3-DHBA) decarboxylase (BDC) from Klebsiella pneumoniae and explore its application on the production of muconic acid. The enzyme properties were systematically studied, including the optimal temperature and pH, kinetic parameters, and substrate specificity. On this basis, we designed an artificial pathway for muconic acid production by connecting 2,3-DHBA biosynthesis with its degradation pathway. Over-expression of entCBA and the key enzymes in the shikimate pathway led to the production of 900 mg L−1 of 2,3-DHBA. Further, expression of the BDC coupled with catechol 1,2-dioxygenase achieved the conversion of 2,3-DHBA into muconic acid. Finally, assembly of the total pathway resulted in the de novo production of muconic acid up to 480 mg L−1.
[Show abstract][Hide abstract] ABSTRACT: 5-Hydroxytryptophan (5-HTP) is a clinically effective drug against depression, insomnia, obesity, chronic headaches, etc. It is only commercially produced by the extraction from the seeds of Griffonia simplicifolia due to lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase (P4H) activity in Escherichia coli. Then, sequence and structure-based protein engineering dramatically shifted its substrate preference, allowing for efficient conversion of tryptophan into 5-HTP. Importantly, E. coli endogenous tetrahydromonapterin (MH4) was able to be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion enabled the high-level production of 5-HTP (1.1-1.2 g l-1) from tryptophan in shake flasks. On its basis, metabolic engineering efforts were further made to achieve the de novo 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy to expand native metabolism of microorganisms.
No preview · Article · Jun 2014 · ACS Synthetic Biology
[Show abstract][Hide abstract] ABSTRACT: cis,cis-Muconic acid (MA) and salicylic acid (SA) are naturally-occurring organic acids having great commercial value. MA is a potential platform chemical for the manufacture of several widely-used consumer plastics; while SA is mainly used for producing pharmaceuticals (for example, aspirin and lamivudine) and skincare and haircare products. At present, MA and SA are commercially produced by organic chemical synthesis using petro-derived aromatic chemicals, such as benzene, as starting materials, which is not environmentally friendly. Here, we report a novel approach for efficient microbial production of MA via extending shikimate pathway by introducing the hybrid of an SA biosynthetic pathway with its partial degradation pathway. First, we engineered a well-developed phenylalanine producing Escherichia coli strain into an SA overproducer by introducing isochorismate synthase and isochorismate pyruvate lyase. The engineered stain is able to produce 1.2 g/L of SA from simple carbon sources, which is the highest titer reported so far. Further, the partial SA degradation pathway involving salicylate 1-monoxygenase and catechol 1,2-dioxygenase is established to achieve the conversion of SA to MA. Finally, a de novo MA biosynthetic pathway is assembled by integrating the established SA biosynthesis and degradation modules. Modular optimization enables the production of 1.5 g/L MA within 48 h in shake flasks. This study not only establishes an efficient microbial platform for the production of SA and MA, but also demonstrates a generalizable pathway design strategy for the de novo biosynthesis of valuable degradation metabolites.
No preview · Article · May 2014 · Metabolic Engineering
[Show abstract][Hide abstract] ABSTRACT: Antioxidants are biological molecules with the ability to protect vital metabolites from harmful oxidation. Due to this fascinating role, their beneficial effects on human health are of paramount importance. Traditional approaches using solvent-based extraction from food/non-food sources and chemical synthesis are often expensive, exhaustive, and detrimental to the environment. With the advent of metabolic engineering tools, the successful reconstitution of heterologous pathways in Escherichia coli and other microorganisms provides a more exciting and amenable alternative to meet the increasing demand of natural antioxidants. In this review, we elucidate the recent progress in metabolic engineering efforts for the microbial production of antioxidant food ingredients — polyphenols, carotenoids, and antioxidant vitamins.
No preview · Article · Apr 2014 · Current Opinion in Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Adipic acid is an important platform chemical that can be used for plastic and nylon production. The global demand for adipic acid is over 2 billion kilograms annually. Currently, the major approach to adipic acid production relies on chemical synthesis using benzene as the starting material. However, this approach is generally considered nonrenewable and environmentally incompatible due to the toxicity of the starting material and intermediates and the release of greenhouse gas N2O. Microbial synthesis from renewable carbon sources provides a feasible and promising alternative in the circumstance of environment deterioration and petroleum depletion. Indeed, adipic acid can be easily generated by hydrogenation of muconic acid, a naturally existing intermediate in aromatic compounds degradation by some soil bacteria. Draths and Frost first reported the constitution of an artificial pathway in Escherichia coli by shunting 3-dehydroshikimate from shikimate pathway, affording the production of muconic acid from glucose. Here we designed a novel muconic acid biosynthetic pathway by connecting anthranilate catabolism with anabolism. Specifically, anthranilate, an E. coli endogenous intermediate in the tryptophan biosynthetic branch was sequentially converted into catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO). First of all, screening for efficient ADO and CDO from different microorganisms led to the convertion of anthranilate to gram per liter-level of muconic acid. Further, to achieve the de novo muconic acid biosynthesis, anthranilate overproducing strains were constructed by blocking tryptophan biosynthesis and over-expressing the key enzymes in shikimate pathway. Interestingly, introduction of a strengthened glutamine regeneration system by over-expressing glutamine synthase dramatically enhanced anthranilate production. Finally, the engineered strain carrying the full pathway produced 390 mg/L of muconic acid from simple carbon sources in shake flask. This approach will demonstrate scale-up potential for microbial production of muconic acid with further condition and pathway optimizations.
[Show abstract][Hide abstract] ABSTRACT: Coumarins are a group of benzopyrone-type natural products that can be classified into four categories: simple coumarins, pyrone-substituted coumarins, furanocoumarins, and pyranocoumarins. Simple coumarins consist of the simplest coumarin skeleton (1,2-benzopyrone) and its hydroxylated, alkylated and glycosylated derivatives. Coumarins and their derivatives have demonstrated a vast array of therapeutical effects, such as antibacterial, anti-inflammatory and anti-coagulant activities. For instance, the well-known synthetic 4-hydroxycoumarin derivatives warfarin, phenprocoumon and acenocoumaroyl are among the most widely prescribed oral anticoagulants over the world. Moreover, extensive research into their pharmacological properties has revealed their therapeutic roles in the treatment of cancer and AIDS. Despite the pharmaceutical importance of coumarins, relatively limited information is available regarding their biosynthesis in nature. Couamrins are usually considered to be derived from the plant phenylpronanoid pathway; while the formation of 4-hydroxycoumarin (the synthetic precursor of warfarin) was prove to be the result of the infection of melilotoside-containing plant materials by molds. Here we present the development of novel biosynthetic mechanisms to produce several pharmaceutically important simple coumarins in Escherchia coli, including umbelliferone, scopoletin and 4-hydroxycoumarin. In this work, artificial biosynthetic pathways were designed, validated and optimized via combinatorial biosynthesis and metabolic engineering approaches. This work not only demonstrates promising application potentials, but also advances understanding of the chemistry in coumarin biosynthesis and lays the foundation for extending the pathways to produce more complicated coumarin derivatives for their biomedical and clinical property study.