Cell-free Biosynthesis of Chlorogenic Acid Using a Mixture of Chassis Cell Extracts and Purified Spy-Cyclized Enzymes.

Fu-Xing Niu, Zhi-Bo Yan, Yuan-Bin Huang, Jian-Zhong Liu

Journal of agricultural and food chemistry 2021 Jul 21

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A novel cell-free biosynthesis system based on a mixture of chassis cell extracts and purified Spy-cyclized enzymes (CFBS-mixture) was developed. As a demonstration, the CFBS-mixture was applied to chlorogenic acid (CGA) biosynthesis. The mix-and-match and Plackett-Burman experiments demonstrated that Lonicera japonica hydroxycinnamate-CoA quinate transferase and p-hydroxyphenylacetate 3-hydroxylase were the key enzymes for the production of CGA. After optimization of the concentrations of the biosynthetic enzymes in the CFBS-mixture reaction using the Plackett-Burman experimental design and the path of the steepest ascent, 711.26 ± 15.63 mg/L CGA was produced after 16 h, which is 71.1-fold the yield obtained using the conventional crude extract-based CFBS and 9.1-fold the reported yield obtained using the living cells. Based on the CFBS-mixture results, the production of CGA was further enhanced in engineered Escherichia coli. The CFBS-mixture strategy is highly effective and will be useful for high-level CFBS of natural products.

Citation

Fu-Xing Niu, Zhi-Bo Yan, Yuan-Bin Huang, Jian-Zhong Liu. Cell-free Biosynthesis of Chlorogenic Acid Using a Mixture of Chassis Cell Extracts and Purified Spy-Cyclized Enzymes. Journal of agricultural and food chemistry. 2021 Jul 21;69(28):7938-7947