In Vitro Metabolic Engineering of Amorpha-4,11-diene Biosynthesis at Enhanced Rate and Specific Yield of Production

ACS Synthetic Biology
Xixian ChenHeng-Phon Too

Abstract

In vitro metabolic engineering is an alternative approach to cell-based biosynthesis. It offers unprecedented flexibility for the study of biochemical pathways, thus providing useful information for the rational design and assembly of reaction modules. Herein, we took the advantage of in vitro metabolic engineering to initially gain insight into the regulatory network of a reconstituted amorpha-4,11-diene (AD) synthetic pathway. Guided by lin-log approximation, we rapidly identified the hitherto unrecognized inhibition of adenosine triphosphate (ATP) on both farnesyl pyrophosphate synthase (FPPS) and amorpha-4,11-diene synthase (ADS). Furthermore, the byproduct, pyrophosphate (PPi), potently inhibits ADS, but not FPPS. To lower the inhibition, an ATP recycling system and pyrophosphatase were used and resulted in a significant (∼3-fold) enhancement in the rate of AD production (∼5.7 μmol L-1min-1). A coimmobilized multienzyme reaction system was then developed to recycle the enzymes. When inhibitory metabolites concentrations were reduced, the specific enzymatic yield of AD was further enhanced (>6-fold). This study demonstrated that mitigating the accumulation of inhibitory metabolites can result in higher yields of AD producti...Continue Reading

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Citations

Aug 29, 2017·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Xixian ChenHeng-Phon Too
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Feb 13, 2020·Current Medicinal Chemistry·Congqiang Zhang, Heng-Phon Too
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Dec 24, 2019·Journal of Agricultural and Food Chemistry·Xixian ChenNicholas D Lindley
Nov 2, 2021·ACS Synthetic Biology·Biaobiao LuoXuebo Hu

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