定向进化植物乳杆菌和痤疮丙酸杆菌高产共轭亚油酸

doi:10.7506/spkx1002-6630-20200305-072

食品科学 ›› 2021, Vol. 42 ›› Issue (12): 99-106.doi: 10.7506/spkx1002-6630-20200305-072

定向进化植物乳杆菌和痤疮丙酸杆菌高产共轭亚油酸

时旭，刘瑛，史海粟，武俊瑞，乌日娜，牛雪晴，洛雪，岳喜庆

（沈阳农业大学食品学院，辽宁沈阳 112000）

出版日期:2021-06-25 发布日期:2021-06-29
基金资助:
国家自然科学基金青年科学基金项目（31701623）；沈阳农业大学2019年大学生创新创业训练计划项目（201910157057）

Directed Evolution of Lactobacillus plantarum and Propionibacterium acnes for Increased Production of Conjugated Linoleic Acid

SHI Xu, LIU Ying, SHI Haisu, WU Junrui, WU Rina, NIU Xueqing, LUO Xue, YUE Xiqing

(School of Food, Shenyang Agriculture University, Shenyang 112000, China)

Online:2021-06-25 Published:2021-06-29

摘要/Abstract

摘要： 为获得高产共轭亚油酸（conjugated linoleic acid，CLA）的菌株，对亚油酸异构酶系植物乳杆菌的肌球蛋白交叉反应抗原（myosin-cross-reactive antigen，MCRA）基因mcra和痤疮丙酸杆菌的亚油酸异构酶基因lai-p进行克隆，克隆成功后连接到骨架质粒pCold-SUMO上构建两种含mcra基因和lai-p基因的大肠杆菌重组菌株，利用异丙基-β-D-硫代半乳糖苷低温诱导阳性重组菌蛋白表达，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳验证得到目的蛋白表达条带，说明两种基因表达成功。采用易错聚合酶链式反应定向进化mcra基因和lai-p基因，构建重组菌突变体库，利用流式细胞术分选获得吸光度提高和CLA产量提高的重组菌株，其中pCold-ycmcra-gfp重组菌243 株，吸光度最高的为155号菌1.050 6±0.000 4，提高了5.02 倍；pCold-yclai-p-gfp重组菌156 株，产量最高的为39号菌（11.62±0.003 6）μg/mL，提高了2.99 倍。本研究为后期深入了解突变菌株CLA产量提高的原因，并获得两种亚油酸异构酶的产酶机制奠定了一定基础。

关键词: 亚油酸异构酶基因；易错聚合酶链式反应；重组质粒；流式细胞术

Abstract: In order to obtain high-yield conjugated linoleic acid (CLA)-producing strains, the myosin-cross-reactive antigen gene (mcra) from Lactobacillus plantarum and the linoleic acid isomerase gene (lai-p) from Propionibacterium acnes were separately cloned and ligated to pCold-SUMO skeleton plasmid to construct recombinant E. coli strains. The expression of the positive recombinant strains was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at low temperature. The target proteins were validated by SDS-PAGE, indicating successful expression of the two genes. Error-prone polymerase chain reaction was employed to establish a library of recombinant mutant strains with mcra or lai-p. Recombinant mutants with higher absorbance and CLA production were identified by flow cytometry, including 243 recombinant pCold-ycmcra-gfp mutants, among which the one numbered 155 displayed the highest absorbance value (1.050 6 ± 0.000 4), 6.02 times higher than that of the original strain; and 156 recombinant pCold-yclai-p-gfp mutants, among which the one numbered 39 showed the highest yield of (11.62 ± 0.003 6) μg/mL, 3.99 times higher than the original strain. This study can be helpful for further understanding the mechanism for increased CLA production in the mutant strains.

Key words: linoleic acid isomerase gene; error-prone polymerase chain reaction; recombinant plasmid; flow cytometry

中图分类号:

TS201.3

时旭，刘瑛，史海粟，武俊瑞，乌日娜，牛雪晴，洛雪，岳喜庆. 定向进化植物乳杆菌和痤疮丙酸杆菌高产共轭亚油酸[J]. 食品科学, 2021, 42(12): 99-106.

SHI Xu, LIU Ying, SHI Haisu, WU Junrui, WU Rina, NIU Xueqing, LUO Xue, YUE Xiqing. Directed Evolution of Lactobacillus plantarum and Propionibacterium acnes for Increased Production of Conjugated Linoleic Acid[J]. FOOD SCIENCE, 2021, 42(12): 99-106.

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定向进化植物乳杆菌和痤疮丙酸杆菌高产共轭亚油酸

Directed Evolution of Lactobacillus plantarum and Propionibacterium acnes for Increased Production of Conjugated Linoleic Acid

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