Physiological And Genomic Characteristics Of Saccharomyces Cerevisiae In Maotai-flavor Liquor Brewing

Saccharomyces cerevisiae is the important microb in liquor brewing, which play important role in the producing of ethanol and volatile substance and then gain strain diversity. Chinese Maotai-flavor liquor adopts unique brewing process which results in specicial fermentation environment with high temperature, high consentration of ethanol, acid pH, etc.. Such a distinct environment then have domesticated role on microbs. Learing physiological characteristics and mechanism of S. cerevisiae in the brewing process will have important practical significance.This research focused on S. cerevisiae MT1 selected from the fermented grains of one famous Maotai-flavor winery in Guizhou. Its physiological characteristics were well studied by comparing with the type strain S288c and other industial yeasts. The physiological metabolic mechanism of S. cerevisiae in Chinese Maotai-flavor liquor brewing was analysised for the first time through genomics and transcriptomics. The main contents and results are as follows:(1) S. cerevisiae MT1 was selected from the fermented grains of Maotai-flavor liquors. By comparing with the type strain S288c and other industrial strains, tolerance of MT1 was well studied. MT1 could tolerate 42℃ temperature,16% ethanol (V/V) and pH 2.0. The maximum specific growth rate and the maximum ethanol production rate of MT1 were as high as 125% and 114% of those of S288c, respectively. Its ethanol conversion rate was also higher than S288c with 6.5 percentage points. Several volatile compounds only can be detected in fermentation liquor of MT1. MT1 could also yield more farnesol, nerolidol, beta damascene and other volatiles. In addition, MT1 could utilize and ferment a variety of carbon source to produce ethanol, such as glucose, sucrose, galactose, xylose, allulose, maltose, melibiose, turanose, trehalose, raffinose, etc..(2) The whole genome of MT1 was sequenced and compared with S288c. Results showed that MT1 has a genome with a size of 11.62 Mb and GC% of 45.7%, which has 5106 ORFs,5 rRNA genes and 312 tRNA genes. Compared with S288c, there were 58960 SNPs, 6474 Indels and 183 CNVs in MT1 genome. Two kinds of chromosome structure variation (inversion and large fragment deletion) were found by colinearity analysis.145 specific genes and 695 missing genes were identified through cluster analysis. Specific genes were mostly related to Stress response, Carbohydrate metabolic process, Biosynthetic process and Organic substance metabolic process, etc., while most missing genes were fell into founctions of DNA binding, RNA binding, Zinc ion binding, Structural constituent of ribosome, etc.. Reliability of the results were also verified by PCR and gene knocking out.(3) Different expressed genes between MTl and S288c were studied by RNA-seq with sorghum juice medium (pH 6.0), Reliability of RNA-seq was verified by fluorescence quantitative PCR. Compared with S288c, most down-regulated genes of MT1 were related to growth and reproduction of cells, including Nucleotide metabolism, Translation, Cell growth and death and others. Especially, Ribosome (ko 03010) had 84 gene down-regulated. While most up-regulated genes were related to carbon metabolism and energy metabolism. Especially, TCA cycle and oxidative phosphorylation had 12 and 18 genes up-regulated, respectively. In conclusion, compared with S288c, MT1 focuses more on metabolism instead of growth, and this is most likely the reason of efficient fermentation performance of MT1.(4) To study mechanism of muti-carbon source assimilation, gene expression of HXT family was compared between MT1 and S288c. Under the condition with glucose concentration was higher than 70 g·L-1, HXT5 and HXT13 whose were inhibit-expressed by high concentration of glucose in S288c still highly expressed in MT1. Expression of HXT5 and HXT13 in MT1 achieved 3 times and 211 times of S288c, respectively. With the single carbon source of glucose as the control group and S288c as the reference strain, multi-carbon source fermentation of MT1 was studied. MT1 have begun to use another carbon source except glucose in the early stage of the logarithmic phase as shown by the dynamic curve of sugar consumption. Especially the galactose, MT1 almost utilized glucose and galactose at the same time; There was no secondary growth in the growth curve of MT1. Indicated by the above analysis, MT1 has a difference mechanism of muti-carbon source assimilationn.(5) Differences between gene expression of MT1 and S288c were studied by RNA-seq under the high acid condition which is the simulation of Maotai-flavor liquor brewing. With acid stress, some genes related to acid pH tolerance were also up-regulated in MT1, including CIK1, HSP30, RPS27B, etc.. In response to the acid stimulation, MT1 major mobilize 15 metabolic pathways. While in the 8 pathways the same as S288c, the number of genes mobilized in MT1 is far more than S288c. For example,71、13 and 10 genes was up-regulated in Translation, Cell growth and death and Nucleotide metabolism, respectively. In addition, HXK2 and FKS1 in the carbon source metabolic of MT1 raised 13.12 times and 10.98 times, respectively. In conclusion, MT1 took a series of positive measures with high acid stress. MT1 would not only boost cell growth and then make up for the damage due to acid stress by actively mobilizing the synthesis of nucleic acid and amino acid, transcription, translation and genes which could repair DNA damage, but also promote carbohydrate metabolism. In addition, under acid condition, MT1 could effectively reduce acid production to improve the acid tolerance.