Construction And Mechanisms Analysis Of High-ethanol-producing Yeasts By Glycerol Metabolic Engineering And Genome Shuffling

Ethanol is one kind of the most important and well-established reproducible biofuel. However, there are several issues and obstacles reminded in the technology of ethanol production. The achievement of fermentation under ultrahigh ethanol concentration opens a new and important avenue to increase the efficiency of ethanol production, and the basic requirement is the development of Saccharomyces cerevisiae strains with high ethanol-production. Random mutagenesis and screening is the leading method for the development of industrial yeast strains. There are many studies which report hybridization, protoplast fusion and genetic engineering for breeding strains, but few can be applied in a large-scale industrial production. Because the industrial yeasts should maintain mutilple performances, such as stress tolerance, fast growth and fermentation rate, which are based on complex metabolic and regulatory networks. It is difficult to improve complex phenotypes of yeasts for industrial production by single technology. Based on previsious studies, we improved and combined hybridization, protoplast fusion, metabolic engineering and whole genome shuffling for strain improvement, and explored the mechanisam. The results were shown as follows:1. Screening industrial S. cerevisiae yeasts as original strains for industrial breeding. Among the ninety haploids of three industrial strains THA, S25 and C87, four high ethanol-producing strains Z1, Z4, Z8 and Z9 were selected as the original strains for industrial breeding. The hybrids of two haploids Zl and Z4 with mating ability were obtained by hybridization. On a sweet distillery mash (230g/L glucose), the hybrid Z14 with the highest ethanol yield (100.05g/L) increased the ethanol production by 1.75% and 3.04%, compared with the parent strain THA and S25 of haploid Z1 and Z4, respectively.2. Development and application of RAPD-SCAR markers to identify hybrids of industrial S. cerevisiae. For the original strain Z8 and Z9 without sporulation and mating ability, RAPD (Random Amplified Polymorphic DNA)-SCAR (Sequence Characterized Amplified Region) markers were developed. Instead of traditional auxotrophic markers, the RAPD-SCAR markers were applied to identify the hybrids from protoplast fusion. On a sweet distillery mash (230g/L glucose), hybrids increased ethanol production by 4.33～8.08%, compared with their parent strain Z8 and Z9. 3. Glycerol metabolic engineering in a laboratory strain. To reduce glycerol yield and increase ethanol production, the laboratory strain 4741 was engineered by expressing GAPN (NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase) of Streptococcus mutans and deleting the FPS1 gene (encoding a channel protein Fps1p for glycerol export). The resulting strain 4FG increased the ethanol production by 9.18%, while decreased the yield of glycerol and acetic acid by 21.47% and 27.09%, respectively. The deletion of FPS1 and expression of gapN could be combined to improve the fermentation of yeast strain.4. Glycerol metabolic engineering in industrial strains. In the original strain Z1 (MATa) and Z4 (MATa), the FPS1 gene was deleted and the gapN gene (encoding GAPN of Streptococcus mutans) was integrated at the locus of the FPS1 gene with kanMX- or Zeocin-resistance marker. The G418-Zeocin drug plate could efficiently select the hybrid FG1 (MATa/αfps1ΔgapN-kanMX fps1ΔgapN-Zeocin) of the engineered strain KFG (MATαfps1ΔgapN-kanMX) and ZFG (MATa fps1ΔgapN-Zeocin). In fermentation medium contaning 250g/L glucose, the ethanol yield of the engineered hybrid FG1 was up to 114.00g/L. Compared with the hybrid Z14(MATa/α) of the original strain Zland Z4, FG1 increased the ethanol production by 4.14%, while decreased the formation of glycerol and acetic acid by 18.14% and 25.04%, respectively.5. Improving ethanol tolerance in S. cerevisiae by the improved whole genome shuffling. Strain A1 was obtained after two rounds of genome shuffling of UV and EMS mutants derived from the engineered strain KFG and ZFG. Under the stress of 8%(v/v) ethanol, maximum specific growth rate of shuffled strain A1 increased 23.83%, compared with the control strain FG1. The protection of cell membrane integrality maybe the reason of the improved ethanol tolerance in strain A1. In fermentation medium contaning 285g/L glucose, ethanol yield rate of the shuffled strain A1 was 1.4-fold than that of the control strain FG1 during the first 30h. Moreover, the ethanol production of the shuffled strain A1 was up to 117.61g/L, and increased by 3.91%, compared with the control strain FG1.