Comparative Functional Genomics And Genetic Breeding Of Saccharomyces Cerevisiae Strains

Bioethanol, one of the most common renewable energy sources, is primarily produced from sugars and starches by industrial yeast Saccharomyces cerevisiae strains. However, there are still many cost restriction factors (i.e. the limited land and water resources to produce materials, high energy consumption, and waste disposal problems) in the application of this biofuel. To improve the economic and social benefit of bioethanol, some novel fermentation technologies, such as fermentation at high temperature, very high gravity fermentation, and cellulosic ethanol. have been proposed and developed. But the use of these technologies will impose more severe stresses or inhibitors (high temperature, osmotic stress, high alcohol concentration, acetic acid, furfural, and phenolic substances) on yeast cells and affect the ethanol fermentation efficiency. Thus, understanding of the mechanisms underlying the stresses tolerance of yeast strains and construction of novel strains with improved stress tolerance and fermentation performances are of great theoretical and realistic significance. In this study, comparative functional genomics were used to reveal the basis of the different phenotypes of some widely used industrial bioethanol strains. At the same time, some breeding strategies based on the genetic information of these strains were also performed. The results were shown as follows:1. Comparison of stresses tolerance and ethanol fermentation of selected bioethanol industrial strains. Different tolerance of various stresses and fermentation performances were observed among the widely used brewing yeast strains Z2-Z7. No single strain was tolerant to all stressors and showed best fermentation performances under all conditions. In contrast, Z5produced highest ethanol yield under high temperature condition; Z7had highest ethanol yield and glucose/ethanol conversion rate under high-gravity conditions; and Z3showed best tolerance of cellulosic hydrolysates.2. Comparative functional genomics to guide the design of personalized breeding strategies. Industrial bioethanol YJS329(Z5) showed much better tolerance of stresses (such as high temperature, oxidative stress, and ethanol) than did BYZ1. Z5also differed from BYZ1in trehalose content, plasma membrane composition, and anti-oxidation factors related to stresses tolerance. Pulse field gel electrophoresis (PFGE) and Array-comparative genomic hybridization (aCGH) showed that Z5is a diploid strain with a relative regular genome. With respect to the S288c genome, a total of64,998SNPs,7,093Indels and11unique genes were identified in the genome of Z5-derived haploid strain YJSH1through whole-genome sequencing. Transcription comparison by RNA-Seq suggested which differently expressed genes were the main contributors to the phenotypic differences of Z5and BYZ1. Combining the genome sequences and the transcriptions, we disclosed how those SNPs, lndels and chromosomal copies variations may affect the mRNA expression profile and phenotypes of yeast strains. Besides, some breeding strategies to improve the adaptabilities of Z5were designed and experimentally verified efficiently. Our work not only enriched the genetic resources of yeast but also suggested how functional genomic study to serve personalized breeding.3. The effects of genomic structure variations (SVs) on the phenotypes of yeast strains. PFGE and aCGH showed that Z3and Z5are diploid strains, whereas Z2. Z4, Z6and Z7are aneuploid strains with some extra chromosomes or large DNA segments in some chromosomes. Evolutionary relationship, phenotype, and physiological and biochemical analysis of Z2-Z7suggested that the genomic structure variations play an important role in the phenotypic diversities of these strains. Genetic manipulation of some specific genes (i.e. the deletion of CUP1and the overexpression of YFL052W) confirmed that copy number variation (CNV) of some specified genes had significant effects on the phenotypes of these strains. In addition, we found that "aneuploid stress response" resulted from SVs acts as an important mechanism to affect the stresses tolerance and fermentation performances of yeast strains.4. Application of genomic structure variations to improve the complex phenotypes of yeast strains. We obtained a series of SVs mutations with improved fermentation performances under very-high-gratuity conditions through three rounds of genomic structure reconstruction of the original strain Z7, suggesting SVs is useful to improve the traits of yeast. One mutant strain. ZTS3. produced6.6%more ethanol and showed better tolerance of ethanol, H2O2, and high temperature than did Z7. Compared with the genome of Z7, there were some SVs cases occurred in the genome of ZTS3, resulting in CNV of some big chromosomal regions. It was proved that the increase of gene YFL052W copy was one reason for the increased tolerance of multiple tolerance of ZTS3. Transcription comparison of these two strains revealed that strain ZTS3suffered less "aneuploid stress response" effect than did Z7, suggested by the up-expressed genes in the synthesis of small molecules such as nucleotide, cell proliferation, trehalose and anti-oxidation factors; and down-regulated genes in protein folding, transportation and positioning, energy metabolism and ionic balance process. We concluded that (1) CNV of some specific genes and (2) the adjustment of aneuploidy are the main molecular basis explaining the complex phenotype of Z7.