构建大肠杆菌合成生物群落利用混合糖同步发酵生产L-乳酸
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(湖北工业大学生命科学与健康工程学院 发酵工程教育部重点实验室工业发酵省部共建协同创新中心 武汉 430068)

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湖北省教育厅科研计划重点项目(D20201403)


Constructing Escherichia coli Synthetic Microbial Communities for Simultaneously Utilizing Mixed Sugars to Product L-Lactic Acid
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(School of Life and Health Sciences, Hubei University of Technology, Key Laboratory of Fermentation Engineering, Ministry of Education, Cooperative Innovation Center of Industrial Fermentation, Wuhan 430068)

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    摘要:

    目的:实现大肠杆菌混合糖发酵产L-乳酸过程中对葡萄糖-木糖的同步利用。方法:以L-乳酸工程菌大肠杆菌JH16(E.coli B,△frdBC△pflB△ackA△adhE,ldhA::ldhL)为出发菌株,通过RED同源重组技术敲除木糖转运及代谢基因xylFGH、xylE和xylA,获得不能利用木糖的菌株大肠杆菌JH16031。以大肠杆菌JH2705(E.coli JH16,△ptsG△mglB)为出发菌株,敲除葡萄糖转运及代谢基因crr、malX和galP,获得不能利用葡萄糖的菌株大肠杆菌JH27071,以构建大肠杆菌合成生物群落。通过摇瓶和5 L发酵罐试验确定该合成生物群落在混合糖发酵时较优的混合接种比例。结果:以60 g/L葡萄糖和40 g/L木糖为碳源进行发酵,当JH16031与JH27071初始接种比为1∶50,初始OD600nm=0.5,混合糖的利用率最高,在84 h内消耗97%的糖,并在96 h内结束发酵。葡萄糖消耗速率为705 mg/(L·h),木糖消耗速率为435 mg/(L·h),L-乳酸产率为951 mg/(L·h),L-乳酸产量达到92 g/L,糖酸转化率为91%。结论:构建大肠杆菌合成生物群落可实现利用混合糖发酵产L-乳酸过程中葡萄糖和木糖的同步利用。研究结果为工业发酵生产中,使用低成本的木质纤维素为原料,降低发酵成本,提高混合糖利用率提供技术支持。

    Abstract:

    Objective: To achieve the simultaneous utilization of glucose-xylose in the fermentative production of L-lactic acid by Escherichia coli. Methods: E.coli JH16 (E.coli B,△frdBC△pflB△ackA△adhE,ldhA::ldhL), an engineered L-lactic acid productive stain was chosen as start strain, and the xylose transport and metabolism relevant genes xylFGH, xylE, xylA were knocked out by Red homologous recombination to get an xylose utilization deficient strain, E.coli JH16031. The glucose transport and metabolism relevant genes crr, malX, galP were also knocked out from E.coli JH2705(E.coli JH16, △ptsG△mglB) to get a glucose utilization deficient strain, E.coli JH27071. The two resulting strains were co-cultured to form an E.coli synthetic microbial community. Shake flasks and 5 L fermenter experiments were carried out to optimize the inoculum ratio of the two strains of this synthetic microbial community for mixed sugar fermentation. Results: When using 60 g/L glucose and 40 g/L xylose for fermentation, with an initial inoculum ratio of 1∶50 between JH16031 and JH27071, initial OD600nm=0.5, the community has the the highest efficiency for sugar utilization, consuming 97% of total sugars in 84 h and ending the fermentation in 96 h. The L-lactic acid yield reaches 92 g/L, and sugar-acid conversion rate is 91%, with glucose consumption rate as 705 mg/(L·h), xylose consumption rate as 435 mg/(L·h), L-lactic acid productivity as 951 mg/(L·h). Conclusion: The constructed E.coli synthetic microbial community enables the simultaneous utilization of glucose and xylose for L-lactic acid fermentative production with mixed sugars as carbon sources, which may benefits the usage of low-cost lignocellulose in industrial fermentative process with improved sugar utilization efficiency.

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邱炜玥,李敏,陈思宇,赵锦芳,王金华,赵筱.构建大肠杆菌合成生物群落利用混合糖同步发酵生产L-乳酸[J].中国食品学报,2024,24(7):208-218

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  • 收稿日期:2023-07-23
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  • 在线发布日期: 2024-08-22
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