维生素B_(12)(钴胺素)具有重要的生理学功能,广泛应用于制药、食品等行业,黏着剑菌(Ensifer adhaerens)是常用生产菌株之一。为进一步提高E.adhaerens生产维生素B_(12)的能力,采用常压室温等离子体(atmospheric and room temperature pl...维生素B_(12)(钴胺素)具有重要的生理学功能,广泛应用于制药、食品等行业,黏着剑菌(Ensifer adhaerens)是常用生产菌株之一。为进一步提高E.adhaerens生产维生素B_(12)的能力,采用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变处理E.adhaerens,并从中筛选出高产维生素B_(12)的诱变菌株。确定了ARTP诱变处理的时间为80 s,输出功率为100 W。应用流式细胞仪、酶标仪、移液工作站等仪器对诱变菌株进行分选、检测。经过2轮ARTP诱变处理、初筛与复筛之后,最终筛选得到1株高产维生素B_(12)的突变菌株46H-1,摇瓶发酵至7.5 d,维生素B_(12)的产量达到110.25 mg/L,相比于原始菌株提高了24.8%,并且通过了遗传稳定性验证。该文所建立的高通量筛选方法能够快速、高效地筛选到高产菌株,有效降低生产成本。展开更多
Cobalamin is an essential human vitamin widely used in the pharmaceutical,food,and feed additive industries and currently produced by bacteria or archaea.Ensifer adhaerens HY-1 is an industrial strain that also produc...Cobalamin is an essential human vitamin widely used in the pharmaceutical,food,and feed additive industries and currently produced by bacteria or archaea.Ensifer adhaerens HY-1 is an industrial strain that also produces cobalamin.However production outputs are poor and the specific synthesis pathways require characterization.In this study,the whole genome sequence of E.adhaerens HY-1 was generated and annotated,and genes associated with cobalamin biosynthesis were identified.Then,three genes,CobSV,CobQ,and CobW were identified as the most efficient ones for enhancing cobalamin synthesis.By transcriptome sequencing of E.adhaerens HY-1 cells at different growth stages,65 endogenous promoters with different gradient strengths were identified.After combined expression of different strength promoters and key genes,a high cobalamin-producing recombinant strain,‘hmm’(genotype:PmetH-CobSV-PibpA-CobQ-Pmdh-CobW),was generated.Cobalamin production was 143.8 mg/L in shaking flasks,which was 41.0%higher than the original strain.Cobalamin production was further enhanced to 171.2 mg/L using fed-batch fermentation.Importantly,our data and novel approach provide important references for the analysis of cobalamin synthesis and other metabolites in complex metabolic pathways.展开更多
文摘维生素B_(12)(钴胺素)具有重要的生理学功能,广泛应用于制药、食品等行业,黏着剑菌(Ensifer adhaerens)是常用生产菌株之一。为进一步提高E.adhaerens生产维生素B_(12)的能力,采用常压室温等离子体(atmospheric and room temperature plasma,ARTP)诱变处理E.adhaerens,并从中筛选出高产维生素B_(12)的诱变菌株。确定了ARTP诱变处理的时间为80 s,输出功率为100 W。应用流式细胞仪、酶标仪、移液工作站等仪器对诱变菌株进行分选、检测。经过2轮ARTP诱变处理、初筛与复筛之后,最终筛选得到1株高产维生素B_(12)的突变菌株46H-1,摇瓶发酵至7.5 d,维生素B_(12)的产量达到110.25 mg/L,相比于原始菌株提高了24.8%,并且通过了遗传稳定性验证。该文所建立的高通量筛选方法能够快速、高效地筛选到高产菌株,有效降低生产成本。
基金supported by the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005)the National Science Fund for Excellent Young Scholars(21822806).
文摘Cobalamin is an essential human vitamin widely used in the pharmaceutical,food,and feed additive industries and currently produced by bacteria or archaea.Ensifer adhaerens HY-1 is an industrial strain that also produces cobalamin.However production outputs are poor and the specific synthesis pathways require characterization.In this study,the whole genome sequence of E.adhaerens HY-1 was generated and annotated,and genes associated with cobalamin biosynthesis were identified.Then,three genes,CobSV,CobQ,and CobW were identified as the most efficient ones for enhancing cobalamin synthesis.By transcriptome sequencing of E.adhaerens HY-1 cells at different growth stages,65 endogenous promoters with different gradient strengths were identified.After combined expression of different strength promoters and key genes,a high cobalamin-producing recombinant strain,‘hmm’(genotype:PmetH-CobSV-PibpA-CobQ-Pmdh-CobW),was generated.Cobalamin production was 143.8 mg/L in shaking flasks,which was 41.0%higher than the original strain.Cobalamin production was further enhanced to 171.2 mg/L using fed-batch fermentation.Importantly,our data and novel approach provide important references for the analysis of cobalamin synthesis and other metabolites in complex metabolic pathways.
