Constructing microbial production platform for the biosynthesis of natural drug candidates-flavonoids
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The current dissertation investigated the construction of production platforms for the synthesis of flavonoids in both prokaryotic host E. coli and eukaryotic host yeast S. cerevisiae. The total flavonoid biosynthetic pathway has been re-assembled starting from the cheap precursor phenylpropanoic acid. Some particular enzymes have been characterized biochemically for the efficient operation of the pathway. We also achieved the efficient production of some specific flavonoids (anthocyanins) by rationally designing the host genotype and intentionally manipulating the metabolic network to support the heterologous biosynthesis. Chapter 2 covers the earliest work in the biosynthesis of natural flavanone in yeast S. cerevisiae by creating an artificial gene cluster. 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. Chapter 3 covers the following work on the biosynthesis of flavones and hydroxylated flavonols in yeast S. cerevisiae. The hydroxylation of flavonoid compounds on the B-ring expands their diversity in nature and their application in pharmaceutical applications. The enzyme in charge of the hydroxylation belong to the type II P450 monooxygenases. Its functional expression in yeast together with other pathway enzymes leads to the biosynthesis of dihydroxylated, trihoxylated flavonols and corresponding intermediates. Furthermore, co-overexpression of its redox partner, cytochrome P450 reductase (CPR) in yeast demonstrated their synergy in hydroxylation reaction. 5-deoxyflavanones are mainly distributed in legumes and involved in the formation of isoflavone daidzein and its derivatives. In chapter 4, we investigated the production of 5-deoxyflavonones in both E. coli and S. cerevisiae. The enzyme CHR from alfalfa was characterized. The species-dependent substrate specificity of enzyme CHI was demonstrated, which led to the optimization of the pathway assembly. The relationship between structure and activity of enzyme CHS was also randomly studied by site mutagenesis. Yield comparison indicated the yeast offer higher productivity than E. coli, which may be due to the fact that yeast allows better protein expression and supply more cofactor malonyl-CoA. As the largest group of flavonoids, anthocyanins are red, purple, or blue plant pigments. In the Chapter 5, we present the work on anthocyanin production in E. coli. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3β-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3- O -glucosyltransferase from Petunia hybrida. Escherichia coli cells expressing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3- O -glucoside or cyanidin 3- O -glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. In the Chapter 6, we present our efforts to enhance anthocyanin production in E. coli through media optimization and various metabolic engineering strategies. With the identification of the limiting step in the biosynthetic pathway, highly active enzymes were introduced for efficient bioconversion. Further studies demonstrated that availability of the glucosyl donor, UDP-glucose, was the key metabolic limitation, while product instability at normal pH was also identified as a barrier for production improvement. Therefore, various optimization strategies were employed for enhancing the intracellular synthesis of UDP-glucose in the host cells while at the same time stabilizing the final anthocyanin product. Such optimizations included culture medium pH adjustment, the creation of fusion proteins and the rational manipulation of E. coli metabolic network for improving the intracellular UDP-glucose metabolic pool. As a result, production of pelargonidin 3- O -glucoside at 78.9 mg/L and cyanidin 3- O -glucoside at 70.7 mg/L was achieved from their precursor flavan-3-ols without supplementation with extracellular UDP-glucose. E. coli as the well-studied and simplest host has been developed and engineered for the production of a diverse array of fine chemicals or high-value compounds. However, in most cases, productivity of natural products from this organism is greatly limited by cofactor and energy availability within the cell. In the Chapter 7, we demonstrate the improvement of intracellular cofactor availability using various systematic approaches which led to the production increase of cyanidin 3- O -glucoside at 113 mg/L from their precursor (+)-catechin.