食品科学 ›› 2021, Vol. 42 ›› Issue (2): 114-120.doi: 10.7506/spkx1002-6630-20191020-200

• 生物工程 • 上一篇    下一篇

代谢工程优化大肠杆菌高效合成L-苯丙氨酸

门佳轩,熊博,郝亚男,李旋,刘益宁,谢希贤   

  1. (天津科技大学生物工程学院,代谢控制发酵技术国家地方联合工程实验室,天津 300457)
  • 出版日期:2021-01-18 发布日期:2021-01-27
  • 基金资助:
    “十三五”国家重点研发计划重点专项(2018YFA0900304)

Metabolic Engineering of Escherichia coli for Efficient Synthesis of L-Phenylalanine

MEN Jiaxuan, XIONG Bo, HAO Yanan, LI Xuan, LIU Yining, XIE Xixian   

  1. (National and Local United Engineering Lab of Metabolic Control Fermentation Technology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
  • Online:2021-01-18 Published:2021-01-27

摘要: 为获得1 株稳定高产的L-苯丙氨酸工程菌株,利用规律间隔成簇短回文重复序列/Cas9技术,在大肠杆菌W3110中引入已解除反馈抑制的分支酸变位酶/预苯酸脱水酶PheA,随后利用不同强度的启动子对莽草酸途径中各反应酶进行调控,并通过摇瓶发酵确定了莽草酸途径各步反应酶的最适表达强度,最后通过增加前体物磷酸烯醇式丙酮酸供应,获得了1 株性能最佳的L-苯丙氨酸工程菌株PHE12。利用该菌株进行摇瓶发酵24 h,L-苯丙氨酸产量达20.5 g/L。利用发酵罐分批补料发酵48 h后,L-苯丙氨酸产量达81.8 g/L,与目前文献报道的最高产量相比产量提高了12.2%,生产强度和糖酸转化率(葡萄糖计)分别为1.7 g/(L·h)和0.24 g/g,工业前景良好。

关键词: L-苯丙氨酸;大肠杆菌;规律间隔成簇短回文重复序列/Cas9;发酵生产

Abstract: In order to obtain an engineered strain with good genetic stability and high ability to produce L-phenylalanine without plasmid, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to introduce the deinhibited bifunctional enzyme (PheA) of chorisate mutase and prephenate dehydratase into E. coli W3110, and then used the promoters with different strengths to regulate the enzymes involved in the shikimate pathway. The optimal expression intensity of the enzymes in the shikimic acid pathway was determined by shaking flask fermentation. Finally, strain PHE12 with the best performance of L-phenylalanine production was obtained by increasing the supply of the precursor phosphoenolpyruvate. The L-phenylalanine titer of PHE12 was 20.5 g/L after 24 h shaking flask fermentation. And after fed-batch fermentation for 48 h, L-phenylalanine production reached 81.8 g/L, which increased by 12.2% as compared with the highest yield reported in the literature. The production intensity and the conversion rate of sugar and acid were 1.7 g/(L·h) and 0.24 g/g glucose, respectively, indicating that this strain is promising for industrial application.

Key words: L-phenylalanine; Escherichia coli; clustered regularly interspaced short palindromic repeats/Cas9; fermentative production

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