| As one of the most ancient wines in the world,huangjiu has been brewed and consumed for more than 5000 of years in China.Huangjiu has been popular in southeast of China due to its unique aroma,subtle flavor and low alcohol content and which has become an important part of wine consumption in China.Multispecies co-fermentation is the core technology of Chinese traditional fermented foods like huangjiu.Microbial interactions play key roles in improving the flavor of fermentation foods,improving process stability,reducing safety risks and so on.The microbial interaction between Saccharomyces cerevisiae HJ and Aspergillus flavus SU-16 creates a unique and efficient process of simultaneous saccharification and fermentation.The unique and efficient simultaneous saccharification and fermentation formed by the interaction between S.cerevisiae HJ and A.flavus SU-16 is the key to a successful fermentation.However,the interaction mechanism of these two fungi is still in a blank stage,which limits the improvement of the fermentation process and quality upgrade of huangjiu.Therefore,taking the interaction mechanism between S.cerevisiae HJ and A.flavus SU-16 during huangjiu brewing as the research object,this study intends to investigate the genomic function,metabolic division of labor and interaction mechanism between SU-16 and HJ from three levels of functional phenotype,metabolic and transcriptional level.(1)SU-16 is a non-aflatoxigenic A.flavus strain.SU-16 was identified as a nonaflatoxigenic A.flavus strain based on the morphology,physiological characteristics and genotyping analysis,as well as the detection of the capability of aflatoxins production.The complete genome sequence of A.flavus SU-16 used for huangjiu brewing was obtained using Nanopore sequencing technology for the first time.The genome size of SU-16 is 37.56 Mb,which contains 12332 protein-encoding genes and 603 genes that encoding the CAZYmes.Although there is only one copy of α-amylase gene in the SU-16 genome,the excellent enzyme activities have been found for SU-16 in the fermentation of wheat qu.Annotation of secondary metabolite gene cluster showed that SU-16 possesses 78 secondary metabolite gene cluster,of which 9 are unique for SU-16 and 21 have potential metabolic activity.Comparative genomic analysis showed that the inactivation of gene afl J,fragment deletion of afl T,Ver-1,ver A and otm B and the loss of promoters and translation start sites in the nor B-cyp A region resulted in the inactivation of the aflatoxin biosynthesis gene cluster in SU-16,and thus none of the aflatoxin and key intermediate products in the aflatoxin biosynthesis process could be produced.(2)The genomic function and variations of S.cerevisiae HJ was carried out using Pacbio sequencing technology and comparative genomics.The genome size of HJ is 37.56 Mb,which contains 5238 protein-encoding genes and 222 transcription factors.142 genes encoding the CAZYmes were found in SU-16 genome,of which only 22 genes related to plant cell wall degradation and starch hydrolysis.Comparative genomic analysis revealed that 34 large regions were identified as synteny,translocation or inversion of the chromosomes.423 genes related to stress-responsive transcription factors(HKR1,MSB2,MSN4,SSK1),alcohol production efficiency(HXT2,HXT9)and aromatic alcohol synthesis(PHA2)evolved rapidly in the adaptation to the huangjiu ecology niche.(3)Metabolites of A.flavus SU-16 in cooked wheat qu is one of the key factors driving the fermentation smoothly.Results based on the Dual-RNA sequencing and fermentation experiments showed that the SU-16 genome were not transcribed during fermentation,the biomass of A.flavus SU-16 in fermentation process was decreased continuously.Whereas,the metabolites of A.flavus SU-16 in cooked wheat qu played the key role in the co-fermentation of A.flavus SU-16 and S.cerevisiae HJ.More than 99% of the genes in S.cerevisiae HJ genome were transcribed and expressed during the co-fermentation,and more than 76% of the genes were at a high expression level.A.flavus SU-16 metabolites significantly affected the expression level of genes related to carbohydrate and amino acid metabolism in HJ genome.The expression levels of the most of genes involved in carbohydrate metabolism were significantly upregulated.The metabolites of A.flavus SU-16 could significantly inhibit the transcriptional level of genes related to amino acid synthesis during fermentation,but promote the expression level of amino acid transporters and genes involved in amino acid catabolism.A.flavus SU-16 metabolites could reduce the synthesis efficiency of higher alcohols by feedback inhibition of the expression levels of genes in Harris pathway and shikimate pathway,but also promote the synthesis efficiency of higher alcohols and aldehydes by regulating the transcription levels of several key genes in Ehrlich pathway.(4)The metabolites of A.flavus SU-16 in cokked wheat qu significantly promoted the formation of flavor compounds during co-fermentation.SU-16 metabolites could regulate the growth of S.cerevisiae HJ,and promote the proliferation of S.cerevisiae HJ during fermentation.SU-16 metabolites in cooked wheat qu significantly affected the nitrogen metabolism of S.cerevisiae HJ,and which were significantly correlated with the production of volatile and non-volatile compounds such as amino acids,organic acids,higher alcohols,aldehydes and acids during fermentation process.The addition of cooked wheat qu significantly improved the content of taste and aroma compounds of huangjiu,but also increased the content of compounds affecting the drinking-comfort of huangjiu.During the co-fermentation,A.flavus SU-16 and S.cerevisiae HJ have a clear metabolic division of labor.A.flavus SU-16 provides130 compounds with potential influence on formation of the huangjiu flavor in wheat qu preparation period,while S.cerevisiae HJ is mainly responsible for the anabolism of 161 compounds in the fermentation process.The mycelium of A.flavus SU-16 plays little role in the fermentation process,but the metabolites of SU-16 could significantly affect the metabolism of S.cerevisiae HJ,and the effect of the macromolecule metabolites of A.flavus SU-16 on the formation of flavor compounds in the fermentation process is greater than that of the small molecule compounds.In conclusion,this study focused on the interaction mechanism between A.flavus SU-16 and S.cerevisiae HJ in the co-fermentation process.A near finished genome sequence of A.flavus SU-16,the first used for traditional fermented food wordwide,was obtained for the first time based on the safety analysis of which using system biology method,and the functional safety and genome structural variations were analyzed from the gene level.Then the key role of the metabolites of A.flavus SU-16 in guarantee of the smooth fermentation of huangjiu,the transcriptional interaction mechanism and metabolic division of labor between A.flavus SU-16 and S.cerevisiae HJ were demonstrated based on the Dual-RNA sequencing and untargeted metabolomics techniques.The results of this study will not only help to improve the scientific understanding of Chinese traditional brewing microorganisms for people,strengthen the protection and utilization of Chinese traditional brewing microorganisms and their genetic resources,but also provide insights for the regulation of key compounds during fermentation. |
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