Pathway Engineering
Increasing flavonoid precursors in biosynthetic pathways.
 

Flavanone Biosynthesis in Recombinant Saccharomyces cerevisiae

Yajun Yan and Joseph Chemler

In order to produce stable flavanones from their precursor phenylcarbonic acids, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: cinnamate 4-hydroxylase (C4H) from Arabidopsis thaliana, 4-coumarate:coenzyme A ligase (4CL) from Petroselinum crispum, and chalcone synthase (CHS) and chalcone isomerase (CHI) from Petunia x hybrida. Using two rounds of Polymerase Chain Reaction, each one of the four genes was first placed under the control of the GAL1 promoter and then cloned sequentially into Saccharomyces cerevisiae vector Yeplac181. S. cerevisiae cells carrying the recombinant plant pathway were able to metabolize cinnamic acid, p-coumaric acid and caffeic acid to their corresponding flavanones pinocembrin, naringenin and eriodictyol. The produced flavanones were present at concentrations significantly higher than what has previously been reported for recombinant prokaryotic strains. The use of recombinant yeast for flavanone biosynthesis opens up the possibility of producing several other high-value flavonoid polyphenols that require the functional expression of P450 monooxygenases that are difficult to express in prokaryotes. [Yan et al., 2005]

Anthocyanin Biosynthesis in Recombinant Escherichia Coli

Yajun Yan, Joseph Chemler, and Amruta Bedekar

Anthocyanins are red, purple or blue plant pigments responsible for the flower and fruit colors together with betalains and carotenoids. They belong to the family of polyphenolic compounds collectively called flavonoids. In the past two decades, anthocyanins received extensive studies for their anti-oxidative, anti-inflammatory, anti-cancer and cardioprotective properties. Their metabolic biosynthetic pathway has been well-investigated with continuously updating. Flavanone 3-hydroxylase (FHT), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (3GT) are involved in the biosynthesis of the first stable glycosylated anthocyanins from flavanones such as naringenin and eriodictyol. In this study, several governing genes of the anthocyanin biosynthesis have been characterized from different plant species and employed for the pathway construct leading from flavanones to anthocyanins in Escherichia coli. The recombinant E. coli cells successfully metabolized both naringenin and eriodictyol. Milligram level production of pelargonidin 3-O-glucoside (0.98mg/L) and cyanidin 3-O-gluside (2.07mg/L) was detected, while intermediates dihydroflavonols and anthocyanidins were also accumulated in the culture with different amounts. The production of cyanidin 3-O-glucoside from (+)-catechin directly by ANS and 3GT gave even high yield of 16.1mg/L. Cofactor effect study shown that UDP-glucose as expensive glucosyl donor contributed the most to the production of anthocyanins in the recombinant strains. Therefore optimization will focus on enhancing the homogenous synthesis of UDP-glucose in the host cells by overexpression corresponding regulatory genes such as galU, pgm and PTS system. [Yan et al., 2005]

Figure: The pH effect (from left to right is 1 to 13) on the color of cyanidin 3-O-glucoside.

5-Deoxyflavanone Biosynthesis in Recombinant Microorganism

Yajun Yan and Joseph Chemler

Leguminous plants have evolved a distinct class of flavanone molecules, known as 5-deoxyflavanones that play important roles in their symbiotic interactions. In this study, a four-step metabolic circuit was constructed in Escherichia coli that contained plant genes from heterologous origins: 4-coumarate:coenzyme A ligase (4CL) from Petroselinum crispum, members of the chalcone synthase (CHS) family from Medicago sativa and Petunia x hybrida and chalcone reductase (CHR) and chalcone isomerase (CHI) from M. sativa. Evaluation of the different recombinant strains in shake flask experiments demonstrated that P. hybrida rather than M. sativa CHS resulted in the highest liquiritigenin production levels in glucose minimal medium, starting from precursor p-coumaric acid. Expression of the same recombinant pathway in Saccharomyces cerevisiae resulted in the accumulation of both 5-hydroxyflavanone and 5-deoxyflavanone as well. The cinnamic acid and caffeic acid can also be metabolized through the artificial pathway, yielding corresponding chalcone and flavanone compounds. The construction of such recombinant strains for 5-deoxyflavanone biosynthesis offers an alternative way to biochemically characterize flavonoid biosynthetic enzymes, valuable insight in protein interactions and promising production platforms for the biosynthesis of such high-value natural products. [submitted]

Biosynthesis of Flavones and Hydroxylated Flavonols in Engineered Saccharomyces cerevisiae

Yajun Yan and Zhen Li

In this study, we present the production of flavones and hydroxylated flavonols from phenylpropanoid acid precursors in recombinant yeast S. cerevisiae cells. We have reported the flavanone biosynthesis in yeast with 4CL from Parsley, CHS from petunia and CHI from petunia before. Basing on that, the introduction of genes controlling flavone and flavonol biosynthesis into yeast cells allowed us to realize the diverse flavonoid production in yeast with substantial amount. Especially with the functional expression of P450 enzyme F3’5’H in yeast, di and tri-hydroxylated flavonols, quercetin and myricetin, were produced from p-coumaric acid in yeast for the first time. Hydroxylated flavonol production was further optimized by overexpressing the P450 redox partner CPR1 together with flavonol biosynthetic enzymes in yeast S. cerevisiae cells. [submitted]