Yajun Yan

University of Georgia, Атина, Georgia, United States

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Publications (11)50.64 Total impact

  • 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; · 3.65 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. · 7.86 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 01/2014; · 6.86 Impact Factor
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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. · 10.02 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; · 3.65 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; · 3.69 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
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    Rachit Jain, Yajun Yan
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    ABSTRACT: ABSTRACT: After publication of this work [Jain R, Yan Y, Microbial Cell Factories 2011, 10:97] we have noticed accidental errors that were introduced during our revision process. In our revision process, to address review comments we reduced the number of significant digits for the results of our enzyme assay experiments. As we reduced the number of significant digits to two in tables 1 and 2, we overlooked the corresponding values mentioned in the text of "Results and Discussion" section discussing the "Methylglyoxal Synthase Assay" and "Secondary Alcohol Dehydrogenase Assay" [Jain R, Yan Y, Microbial Cell Factories 2011, 10:97]. In order to maintain consistency between the text and the tables, we would like to correct tables 1 and 2 by increasing the number of significant digits up to four, which were used for our original submission. These changes will in no manner affect the outcome/interpretation of the experiments as described in the original publication and will not affect the merit of this work. In addition, we would like to modify the "Competing Interests" as below. The authors apologize for any inconvenience caused thereof. Competing Interests The University of Georgia has filed a United States provisional patent on this technology.
    Microbial Cell Factories 03/2012; 11(1):38. · 3.31 Impact Factor
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    Rachit Jain, Yajun Yan
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    ABSTRACT: With the increasing consumption of fossil fuels, the question of meeting the global energy demand is of great importance in the near future. As an effective solution, production of higher alcohols from renewable sources by microorganisms has been proposed to address both energy crisis and environmental concerns. Higher alcohols contain more than two carbon atoms and have better physiochemical properties than ethanol as fuel substitutes. We designed a novel 1-propanol metabolic pathway by expanding the well-known 1,2-propanediol pathway with two more enzymatic steps catalyzed by a 1,2-propanediol dehydratase and an alcohol dehydrogenase. In order to engineer the pathway into E. coli, we evaluated the activities of eight different methylglyoxal synthases which play crucial roles in shunting carbon flux from glycolysis towards 1-propanol biosynthesis, as well as two secondary alcohol dehydrogenases of different origins that reduce both methylglyoxal and hydroxyacetone. It is evident from our results that the most active enzymes are the methylglyoxal synthase from Bacillus subtilis and the secondary alcohol dehydrogenase from Klebsiella pneumoniae, encoded by mgsA and budC respectively. With the expression of these two genes and the E. coli ydjG encoding methylglyoxal reductase, we achieved the production of 1,2-propanediol at 0.8 g/L in shake flask experiments. We then characterized the catalytic efficiency of three different diol dehydratases on 1,2-propanediol and identified the optimal one as the 1,2-propanediol dehydratase from Klebsiella oxytoca, encoded by the operon ppdABC. Co-expressing this enzyme with the above 1,2-propanediol pathway in wild type E. coli resulted in the production of 1-propanol at a titer of 0.25 g/L. We have successfully established a new pathway for 1-propanol production by shunting the carbon flux from glycolysis. To our knowledge, it is the first time that this pathway has been utilized to produce 1-propanol in E. coli. The work presented here forms a basis for further improvement in production. We speculate that dragging more carbon flux towards methylglyoxal by manipulating glycolytic pathway and eliminating competing pathways such as lactate generation can further enhance the production of 1-propanol.
    Microbial Cell Factories 11/2011; 10:97. · 3.31 Impact Factor