维生素B12(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B12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B12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B12的突变菌株46H-1,摇瓶发酵至7.5 d,维生素B12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。
Vitamin B12 (cobalamin) has important physiological functions, which is widely used in pharmaceutical, food and other industries. Ensifer adhaerens is one of the commonly used strains for the production of vitamin B12. The processing time of atmospheric and room temperature plasma (ARTP) was 80 s, and the output power was 100 W. Through two rounds of ARTP mutagenesis, preliminary screening and re-screening, a genetic stability mutant strain 46H-1 with high vitamin B12 production was screened. By fermentation in shaking flask for 7.5 d, the titer of vitamin B12 reached 110.25 mg/L, which increased by 24.8% compared with E. adhaerens. The established high-throughput screening method could apply to harvest the excellent performance mutant strains quickly, thus reducing the cost of production.
[1] MINOT G R,MURPHY W P.Treatment of pernicious anemia by a special diet[J].Jama,1983,250:3 328-3 335.
[2] SMITH E L.Presence of cobalt in the anti-pernicious anaemia factor[J].Nature,1948,162(4 108):144.
[3] 马蕙, 王丽丽,张春晓,等.维生素B12的生物合成、发酵生产与应用[J].生物工程学报,2008,24(6):927-932.
MA H,WANG L L,ZHANG C X,et al.Biosynthesis,fermentation and application of vitamin B12-A review[J].Chinese Journal of Biotechnology,2008,24(6):927-932.
[4] WARREN M J,RAUX E,SCHUBERT H L,et al.The biosynthesis of adenosylcobalamin (vitamin B-12)[J].Natural Product Reports,2002,19(4):390-412.
[5] CHAN C W,MONDRAGÓN A.Crystal structure of an atypical cobalamin riboswitch reveals RNA structural adaptability as basis for promiscuous ligand binding[J].Nucleic Acids Research,2020,48(13):7 569-7 583.
[6] MARSH E N G,PATTERSON D P,LI L,et al.Adenosyl radical:Reagent and catalyst in enzyme reactions[J].Chembiochem,2010,11:604-621.
[7] XIA W,CHEN W,PENG W F,et al.Industrial vitamin B12 production by Pseudomonas denitrificans using maltose syrup and corn steep liquor as the cost-effective fermentation substrates[J].Bioprocess and Biosystems Engineering,2015,38(6):1 065-1 073.
[8] WANG P,ZHANG Z W,JIAO Y J,et al.Improved propionic acid and 5,6-dimethylbenzimidazole control strategy for vitamin B12 fermentation by Propionibacterium freudenreichii[J].Journal of Biotechnology,2015,193:123-129.
[9] THI VU H,ITOH H,ISHII S,et al.Identification and phylogenetic characterization of cobalamin biosynthetic genes of Ensifer adhaerens[J].Microbes and Environments,2013,28(1):153-155.
[10] RYCHEN G,AQUILINA G,AZIMONTI G,et al.Safety and efficacy of vitamin B12 (in the form of cyanocobalamin) produced by Ensifer spp.as a feed additive for all animal species based on a dossier submitted by VITAC EEIG[J].Efsa Journal,2018,16:e05336.
[11] FANG H,LI D,KANG J,et al.Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12[J].Nature Communications,2018,9(1):4 917.
[12] VITRESCHAK A G,RODIONOV D A,MIRONOV A A,et al.Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural element[J].RNA,2003,9(9):1 084-1 097.
[13] RATHORE D S,LOPEZ-VERNAZA M A,DOOHAN F,et al.Profiling antibiotic resistance and electrotransformation potential of Ensifer adhaerens OV14;a non-Agrobacterium species capable of efficient rates of plant transformation[J].FEMS Microbiology Letters,2015,362(17):fnv126.
[14] ZHANG X,ZHANG X F,LI H P,et al.Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool[J].Applied Microbiology and Biotechnology,2014,98(12):5 387-5 396.
[15] ALVAREZ J C.High-throughput docking as a source of novel drug leads[J].Current Opinion in Chemical Biology,2004,8:365-370.
[16] LIANG Y,ZHU Y,FANG Q,et al.Advances in high-throughput,automated techniques and instrumention for protein crystallization screening[J].Chinese Journal of Chromatography,2016,34:1 137-1 144.
[17] DU G S,FANG Q,DEN TOONDER J M J.Microfluidics for cell-based high throughput screening platforms-A review[J].Analytica Chimica Acta,2016,903:36-50.
[18] LUO Z S,ZENG W Z,DU G C,et al.A high-throughput screening procedure for enhancing pyruvate production in Candida glabrata by random mutagenesis[J].Bioprocess abd Biosystems Engineering,2017,40(5):693-701.
[19] CAO X,LUO Z,ZENG W,et al.Enhanced avermectin production by Streptomyces avermitilis ATCC 31267 using high-throughput screening aided by fluorescence-activated cell sorting[J].Applied Microbiology Biotechnolgy,2018,102:703-712.
[20] CAI Y Y,XIA M M,DONG H N,et al.Engineering a vitamin B-12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti[J].BMC Biotechnology,2018,18:27.
[21] ZENG W,GUO L,XU S,et al.High-throughput screening technology in industrial biotechnology[J].Trends in Biotechnology,2020,38:888-906.
