Mining And Application Of Dynamic Regulatory Promoters In Baijiu Brewing

Synthetic biology is an interdisciplinary discipline that has emerged in recent years and has shown great promise in a wide range of applications,including microbial cell factories and gene editing.The creation of finely regulated gene circuits is the core foundation of synthetic biology,and promoters are one of the most critical components to achieving efficient and controlled expression of target genes at the transcriptional level.As one of the four major flavors of Chinese baijiu,ethyl acetate has a pivotal role in the presentation of aroma in Chinese baijiu and is one of the most important flavor compounds in white wine.In Saccharomyces cerevisiae,esters are mainly produced by acetyl-Co A and ethanol as substrates catalyzed by alcohol acyltransferase(AATase).Therefore,the synthesis of ethyl acetate can be dynamically regulated by AATase through the promoter,which in turn dynamically regulates the synthesis of ethyl acetate to keep the cell in an optimal state during the production phase,thus increasing productivity,enhancing the robustness of the host and creating an efficient cell factory.To screen for promoters that can dynamically regulate ethyl acetate in Saccharomyces cerevisiae,this experiment used haploid a45 of industrial brewer’s yeast AY12 as the parental strain monitored changes in gene transcript levels at the genome-wide level during simulated white wine fermentation,screened for promoter elements with dynamic regulatory properties,and used the green fluorescent protein gene GFP to determine the expression intensity of dynamically regulated promoters,and finally,In addition,this paper also provides a preliminary investigation of the dynamically regulated promoter elements.In addition,this paper also preliminarily investigated the causes of dynamic changes in promoter HSP26p expression levels with the fermentation process,as follows:Based on the RNA-Seq data,18 yeast promoter elements with dynamic regulatory properties were initially screened and obtained.The transcriptome data were obtained at different time points during the fermentation process by simulating white wine fermentation,and the genes were screened according to the requirement that the FPKM value gradually increased and that the FPKM at 12 h of fermentation was below 200 and above 500 at 36 h.Expression of the green fluorescent protein gene GFP was achieved using a dynamically regulated promoter.The recombinant strain a45-EPromoter was obtained by integrating overexpression of the green fluorescent protein gene GFP at the Gal80 locus of the starting strain a45 using the dynamically regulated promoter,the strong constitutive promoter PGK1p,the ethanol-inducible promoter BTN2p,the glucose repressor promoter ADH2p,and the terminator PGK1t,respectively.The fluorescence values of the parental strain and the recombinant strain were measured by YPD fermentation.The fluorescence values of the recombinant strain were correlated with the FPKM values of the pair of genes used.The results of R~2=0.7046 showed a high correlation between the fluorescence intensity of the promoter and the FPKM of the corresponding gene,except for the promoter HSP26p.Gradient synthesis of ethyl acetate using dynamic regulation of the promoter.The recombinant strain a45-APromoter was obtained by integrating overexpression of the alcohol acetyltransferase gene ATF1 at the Gal80 locus of the departure strain a45 using the dynamically regulated promoter,the strong constitutive promoter PGK1p,the ethanol-inducible promoter BTN2p,the glucose repressor promoter ADH2p,and the terminator PGK1t,respectively.The results of R~2=0.8071 showed that there was a high correlation between the promoter ethyl acetate production and the FPKM of the corresponding gene.To find the reason for the low correlation between the fluorescence intensity of the promoter HSP26p and the FPKM of the corresponding gene,its transcription at the m RNA level under different stress conditions was explored.The YPD fermentation of strain a45was carried out under different ethanol concentrations,carbon starvation,and high concentration of both ethanol and carbon starvation,and RNA was extracted and RT-PCR was performed to investigate the changes of promoter HSP26p in response to different stress conditions at the m RNA level.The results showed that the transcript level of promoter HSP26p was significantly increased under a high concentration of ethanol and carbon starvation stress conditions.Construction of a dynamically regulated promoter-based lactate cell factory.A recombinant strain overexpressing the lactate dehydrogenase gene LDHL1 was constructed.The recombinant strain a45-LPromoter was obtained by integrating overexpression of the lactate dehydrogenase gene LDHL1 at the Gal80 site of the starting strain a45 using HSP82p,HSP26p,RTS3p,the strong constitutive promoter PGK1p,and the terminator PGK1t,respectively,of the dynamically regulated promoter.The L-lactate yields of the parental and recombinant strains were 30.08 g/L,21.6 g/L,17.57 g/L,and 31.92 g/L,respectively,after high sugar YPD fermentation and testing.The results indicate that the dynamically regulated promoter obtained by the screening has a wide application.