Physiological And Adaptive Evolution Of Acetobacter Pasterianus Under Acetic Acid Stress

High-efficiency acetic acid fermentation requires acetic acid bacteria with high acetic acid resistance.In this paper,the physiological and intracellular microenvironment of the parental strain Acetobacter pasteurianus CICIM B7003 and the mutant strain Acetobacter pasteurianus CICIM B7003-02 were compared to reveal the acetic acid resistance mechanism during the fermentation process of Acetobacter pasteurianus at physiological level.In addition,the culture conditions and fermentation process of the mutant strain were optimized,and strains with higher acetic acid tolerance involved mutations in multiple genes of pathways.Finally,comparative genomics and evolutionary analysis of the parental and mutant strain was carried out under acetic acid stress.?1?The changes of intracellular microenvironment between the parental strain Acetobacter pasteurianus CICIM B7003 and its mutant strain Acetobacter pasteurianus CICIM B7003-02 to reveal the acid resistance mechanism of Acetobacter pasteurianus.Comparison of fermentation kinetics during batch and semi-continuous fermentation in 7.5 L fermenter.With the presence of initial 10 g?L^-1 acetic acid,the start time of the mutant strain in batch and semi-continuous fermentation was significantly shortened,the acid production rate of the mutant was slightly higher,and the acid-resistance process and acid-producing period of the two strains were relatively divided.Enzymes directly related to acid production such as ADH and ALDH,the highest activity of which increased by 27.0%and 15.2%,respectively.Coenzyme Q9 content also increased by 69.5%.Intracellular microenvironment analysis showed octadecenoic acid in the mutant strain was 31.5%higher than the parental strain.The maximum intracellular ATP content of the mutant strain was 2.33 fold of the parental strain,which was positively correlated with specific growth rate.Intracellular glutamate and aspartic acid also increased by 10.7%and 18.3%.The mutant strain was mainly relied on the synergistic effect of strengthening ethanol respiratory chain,ATP synthesis and critical amino acid metabolism to improve acid resistance.?2?To achieve the coordination of acid resistance and acid production ability,taking A.pasteurianus CICIM B7003-02 as the research object,the acid production optimization was carried out based on alcohol respiratory chain pathway from two aspects respectively,the optimization of aeration rate and strengthening the synthesis of coenzyme Q9.Firstly,fermentation kinetics of different aeration rates were compared,a two-stage oxygen-fed fermentation strategy was established,and then based on the regulation of exogenous Fe²⁺and isoprenol,the enhanced fermentation strategy of alcohol respiratory chain trophic factor under two-stage aeration fermentation strategy was established.The results showed that the aeration rate of 0.25 vvm significantly increased the initial biomass accumulation,the aeration rate of0.35 vvm increased the acid production rate in the later stage,when the critical dissolved oxygen decreased to about 10%,exogenous addition of prenol and Fe²⁺significantly increased the fermentation efficiency,the fermentation process ended at 35 h,the final acidity reached to 52.80 g?L^-1,and the acid production rate reached to 1.51 g?L^-1?h^-1.The average acid production rate in semi-continuous fermentation was 1.67 g?L^-1?h^-1,which was 65.01%higher than the unoptimized group 1.01 g?L^-1?h^-1.?3?To explore the acid resistance mechanism of the mutant at the genomic and transcriptional level of A.pasteurianus,genome sequence of A.pasteurianus CICIM B7003was sequenced.It encoded all the genes that reported to be associated with acetic acid resistance,comparative analysis with other intra-species strains showed that genes related to alcohol respiratory chain and the tricarboxylic acid cycle pathway are relatively conserved.Compared with A.pasteurianus CICIM B7003-02,a few mutations occurred in respiratory chain and metabolic genes including the subunit of F1F0 ATPase,PQQ-glucose dehydrogenase,ubiquinone cytochrome c reductase and niacin dehydrogenase subunit B,the transcriptional level of the genes related to the respiratory chain,tricarboxylic acid cycle and the stress response genes had a large up-regulation with different initial acidity.The transcriptional analysis of the mutant genes in the respiratory chain and metabolic pathway showed that transcription level of fapA,cyc1,gcd,nhaP,marR,mp1,pdc of the mutant strain was increased with 0,10 g?L^-1,20 g?L^-1,30 g?L^-1 initial acetic acid,mutS showed a downward trend,and its inactivation led to a significant increase of mutation rate.Transcriptional regulator marR and membrane protein of mp1 with Indels may be related to acetic acid tolerance.?4?To explore the adaptive evolution mechanism of Acetobacter pasterianus under acetic acid stress.The parental strain A.pasteurianus CICIM B7003,the mutant strain A.pasteurianus CICIM B7003-02,and the type strain A.pasteurianus ATCC 33445 were used as the parental strain,we carried out the adaptive evolution experiment for four months at two start-up acetic acid concentration of 20 g·L^-1 and 30 g·L^-1 separately.Six evolutionary strains P20,M20,T20,P30,M30,T30 were obtained.Enrichment analysis of the shared mutant genes showed that it mainly concentrated on transcriptional regulators and membrane proteins,and the metabolic pathway enzymes were relatively conservative.Interspecies comparison of the evolutionary strains showed that non-synonymous mutation rate was M20>P20>T20,M30>P30>T30,and mutation frequency was T20>P20>M20,T30>P30>M30.In addition,M20 and M30 had least mutation sites,and higher acid resistance of A.pasteurianus CICIM B7003-02 may limit this excess acid-resistant mutation.Finally,the acid-adaptive mutant genes were screened?which contained more than 2 types of mutations?,including sulfonate ATP-binding ABC transporter gene?APA01_RS06905?,lactate dehydrogenase?APA01_RS15365?,DEAD/DEAH-box helicase gene?APA01_RS14860?,XRE family transcriptional regulator?APA01_RS15625,APA01_RS15630? and so on.