Yajun Yan

University of Georgia, Атина, Georgia, United States

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Publications (22)75.82 Total impact

  • [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.
    ACS Synthetic Biology 12/2014; · 3.95 Impact Factor
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    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.
    14 AIChE Annual Meeting; 11/2014
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    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.
    14 AIChE Annual Meeting; 11/2014
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    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.
    14 AIChE Annual Meeting; 11/2014
  • Yaping Yang, Yuheng Lin, Yajun Yan
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    ABSTRACT: Malonyl-CoA is not only a critical precursor for fatty acid biosynthesis, but also serves as an essential building block of natural products such as coumarins, flavonoids, and other polyketides. Its biosynthesis is tightly regulated and its intracellular concentration is usually controlled at low levels, which poses a great challenge to biosynthesize malonyl-CoA derived molecules via metabolic engineering. Previous efforts to increase malonyl-CoA availability were mainly focused on the overexpression of acetyl-CoA carboxylase, the enzyme responsible for the conversion of acetyl-CoA into malonyl-CoA. However, inactivation of its consumption pathway has not been achieved since the disruption of fatty acid biosynthesis would be lethal to the host cells. To overcome this limitation, we report an antisense RNA strategy to down-regulate fatty acid biosynthesis and reduce undesired malonyl-CoA consumption to support heterologous biosynthesis. To increase the stability of the antisense RNAs, we employed an artificial loop-stem structure, which was further optimized in terms of its length and binding position to maximize the interference efficiency. We observed the interference effects at transcriptional, translational, and metabolic levels. Furthermore, we used three heterologous biosynthetic pathways of polyketide compounds to test the applicability of this RNA-mediated down-regulating strategy. The results showed that this strategy greatly improved the biosynthesis of malonyl-CoA derived natural products. This work demonstrates that antisense RNA is an efficient synthetic biology tool to increase microbial production of economically and pharmaceutically valued compounds.
    14 AIChE Annual Meeting; 11/2014
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    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.
    ACS Synthetic Biology 10/2014; · 3.95 Impact Factor
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    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.
    ChemSusChem 07/2014; · 7.48 Impact Factor
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    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.
    ACS Synthetic Biology 06/2014; · 3.95 Impact Factor
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    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.
    Metabolic Engineering 05/2014; · 6.86 Impact Factor
  • Yuheng Lin, Yajun Yan
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    ABSTRACT: Hydroxylated phenylpropanoid compounds (e.g., esculetin, piceatannol, and eriodictyol) have been proved to possess important biological activities and pharmacological properties. These compounds exist at low abundance in nature, which hampers their cost-effective isolation, and broad application. Meanwhile, regiospecific hydroxylation of complex aromatic compounds is still quite challenging for chemical synthesis. In past decades, biocatalytic hydroxylation of plant phenylpropanoids was achieved due to the identification and engineering of some cytochrome P450 hydroxylases; however, the conversion efficiency was still too low for scale-up production use. In this work, we identify a non-P450 monooxygenase (HpaBC) from Escherichia coli, which is able to catalyze the efficient ortho-hydroxylation towards plant phenylpropanoids umbelliferone and resveratrol; meanwhile it also exhibits activity towards naringenin. On this basis, whole-cell biocatalysis enables the production of esculetin and piceatannol at high titers (2.7 and 1.2 g/L, respectively, in shake flasks) and high yields (close to 100%). To our knowledge, this work reports the highest titers and yields for biotechnological production of esculetin and piceatannol, representing a promising hydroxylation platform. Biotechnol. Bioeng. 2014;9999: 1-5. © 2014 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 04/2014; · 4.16 Impact Factor
  • Yuheng Lin, Rachit Jain, Yajun Yan
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    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.
    Current Opinion in Biotechnology 01/2014; 26:71–78. · 8.04 Impact Factor
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    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.
    13 AIChE Annual Meeting; 11/2013
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    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.
    13 AIChE Annual Meeting; 11/2013
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    ABSTRACT: 4-Hydroxycoumarin (4HC) type anticoagulants (for example, warfarin) are known to have a significant role in the treatment of thromboembolic diseases-a leading cause of patient morbidity and mortality worldwide. 4HC serves as an immediate precursor of these synthetic anticoagulants. Although 4HC was initially identified as a naturally occurring product, its biosynthesis has not been fully elucidated. Here we present the design, validation, in vitro diagnosis and optimization of an artificial biosynthetic mechanism leading to the microbial biosynthesis of 4HC. Remarkably, function-based enzyme bioprospecting leads to the identification of a characteristic FabH-like quinolone synthase from Pseudomonas aeruginosa with high efficiency on the 4HC-forming reaction, which promotes the high-level de novo biosynthesis of 4HC in Escherichia coli (~500 mg l(-1) in shake flasks) and further in situ semisynthesis of warfarin. This work has the potential to be scaled-up for microbial production of 4HC and opens up the possibility of biosynthesizing diverse coumarin molecules with pharmaceutical importance.
    Nature Communications 10/2013; 4:2603. · 10.74 Impact Factor
  • Qin Huang, Yuheng Lin, Yajun Yan
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    ABSTRACT: Caffeic acid is a plant-specific phenylpropanoic acid with multiple health-improving effects reported, and its therapeutic derivatives have also been studied throughout the last decade. To meet its market need and achieve high-level production, microbial production of caffeic acid approaches have been developed in metabolically engineered Escherichia coli. In our previous work, we have established the first artificial pathway that realized de novo production of caffeic acid using E. coli endogenous 4-hydroxyphenylacetate 3-hydroxylase (4HP3H). In this work, we exploited the catalytic potential of 4HPA3H in the whole-cell bioconversion study and produced 3.82 g/L (461.12 mg/L/OD) caffeic acid from p-coumaric acid, a direct precursor. We further engineered a phenylalanine over-producer into a tyrosine over-producer and then introduced the artificial pathway. After adjusting the expression strategy and optimizing the inoculants timing, de novo production of caffeic acid reached 766.68 mg/L. Both results from the direct precursor and simple carbon sources represent the highest titers of caffeic acid from microbial production so far. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 06/2013; · 4.16 Impact Factor
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    ABSTRACT: Coumarins are plant secondary metabolites that have demonstrated a variety of important therapeutic properties, such as antibacterial, anti-inflammatory, and anti-coagulant effects, as well as anti-cancer and anti-AIDS activities. However, knowledge regarding their biosynthesis is relatively limited even for the simplest coumarin molecule, which serves as the gateway molecule to many pharmaceutically-important coumarin derivatives. Here we reported the design and validation of artificial pathways leading to the biosynthesis of plant-specific simple coumarins in bacteria. First, Escherichia coli strains were engineered to convert inexpensive phenylpropanoid acid precursors, 4-coumarate and ferulate to simple coumarins, umbelliferone (4.3mg/L) and scopoletin (27.8mg/L), respectively. Further, we assembled the complete artificial pathways in E. coli and achieved de novo biosynthesis of umbelliferone and scopoletin without addition of precursors. This study lays the foundation for microbial production of more diverse coumarin compounds.
    Metabolic Engineering 04/2013; · 6.86 Impact Factor
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    ABSTRACT: Muconic acid is the synthetic precursor of adipic acid and the latter is an important platform chemical that can be used for the production of nylon-6, 6 and polyurethane. Currently, the production of adipic acid mainly relies on chemical processes utilizing petrochemicals such as benzene as starting materials, which are generally considered environmentally unfriendly and nonrenewable. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway in Escherichia coli for the biosynthesis of muconic acid, in which anthranilate, the first intermediate in tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-level muconic acid from supplemented anthranilate in 5 hours. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by over-expressing the key enzymes in shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by over-expressing glutamine synthase significantly improved anthranilate production. Finally, the engineered E. coli strain carrying the full pathway produced 389.96 ± 12.46 mg/L muconic acid from simple carbon sources in shake flask experiments, which demonstrates scale-up potential for microbial production of muconic acid.
    Applied and Environmental Microbiology 04/2013; · 3.95 Impact Factor
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    ABSTRACT: The genome sequencing of Streptomyces coelicolor A3(2) has lead to the identification of numerous cryptic gene clusters involved in the biosynthesis of secondary metabolites; throwing open the challenge of identifying the enzymatic functions that the gene clusters are associated with. In this work, we report the biochemical characterization of one such cryptic gene, SCO7467 from S. coelicolor A3(2), which is annotated as a prenyltransferase. Based on LC–MS and 2D-NMR studies, we show that SCO7467 acts as a 5-dimethylallyl tryptophan synthase (5-DMATS), and catalyzes the transfer of a dimethylallyl group to the C-5 position of the indole ring of l-tryptophan. The studies indicate that SCO7467 could be involved in the synthesis of C-5 prenylated indole alkaloids, which may exhibit unique pharmacological and biological properties.
    Process Biochemistry. 09/2012; 47(9):1419–1422.
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    ABSTRACT: This work describes the production of (R,R)-2,3-butanediol in Escherichia coli using glycerol by metabolic engineering approaches. The introduction of a synthetic pathway converting pyruvate to (R,R)-2,3-butanediol into wild-type E. coli strain BW25113 led to the production of (R,R)-2,3-butanediol at a titer of 3.54 g/l and a yield of 0.131 g product/g glycerol (26.7 % of theoretical maximum) with acetate (around 3.00 g/l) as the dominant by-product. We therefore evaluated the impacts of deleting the genes ackA or/and poxB that are responsible for the major by-product, acetate. This increased production of (R,R)-2,3-butanediol to 9.54 g/l with a yield of 0.333 g product/g glycerol (68.0 % of theoretical maximum) in shake flask studies. The utilization of low-priced crude glycerol to produce value-added chemicals is of great significance to the economic viability of the biodiesel industry.
    Journal of Industrial Microbiology 07/2012; 39(11):1725-9. · 1.80 Impact Factor
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    Yuheng Lin, Yajun Yan
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    ABSTRACT: Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE) have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H), is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs) from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc) possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L) in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from caffeic acid. In addition, we have identified that TAL is the rate-limiting enzyme in this pathway. Thus, exploration for more active TALs via bio-prospecting and protein engineering approaches is necessary for further improvement of caffeic acid production.
    Microbial Cell Factories 04/2012; 11:42. · 3.31 Impact Factor

Publication Stats

55 Citations
75.82 Total Impact Points

Institutions

  • 2011–2014
    • University of Georgia
      • College of Engineering
      Атина, Georgia, United States
  • 2012–2013
    • Beijing University of Chemical Technology
      Peping, Beijing, China