Physical And Chemical Mutagenesis Combined With Adaptive Laboratory Evolution As A Strategy To Improve The High Temperature Resistance Of Pseudomonas Plecoglossicida JUIM01

2-ketogluconic acid(2KGA)is an organic acid with a wide range of applications,which has been used as the precursor of synthetic food antioxidant D-isoascorbic acid and its sodium salt.Generally,2KGA is produced by microbial fermentation genus Pseudomonas,using D-glucose as the substrate to the conversion of 2KGA.Under high temperature stress,the production of fermentation product decrease significantly due to the shifting in glucose metabolism from extracellular oxidation pathway to intracellular phosphorylation pathway.Therefore,the breeding of high temperature resistant strains of Pseudomonas has a significant research value both in theory and application.In this study,P.plecoglossicida JUIM01,a major industrial strain of 2KGA production was utilized as initial strain.By using ultraviolet(UV),diethyl sulfate(DES)and atmospheric room temperature plasma(ARTP)mutagenesis to developed high temperature resistant mutant strain.Moreover,the adaptive evolution technology of the mutagenised strain was applied to improve the tolerance of the high temperature,and obtained high-temperature resistant 2KGA high-yield strains with industrial application potential.The response of glucose metabolism to high temperature stress in Pseudomonas was investigated by comparing the differences in the transcription levels of 2KGA synthesis and metabolism related genes at different temperatures between the parent strain and the evolved strain.The main conclusions are as follows:(1)The P.plecoglossicida JUIM01 suspension during mid-logarithmic growth(14h)was exposed to UV,DES and ARTP as mutagenesis treatment,respectively.High throughput screening by calcium carbonate bromocresol green to filter the high acid production by high temperature resistant strains,while further screening steps by high temperature fermentation test to evaluate the higher acid production level and genetic stable traits.Eventually,three high temperature resistant with high yield 2KGA strains,UV mutation strain JUIM01-UV14,UV-DES complex mutant JUIM01-UD4 and ARTP mutant JUIM01-A25 were obtained.The fermentation conversion rates at 40?were84.68%,84.70%and 85.67%,respectively,which improved by 5.08%,5.11%and 6.32%relative to the parental strain.(2)Out of 3 mutant strains JUIM01-UV14,JUIM01-UD4 and JUIM01-A25variance in high thermal-tolerance and high thermal fermentation acid production level,JUIM01-A25 opted for adaptive evolution initial strain.Based on the JUIM01-A25growth condition under various temperatures,the passage was done once in every 12 h during its adaptive evolution phase.After 40 generations of adaptive evolution at 40?,an evolutionary strain JUIM01-T1 was obtained which could grow normally at 40?.The fermentation under high temperature results showed that the maximum cell concentration(OD_650nm)of JUIM01-T1 was 9.56,as opposed to 8.29 of its JUIM01parental strain;The conversion rate of JUIM01-T1 fermentation was 88.10%,an increase of 9.34%from its parental strain,which was approximate to the fermentation conversion rate(89.70%)of its parental strain at 32?;The JUIM01-T1 fermentation intensity was 2.77 g/L·h,which improved by 16.88%of its parental strain and an increase of 18.88%in production intensity relative to its parental strain at 32?.(3)Under high temperature stress,the expression of genes associated with extracellular oxidation pathway(2KGA synthesis pathway)of adaptive evolved strain JUIM01-T1 and the parent strain down-regulated to different degrees,however,the expression of glucose dehydrogenase genes(gcd)and gluconate dehydrogenase large subunit gene(gnd L)of JUIM01-T1 were higher than parental strain,which demonstrated that JUIM01-T1 had a relatively high level of 2KGA biosynthesis.Moreover,the expression of glucose kinase gene(glk)and glucose 6-phosphate dehydrogenase gene(zwf)in the glucose phosphorylation pathway was up-regulated;the expression of 2KGA transporter gene(kgu T),2KGA kinase gene(kgu K),2-Keto-6-phosphate gluconate reductase gene(kgu D)in the 2KGA phosphorylation pathway(2KGA catabolism pathway)was down-regulated.However,the zwf expression level of JUIM01-T1 was higher than parent strain,and the expression level of each gene in the 2KGA phosphorylation pathway was significantly lower than that of the parent strain,which indicated that the glucose phosphorylation pathway of JUIM01-T1 was more active than that of the parent strain(favorable for bacterial growth),and the 2KGA catabolism pathway was less active than the parent strain(facilitates the accumulation of 2KGA).Corresponding to the up-regulation of the glucose phosphorylation pathway glk and zwf genes,the expression of transcription factor Hex R was down-regulated;while the up-regulated expression of transcription factor Ptx S was in accordance with the down-regulation of the expression of each structural gene in the kgu operon.