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.     

Heterologous production of flavanones in<Emphasis Type="Italic"> Escherichia coli</Emphasis>: potential for combinatorial biosynthesis of flavonoids in bacteria

Chalcones, the central precursor of flavonoids, are synthesized exclusively in plants from tyrosine and phenylalanine via the sequential reaction of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL) and chalcone synthase (CHS). Chalcones are converted into the corresponding flavanones by the action of chalcone isomerase (CHI), or non-enzymatically under alkaline conditions. PAL from the yeast Rhodotorula rubra, 4CL from an actinomycete Streptomyces coelicolor A3(2), and CHS from a licorice plant Glycyrrhiza echinata, assembled as artificial gene clusters in different organizations, were used for fermentation production of flavanones in Escherichia coli. Because the bacterial 4CL enzyme attaches CoA to both cinnamic acid and 4-coumaric acid, the designed biosynthetic pathway bypassed the C4H step. E. coli carrying one of the designed gene clusters produced about 450 μg naringenin/l from tyrosine and 750 μg pinocembrin/l from phenylalanine. The successful production of plant-specific flavanones in bacteria demonstrates the usefulness of combinatorial biosynthesis approaches not only for the production of various compounds of plant and animal origin but also for the construction of libraries of "unnatural" natural compounds. Dedicated to Professor Sir David Hopwood.     

Investigation of two distinct flavone synthases for plant-specific flavone biosynthesis in Saccharomyces cerevisiae

Flavones are plant secondary metabolites that have wide pharmaceutical and nutraceutical applications. We previously constructed a recombinant flavanone pathway by expressing in Saccharomyces cerevisiae a four-step recombinant pathway that consists of cinnamate-4 hydroxylase, 4-coumaroyl:coenzyme A ligase, chalcone synthase, and chalcone isomerase. In the present work, the biosynthesis of flavones by two distinct flavone synthases was evaluated by introducing a soluble flavone synthase I (FSI) and a membrane-bound flavone synthase II (FSII) into the flavanone-producing recombinant yeast strain. The resulting recombinant strains were able to convert various phenylpropanoid acid precursors into the flavone molecules chrysin, apigenin, and luteolin, and the intermediate flavanones pinocembrin, naringenin, and eriodictyol accumulated in the medium. Improvement of flavone biosynthesis was achieved by overexpressing the yeast P450 reductase CPR1 in the FSII-expressing recombinant strain and by using acetate rather than glucose or raffinose as the carbon source. Overall, the FSI-expressing recombinant strain produced 50% more apigenin and six times less naringenin than the FSII-expressing recombinant strain when p-coumaric acid was used as a precursor phenylpropanoid acid. Further experiments indicated that unlike luteolin, the 5,7,4'-trihydroxyflavone apigenin inhibits flavanone biosynthesis in vivo in a nonlinear, dose-dependent manner.     

Investigation of Two Distinct Flavone Synthases for Plant-Specific Flavone Biosynthesis in Saccharomyces cerevisiae

Flavones are plant secondary metabolites that have wide pharmaceutical and nutraceutical applications. We previously constructed a recombinant flavanone pathway by expressing in Saccharomyces cerevisiae a four-step recombinant pathway that consists of cinnamate-4 hydroxylase, 4-coumaroyl:coenzyme A ligase, chalcone synthase, and chalcone isomerase. In the present work, the biosynthesis of flavones by two distinct flavone synthases was evaluated by introducing a soluble flavone synthase I (FSI) and a membrane-bound flavone synthase II (FSII) into the flavanone-producing recombinant yeast strain. The resulting recombinant strains were able to convert various phenylpropanoid acid precursors into the flavone molecules chrysin, apigenin, and luteolin, and the intermediate flavanones pinocembrin, naringenin, and eriodictyol accumulated in the medium. Improvement of flavone biosynthesis was achieved by overexpressing the yeast P450 reductase CPR1 in the FSII-expressing recombinant strain and by using acetate rather than glucose or raffinose as the carbon source. Overall, the FSI-expressing recombinant strain produced 50% more apigenin and six times less naringenin than the FSII-expressing recombinant strain when p-coumaric acid was used as a precursor phenylpropanoid acid. Further experiments indicated that unlike luteolin, the 5,7,4′-trihydroxyflavone apigenin inhibits flavanone biosynthesis in vivo in a nonlinear, dose-dependent manner.     

Optimization of a heterologous pathway for the production of flavonoids from glucose

The development of efficient microbial processes for the production of flavonoids has been a metabolic engineering goal for the past several years, primarily due to the purported health-promoting effects of these compounds. Although significant strides have been made recently in improving strain titers and yields, current fermentation strategies suffer from two major drawbacks-(1) the requirement for expensive phenylpropanoic precursors supplemented into the media and (2) the need for two separate media formulations for biomass/protein generation and flavonoid production. In this study, we detail the construction of a series of strains capable of bypassing both of these problems. A four-step heterologous pathway consisting of the enzymes tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) was assembled within two engineered l-tyrosine Escherichia coli overproducers in order to enable the production of the main flavonoid precursor naringenin directly from glucose. During the course of this investigation, we discovered that extensive optimization of both enzyme sources and relative gene expression levels was required to achieve high quantities of both p-coumaric acid and naringenin accumulation. Once this metabolic balance was achieved, however, such strains were found to be capable of producing 29 mg/l naringenin from glucose and up to 84 mg/l naringenin with the addition of the fatty acid enzyme inhibitor, cerulenin. These results were obtained through cultivation of E. coli in a single minimal medium formulation without additional precursor supplementation, thus paving the way for the development of a simple and economical process for the microbial production of flavonoids directly from glucose.     

Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy

Flavonoids are valuable natural products widely used in human health and nutrition. Recent advances in synthetic biology and metabolic engineering have yielded improved strain titers and yields. However, current fermentation strategies often require supplementation of expensive phenylpropanoic precursors in the media and separate evaluation of each strategy in turn as part of the flavonoid pathway, implicitly assuming the modifications are additive. In this study, an Escherichia coli fermentation system was developed to bypass both of these problems. An eight-step pathway, consisting of 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydratase (CM/PDT), phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was assembled on four vectors in order to produce the flavonoid precursor (2S)-pinocembrin directly from glucose. Furthermore, a modular metabolic strategy was employed to identify conditions that optimally balance the four pathway modules. Once this metabolic balance was achieved, such strains were capable of producing 40.02 mg/L (2S)-pinocembrin directly from glucose. These results were attained by culturing engineered cells in minimal medium without additional precursor supplementation. The fermentation platform described here paves the way for the development of an economical process for microbial production of flavonoids directly from glucose.     

柚皮素合成关键基因表达水平对目标产物积累水平的量化影响

柚皮素是一种天然黄酮类化合物,具有抗炎、抗氧化、抗病毒、预防动脉粥样硬化等多种药理活性,也是其他黄酮类化合物合成的重要前体,具有重要的应用价值。目前,微生物法生产柚皮素等黄酮类化合物由于代谢通路不平衡等原因导致产量较低,在很大程度上限制了其工业应用。文中以一株产柚皮素的酿酒酵母菌株Y-01为研究对象,利用启动子和拷贝数控制柚皮素合成代谢途径关键酶4-香豆酸:CoA连接酶(4CL)、查尔酮合成酶(CHS)和查尔酮异构酶(CHI)编码基因的表达水平,考察这些基因的表达水平对目标产物积累水平的量化影响。结果表明,柚皮素产量与4CL或CHI编码基因的表达量之间关联性较低,而与chs基因的表达量存在显著的正相关性。通过调控chs基因的表达水平,获得一株高产柚皮素的酿酒酵母工程菌株Y-04,产量较出发菌株Y-01提高了4.1倍。研究结果表明,CHS是柚皮素合成过程的关键调控靶点,合理调控CHS表达可以显著促进酿酒酵母积累柚皮素。相关结果为采用代谢工程强化微生物合成柚皮素等重要黄酮类化合物提供了重要的理论参考。     

Accumulation of phenylpropanoid derivatives in chitosan-induced cell suspension culture of Cocos nucifera

Chitosan-induced elicitation responses of dark-incubated Cocos nucifera (coconut) endosperm cell suspension cultures led to the rapid formation of phenylpropanoid derivatives, which essentially mimics the defense-induced biochemical changes in coconut palm as observed under in vivo conditions. An enhanced accumulation of p-hydroxybenzoic acid as the major wall-bound phenolics was evident. This was followed by p-coumaric acid and ferulic acid. Along with enhanced peroxidases activities in elicited lines, the increase in activities of the early phenylpropanoid pathway enzymes such as, phenylalanine ammonia lyase (PAL), p-coumaroyl-CoA ligase (4CL) and p-hydroxybenzaldehyde dehydrogenase (HBD) in elicited cell cultures were also observed. Furthermore, supplementation of specific inhibitors of PAL, C4H and 4CL in elicited cell cultures led to suppressed accumulation of p-hydroxybenzoic acid, which opens up interesting questions regarding the probable route of the biosynthesis of this phenolic acid in C. nucifera.     

Distribution of Phenylalanine Ammonia Lyase and Chalcone Synthase within Trunks of Robinia pseudoacacia L.

During heartwood formation, a kind of apoptosis in the inner parts of woody axes, phenolic substances are accumulated by in situ biosynthesis. In Robinia pseudoacacia L, these compounds are mainly flavonoids. In the present work, we performed a study to show if there is a correlation between measurable activities and detectable protein levels of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and chalcone synthase (CHS; EC 2.3.1.74), key enzymes of general phenylpropanoid metabolism and flavonoid biosynthesis, respectively. After separation of total protein extracts by one-dimensional micro-gel electrophoresis, newly emerging polypeptides were detectable within the sapwood-heartwood transition zone, pointing toward a transient activation of metabolism shortly before cell death occurs. Most prominent was a polypeptide around 46 kDa. By immunoblotting, this band was identified as a CHS subunit. Thus, the exclusive presence of both enzyme protein and extractable enzyme activity of CHS in the heartwood bordering tissue was shown. In contrast, levels of PAL protein were similar in all xylem tissues which contain living cells. PAL activity, however, was measurable only in the differentiating xylem and the sapwood-heartwood transition zone. From these results we conclude that during heartwood formation, CHS and PAL differ in their mode of regulation. It seems likely that CHS activity is regulated at the level of enzyme protein while PAL regulation is most probably post-translational.     

In Situ Localization of Phenylpropanoid Biosynthetic mRNAs and Proteins in Parsley (Petroselinum crispum)

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together consituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence.     

Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli

Flavonols are plant polyphenolic compounds that belong to the class of molecules collectively known as flavonoids. Because of their demonstrated health benefits towards a wide array of human pathological conditions, a great interest has emerged for their biosynthesis from well-characterized microbial hosts. We present the functional expression in Escherichia coli of a plant P450 flavonoid 3', 5'-hydroxylase (F3'5'H) as a fusion protein with a P450 reductase. This expression allowed metabolic engineering of E. coli to produce the flavonol kaempferol and the 3', 4' B-ring hydroxylated flavonol quercetin from the p-coumaric acid precursor by simultaneously co-expressing the fusion protein with 4-coumaroyl:CoA-ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3beta-hydroxylase (FHT) and flavonol synthase (FLS). Biosynthesis of the B-ring tri-hydroxylated flavonol myricetin from the engineered strains was accomplished when flavanones rather than phenylpropanoid acids were used as precursor molecules. Cultivation of the recombinant strains in rich medium increased the synthesis of all flavonoids with the exception of myricetin. The present work opens the possibility of the future production of several other hydroxylated flavonoid molecules in E. coli.     

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.     

Multiple phytohormones and phytoalexins are involved in disease resistance to Magnaporthe oryzae invaded from roots in rice

Blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. Phenylalanine ammonia lyase (PAL) is a key enzyme in the phenylpropanoid pathway, which leads to the biosynthesis of defense‐related phytohormone salicylic acid (SA) and flavonoid‐type phytoalexins sakuranetin and naringenin. However, the roles and biochemical features of individual rice PALs in defense responses to pathogens remain unclear. Here, we report that rice OsPAL06, which can catalyze the formation of trans‐cinnamate using l ‐phenylalanine, is involved in rice root–M. oryzae interaction. OsPAL06‐knockout mutant showed increased susceptibility to M. oryzae invaded from roots and developed typical leaf blast symptoms, accompanied by nearly complete disappearance of sakuranetin and naringenin and a two‐third reduction of the SA level in roots. This mutant also showed compensatively induced expression of chalcone synthase, which is involved in flavonoid biosynthesis, isochorismate synthase 1, which is putatively involved in SA synthesis via another pathway, reduced jasmonate content and increased ethylene content. These results suggest that OsPAL06 is a positive regulator in preventing M. oryzae infection from roots. It may regulate defense by promoting both phytoalexin accumulation and SA signaling that synergistically and antagonistically interacts with jasmonate‐ and ethylene‐dependent signaling, respectively.     

Modular Optimization of Heterologous Pathways for De Novo Synthesis of (2S)-Naringenin in Escherichia coli

Due to increasing concerns about food safety and environmental issues, bio-based production of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention. Here, the complete biosynthetic pathway, consisting of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydrogenase (CM/PDH), tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was constructed using pre-made modules to overproduce (2S)-naringenin from D-glucose. Modular pathway engineering strategies were applied to the production of the flavonoid precursor (2S)-naringenin from L-tyrosine to investigate the metabolic space for efficient conversion. Modular expression was combinatorially tuned by modifying plasmid gene copy numbers and promoter strengths to identify an optimally balanced pathway. Furthermore, a new modular pathway from D-glucose to L-tyrosine was assembled and re-optimized with the identified optimal modules to enable de novo synthesis of (2S)-naringenin. Once this metabolic balance was achieved, the optimum strain was capable of producing 100.64 mg/L (2S)-naringenin directly from D-glucose, which is the highest production titer from D-glucose in Escherichia coli. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.