Efficient production of salvianic acid A from l-dihydroxyphenylalanine through a tri-enzyme cascade

Abstract

Salvianic acid A (SAA), used for treating cardiovascular and cerebrovascular diseases, possesses several pharmacological properties. However, the current methods for the enzymatic synthesis of SAA show low efficiency. Here, we constructed a three-enzyme cascade pathway in Escherichia coli BL21 (DE3) to produce SAA from l-dihydroxyphenylalanine (L-DOPA). The phenylpyruvate reductase (LaPPR) from Lactobacillus sp. CGMCC 9967 is a rate-limiting enzyme in this process. Therefore, we employed a mechanism-guided protein engineering strategy to shorten the transfer distances of protons and hydrides, generating an optimal LaPPR mutant, LaPPR^Mu2 (H89M/H143D/P256C), with a 2.8-fold increase in specific activity and 9.3-time increase in k_cat/K_m value compared to that of the wild type. Introduction of the mutant LaPPR^Mu2 into the cascade pathway and the optimization of enzyme levels and transformation conditions allowed the obtainment of the highest SAA titer (82.6 g L⁻¹) ever reported in vivo, good conversion rate (91.3%), excellent ee value (99%) and the highest productivity (6.9 g L⁻¹ h⁻¹) from 90 g L⁻¹ L-DOPA in 12 h. This successful strategy provides a potential new method for the industrial production of SAA.

Keywords

Salvianic acid A / Tri-enzyme cascade / Whole-cell biotransformation / Protein engineering

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Funding

the National Key R & D Program of China (2021YFC2100100)

he General Program of National Natural Science Foundation of China(22178146)

Natural Science Foundation for Young Scientists of Shanxi Province(22008089)