Rational modular design of metabolic network for efficient production of plant polyphenol pinosylvin

Junjun Wu,Mingsheng Dong,Xia Zhang,Jiacheng He,Qinyu Tan,Yingjie Zhu

doi:10.1038/s41598-017-01700-9

Abstract

Efficient biosynthesis of the plant polyphenol pinosylvin, which has numerous applications in nutraceuticals and pharmaceuticals, is necessary to make biological production economically viable. To this end, an efficient Escherichia coli platform for pinosylvin production was developed via a rational modular design approach. Initially, different candidate pathway enzymes were screened to construct de novo pinosylvin pathway directly from D-glucose. A comparative analysis of pathway intermediate pools identified that this initial construct led to the intermediate cinnamic acid accumulation. The pinosylvin synthetic pathway was then divided into two new modules separated at cinnamic acid. Combinatorial optimization of transcriptional and translational levels of these two modules resulted in a 16-fold increase in pinosylvin titer. To further improve the concentration of the limiting precursor malonyl-CoA, the malonyl-CoA synthesis module based on clustered regularly interspaced short palindromic repeats interference was assembled and optimized with other two modules. The final pinosylvin titer was improved to 281 mg/L, which was the highest pinosylvin titer even directly from D-glucose without any additional precursor supplementation. The rational modular design approach described here could bolster our capabilities in synthetic biology for value-added chemical production.

Highlights

The polyphenol pinosylvin is a plant secondary metabolite fulfilling several functions including protection from attack by microbes or insects[1]
In E. coli, two rate-limiting steps exist toward the synthesis of L-phenylalanine
By a comparative analysis of pathway intermediate concentrations (Table 1), we found that these two combinations both led to high accumulation of cinnamic acid

Summary

Introduction

The polyphenol pinosylvin (trans-3,5-dihydroxystilbene) is a plant secondary metabolite fulfilling several functions including protection from attack by microbes or insects[1]. Relevant study reported that supplementation of 15 mM p-coumaric acid led to a high product titer of 2.3 g/L resveratrol in Escherichia coli[6]. These strategies rely heavily on supplementation of phenylpropanoids as stilbene precursors, which is costly and present in few industrial processes. As cerulenin is expensive and cost prohibitive for large-scale fermentation process, another study examined using clustered regularly interspaced short palindromic repeats interference (CRISPRi) system to repress fadD gene, and product titer of 47.5 mg/L was obtained from glycerol[5]. Corynebacterium glutamicum has been engineered and 121 mg/L of pinosylvin and 158 mg/L of resveratrol was achieved in the presence of 25 μM cerulenin[8]

Methods

Results

Conclusion