Study On The Selection Of High Acid-yielding Acetic Acid Bacteria By He-Ne Laser And Intense Pulsed Light Mutagenesis And Its Mechanisms

As a traditional condiment in China,vinegar is favored by the public.Acetic acid bacteria are indispensable in the vinegar brewing process,and their fermentation performance is closely related to the production efficiency of vinegar.At present,acetic acid bacteria strains of Pasteurella subspecies and Turbid variants have been mostly used for vinegar brewing production in China,but their fermentation performance still required to be improved,therefore,mutagenesis is often used to select mutant strains with excellent performance.As novel mutagenesis techniques,the He-Ne laser and intense pulsed light have been used in microbial mutagenesis breeding,whereas there are few reports on the selection of acetic acid bacteria with the mentioned two novel mutagenesis techniques.In addition,the molecular biological mechanisms of the high acid production improvement of acetic acid bacteria mutant strains by He-Ne laser and intense pulsed light mutagenesis technology need to be further explored.This study firstly aims to isolate and screen high acid-yielding acetic acid bacteria with good tolerance and heritability from vinegar grains and to identify their species status based on morphological observation,physicochemical characteristics,and 16 S r DNA test.Then,using He-Ne laser and intense pulsed light to mutagenize acetic acid bacteria,with the combination of high-throughput screening technology to screen out high acid-yielding mutants,and use response surface to optimize the fermentation conditions of high acid-yielding mutant strains.Finally,the biological mechanisms of the He-Ne laser and intense pulsed light mutagenesis will be explored with genome and transcriptomics analysis.The main research contents and results of this paper are as follows:(1)Isolation,screening,and identification of high-quality acetic acid bacteria from vinegar grains.By using the bromocresol violet chromatic circle method,98 strains of acid-producing bacteria were isolated from the vinegar grains,and 5 strains with high acid production were obtained by pre-screening and re-screening.Then,the best-performing strain H3 was identified as Acetobacter pomorum by morphological observation,physiological biochemistry,and 16 S r DNA biology.In addition,according to the determination of the growth and fermentation performance of strain H3,it showed that in growth performance,strain H3 was in the exponential growth period at 10～20 h;in fermentation performance,the fermentation acid production of strain H3 could reach 44.16 g/L at the 5th d;in tolerance performance,strain H3 could tolerate 9%(v/v)ethanol and 3%(v/v)acetic acid.(2)Study on the mutation breeding and fermentation conditions optimization of high-yield acetic acid bacteria by He-Ne laser and intense pulsed light mutagenesis.Considering that the strain H3 obtained through screening has been identified as Acetobacter pomorum,which is generally not used for vinegar fermentation in production,it may be due to its potential food safety and other issues.Therefore,in this study,the commercial Acetobacter pasteurianus CGMCC 1.59 was selected as the starting strain,which was mutagenized by using the novel mutagenesis techniques of He-Ne laser and intense pulsed light,and combined with the high-throughput screening technology to screen out the mutants with better performance.Under the conditions of He-Ne laser wavelength of 633 nm,power of 10 MW,and treatment time of 50 min,mutant strain 1-F-6 with a significantly acid yield increase of 18.21%and stable heritability was screened;under the conditions of pulsed intense light power of 5 J/s and treatment time of 60 min,mutant strain 1-C-1 with a significant higher acid yield of 8.41% and stable heritability was screened.Subsequently,using acid yield as an indicator,the fermentation conditions of mutant 1-F-6 with high acid yield were optimized by response surface method,and the highest acid yield of 85.68g/L was obtained at the conditions as: 8.46% anhydrous ethanol,30.54°C fermentation temperature,and 5.85% inoculum level.And conduct fermentation validation under optimal conditions resulted in an average acid production of 85.52g/L,which was 1.23 times as high as the mutant strain 1-F-6(69.45 g/L)and 1.46 times as high as the original strain Acetobacter pasteurianus CGMCC 1.59(58.75g/L).(3)Genomics was used to investigate the influence of the He-Ne laser and intense pulsed light mutagenesis on the acid production mechanisms of acetic acid bacteria mutant strains.The mutant strain 1-C-1 obtained by intense pulsed light mutagenesis had a total of 32,604 single nucleotide polymorphism(SNP)sites and297 insertion/deletion(In Del)sites compared to the reference genome(model strain),while the mutant strain 1-F-6 obtained by He-Ne laser mutagenesis had a total of32,609 SNP sites and 298 In Del sites.Then,GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)indicated that the genes with SNP and In Del mutation sites in the two mutants were mainly involved in ABC transporter,nicotinic acid and nicotinamide metabolism,RNA degradation,and cell cycle pathways,and there were 28,9,11,and 9 effective mutations in the two mutants.Therefore,it was hypothesized that the increased acid yield of the mutant 1-F-6 and 1-C-1 might be related to the genes with SNP and In Del mutations in the ABC transporter,nicotinic acid and nicotinamide metabolism,and the increased growth might be related to the genes with SNP and In Del mutations in the RNA degradation and cell cycle pathways.(4)Transcriptomics was used to further investigate the biological mechanisms underlying the high acid yield of acetic acid mutant strains from the m RNA level.The sequencing results showed that mutant strain 1-C-1 had 17 differentially expressed genes(15 up-regulated,2 down-regulated)compared to the original strain Acetobacter pasteurianus CGMCC 1.59;mutant strain 1-F-6 had 132 differentially expressed genes(58 up-regulated,74 down-regulated)compared to the original strain;mutant strain 1-F-6 had 156 differentially expressed genes(63 up-regulated,93down-regulated)compared to mutant strain 1-C-1(P < 0.05).Then,GO and KEGG analysis of the screened genes indicated that the mutant strain 1-C-1 obtained by intense pulsed light mutagenesis only up-regulated the expression of genes involved in pyruvate metabolism and TCA cycle(ghr A,acs A,prp C,prp D)by 2.41,2.07,2.08,and 2.21-fold,respectively,while the mutant strain 1-F-6 obtained by He-Ne laser mutagenesis up-regulated the expression of ethanol oxidation respiratory chain pathway-related genes(adh,aldh,cyd A,cyd B)by 2.24,2.53,3.67,and 2.42 fold,respectively,in addition to ghr A,acs A,prp C,and prp D(1.45,2.81,3.33,and 3.67fold).Among them,acetaldehyde dehydrogenase(aldh)and alcohol dehydrogenase(adh)as the most critical enzyme systems in acetic acid fermentation,were up-regulated in favor of the increase of acid production in acetic acid fermentation,which was consistent with the result that the acid production in fermentation of mutant strain 1-F-6 was higher than that of mutant strain 1-C-1,verifying that He-Ne laser was more favorable for mutagenesis selection of high acid-producing acetic acid bacteria.By mining the information of differentially expressed genes,the biological mechanisms of high acid production by He-Ne laser and intense pulsed light mutagenesis in acetic acid bacteria mutants were investigated,which is of great reference value for improving the fermentation acid production of acetic acid bacteria.