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.     

红树植物秋茄类黄酮代谢及其抗氧化活性对高盐胁迫的响应

以红树植物秋茄为试验材料,设置不同浓度NaCl(0、200和500mmol·L-1)处理的砂培实验,应用qRTPCR分析秋茄叶片中类黄酮物质合成上游的4个关键酶基因——苯丙氨酸解氨酶基因(PAL)、肉桂酸羟化酶基因(C4 H)、4-香豆酰辅酶A连接酶基因(4CL)和查尔酮合成酶基因(CHS)的转录水平,并对关键酶活性进行了分析,同时测定了幼苗生物量、钾钠离子含量、类黄酮含量及其抗氧化活性,以探讨秋茄耐盐性与类黄酮物质的关系,为揭示木本植物耐盐机制奠定基础。结果显示:(1)在盐处理条件下,秋茄叶片中PAL、4CL、C4 H和CHS4个关键酶基因的转录水平显著上调,PAL、4CL、C4H酶活性和CHS含量随着盐浓度的增加而明显上升。(2)与对照相比,秋茄根、茎、叶干重在盐处理3d和15d后均无显著变化,而秋茄株高仅在200mmol·L-1盐处理15d后显著增加,其余浓度和时间均未发现有显著性变化。(3)随着盐浓度的升高,秋茄叶片类黄酮含量显著增加,K+/Na+明显下降,丙二醛含量显著降低,活性氧自由基清除率显著增加。研究表明,盐处理加强了秋茄叶片中类黄酮代谢过程中相关酶基因的表达,类黄酮物质的累积有助于其抗氧化能力的提高,进而提高秋茄的抗盐性,维持盐胁迫下秋茄的正常生长。     

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.     

Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster

For the fermentative production of plant-specific flavanones (naringenin, pinocembrin) by Escherichia coli, a plasmid was constructed which carried an artificial biosynthetic gene cluster, including PAL encoding a phenylalanine ammonia-lyase from a yeast, ScCCL encoding a cinnamate/coumarate:CoA ligase from the actinomycete Streptomyces coelicolor A3(2), CHS encoding a chalcone synthase from a licorice plant and CHI encoding a chalcone isomerase from the Pueraria plant. The recombinant E. coli cells produced (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. When the two subunit genes of acetyl-CoA carboxylase from Corynebacterium glutamicum were expressed under the control of the T7 promoter and the ribosome-binding sequence in the recombinant E. coli cells, the flavanone yields were greatly increased, probably because enhanced expression of acetyl-CoA carboxylase increased a pool of malonyl-CoA that was available for flavanone synthesis. Under cultural conditions where E. coli at a cell density of 50 g/l was incubated in the presence of 3 mM tyrosine or phenylalanine, the yields of naringenin and pinocembrin reached about 60 mg/l. The fermentative production of flavanones in E. coli is the first step in the construction of a library of flavonoid compounds and un-natural flavonoids in bacteria.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

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.     

Comparative metabolic activity related to flavonoid synthesis in leaves and flowers of Chrysanthemum morifolium in response to K deficiency

K limitation could decrease the flavonoids content in many Chinese traditional herbs. These results may be caused by the influence of K deficiency on the synthesis pathway of flavonoids. In this paper, we aim to study the influence of K deficiency on secondary metabolites and activities of phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4CL) and cinnamate 4-hydroxylase (C4H) enzymes in the process of flavonoid synthesis in Chrysanthemum morifolium Ramat. The results show that K deficiency decreased the flavonoid and chlorogenic acid contents slightly in flower of C. morifolium while the same effect was obtained on C4H and PAL. Total flavonoids content increased with the content of cinnamic acid and p-coumaric acid in the plant under K deficiency, respectively. The regression equations between content of flavonoids and cinnamic acid/ p-coumaric acid should be expressed in term of linear effects (R²?=?0.9809, P?<?0.001 for cinnamic acid and R²?=?0.9929, P?<?0.0001 for p-coumaric acid, respectively). But the effect of phenylalanine on flavonoids should be expressed in term of quadratic pattern (R²?=?0.9375, P?<?0.05). There were two kinds principal components from chorismate to coumaryl CoA synthesis process, principal 1 was the substrate (phenylalanine, cinnamic acid, p-coumaric acid and PAL) and principal 2 was the enzyme (4CL, C4H), the principal 1 was the domination principal in both K deficient (88.36% and 10.57%) and sufficient treatments (88.17% and 9.64%), however, the factor loading (correlation coefficient of measured targets and the corresponding principal component) in principal 1 was opposite under different K application.     

Metabolism of aromatic acids by Polyporus hispidus

When grown on glucose as principal carbon source the culture medium of Polyporus hispidus was found to contain phenolic acids, including p-coumaric and caffeic acids. ¹⁴C-Studies indicated that phenylalanine is converted to cinnamic acid as well as to phenylpyruvic acid and tyrosine in cultures. Cell-free preparations of mycelium contained phenylalanine and tyrosine ammonia-lyse activities and were capable of effecting the hydroxylation of cinnamic, p-coumaric and benzoic acids.     

Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits

Flavonoids possess diverse health‐promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone‐3‐hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm‐specific GluB‐1 promoter or embryo‐ and aleurone‐specific 18‐kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB‐II‐type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes.     

Bioconversion of Pinoresinol Diglucoside and Pinoresinol from Substrates in the Phenylpropanoid Pathway by Resting Cells of Phomopsis sp.XP-8

Pinoresinol diglucoside (PDG) and pinoresinol (Pin) are normally produced by plant cells via the phenylpropanoid pathway. This study reveals the existence of a related pathway in Phomopsis sp. XP-8, a PDG-producing fungal strain isolated from the bark of the Tu-chung tree (Eucommiaulmoides Oliv.). After addition of 0.15 g/L glucose to Phomopsis sp. XP-8, PDG and Pin formed when phenylalanine, tyrosine, leucine, cinnamic acid, and p-coumaric acid were used as the substrates respectively. No PDG formed in the absence of glucose, but Pin was detected after addition of all these substrates except leucine. In all systems in the presence of glucose, production of PDG and/or Pin and the accumulation of phenylalanine, cinnamic acid, or p-coumaric acid correlated directly with added substrate in a time- and substrate concentration- dependent manner. After analysis of products produced after addition of each substrate, the mass flow sequence for PDG and Pin biosynthesis was defined as: glucose to phenylalanine, phenylalanine to cinnamic acid, then to p-coumaric acid, and finally to Pin or PDG. During the bioconversion, the activities of four key enzymes in the phenylpropanoid pathway were also determined and correlated with accumulation of their corresponding products. PDG production by Phomopsis sp. exhibits greater efficiency and cost effectiveness than the currently-used plant-based system and will pave the way for large scale production of PDG and/or Pin for medical applications.     

Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones

Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35 mg/L naringenin and 37 mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158 mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.