Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.     

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.     

Modulation of the central carbon metabolism of Corynebacterium glutamicum improves malonyl-CoA availability and increases plant polyphenol synthesis

In recent years microorganisms have been engineered towards synthesizing interesting plant polyphenols such as flavonoids and stilbenes from glucose. Currently, the low endogenous supply of malonyl-CoA, indispensable for plant polyphenol synthesis, impedes high product titers. Usually, limited malonyl-CoA availability during plant polyphenol production is avoided by supplementing fatty acid synthesis-inhibiting antibiotics such as cerulenin, which are known to increase the intracellular malonyl-CoA pool as a side effect. Motivated by the goal of microbial polyphenol synthesis being independent of such expensive additives, we used rational metabolic engineering approaches to modulate regulation of fatty acid synthesis and flux into the tricarboxylic acid cycle (TCA cycle) in Corynebacterium glutamicum strains capable of flavonoid and stilbene synthesis. Initial experiments showed that sole overexpression of genes coding for the native malonyl-CoA-forming acetyl-CoA carboxylase is not sufficient for increasing polyphenol production in C. glutamicum. Hence, the intracellular acetyl-CoA availability was also increased by reducing the flux into the TCA cycle through reduction of citrate synthase activity. In defined cultivation medium, the constructed C. glutamicum strains accumulated 24 mg·L ⁻¹ (0.088 mM) naringenin or 112 mg·L ⁻¹ (0.49 mM) resveratrol from glucose without supplementation of phenylpropanoid precursor molecules or any inhibitors of fatty acid synthesis.     

Engineering a bacterial platform for total biosynthesis of caffeic acid derived phenethyl esters and amides

Caffeic acid has been widely recognized as a versatile pharmacophore for synthesis of new chemical entities, among which caffeic acid derived phenethyl esters and amides are the most extensively-investigated bioactive compounds with potential therapeutical applications. However, the natural biosynthetic routes for caffeic acid derived phenethyl esters or amides remain enigmatic, limiting their bio-based production. Herein, product-directed design of biosynthetic schemes allowed the development of thermodynamically favorable pathways for these compounds via acyltransferase (ATF) mediated trans-esterification. Production based screening identified a microbial O-ATF from Saccharomyces cerevisiae and a plant N-ATF from Capsicum annuum capable of forming caffeic acid derived esters and amides, respectively. Subsequent combinatorial incorporation of caffeic acid with various aromatic alcohol or amine biosynthetic pathways permitted the de novo bacterial production of a panel of caffeic acid derived phenethyl esters or amides in Escherichia coli for the first time. Particularly, host strain engineering via systematic knocking out endogenous caffeoyl-CoA degrading thioesterase and pathway optimization via titrating co-substrates enabled production enhancement of five caffeic acid derived phenethyl esters and amides, with titers ranging from 9.2 to 369.1 mg/L. This platform expanded the capabilities of bacterial production of high-value natural aromatic esters and amides from renewable carbon source via tailoring non-natural biosynthetic pathways.     

Lack of involvement of polyphenol oxidase in ortho-hydroxylation of phenolic compounds in mung bean seedlings

Complete elimination of polyphenol oxidase activity in hypocotyls and leaves of developing mung bean ( Vigna radiata L. Wilczek cv. Berkin) seedlings by tentoxin had no effect on the content of the ortho-hydroxylated flavonoids delphinidin and rutin. Tentoxin completely eliminated polyphenol oxidase-mediated ortho-hydroxylation of p -coumaric acid to caffeic acid. Despite this, tentoxin had no effect on caffeic acid derivative contents in the seedlings. High performance liquid chromatography profiles indicated that elimination of polyphenol oxidase had no effect on either the quality or the quantity of soluble phenolic compounds, These data strongly indicate that polyphenol oxidase is not involved in metabolism of phenolic compounds in developing plant tissues.     

Engineering E. coli for caffeic acid biosynthesis from renewable sugars

Caffeic acid is a valuable aromatic compound that possesses many important pharmacological activities. In structure, caffeic acid belongs to the hydroxycinnamic acid family and can be biosynthesized from the aromatic amino acid tyrosine. In the present paper, the caffeic acid biosynthesis pathway was reconstituted in engineered Escherichia coli to produce caffeic acid from simple biomass sugar glucose and xylose. Different engineering approaches were utilized to optimize the production. Specifically, two parallel biosynthesis routes leading from tyrosine to caffeic acid were studied. The copy number of the intermediate biosynthesis genes was varied to find appropriate gene doses for caffeic acid biosynthesis. Three different media, including a MOPS medium, a synthetic medium, and a rich medium, were also examined to improve the production. The highest specific caffeic acid production achieved was 38 mg/L/OD. Lastly, cultivation of engineered E. coli in a bioreactor resulted in a production of 106 mg/L caffeic acid after 4 days.     

Biosynthesis of plant-specific phenylpropanoids by construction of an artificial biosynthetic pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biological synthesis of plant secondary metabolites has attracted increasing attention due to their proven or assumed beneficial properties and health-promoting effects. Phenylpropanoids are the precursors to a range of important plant metabolites such as the secondary metabolites belonging to the flavonoid/stilbenoid class of compounds. In this study, engineered Escherichia coli containing artificial phenylpropanoid biosynthetic pathways utilizing tyrosine as the initial precursor were established for production of plant-specific metabolites such as ferulic acid, naringenin, and resveratrol. The construction of the artificial pathway utilized tyrosine ammonia lyase and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis, cinnamate/4-coumarate:coenzyme A ligase from Streptomyces coelicolor, caffeic acid O-methyltransferase and chalcone synthase from Arabidopsis thaliana, and stilbene synthase from Arachis hypogaea.     

Metabolic Engineering for Resveratrol Derivative Biosynthesis in Escherichia coli

We previously reported that the SbROMT3syn recombinant protein catalyzes the production of the methylated resveratrol derivatives pinostilbene and pterostilbene by methylating substrate resveratrol in recombinant E. coli. To further study the production of stilbene compounds in E. coli by the expression of enzymes involved in stilbene biosynthesis, we isolated three stilbene synthase (STS) genes from rhubarb, peanut, and grape as well as two resveratrol O-methyltransferase (ROMT) genes from grape and sorghum. The ability of RpSTS to produce resveratrol in recombinant E. coli was compared with other AhSTS and VrSTS genes. Out of three STS, only AhSTS was able to produce resveratrol from p-coumaric acid. Thus, to improve the solubility of RpSTS, VrROMT, and SbROMT3 in E. coli, we synthesized the RpSTS, VrROMT and SbROMT3 genes following codon-optimization and expressed one or both genes together with the cinnamate/4-coumarate:coenzyme A ligase (CCL) gene from Streptomyces coelicolor. Our HPLC and LC-MS analyses showed that recombinant E. coli expressing both ScCCL and RpSTSsyn led to the production of resveratrol when p-coumaric acid was used as the precursor. In addition, incorporation of SbROMT3syn in recombinant E. coli cells produced resveratrol and its mono-methylated derivative, pinostilbene, as the major products from p-coumaric acid. However, very small amounts of pterostilbene were only detectable in the recombinant E. coli cells expressing the ScCCL, RpSTSsyn and SbROMT3syn genes. These results suggest that RpSTSsyn exhibits an enhanced enzyme activity to produce resveratrol and SbROMT3syn catalyzes the methylation of resveratrol to produce pinostilbene in E. coli cells.     

Co-production of caffeic acid and p-hydroxybenzoic acid from p-coumaric acid by Streptomyces caeruleus MTCC 6638

In a culture medium of Streptomyces caeruleus MTCC 6638 grown with p-coumaric acid (5 mM) as the sole source of carbon, co-production of caffeic acid and p-hydroxybenzoic acid was observed. Both caffeic acid and p-hydroxybenzoic acid are important phenolic compounds with pharmaceutical importance. These biotransformed products were identified by high-performance liquid chromatography and electrospray ionization mass spectrometry. Obtained data suggest that p-coumaric acid was possibly utilized by two different routes, resulting in the formation of a hydroxycinnamate and a hydroxybenzoate compound. However, higher concentration of p-coumaric acid (10 mM) favoured caffeic acid formation. Addition of 5 mM p-coumaric acid into S. caeruleus cultures pre-grown on minimal medium with 1.0 g/l glucose resulted in the production of 65 mg/l caffeic acid. Furthermore, S. caeruleus cells were able to produce the maximum amount of caffeic acid when pre-grown on nutrient broth for 16 h. Under this condition, the addition of 5 mM p-coumaric acid was sufficient for the S. caeruleus culture to produce 150 mg/l caffeic acid, with a molar yield of 16.6% after 96 h of incubation.     

Effects of elevated CO2 on the vasculature and phenolic secondary metabolism of Plantago maritima

We have examined the effect of elevated CO₂ on the vasculature and phenolic secondary metabolism on clones of the maritime plant Plantago maritima (L.). Plants were exposed to either ambient (360 μmol CO₂ mol⁻¹) or elevated (600 μmol CO₂ mol⁻¹) atmospheric CO₂ within a Solardome facility and harvested after 12 months' growth. Histochemical analysis of the leaves identified increases in the diameter of the minor leaf vein and associated lignified vessels in plants exposed to elevated CO₂. In the roots the number of lignified root vessels and stele width were also increased, but overall the lignified vessel-wall thickness was reduced in plants exposed to elevated CO₂, compared to those grown under ambient CO₂. To investigate whether or not these subtle changes in lignification were associated with perturbations in phenolic metabolism, aromatic natural products were analysed by HPLC-MS after treatment with cellulase to hydrolyse the respective glycosidic conjugates. The phenylpropanoids p-coumaric acid, caffeic acid, ferulic acid and the flavone luteolin were identified, together with the caffeoyl phenylethanoid glycosides, verbascoside and plantamajoside which were resistant to enzymatic digestion. Exposure to enhanced CO₂ resulted in subtle changes in the levels of individual metabolites. In the foliage a one-year exposure to enhanced CO₂ resulted in an increased accumulation of caffeic acid, whilst in the roots p-coumaric acid and verbascoside were enhanced. Our results suggest that significant changes in the vasculature of P. maritima on exposure to increased CO₂ are associated with only minor changes in the leaves of specific lignin-related metabolites.     

Enhanced Lignin Monomer Production Caused by Cinnamic Acid and Its Hydroxylated Derivatives Inhibits Soybean Root Growth

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.     

Induction of phenolic acids and metals in Arachis hypogaea L. plants due to feeding of three lepidopteran pests

The feeding of lepidopteran pests, Amsacta albistriga W., Aproaerema modicella D. and Spilosoma obliqua W., caused feeding stress in the Arachis hypogaea L. plants. As a common defensive response, the plant exhibited variations in the primary and secondary metabolic contents. Groundnut plant antioxidative enzymes such as peroxidase, superoxide dismutase, polyphenol oxidase, catalase and phenyl ammonia lyase exhibited alterations to confer resistance against the pest. Phenolic compounds, namely coumaric acid, chlorogenic acid, resveratrol, epicatechin, ferulic acid and caffeic acid, showed active defensive role in groundnut plant against the pest feeding through their significant presence in Fourier transform infrared spectroscopy, high-performance liquid chromatography and gas chromatography studies. Changes in metal contents like iron, magnesium, zinc, potassium and calcium were also reported through atomic absorption studies, indicating their defensive role against the biotic stress caused on groundnut by the three lepidopteran pests. The present study can further assist in recognizing the importance of these specific phenolic acids and metals of groundnut in its pest management.     

毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸*

目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。     

Further studies on the biosynthesis of mangiferin in Anemarrhena asphodeloides: hydroxylation of the shikimate-derived ring

The hydroxylation at C-3′ of maclurin, an intermediate in mangiferin biosynthesis, has been studied. Labelled cinnamic acid, p-coumaric acid, caffeic acid, iriflophenone and maclurin were fed to Anemarrhena asphodeloides. Cinnamic acid and p-coumaric acid were better precursors than caffeic acid for mangiferin, and iriflophenone as well as maclurin was effectively incorporated into mangiferin and isomangiferin. These results show that maclurin is biosynthesized via hydroxylation of iriflophenone derived from p-coumarate in this plant.     

Interaction of Moisture Stress and Three Phenolic Compounds on Lettuce Seed Germination

No interactions between water stress and three phenolic acids(p-coumaric, caffeic and ferulic acids) on lettuce (Lactucasativa L. var. Grand Rapids) seed germination were found. Probitanalysis indicated that mechanisms of action of water stressand the phenolic inhibitors were similar. The relative effectivenessof the compounds was p-coumaric > ferulic > caffeic. Nointeraction was found between p-coumaric and ferulic acid, whereasantagonism was found between caffeic acid and each of the othertwo phenolic acids. Lactuca sativa L., lettuce, germination, phenolic compounds, moisture stress, allelopathy, seed     

Fungal biotransformation of p-coumaric acid into caffeic acid by Pycnoporus cinnabarinus: an alternative for producing a strong natural antioxidant

Fungal biotransformation of p-coumaric acid into caffeic acid, potentially a strong antioxidant, was evidenced in Pycnoporus cinnabarinus cultures grown with high feeding of p-coumaric acid. Preliminary experiments showed no toxicity of both p-coumaric and caffeic acids at concentrations ranging from 0 to 500 mg l^–1. Feeding 450 mg p-coumaric acid l^–1 into P. cinnabarinus cultures grown on 20 g l^–1 glucose medium resulted in the production of 257 mg caffeic acid l^–1with a molar yield of 21%.     

Production of Plant-Specific Flavanones by Escherichia coli Containing an Artificial Gene Cluster

In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically. Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS). For the purpose of production of flavanones in Escherichia coli, three sets of an artificial gene cluster which contained three genes of heterologous origins—PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor A3(2), and CHS from the licorice plant Glycyrrhiza echinata—were constructed. The constructions of the three sets were done as follows: (i) PAL, 4CL, and CHS were placed in that order under the control of the T7 promoter (P_T7) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of 4CL and CHS were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single P_T7 in front of PAL, and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both P_T7 and the RBS. These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid. E. coli cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid. Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones. This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin.     

Inhibition by phenolic compounds of cytokinin-stimulated betacyanin synthesis inAmaranthus caudatus

A number of phenolic compounds have been tested for ability to inhibit the cytokinin-induced synthesis of betacyanin inAmaranthus caudatus cotyledons. Under the conditions employed, the compounds responded thus: (1) no inhibition with up to 1 mg ml^-1 (quercetin 3-rhamnosylglucoside and chlorogenic acid), (2) partial or no inhibition up to 0.1 mg ml^-1 with greater inhibition at 1 mg ml^-1 (phloridzin, arbutin, caffeic acid, p-hydroxybenzoic acid and 3,4-dihydroxyphenylalanine) and (3) partial or no inhibition up to 0.1 mg ml^-1 with complete, inhibition at 1 mg ml^-1 (o-coumaric,m-coumaric,p-coumaric, protocatechuic and ferulic acids and phenylalanine). The results show that if theAmaranthus betacyanin bioassay for cytokinins is to give reliable results, then certain contaminating phenolics must be removed beforehand; some difficulties involved in this are briefly discussed.     

Substrates and enzyme activities related to biotransformation of resveratrol from phenylalanine by Alternaria sp. MG1

To identify the substrates and enzymes related to resveratrol biosynthesis in Alternaria sp. MG1, different substrates were used to produce resveratrol, and their influence on resveratrol production was analyzed using high performance liquid chromatography (HPLC). Formation of resveratrol and related intermediates was identified using mass spectrum. During the biotransformation, activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), were analyzed and tracked. The reaction system contained 100 mL 0.2 mol/L phosphate buffer (pH 6.5), 120 g/L Alternaria sp. MG1 cells, 0.1 g/L MgSO₄, and 0.2 g/L CaSO₄ and different substrates according to the experimental design. The biotransformation was carried out for 21 h at 28 °C and 120 rpm. Resveratrol formation was identified when phenylalanine, tyrosine, cinnamic acid, and p-coumaric acid were separately used as the only substrate. Accumulation of cinnamic acid, p-coumaric acid, and resveratrol and the activities of PAL, C4H, and 4CL were identified and changed in different trends during transformation with phenylalanine as the only substrate. The addition of carbohydrates and the increase of phenylalanine concentration promoted resveratrol production and yielded the highest value (4.57 μg/L) when 2 g/L glucose, 1 g/L cyclodextrin, and phenylalanine (4.7 mmol/L) were used simultaneously.     

Degradation of phenylalanine and tyrosine by Sporobolomyces roseus

Ammonia-lyase activity for l-phenylalanine, m-hydroxyphenylalanine and l-tyrosine was demonstrated in cell-free extracts of Sporobolomyces roseus. Cultures of this organism converted dl-[ring-¹⁴C]phenylalanine and l-[U-¹⁴C]tyrosine into the corresponding cinnamic acid. Tracer studies showed that these compounds were further metabolized to [¹⁴C]protocatechuic acid. Benzoic acid and p-hydroxybenzoic acid were intermediates in this pathway. Washed cells of the organism readily utilized cinnamic acid, p-coumaric acid, caffeic acid, benzoic acid and p-hydroxybenzoic acid. Protocatechuic acid was the terminal aromatic compound formed during the metabolism of these compounds. The cells of S. roseus were able to convert m-coumaric acid into m-hydroxybenzoic acid, but the latter compound, which accumulated in the medium, was not further metabolized. 4-Hydroxycoumarin was identified as the product of o-coumaric acid metabolism by this organism.