Multienzyme Biosynthesis of Dihydroartemisinic Acid.

Xixian Chen,Heng-Phon Too,Congqiang Zhang

doi:10.3390/molecules22091422

Abstract

One-pot multienzyme biosynthesis is an attractive method for producing complex, chiral bioactive compounds. It is advantageous over step-by-step synthesis, as it simplifies the process, reduces costs and often leads to higher yield due to the synergistic effects of enzymatic reactions. In this study, dihydroartemisinic acid (DHAA) pathway enzymes were overexpressed in Saccharomyces cerevisiae, and whole-cell biotransformation of amorpha-4,11-diene (AD) to DHAA was demonstrated. The first oxidation step by cytochrome P450 (CYP71AV1) is the main rate-limiting step, and a series of N-terminal truncation and transcriptional tuning improved the enzymatic activity. With the co-expression of artemisinic aldehyde dehydrogenase (ALDH1), which recycles NADPH, a significant 8-fold enhancement of DHAA production was observed. Subsequently, abiotic conditions were optimized to further enhance the productivity of the whole-cell biocatalysts. Collectively, approximately 230 mg/L DHAA was produced by the multi-step whole-cell reaction, a ~50% conversion from AD. This study illustrates the feasibility of producing bioactive compounds by in vitro one-pot multienzyme reactions.

Highlights

Terpenoids are an important class of natural products that play a critical role in many industrial sectors, such as healthcare, flavor and fragrance, and biofuels [1,2]
They are synthesized from the two C5 molecules, namely isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP)
In contrast to the previous report [7], our initial attempt to use CYP71AV1-overexpressing E. coli as the biocatalyst did not produce detectable levels of artemisinic acid (AA) from extracellularly-fed AD; only a trace amount of artemisinin alcohol was detected. This may be due to the lack of internal membrane structures in E. coli, as P450 enzymes are known to be targeted to the endoplasmic reticulum

Summary

Introduction

Terpenoids are an important class of natural products that play a critical role in many industrial sectors, such as healthcare, flavor and fragrance, and biofuels [1,2] They are synthesized from the two C5 molecules, namely isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In contrast to the previous report [7], our initial attempt to use CYP71AV1-overexpressing E. coli as the biocatalyst did not produce detectable levels of AA from extracellularly-fed AD; only a trace amount of artemisinin alcohol was detected This may be due to the lack of internal membrane structures in E. coli, as P450 enzymes are known to be targeted to the endoplasmic reticulum. Since all the terpenoids were formed from the two precursors (IPP and DMAPP), our system presents a rapid and easy way to test and optimize the production of oxygenated terpenoids

Enhancing CYP71AV1 Activity by Enzyme Engineering and Transcription Tuning

Dihydroartemisinic Acid Production and Reaction Condition Optimization

In Situ Production of Amorphadiene and Dihydroartemisinic Acid

Strains and Plasmids

Yeast Growth and Protein Expression

Yeast Whole Cell Biocatalysis and Product Extraction