摘要
【背景】大肠杆菌(Escherichia coli)由于生长性能优良、遗传背景清楚、遗传操作手段成熟,是合成β-法尼烯的合适生产菌,但其合成β-法尼烯的产量目前仍不能满足工业化生产的需求。【目的】通过诱变筛选技术选育β-法尼烯高产突变株。【方法】采用常压室温等离子体(atmosphericand room temperature plasma,ARTP)诱变技术和紫外线照射对出发菌株大肠杆菌EC-16进行复合诱变,并以异戊烯焦磷酸耐受性为选择压力进行平板初筛,之后进行摇瓶复筛,最后进行发酵罐验证。通过连续多代培养筛选到的高产突变菌株,观察其遗传稳定性。【结果】经复合诱变选育筛选出一株β-法尼烯高产突变株E.coliHVK-9,其产量高达22.1g/L,相比出发菌株提高了168.74%。【结论】采用ARTP-紫外复合诱变,再结合异戊烯焦磷酸抗性筛选的集成方法,使得诱变菌株的正突变率大大提高,可以有效地提高诱变菌株的β-法尼烯产量。突变株HVK-9作为工业化发酵生产菌种具有较好的遗传稳定性,为β-法尼烯的工业化生产和应用奠定了良好的基础。
[Background]Due to excellent growth performances,clear genetic backgrounds and mature genetic manipulations,Escherichia coli typical strains are commonly selected for producingβ-farnesene,but the yields ofβ-farnesene still cannot meet the needs of industrial production until now.[Objective]Combined mutagenesis breeding techniques were used to obtainβ-farnesene producing strains with high yields.[Methods]Escherichia coli EC-16 was bred by combination mutation methods of atmospheric and room temperature plasma(ARTP)and ultraviolet(UV),through the first screening with isopentenyl pyrophosphate(IPP)screening plate as selection pressure and the following second screening step with flask fermentation culture,finally subjected to fermentation tank verification.The genetic stability of the highly productive mutant strains was carefully comparatively tested by continuous multi-generation cultured.[Results]A mutant strain named E.coli HVK-9 was obtained and the yield ofβ-farnesene reached up to 22.1 g/L,which was 168.74%higher than that of the original strain.[Conclusion]The combinational mutations by virtue of ARTP-UV with isopentenyl pyrophosphate resistance screening model in this study can significantly improve the positive mutation rates,which can finally confirm the effectively increase with the yield ofβ-farnesene.The mutant strain HVK-9 has good genetic stability as an industrial fermentation strain,which lays a good foundation for the industrial production and application ofβ-farnesene.
作者
潘俊潼
寇凤雨
刘瑞艳
王一然
赵辉
PAN Jun-Tong;KOU Feng-Yu;LIU Rui-Yan;WANG Yi-Ran;ZHAO Hui(Bioresources Laboratory,Department of Bioengineering,College of Chemical and Environmental Engineering,Shandong University of Science and Technology,Qingdao,Shandong 266590,China;Shandong Hongda Biotechnology Co.Ltd.,Linyi,Shandong 276400,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2020年第2期542-551,共10页
Microbiology China
基金
国家自然科学基金(41772095,41302079).
关键词
法尼烯
常压室温等离子体(ARTP)
紫外线
异戊烯焦磷酸抗性筛选
复合诱变
farnesene
Atmospheric and room temperature plasma(ARTP)
Ultraviole(UV)
Isopentenyl pyrophosphate(IPP)resistance screening
Combined mutagenesis
