Metabolic engineering of Escherichia coli for high-level production of benzyl acetate from glucose.

BACKGROUND: Benzyl acetate is an aromatic ester with a jasmine scent. It was discovered in plants and has broad applications in food, cosmetic, and pharmaceutical industries. Its current production predominantly relies on chemical synthesis. In this study, Escherichia coli was engineered to produce benzyl acetate. RESULTS: Two biosynthetic routes based on the CoA-dependent ß-oxidation pathway were constructed in E. coli for benzyl acetate production. In route I, benzoic acid pathway was extended to produce benzyl alcohol by combining carboxylic acid reductase and endogenous dehydrogenases and/or aldo-keto reductases in E. coli. Benzyl alcohol was then condensed with acetyl-CoA by the alcohol acetyltransferase ATF1 from yeast to form benzyl acetate. In route II, a plant CoA-dependent ß-oxidation pathway via benzoyl-CoA was assessed for benzyl alcohol and benzyl acetate production in E. coli. The overexpression of the phosphotransacetylase from Clostridium kluyveri (CkPta) further improved benzyl acetate production in E. coli. Two-phase extractive fermentation in situ was adopted and optimized for benzyl acetate production in a shake flask. The most optimal strain produced 3.0 ± 0.2 g/L benzyl acetate in 48 h by shake-flask fermentation. CONCLUSIONS: We were able to establish the whole pathway for benzyl acetate based on the CoA-dependent ß-oxidation in single strain for the first time. The highest titer for benzyl acetate produced from glucose by E. coli is reported. Moreover, cinnamyl acetate production as an unwanted by-product was very low. Results provided novel information regarding the engineering benzyl acetate production in microorganisms.

Identification of a highly promiscuous glucosyltransferase from Penstemon barbatus for natural product glycodiversification.

Glycosylation reactions mediated by UDP-glycosyltransferases (UGTs) are common post-modifications involved in plant secondary metabolism and significantly improve the solubility and bioactivity of aglycones. Penstemon barbatus is rich in phenylethanoid glycosides (PhGs), such as echinacoside and verbascoside. In this study, a promiscuous glycosyltransferase UGT84A95 was identified from P. barbatus. In vitro enzyme assays showed that UGT84A95 catalyzed the glucosylation of the phenol hydroxyl group of PhGs efficiently as well as other structurally diverse phenolic glycosides, including flavonoids, terpenoids, stilbene glycosides, coumarins, and simple polyphenols. By using UGT84A95, 12 glycosylated products were prepared and structurally identified by NMR spectroscopy, among which 7 are new compounds. These findings suggest that UGT84A95 could be a potential biocatalyst to synthesize multi-glycosylated glycosides.

Biosynthesis of a rosavin natural product in Escherichia coli by glycosyltransferase rational design and artificial pathway construction.

Phytochemicals are rich resources for pharmaceutical and nutraceutical agents. A key challenge of accessing these precious compounds can present significant bottlenecks for development. The cinnamyl alcohol disaccharides also known as rosavins are the major bioactive ingredients of the notable medicinal plant Rhodiola rosea L. Cinnamyl-(6'-O-ß-xylopyranosyl)-O-ß-glucopyranoside (rosavin E) is a natural rosavin analogue with the arabinopyranose unit being replaced by its diastereomer xylose, which was only isolated in minute quantity from R. rosea. Herein, we described the de novo production of rosavin E in Escherichia coli. The 1,6-glucosyltransferase CaUGT3 was engineered into a xylosyltransferase converting cinnamyl alcohol monoglucoside (rosin) into rosavin E by replacing the residue T145 with valine. The enzyme activity was further elevated 2.9 times by adding the mutation N375Q. The synthesis of rosavin E from glucose was achieved with a titer of 92.9 mg/L by combining the variant CaUGT3T145V/N375Q, the UDP-xylose synthase from Sinorhizobium meliloti 1021 (SmUXS) and enzymes for rosin biosynthesis into a phenylalanine overproducing E. coli strain. The production of rosavin E was further elevated by co-overexpressing UDP-xylose synthase from Arabidopsis thaliana (AtUXS3) and SmUXS, and the titer in a 5 L bioreactor with fed-batch fermentation reached 782.0 mg/L. This work represents an excellent example of producing a natural product with a disaccharide chain by glycosyltransferase engineering and artificial pathway construction.

Two O-Methyltransferases Mediate Multiple Methylation Steps in the Biosynthesis of Coumarins in Cnidium monnieri.

Coumarins with methoxy groups such as osthole (1), xanthotoxin (2), bergapten (3), and isopimpinellin (4) are typical bioactive ingredients of many medicinal plants. The methylation steps remain widely unknown. Herein, we report the discovery of two methyltransferases in the biosynthesis of O-methyl coumarins in Cnidium monnieri by transcriptome mining, heterologous expression, and in vitro enzymatic assays. The results reveal that (i) CmOMT1 catalyzes the methylation of osthenol (8) as the final step in the biosynthesis of 1, (ii) CmOMT2 shows the highest efficiency and preference for methylating xanthotoxol (11) to form 2, and (iii) CmOMT1 and CmOMT2 also efficiently transform bergaptol (10) and 8-hydroxybergapten (13) into 3 or 4, suggesting the CmOMTs mediate multistep methylations in the biosynthesis of linear furanocoumarins in C. monnieri.

Metabolic engineering of Saccharomyces cerevisiae for high-level production of gastrodin from glucose.

BACKGROUND: The natural phenolic glycoside gastrodin is the major bioactive ingredient in the well-known Chinese herb Tianma and is widely used as a neuroprotective medicine in the clinic. Microbial production from sustainable resources is a promising method to replace plant extraction and chemical synthesis which were currently used in industrial gastrodin production. Saccharomyces cerevisiae is considered as an attractive host to produce natural plant products used in the food and pharmaceutical fields. In this work, we intended to explore the potential of S. cerevisiae as the host for high-level production of gastrodin from glucose. RESULTS: Here, we first identified the plant-derived glucosyltransferase AsUGT to convert 4-hydroxybenzyl alcohol to gastrodin with high catalytic efficiency in yeast. Then, we engineered de novo production of gastrodin by overexpressing codon-optimized AsUGTsyn, the carboxylic acid reductase gene CARsyn from Nocardia species, the phosphopantetheinyl transferase gene PPTcg-1syn from Corynebacterium glutamicum, the chorismate pyruvate-lyase gene UbiCsyn from Escherichia coli, and the mutant ARO4K229L. Finally, we achieved an improved product titer by a chromosomal multiple-copy integration strategy and enhancement of metabolic flux toward the aglycon 4-hydroxybenzyl alcohol. The best optimized strain produced 2.1 g/L gastrodin in mineral medium with glucose as the sole carbon source by flask fermentation, which was 175 times higher than that of the original gastrodin-producing strain. CONCLUSIONS: The de novo high-level production of gastrodin was first achieved. Instead of chemical synthesis or plants extraction, our work provides an alternative strategy for the industrial production of gastrodin by microbial fermentation from a sustainable resource.

Production of caffeoylmalic acid from glucose in engineered Escherichia coli.

OBJECTIVES: To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli. RESULTS: We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli-E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L. CONCLUSIONS: Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

De novo biosynthesis of Gastrodin in Escherichia coli.

Gastrodin, a phenolic glycoside, is the key ingredient of Gastrodia elata, a notable herbal plant that has been used to treat various conditions in oriental countries for centuries. Gastrodin is extensively used clinically for its sedative, hypnotic, anticonvulsive and neuroprotective properties in China. Gastrodin is usually produced by plant extraction or chemical synthesis, which has many disadvantages. Herein, we report unprecedented microbial synthesis of gastrodin via an artificial pathway. A Nocardia carboxylic acid reductase, endogenous alcohol dehydrogenases and a Rhodiola glycosyltransferase UGT73B6 transformed 4-hydroxybenzoic acid, an intermediate of ubiquinone biosynthesis, into gastrodin in Escherichia coli. Pathway genes were overexpressed to enhance metabolic flux toward precursor 4-hydroxybenzyl alcohol. Furthermore, the catalytic properties of the UGT73B6 toward phenolic alcohols were improved through directed evolution. The finally engineered strain produced 545mgl(-1) gastrodin in 48h. This work creates a new route to produce gastrodin, instead of plant extractions and chemical synthesis.

Biosynthesis of phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone in Escherichia coli from glucose.

BACKGROUND: Type III polyketide synthases (PKSs) contribute to the synthesis of many economically important natural products, which are typically produced by direct extraction from plants or synthesized chemically. For example, humulone and lupulone (Fig. 1a) in hops (Humulus lupulus) account for the characteristic bitter taste of beer and display multiple pharmacological effects. 4-Hydroxy-6-methyl-2-pyrone is a precursor of parasorboside contributing to insect and disease resistance of plant Gerbera hybrida, and was recently demonstrated to be a potential platform chemical. Fig. 1 Examples of phloroglucinols (a) and 2-pyrones (b) synthesized by type III PKS. PIBP phlorisobutyrophenone; PIVP phlorisovalerophenone; TAL 4-hydroxy-6-methyl-2-pyrone (triacetic acid lactone); HIPP 4-hydroxy-6-isopropyl-2-pyrone; HIBP 4-hydroxy-6-isobutyl-2-pyrone RESULTS: In this study, we achieved simultaneous biosynthesis of phlorisovalerophenone, a key intermediate of humulone biosynthesis and 4-hydroxy-6-isobutyl-2-pyrone in Escherichia coli from glucose. First, we constructed a biosynthetic pathway of isovaleryl-CoA via hydroxy-3-methylglutaryl CoA followed by dehydration, decarboxylation and reduction in E. coli. Subsequently, the type III PKSs valerophenone synthase or chalcone synthase from plants were introduced into the above E. coli strain, to produce phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone at the highest titers of 6.4 or 66.5 mg/L, respectively. CONCLUSIONS: The report of biosynthesis of phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone in E. coli adds a new example to the list of valuable compounds synthesized in E. coli from renewable carbon resources by type III PKSs.

Synthesis of rosmarinic acid analogues in Escherichia coli.

OBJECTIVES: To produce rosmarinic acid analogues in the recombinant Escherichia coli BLRA1, harboring a 4-coumarate: CoA ligase from Arabidopsis thaliana (At4CL) and a rosmarinic acid synthase from Coleus blumei (CbRAS). RESULTS: Incubation of the recombinant E. coli strain BLRA1 with exogenously supplied phenyllactic acid (PL) and analogues as acceptor substrates, and coumaric acid and analogues as donor substrates led to production of 18 compounds, including 13 unnatural RA analogues. CONCLUSION: This work demonstrates the viability of synthesizing a broad range of rosmarinic acid analogues in E. coli, and sheds new light on the substrate specificity of CbRAS.

Engineered synthesis of rosmarinic acid in Escherichia coli resulting production of a new intermediate, caffeoyl-phenyllactate.

OBJECTIVES: To achieve high production of rosmarinic acid and derivatives in Escherichia coli which are important phenolic acids found in plants, and display diverse biological activities. RESULTS: The synthesis of rosmarinic acid was achieved by feeding caffeic acid and constructing an artificial pathway for 3,4-dihydroxyphenyllactic acid. Genes encoding the following enzymes: rosmarinic acid synthase from Coleus blumei, 4-coumarate: CoA ligase from Arabidopsis thaliana, 4-hydroxyphenyllactate 3-hydroxylase from E. coli and D-lactate dehydrogenase from Lactobacillus pentosus, were overexpressed in an L-tyrosine over-producing E. coli strain. The yield of rosmarinic acid reached ~130 mg l(-1) in the recombinant strain. In addition, a new intermediate, caffeoyl-phenyllactate (~55 mg l(-1)), was also produced by the engineered E. coli strain. CONCLUSION: This work not only leads to high yield production of rosmarinic acid and analogues, but also sheds new light on the construction of the pathway of rosmarinic acid in E. coli.

Heterologous biosynthesis of costunolide in Escherichia coli and yield improvement.

Costunolide, the main bioactive compound of the medicinal plant, Radix Aucklandiae, is a sesquiterpene lactone (SL) and has a broad range of biological activities. It is also a precursor of many biologically-active SLs and is a branching point in the biosynthesis of SLs. Here we have reconstituted the costunolide biosynthetic pathway in Escherichia coli by co-expression of three genes (GAS, GAO, LsCOS) involved in costunolide biosynthesis and eight genes involved in converting acetyl-CoA into farnesyl diphosphate from mevalonate pathway. Costunolide production was then detected. By screening and optimization of cultured medium and inducing temperature, costunolide yield was up to 100 mg l(-1) in E. coli.

Phenolic polyketides from the co-cultivation of marine-derived Penicillium sp. WC-29-5 and Streptomyces fradiae 007.

Penicillium sp. WC-29-5 was co-cultured with Streptomyces fradiae 007 to produce five natural products (1-3, 4a and 4b) that were isolated and characterized by spectroscopic analysis. Interestingly, these compounds were found to be different from those produced in discrete fungal and bacterial controls. Among these compounds, the absolute configurations of compounds 4a and 4b were determined for the first time by X-ray single crystal diffraction experiments and electronic circular dichroism (ECD) calculations. An evaluation of the cytotoxic activities of these compounds revealed that 4b was moderately cytotoxic towards HL-60 and H1975 tumor cells with IC50 values of 3.73 and 5.73 µM, respectively, whereas compound 4a was only moderately cytotoxic towards H1975 cells with an IC50 value of 3.97 µM.

Engineered short branched-chain acyl-CoA synthesis in E. coli and acylation of chloramphenicol to branched-chain derivatives.

Short branched-chain acyl-CoAs are important building blocks for a wide variety of pharmaceutically valuable natural products. Escherichia coli has been used as a heterologous host for the production of a variety of natural compounds for many years. In the current study, we engineered synthesis of isobutyryl-CoA and isovaleryl-CoA from glucose in E. coli by integration of the branched-chain α-keto acid dehydrogenase complex from Streptomyces avermitilis. In the presence of the chloramphenicol acetyltransferase (cat) gene, chloramphenicol was converted to both chloramphenicol-3-isobutyrate and chloramphenicol-3-isovalerate by the recombinant E. coli strains, which suggested successful synthesis of isobutyryl-CoA and isovaleryl-CoA. Furthermore, we improved the α-keto acid precursor supply by overexpressing the alsS gene from Bacillus subtilis and the ilvC and ilvD genes from E. coli and thus enhanced the synthesis of short branched-chain acyl-CoAs. By feeding 25 mg/L chloramphenicol, 2.96 ± 0.06 mg/L chloramphenicol-3-isobutyrate and 3.94 ± 0.06 mg/L chloramphenicol-3-isovalerate were generated by the engineered E. coli strain, which indicated efficient biosynthesis of short branched-chain acyl-CoAs. HPLC analysis showed that the most efficient E. coli strain produced 80.77 ± 3.83 nmol/g wet weight isovaleryl-CoA. To our knowledge, this is the first report of production of short branched-chain acyl-CoAs in E. coli and opens a way to biosynthesize various valuable natural compounds based on these special building blocks from renewable carbon sources.

Metabolic Engineering of Escherichia coli for High-Level Production of Salicin.

Salicin is a notable phenolic glycoside derived from plants including Salix and Populus genus and has multiple biological activities such as anti-inflammatory and antiarthritic, anticancer, and antiaging effects. In this work, we engineered production of salicin from cheap renewable carbon resources in Escherichia coli (E. coli) by extending the shikimate pathway. We first investigated enzymes synthesizing salicylate from chorismate. Subsequently, carboxylic acid reductases (CARs) from different resources were screened to achieve efficient reduction of salicylate. Third, glucosyltransferases from different sources were selected for constructing cell factories of salicin. The enzymes including salicylate synthase AmS from Amycolatopsis methanolica, carboxylic acid reductase CARse from Segniliparus rotundus, and glucosyltransferase UGT71L1 from Populous trichocarpa were overexpressed in a modified E. coli strain MG1655-U7. The engineered strain produced 912.3 ± 12.7 mg/L salicin in 72 h of fermentation. These results demonstrated the production of salicin in a microorganism and laid significant foundation for its commercialization for pharmaceutical and nutraceutical applications.

In Vitro and In Silico Analyses of New Cinnamid and Rosmarinic Acid-Derived Compounds Biosynthesized in Escherichia coli as Leishmania amazonensis Arginase Inhibitors.

Arginase is a metalloenzyme that plays a central role in Leishmania infections. Previously, rosmarinic and caffeic acids were described as antileishmanial agents and as Leishmania amazonensis arginase inhibitors. Here, we describe the inhibition of arginase in L. amazonensis by rosmarinic acid analogs (1-7) and new caffeic acid-derived amides (8-10). Caffeic acid esters and amides were produced by means of an engineered synthesis in E. coli and tested against L. amazonensis arginase. New amides (8-10) were biosynthesized in E. coli cultured with 2 mM of different combinations of feeding substrates. The most potent arginase inhibitors showed Ki(s) ranging from 2 to 5.7 µM. Compounds 2-4 and 7 inhibited L. amazonensis arginase (L-ARG) through a noncompetitive mechanism whilst compound 9 showed a competitive inhibition. By applying an in silico protocol, we determined the binding mode of compound 9. The competitive inhibitor of L-ARG targeted the key residues within the binding site of the enzyme, establishing a metal coordination bond with the metal ions and a series of hydrophobic and polar contacts supporting its micromolar inhibition of L-ARG. These results highlight that dihydroxycinnamic-derived compounds can be used as the basis for developing new drugs using a powerful tool based on the biosynthesis of arginase inhibitors.

[Advances in microbial synthesis of plant natural products].

Plant natural products are one of the main sources of small molecule drugs, nutraceuticals, cosmetics and fragrances, and play an important role in economy development. At present, the way of obtaining plant natural products mainly depends on direct extraction from plants, which is farm land occupying and time consuming. The contents of active ingredients in plants are usually low, and thus the production cost is high. By elucidating the biosynthetic pathways and reconstructing the pathways in microbial cells, plant natural products can be produced by fermentation using renewable raw materials. Microbial biosynthesis provides a new route for the supply of plant natural products. This review summarizes the research progress of microbial synthesis of terpenoids, flavonoids, phenylpropanoids and other important natural products of plants in Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Current research challenges and future prospects are also briefly discussed.

CYP82AR Subfamily Proteins Catalyze C-1' Hydroxylations of Deoxyshikonin in the Biosynthesis of Shikonin and Alkannin.

Shikonin and S-enantiomer alkannin are important naphthoquinone derivatives present in many Boraginaceae species. We report that cytochrome P450 monooxygenases (CYPs) from a new CYP82AR subfamily catalyzed hydroxylations of deoxyshikonin at C-1' position of isoprenoid side chain. Two homologues were discovered from each species of the four Boraginaceae plants. One CYP preferred converting deoxyshikonin into shikonin, and the other stereoselectively hydroxylated deoxyshikonin into alkannin. The discovery might be a general feature of shikonin/alkannin-producing Boraginaceae plants.

Discovery of Glycosyltransferases Involved in the Biosynthesis of Ligupurpuroside B.

In this study, we report the characterization of three glycosyltransferases involved in the biosynthesis of ligupurpuroside B, a complex acylated phenolic glycoside in Ligustrum robustum. UGT85AF8 catalyzed the formation of salidroside from tyrosol. UGT79G7, an osmanthuside A 1,3-rhamnosyltransferase, and UGT79A19, an osmanthuside B 1,4-rhamnosyltransferase, sequentially converted osmanthuside A into ligupurpuroside B. Orthologs of UGT79G7 were also discovered from other plants producing verbascoside. These rhamnosyltransferases expand the toolbox for the biosynthesis of natural products with various sugar chains.

[Advances in the microbial synthesis of aromatic fragrance molecules].

Aromatic compounds make up a large part of fragrances and are traditionally produced by chemical synthesis and direct extraction from plants. Chemical synthesis depends on petroleum resources and has disadvantages such as causing environment pollutions and harsh reaction conditions. Due to the low content of aromatic compounds in plants and the low yield of direct extraction, plant extractions require large amounts of plant resources that occupy arable land. In recent years, with the development of metabolic engineering and synthetic biology, microbial synthesis of aromatic compounds from renewable resources has become a promising alternative approach to traditional methods. This review describes the research progress on the synthesis of aromatic fragrances by model microorganisms such as Escherichia coli or yeast, including the synthesis of vanillin through shikimic acid pathway and the synthesis of raspberry ketone through polyketide pathway. Moreover, this review highlights the elucidation of native biosynthesis pathways, the construction of synthetic pathways and metabolic regulation for the production of aromatic fragrances by microbial fermentation.