Directive Breeding Of Bacitracin-high-producing Strains

Bacitracin is a widely used peptide broad-spectrum antibiotic produced by Bacillus subtilis and Bacillus licheniformis.Microbial breeding technology has experienced rapid development in several stages including natural breeding,mutation breeding,hybrid breeding,metabolic engineering breeding,genetic engineering breeding,and synthetic biology breeding,However,bacitracin is a secondary metabolite with the unclear mechanism of biosynthesis and regulation,and random mutation combined with directional screening for its excellent production strains will still occupy an advantage.In this study,an industrial strain for bacitracin production,Bacillus licheniformis DW2 was treated with random mutation through physical and chemical mutagenesis,the use of high-throughput screening methods based on 96/48-well plates,combined with flow cytometry sorting technology,to achieve the purpose of large-scale mutant individuals identified and screened rapidly.The fermentation conditions were then optimized for the obtained high-yield strain by mutagenesis to improve the level of potency in the 5-L fermenter.By comparing the genome from mutagenized the high-yield strain parsed by de novo sequencing with the reference genome in the database and the mutation information was seized,and by comparing with the genome providing with the bacitracin-synthesized gene cluster,common and unique genes were obtained,and the metabolic characteristics of the DW2 strain were analyzed.The research results of this article are as follows:(1)High-throughput screening of high-producing-bacitracin strains.This study established a process for compound mutagenesis that atmospheric pressure room temperature plasma(ARTP)mutagenesis was foremost,and ethyl methanesulfonate(EMS)mutagenesis,nitrosoguanidine(NTG)mutagenesis and ultraviolet(UV)mutagenesis were auxiliary.Based on the chemical properties of the amide bond in the bacitracin molecule to react with copper ions in an alkaline environment,a high-throughput screening method that biuret reaction combined with trichloroacetic acid(TCA)precipitation was established.With the help of flow cytometry and microplate reader,the purpose of rapid sorting and identification from mutant high-yield strains was achieved.Through shake flask rescreening,excellent strains with a 22.2% increase in bacitracin production were obtained,which proved the feasibility of methods about mutagenesis and screening;(2)The potency of bacitracin was increased in the 5-L fermenter by fermentation optimization.The difference of fermentation process between 5-L fermenter and 250-ml shake flask was compared under the same fermentation medium,and a series of optimization strategies were developed to make bacitracin production reach a reasonable level in the 5-L fermenter.By optimizing the composition of fermentation medium,the potency of bacitracin was increased by 14.9%.The potency of bacitracin was increased by 26.3% through adding sodium acetate in the medium.Employing the strategy that fed-batch by one-time and at the constant speed for sodium acetate,the yield of bacitracin was increased by 27.3% and 28.1%,respectively.Compared with shake flask fermentation,the potency of bacitracin in the 5-L fermenter,especially bacitracin A,was markedly increased,and the potency of bacitracin A was increased by 25.7% after optimization.(3)The genetic information of DW2 was mined by comparative genomics.Second-generation sequencing with Illumina HiSeq and third-generation sequencing with PacBio were used to de novo sequencing for the genome in mutant high-yield strain 1#7E.The corresponding genomes from the National Genomic Data Center(NGDC)and the National Center for Biotechnology Information(NCBI)were used as a reference to obtain the mutation site of high-yield mutant strains by comparative genomics;Meanwhile,the genomes taking along with the bacitracin-synthesized gene cluster were analyzed,and the common and unique genes of DW2 were obtained.These genes were mostly related to amino acids and derivatives metabolism,protein metabolism and carbohydrate metabolism,which was instrumental in explaining the reason that Bacillus licheniformis DW2 has provided with a high-production bacitracin,so as to provide breeding at the molecular level for strains of high-production bacitracin with reference.