透明颤菌血红蛋白基因在地衣芽孢杆菌ATCC9945a中的表达及作用  

Expression of Vitreoscilla Hemoglobin Gene in Bacillus Licheniformis ATCC9945a and Its Effects on Fermentation Characters

作　　者：孙廷丽[1] 施庆珊[1] 欧阳友生[1] 谢小保[1] 陈仪本[1] 

机构地区：[1]广东省微生物研究所广东省菌种保藏与应用重点实验室广东省微生物应用新技术公共实验室广东省华南应用微生物重点实验室-省部共建国家重点实验室培育基地,广东广州510070

出　　处：《生物技术》2011年第5期16-22,共7页Biotechnology

基　　金：广东省高新技术产业化项目("生物基可降解高分子材料生物转化关键技术研究";2009A010700005)资助

摘　　要：目的:研究透明颤菌血红蛋白基因(vgb)在产聚γ-谷氨酸(γ-PGA)的地衣芽孢杆菌ATCC9945a中的表达及对其生物量和产量的影响。方法:以大肠杆菌-枯草芽孢杆菌的穿梭表达载体pUBC19为骨架,构建含有枯草芽孢杆菌的组成型启动子P43和透明颤菌血红蛋白结构基因的穿梭表达载体pUBC19-PV,并通过电击转化得到重组的产γ-PGA的地衣芽孢杆菌(B.licheniformis)。一氧化碳差光谱验证重组B.licheniformis中是否表达了有活性的血红蛋白。摇瓶发酵试验研究重组菌株和对照菌株生物量和发酵产物产量的变化。结果表明,重组菌株的生长明显比对照菌株快,但是γ-PGA的产量却比原始菌株低:在正常供氧时,其产量12.8g/L,比亲株产量21.7g/L下降了41%,贫氧环境下产量8.8g/L,比亲株产量12.8g/L降低了31%。结论:vgb在重组菌株中表达了有活性的的透明颤菌血红蛋白,并可以促进细胞的生长,但聚谷氨酸的产量却有所下降。文章针对聚γ-谷氨酸产量的下降原因进行了讨论,并对下一步的工作提出了建议。Objective：To investigate the expression of Vitrescilla hemoglobin gene（vgb） in Bacillus licheniformis and its effect on the biomass and the production of Poly γ-glutamate（γ-PGA）.Method： A new vector pUBC19-PV was constructed by using pUBC19,a shuttle vector between Gram negative and positive,in which vgb gene including P43 promoter was inserted.Then transform it into Bacillus licheniformis ATCC9945a by electroporation.The expression of VHb was confirmed by CO-difference spectra analysis.Shake flask fermentation was used to determine the biomass and production of γ-PGA in both of the control strains and recombinant strains.Result： The expression of the intracellular VHb in the recombinant B.licheniformis ATCC9945a improved its biomass under both normal and limited oxygen supply conditions,but the production of γ-PGA under normal and limited aeration were both decreasing.The yield of r-PGA of the recombinant strain was 12.8g/L at normal aeration conditions and showed a decreasing of 41% because of the yield of parent strain was 21.7g/L.Under limited aeration conditions,the yield of recombinant strain turned out to be 8.8g/L and decreased 31% compared to the yield 12.8g/L obtained of the parent strain at the same conditions.Conclusion： The expression of Vitrescilla hemoglobin（VHb） in the B.licheniformis can improve the growth of cells,while the production of γ-PGA was poor performed.There also gives a discussion about this decrease of γ-PGA and several suggestions for the further research.

关 键 词：地衣芽胞杆菌 透明颤菌血红蛋白 穿梭表达载体 聚Γ-谷氨酸 

分 类 号：Q812[生物学—生物工程]