Functional analysis of flavonoid 3',5'-hydroxylase from tea plant (Camellia sinensis): critical role in the accumulation of catechins.

BACKGROUND: Flavonoid 3',5'-hydroxylase (F3'5'H), an important branch point enzyme in tea plant flavan-3-ol synthesis, belongs to the CYP75A subfamily and catalyzes the conversion of flavones, flavanones, dihydroflavonols and flavonols into 3',4',5'-hydroxylated derivatives. However, whether B-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo remains unknown. RESULTS: The Camellia sinensis F3'5'H (CsF3'5'H) gene was isolated from tea cDNA library. Expression pattern analysis revealed that CsF3'5'H expression was tissue specific, very high in the buds and extremely low in the roots. CsF3'5'H expression was enhanced by light and sucrose. Over-expression of CsF3'5'H produced new-delphinidin derivatives, and increased the cyanidin derivative content of corollas of transgenic tobacco plants, resulting in the deeper transgenic plant flower color. Heterologous expressions of CsF3'5'H in yeast were carried out to demonstrate the function of CsF3'5'H enzyme in vitro. Heterologous expression of the modified CsF3'5'H (CsF3'5'H gene fused with Vitis vinifera signal peptide, FSI) revealed that 4'-hydroxylated flavanone (naringenin, N) is the optimum substrate for CsF3'5'H, and was efficiently converted into both 3'4'- and 3'4'5'-forms. The ratio of 3'4'5'- to 3'4'-hydroxylated products in FSI transgenic cells was significantly higher than VvF3'5'H cells. CONCLUSIONS: CsF3'5'H is a key controller of tri-hydroxyl flavan-3-ol synthesis in tea plants, which can effectively convert 4'-hydroxylated flavanone into 3'4'5'- and/or 3'4'-hydroxylated products. These findings provide animportant basis for further studies of flavonoid biosynthesis in tea plants. Such studies would help accelerate flavonoid metabolic engineering in order to increase B-ring tri-hydroxyl product yields.

A plant malonyl-CoA synthetase enhances lipid content and polyketide yield in yeast cells.

Malonyl-CoA is the essential building block of natural products such as fatty acids, polyketides, and flavonoids. Engineering the biosynthesis of fatty acids is important for biofuel production while that of polyketides provides precursors of medicines and nutritional supplements. However, microorganisms maintain a small amount of cellular malonyl-CoA, which could limit production of lipid and polyketides under certain conditions. Malonyl-CoA concentration is regulated by multiple pathways and signals, and changes in intracellular malonyl-CoA often lead to complex alterations in metabolism. In the present work, overexpression of a plant malonyl-CoA synthetase gene (AAE13) in Saccharomyces cerevisiae resulted in 1.6- and 2.4-fold increases in lipid and resveratrol accumulation simultaneously. We also demonstrated that AAE13 partially complemented the temperature-sensitive acc1 mutant, replacing this key enzyme in central metabolism. Mechanistic analysis by CoA quantification and transcriptomic measurement suggested that increases in malonyl-CoA concentration were coupled with drastic reductions in other major CoA compounds and clear suppression of tricarboxylic acid cycle-related genes. These results suggest that malonyl-CoA is a critical target for fatty acid and polyketide engineering and that overexpression of malonyl-CoA synthetic enzymes needs to be combined with upregulation of CoA synthesis to maintain metastasis of central metabolism.

Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells.

Resveratrol is a polyphenolic compound produced by a few higher plants when under attack by pathogens such as bacteria or fungi. Besides antioxidant benefits to humans, this health-promoting compound has been reported to extend longevity in yeasts, flies, worms, fishes and obesity mice. Here we utilized the synthetic scaffolds strategy to improve resveratrol production in Saccharomyces cerevisiae. We observed a 5.0-fold improvement over the non-scaffolded control, and a 2.7-fold increase over the previous reported with fusion protein. This work demonstrated the synthetic scaffolds can be used for the optimization of engineered metabolic pathway.

Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering.

Resveratrol is a unique, natural polyphenolic compound with diverse health benefits. In the present study, we attempted to improve resveratrol biosynthesis in yeast by different methods of metabolic engineering. We first mutated and then re-synthesized tyrosine ammonia lyase (TAL) by replacing the bacteria codons with yeast-preferred codons, which increased translation and improved p-coumaric acid and resveratrol biosynthesis drastically. We then demonstrated that low-affinity, high-capacity bacterial araE transporter could enhance resveratrol accumulation, without transporting resveratrol directly. Yeast cells carrying the araE gene produced up to 2.44-fold higher resveratrol than control cells. For commercial applications, resveratrol biosynthesis was detected in sucrose medium and fresh grape juice using our engineered yeast cells. In collaboration with the Chaumette Winery of Missouri, we were able to produce resveratrol-containing white wines, with levels comparable to the resveratrol levels found in most red wines.

Regulation of growth response to water stress in the soybean primary root. I. Proteomic analysis reveals region-specific regulation of phenylpropanoid metabolism and control of free iron in the elongation zone.

In water-stressed soybean primary roots, elongation was maintained at well-watered rates in the apical 4 mm (region 1), but was progressively inhibited in the 4-8 mm region (region 2), which exhibits maximum elongation in well-watered roots. These responses are similar to previous results for the maize primary root. To understand these responses in soybean, spatial profiles of soluble protein composition were analysed. Among the changes, the results indicate that region-specific regulation of phenylpropanoid metabolism may contribute to the distinct growth responses in the different regions. Several enzymes related to isoflavonoid biosynthesis increased in abundance in region 1, correlating with a substantial increase of isoflavonoid content in this region which could contribute to growth maintenance via various potential mechanisms. In contrast, caffeoyl-CoA O-methyltransferase, which is involved in lignin synthesis, was highly up-regulated in region 2. This response was associated with enhanced accumulation of lignin, which may be related to the inhibition of growth in this region. Several proteins that increased in abundance in both regions of water-stressed roots were related to protection from oxidative damage. In particular, an increase in the abundance of ferritin proteins effectively sequestered more iron and prevented excess free iron in the elongation zone under water stress.

Nature's assembly line: biosynthesis of simple phenylpropanoids and polyketides.

Plants produce large amounts of phenylpropanoids, both in terms of molecular diversity and absolute quantity of these compounds. The phenylpropanoids, and the related plant polyketides, have multiple biological functions. They serve to attract pollinators, support secondary cell-wall growth, provide protection against various plant diseases, and interact with beneficial soil microbes. Their basic chemical properties also make them useful in the biofuel and biomaterial industries. Phenylpropanoid metabolism begins with the amino acid phenylalanine, which feeds into various biosynthetic pathways that generate a wide range of structurally related polyphenolic compounds. This review focuses on four sub-groups of these polyphenolic compounds - polyketides, stilbenes, isoflavones and catechins. We discuss the biosynthesis of these molecules, their physiological role in plants, and their striking pharmacological and physiological effects on humans. This review also highlights metabolic engineering efforts aimed at increasing or decreasing the amounts of each class of compound in various model plants and crops.

Potential for metabolic engineering of resveratrol biosynthesis.

Resveratrol, an interesting plant phenolic compound, is found in red wine but is not widely distributed in other common food sources. Health benefits of resveratrol include prevention of cardiovascular diseases and cancers, and--as discovered more recently--promotion of longevity in several animal systems. The pathway and enzymes for resveratrol biosynthesis are well characterized. Furthermore, metabolic engineering of this compound has been achieved in plants, microbes and animals. This review attempts to summarize current understanding of resveratrol pathway-engineering in various systems, to outline the challenges in commercial applications and to identify future opportunities for resveratrol bioengineering.

Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases.

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4'-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3beta-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.