摘要
【目的】地衣芽孢杆菌(Bacillus licheniformis)是发酵工业中表达异源蛋白的重要底盘细胞,筛选易转化和高生物量的B.licheniformis菌株,为有效提升宿主的改造效率和蛋白表达水平提供菌种资源。【方法】经过富集、gyrB扩增与16S rDNA序列分析从土壤中筛选与地衣芽孢杆菌同源性较高的菌株,将所筛菌株的电转效率和生物量与工业常用底盘细胞B.licheniformis 2709进行比较分析。对筛选出的易电转和高生物量菌株进行形态学与生理生化鉴定,并进行抗生素敏感性测定和高温碱性环境生长情况测定。【结果】筛选出10株与地衣芽孢杆菌同源性较高的菌株,其中菌株1-33电转化效率为6700 CFU/μg DNA,是B.licheniformis 2709的13.7倍。在豆粕玉米粉半固体培养基和SR液体培养基中培养,菌株1-33最大生物量达到5.2×10^(10) CFU/mL和6.8 g/L,分别为B.licheniformis 2709的1.71倍和1.3倍。进一步经过形态学、生理生化特性实验将菌株1-33最终鉴定为B.licheniformis,并表现出多种常用抗生素的敏感性和良好的耐碱与耐高温的能力。【结论】筛选出一株易电转和高生物量的B.licheniformis 1-33,为开发成高效表达外源蛋白的宿主菌奠定基础。
【Objective】Bacillus licheniformis is an important chassis cell for expressing heterologous proteins in the fermentation industry.Screening B.licheniformis strains with easy electro-transfection and high biomass provides strain resources for effectively improving the host modification efficiency and protein expression level.【Method】Screening of strains with high homology to B.licheniformis from soil was carried out through enrichment,gyrB amplification,and 16S rDNA sequence analysis.The electro-transfection efficiency and biomass of the screened strains were compared and analyzed with the commonly used chassis cell B.licheniformis 2709 in the industry.The screened strains with easy electro-transfection and high biomass were identified morphologically and physiologically and biochemically as well as were subjected to antibiotic susceptibility assay and growth in high temperature and alkaline environment.【Result】Ten strains with high homology to B.licheniformis were screened.Among them,the strain 1-33 presented the highest electro-transfection efficiency with a maximum value of 6700 transformants perμg DNA,which was 13.7 times higher than that of B.licheniformis 2709.Cultured in semi-solid soymeal-corn flour medium and SR liquid medium,the maximum biomass of strain 1-33 was found to be 5.2×10^(10) CFU/mL and 6.8 g/L,respectively,surpassing B.licheniformis 2709 by 1.71 and 1.3-fold.Further morphological and physiological experiments identified strain 1-33 as Bacillus licheniformis,which had sensitivity to various commonly used antibiotics and good alkaline and heat resistance.【Conclusion】A B.licheniformis strain 1-33 with high electro-transfection efficiency and elevated biomass is screened,thereby foundation for its further development as an efficient host for exogenous protein expression in industrial process is established.
作者
杜薇
李志敏
邢晏铭
刘蒲临
缪礼鸿
DU Wei;LI Zhi-min;XING Yan-ming;LIU Pu-lin;MIAO Li-hong(College of Life Science and Technology,Wuhan Polytechnic University,Wuhan 430023)
出处
《生物技术通报》
CAS
CSCD
北大核心
2024年第9期181-189,共9页
Biotechnology Bulletin
基金
国家重点研发计划(2021YFC2100202)。
关键词
地衣芽孢杆菌
易转化
高生物量
筛选
鉴定
Bacillus licheniformis
easy electro-transfection
high biomass
screening
identification
