摘要
酪醇是一种多酚类天然产物,广泛应用于化工、医药和食品等领域。目前大肠杆菌(Escherichia coli)从头合成酪醇存在发酵菌体密度低和产量低等问题。为此,本研究将前期获得苯丙酮酸脱羧酶突变体ARO10F138L/D218G与不同来源的醇脱氢酶融合表达,最优组合ARO10F138L/D218G-L-YahK酪醇产量达到1.09 g/L。为进一步提高酪醇产量,敲除了4-羟基苯乙酸竞争途径关键基因feaB,使酪醇产量提高了21.15%,达到1.26g/L。针对酪醇发酵菌体密度低的问题,通过群体感应系统动态调控酪醇合成途径,减轻酪醇对底盘细胞的毒性作用,缓解生长抑制,使其产量提高了33.82%,达到1.74 g/L。在2 L发酵罐中,群体感应动态调控工程菌TRFQ5的酪醇产量达到4.22g/L,OD600值达到42.88,分别较静态诱导表达工程菌TRF5提高了38.58%和43.62%。本研究应用基因敲除技术,阻断了酪醇合成竞争途径;同时结合群体感应动态调控策略,减轻了酪醇毒性对底盘细胞的生长抑制,从而有效地提高了酪醇产量。本研究对其他高毒性化学品的生物合成具有良好的借鉴和应用价值。
Tyrosol is a natural polyphenolic product that is widely used in chemical,pharmaceutical and food industries.Currently,the de novo synthesis of tyrosol by Escherichia coli suffers from issues such as low cell density and poor yield.Therefore,the phenylpyruvate decarboxylase mutant ARO10F138L/D218G obtained in our previous study was fused with an alcohol dehydrogenase from different microorganisms for fusion expression,and the optimal ARO10F138L/D218G-L-YahK produced 1.09 g/L tyrosol in shake flasks.In order to further improve tyrosol production,feaB,a key gene in the competing pathway of 4-hydroxyphenylacetic acid,was knocked out,and the resulted strain produced 1.26 g/L tyrosol with an increase of 21.15%compared to that of the control.To overcome the low cell density in tyrosol fermentation,the quorum-sensing circuit was used to dynamically regulate the tyrosol synthesis pathway,so as to alleviate the toxic effect of tyrosol on chassis cells and relieve the growth inhibition.Using this strategy,the yield of tyrosol was increased to 1.74 g/L,a 33.82%increase.In a 2 L fermenter,the production of tyrosol in the engineered strain TRFQ5 dynamically regulated by quorum-sensing reached 4.22 g/L with an OD600 of 42.88.Compared with those in the engineered strain TRF5 statically regulated by induced expression,the yield was increased by 38.58%and the OD600 was enhanced by 43.62%.The combination of blocking the competing pathway using gene knockout technology,and reducing the inhibitory effect of tyrosol toxicity on chassis cells through quorum-sensing dynamic regulation increased the production of tyrosol.This study may facilitate the biosynthesis of other chemicals with high toxicity.
作者
沈玉平
周紫微
贺茜
尹乐义
何春兰
张祖姣
SHEN Yuping;ZHOU Ziwei;HE Xi;YIN Leyi;HE Chunlan;ZHANG Zujiao(College of Chemistry and Bioengineering,Hunan University of Science and Engineering,Yongzhou 425199,Hunan,China;Hunan Engineering Research Center for Research and Development of Plant Resources in Nanling Area,Yongzhou 425199,Hunan,China;Hunan Provincial Engineering Research Center for Ginkgo biloba,Yongzhou 425199,Hunan,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2023年第8期3379-3393,共15页
Chinese Journal of Biotechnology
基金
湖南省自然科学基金(2022JJ30273,2020JJ5204)
湖南省教育厅科学研究项目(22B0804)
湖南省应用特色学科建设项目。
关键词
酪醇
大肠杆菌
群体感应
动态调控
tyrosol
Escherichia coli
quorum sensing
dynamic control