Engineering of Microbial Substrate Promiscuous CYP105A5 for Improving the Flavonoid Hydroxylation

Abstract

Bacterial cytochrome P450 (CYP) enzymes are versatile biocatalysts that are responsible for the biotransformation of diverse endogenous substances. CYP105A5 from Streptomyces sp. showed substrate flexibility with different flavonoids and was able to catalyze O-demethylation of biochanin A, regioselective C3′-hydroxylation of daidzein, genistein, and naringenin, and additional C8-hydroxylation for daidzein using heterologous redox partners putidaredoxin and putidaredoxin reductase. By rational design of substrate-binding pocket based on experimental data, homology modeling, and molecular docking analysis, we enhanced the product formation rate of flavonoids. The double mutant L100A/I302A and L100A/I408N exhibited greatly enhanced in vivo conversion rates for flavonoid hydroxylation. Particularly, the L100A/I302A mutant’s kcat/Kmvalues and in vivo conversion rate increased by 1.68-fold and 2.57-fold, respectively, for naringenin. Overall, our result might facilitate the potential use of CYP105A5 for future modification and application in whole-cell biocatalysts for the production of valuable polyphenols.

Efficient hydroxylation of flavonoids by using whole-cell P450 sca-2 biocatalyst in Escherichia coli

“…Currently, the hydroxylation of flavonoids has not been well achieved in bacteria. Several bacterial P450s, including P450 BM3 ( Chu et al, 2016 ), CYP105D7 ( Liu et al, 2016 ), CYP107P2 ( Pandey et al, 2011 ), CYP107Y1 ( Pandey et al, 2011 ), and CYP105A5 ( Subedi et al, 2022 ), have been explored to hydroxylate selected flavonoids with a low conversion rate.…”