Optimization of Phenylpropanoid Production in Saccharomyces Cerevisiae through Disruption of Double Bound Reductase Activity

Flavonoids are secondary plant metabolites derived from the phenylpropanoid pathway. These bioactive compounds are of great commercial interest due to their varied properties, such as anti-oxidative, anti-tumor or antibacterial activities. However, mass production based on purification of flavonoids from plants can be problematic due to slow growth or limited access.

The objective of this study is to produce naringenin (as a model flavonoid) in Saccharomyces cerevisiae, without accumulating the undesired side product phloretic acid (dihydrocoumaric acid). Accumulation of phloretic acid results from the reduction of coumaroyl-CoA by an unknown endogenous yeast reductase activity.

In order to identify said reductases, 26 putative double-bond reductase knockout strains (KO) were transformed with plant genes coding for active 4-coumarate-CoA ligase (4CL) , then fed with p-coumaric acid (the strains were homozygous diploids for nonessential genes whereas in case of the essential genes the strains were heterologous diploids). Two of the KO strains consumed less p-coumaric acid, and produced less phloretic acid. To confirm their activity on coumaroyl-CoA, the two reductases were overexpressed in a naringenin producing strain: This revealed that one of the reductases had a high catalytic activity towards coumaroyl-CoA while the second had had only marginal activity. The identified gene encodes an essential enzyme. Therefore the next step was to find an alternative version of the enzyme that can perform its normal function in the cell but that cannot reduce coumaroyl-CoA. Different versions of the identified enzyme were obtained by constructing a site-saturated mutagenesis library focusing on the residues around the active site and the substrate binding pocket. The mutant library was introduced in a wild type yeast strain, also containing naringenin biosynthesis machinery, and afterwards the reductase gene in question was deleted. The resulting cells were analysed for their growth, production of coumaric acid, naringenin and phloretic acid. While some of the mutant reductase enzymes had significantly lower activity towards coumaroyl-CoA, their growth was negatively affected. We therefore looked at alternative strategies to the same end, some of which were successful – results and conclusions of these experiments will be discussed.