| The production of Chinese Baijiu is carried by a solid state fermentation in an open environment,which is greatly affected by environmental factors.Among the environmental factors,the high acidity of fermented grains is an important factor to inhibit the physiological activities of brewing microbes,which seriously affects the yield and quality of Baijiu.The yeasts are the most important ethanol-producing microorganisms in the brewing process,and its stress resistance and fermentation performance determine the yield and quality of base liquor.In the brewing process of sauce-flavor liquor,the domestication of long-term high acid environment enables some yeasts to have good stress resistance,which ensures the normal progress of the later round in-pit fermentation.The screening of acid-tolerant yeasts and the analysis of acid tolerance mechanism will help to scientifically understand the brewing mechanism of Baijiu,and provide a new regulatory strategy for improving the quality and production stability of base liquor.(1)In this study,the Schizosaccharomyces pombe Sp.65 was screened from the acidic fermented grains of sauce-flavor Baijiu and the strain grew well in YPD medium containing20 g/L acetic acid or 60 g/L lactic acid,as well as under the condition of 40°C.With the initial sugar content of 16°Bx sorghum hydrolyzate to simulate liquid fermentation,the results showed that the final ethanol content produced by the strain reached 7.24±0.23%vol under the simultaneous stress of 6 g/L acetic acid and 30 g/L lactic acid.However,the growth of S.pombe type strain ATCC16979 and Saccharomyces cerevisiae AY12 were completely inhibited under this stress condition,resulting in the failure of fermentation.The results of the simulated solid-state fermentation showed that the final ethanol content of Sp.65 was47.41±2.72 g/kg fermented grains.And the value was 41.53±1.24 g/kg,41.8±1.41 g/kg,41.5±0.6 g/kg under the conditions of inoculated with AY12,ATCC16979 and without yeast inoculated,respectively.The results suggested that the strain Sp.65 had a promoting effect on the improvement of ethanol production under acid stress,and provided some advantages for potential application.(2)In order to analyze the acid tolerance mechanism of S.pombe,the acid-tolerant strain Sp.65 was used as the research object,and the acid-sensitive strain ATCC16979 was used as the control.The response of S.pombe to acetic acid and lactic acid stress was investigated by comparative transcriptomics.The results of growth curves and RT-q PCR analysis under different concentrations of acid stress confirmed that the optimal acid treatment concentrations for transcriptome sampling were 8 g/L acetic acid and 40 g/L lactic acid,and the optimal sampling time was 4 hours under acid stress.From the perspective of adaptive evolution,it was found that genes related to carboxylic acid metabolic process,nucleoside metabolic process,hexose catabolic process,and NADH regeneration had higher expression levels in strain Sp.65.This result indicated that strain Sp.65 has stronger acid ion metabolism,energy supply and antioxidant capacity,which was a significant guarantee for its high acid resistance.The intracellular ROS detection showed that the intracellular ROS level of ATCC16979 under acetic stress was 2.86 times higher than that of the Sp.65,which was inseparable from the ROS scavenging ability of Sp.65.(3)Comparative analysis found that the responses of S.pombe to acetic and lactic stress were different.The specific growth rate of strain Sp.65 under 8 g/L acetic acid stress was close to that under 40 g/L lactic acid stress,indicating that acetic acid had a stronger inhibitory effect on strain Sp.65.The stress effect of lactic acid on the strain might be more dependent on the way of cytoplasmic acidification caused by H~+accumulation,while acetic acid caused more serious toxicity to the strain,which was inseparable from the effect of CH3COO~-.The results of intracellular ROS analysis showed that acetic acid was more likely to induce the production of intracellular ROS than lactic acid,resulting in more severe oxidative stress.The results of intracellular acetaldehyde analysis showed that acetic acid caused more accumulation of acetaldehyde,making acetaldehyde one of the intermediates for the cytotoxic effect of acetic acid.As a response to acetic acid stress,genes involved in response to oxidative stress and cellular aldehyde metabolic process had higher expression levels.In addition,genes related to copper ion transport,zinc ion transport and trehalose synthesis were significantly enriched only under acetic acid stress.However,the genes related to energy synthesis and metabolism and the proton pump protein-encoding gene PMA1 had higher expression levels under 40 g/L lactic acid stress to alleviate the excessive acidification of the cytoplasm.(4)Transcription factors are responsible for the regulation of multiple genes and play an important role in microbial stress resistance.The correlation between gene expression changes and strain phenotype was analyzed by WGCNA,and 115 genes were found to be significantly correlated with acid resistance of Sp.65.Furthermore,the YEASTRACT database was used to predict the transcription factors regulating 115 genes,and it was found that the genes pap1,SPAC3F10.12c and rsv2 regulated 108,94 and 87 of them,accounting for 93.90%,81.66 and 75.65%,respectively.The results of semi-quantitative spot assay and growth curve detection showed that the knockout of genes pap1,SPAC3F10.12c and rsv2decreased acid resistance.These results indicate that these three transcription factors are the key elements to maintain the acid-tolerance of S.pombe.(5)Transcriptome analysis showed that the genes related to eisosome assembly were significantly different between the acid-tolerant strain Sp.65 and the acid-sensitive strain ATCC16979,suggesting that the eisosome may be related to the acid resistance of the strain.By laser scanning confocal microscope analysis,pil1,fhn1 and sle1 were identified as the key genes to maintain the eisosome structure in S.pombe.The results of stress resistance analysis of the recombinant strains showed that the disruption of the eisosomes resulted in a decrease in the tolerance of Sp.65 to acetic acid and lactic acid.Further analysis found that eisosomes disruption also leaded to the increase of cell membrane permeability,resulting in more intracellular accumulation of organic acids and higher ROS levels under acid stress conditions.Lipidomic profiles showed that eisosome disruption not only decreased the content of cylindrical glycerophospholipids,sphingolipids and sterols,but also hindered the occurrence of desaturation and chain extension of lipid acyl chains,which explained the intrinsic reason why eisosomes affected membrane properties.The cell membrane conducts information transmission with endoplasmic reticulum through the interaction between eisosomes and VAPs(Vesicle-associated membrane protein(VAMP)-associated proteins,VAPs).The deletion of VAPs coding genes scs2 and scs22 also caused the decrease of acid tolerance of the strains.Therefore,we speculated that the communication barrier between cell membrane and endoplasmic reticulum caused by the disruption of eisosomes was the basic reason for the decreased acid-tolerance.The results here provide a theoretical basis for the functional role of eisosomes in the regulation of stress resistance. |
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