| Organic acids are widely used as platform chemicals in chemical,pharmaceutical,food and other fields.Microbial fermentation is one of the main methods of production.However,industrial fermentation is generally faced with acid inhibition,which not only affects the normal growth and metabolism of cells and leads to low production efficiency.In addition,the adjustment of acidity by NaOH results in an increase in the osmotic pressure and difficulty in the downstream separation.There are few promoters that use the product organic acid as the inducer in commonly used expression systems.The approach to solve the problem of acid fermentation can be applied to the application of strong acid-resistant industrial microorganisms combined with acid-induced promoters.Candida glycerinogenes is an excellent industrial strain that is resistant to hyperosmotic and acid-tolerant properties.The study on the novel acid-modulating promoters of this strain will provide the basis for gene transformation and organic acid production in industrial yeasts.The specific study is as follows:The differences in pH and organic acid tolerance between C.glycerinogenes and S.cerevisiae were analyzed,and relative expression levels of acid stress response genes were determined at pH 2.5 using qRT-PCR.The acid stress response genes GMT,GUK,ADH3and TPO2 with relative expression levels greater than 5 were selected,among which the GMT gene had the most significant change.The promoters PCgGMT,PCgGUK,PCgADH3,and PCgTPO2 were obtained by cloning technology,and the potential acid response-related transcription factor binding sites in the promoter sequences were analyzed using MatInspector software for bioinformatics analysis.In order to investigate the properties of acid-regulated promoters,integrated expression vectors with PCgGMT,PCgGUK,PCgADH3,and PCgTPO2 as promoters and the green fluorescent protein gene GFP as the reporter gene were constructed and introduced into C.glycerinogenes using lithium acetate transformation method.The expression fluorescence of GFP in different recombination cells under different pH conditions was observed.The fluorescence intensity was measured by the optical density?IOD?analysis of Image-Pro Plus to evaluate the promoter strength.The results showed that PCgGMT,PCgGUK,PCgADH3 and PCgTPO2 could initiate the downstream GFP gene expression,and the fluorescence intensity increased with the decrease of pH.It was confirmed that the selected promoters were all regulated by acid.In order to investigate the versatility of acid-induced promoters,S.cerevisiae strains were used as host cells.GFP fluorescence was detected at different pH conditions and analyzed.The results showed that promoters all can initiate GFP gene expression in S.cerevisiae.Phosphoric acid,hydrochloric acid,citric acid and acetic acid were used to further investigate the performance of PCgGMT,PCgGUK and PCgADH3 induced by different acids to express GFP gene.From the results,the inorganic acid-inducing ability was slightly stronger than that of the organic acid,but the difference was not significant,indicating that the promoter inducibility was mainly regulated by H+and less affected by acid radicals.In order to study the industrial application of acid-induced promoters,lactic acid was produced in C.glycerinogenes using the PCgGMT promoter with the most acid-inducing level.The key gene ldhA for lactic acid produce was cloned with the filamentous fungus Rhizopus oryzae cDNA as a template.Connected to the expression vector with PCgGMT as promoter,the expression vector was introduced into C.glycerinogenes to obtain lactic acid-producing recombinant strains and the fermentation performance was examined.From the results,it was found that the recombinant strains had higher specific enzyme activity at pH 2.5,which was 13.83.mU/mg,lactic acid accumulation of 12.32 g·L-1,is 3.18 times higher than pH 5.5,indicating that PCgGMT can enhance the expression of lactate dehydrogenase gene under low pH conditions,giving the transformant a stronger ability to synthesize lactic acid. |
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