Optimization Of Fermentation Conditions Of Bacillus Subtilis Producing Pulcherriminic Acid And Expression And Purification Of Key Enzymes

Pulcherriminic acid is derived from the second ring shrinkage leucine(Cyclo(L-Leu-L-Leu),cLL),is a kind of synthesized from Bacillus and yeast ring dipeptide.The structure of different hydroxyl oxime acid groups can chelate four ferric iron ions to form red brown pulcherrimin.It makes pulcherriminic acid production in the microbes,which can compete with other microbes around iron ions,thus to obtain competitive advantage and achieve bacteriostatic action.Based on the bacteriostatic effect of pulcherriminic acid,pulcherriminic acid can be a beneficial substance or a functional factor.Members of my research group have detected pulcherriminic acid and its intermediates or analogs in Chinese liquor(unpublished data).In this paper,Daqu,fermented grains and pit mud were used as samples,and the target strains were isolated and identified by preliminary screening,re-screening,morphological,physicochemical properties and molecular biology methods.Taking the target strain as the research object of genetic engineering technology,the key enzyme genes in the pathway of pulcherriminic acid production,namely cyclic dipeptide synthase(YvmC)and cytochrome oxidase P450(CypX),were amplified by PCR,cloned and heterologously expressed,and the crystallization conditions of the two key enzymes were preliminarily discussed.The main research results of this paper are as follows:1.Screening of target strains for producing pulcherriminic acid: Daqu,fermented grains and pit mud of a Luzhou-flavor liquor were selected as the screening objects to obtain the strains capable of producing pulcherriminic acid;Single strain fermentation was conducted on the target strain,fermentation products were collected,identified and analyzed to obtain a strain Y1 with the highest yield.The strain was identified as Bacillus subtilis Y1 by molecular biological methods.2.Optimization of the fermentation conditions of the target strain producing pulcherrimin: strain Y1 and the basic fermentation medium were used for fermentation,and the optimal fermentation medium composition was determined by single factor experiment and orthogonal experiment.The yield of strain Y1 pulcherrimin was1230.32mg/L by orthogonal experiment.The optimal fermentation conditions of strain Y1 were as follows: temperature 40℃,inoculation amount 2%,fermentation time 36 h,2L fermentation experiment,and the yield of pulcherriminic acid was 1332.12mg/L.3.Acquisition of YvmC and CypX gene fragments and construction of vector:YvmC and CypX genes were obtained by PCR amplification,and the recombinant plasmids of YvmC and CypX genes were successfully constructed with pET-28 a plasmid respectively.4.Heterologous expression and purification of YvmC and CypX genes: 1)Transformation: the recombinant plasmids of YvmC and CypX genes were transformed into E.coli BL21(DE3)for heterologous soluble expression;2)The optimal expression conditions of the recombinant strain: YvmC was 18℃,12 h,and the final IPTG concentration was 1mmol/L;CypX was 18℃,10 h,and the final IPTG concentration was 0.1mmol/L.5.Preliminary study on crystallization conditions of YvmC protein and CypX protein: 1)YvmC was concentrated at a concentration of 10mg/mL,and the concentrated protein was initially screened by sitting drop method under crystallization conditions.The initial crystallization conditions were 1.0M Sodium citrate-0.1M CHES PH9.5,24% PEG 1500-20% Glycerol,0.1M CHES PH9.5-50% PEG 200;2)The crystallization conditions of CypX were explored at 8.9mg/mL,but no crystal results were obtained.