大肠杆菌高密度发酵表达4-羟基苯乙酸酯3-羟化酶及咖啡酸的高效生物合成

High-density fermentation of Escherichia coli to express 4-hydroxyphenylacetate 3-hydroxylase and efficient biosynthesis of caffeic acid

摘要:

来源于大肠杆菌的4-羟基苯乙酸酯3-羟化酶(4-hydroxyphenylacetate 3-hydroxylase,4HPA3H)可以催化对香豆酸生物合成咖啡酸。为了实现4HPA3H的扩大生产和咖啡酸的高效生物合成，首先构建过表达4HPA3H的大肠杆菌工程菌，其次使用5 L发酵罐进行高密度发酵生产4HPA3H，再而优化采用工程菌株进行全细胞催化产咖啡酸的条件。最终实现了在5 L发酵罐中发酵，工程菌株生物量达到干重34.80 g/L。通过使用5 L发酵罐作为生物反应器进行全细胞催化，经过6 h的催化可产生18.74 g/L (0.85 g/(L·OD₆₀₀))咖啡酸，摩尔转化率为78.81%，是目前文献报道4HPA3H以对香豆酸为底物合成咖啡酸的最高水平。初步实现了高密度培养大肠杆菌表达4HPA3H并高效生物合成咖啡酸，为工业化生产奠定了基础。

关键词:4-羟基苯乙酸酯3-羟化酶;咖啡酸;高密度发酵;全细胞催化

Abstract:

The 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H), originated from Escherichia coli, converts p-coumaric acid to caffeic acid. In order to improve the efficiency of caffeic acid biosynthesis, we engineered E. coli for overexpression of 4HPA3H. The high-density fermentation of the engineered E. coli was conducted in a 5 L bioreactor. Subsequently, the conditions for whole-cell biocatalysis were optimized. The dry cell weight of the 4HPA3H-expressed strain reached 34.80 g/L. After incubated in the bioreactor for 6 h, 18.74 g/L (0.85 g/(L·OD₆₀₀)) of caffeic acid was obtained, with a conversion rate of 78.81% achieved. To the best of our knowledge, the titer of caffeic acid is the highest reported to date. The high-density fermentation of E. coli for overexpression of 4HPA3H and the efficient biosynthesis of caffeic acid may facilitate future large-scale production of caffeic acid.

Key words:4-hydroxyphenylacetate 3-hydroxylase;caffeic acid;high-density fermentation;whole-cell biocatalysis