Isolation And Functional Analysis Of Flocculation Genes From The Flocculating Yeast SPSC01

Yeast flocculation is cost-effective for biomass recovery, through which capital investment on centrifuges and energy consumption for centrifuge running can be saved. Meanwhile, when yeast cells self-flocculate into flocs with desirable size distribution, they can be self-immobilized within fermentors for high density culture and fermentation to improve productivity. Moreover, stress tolerance can be significantly improved when cells self-flocculated due to enhanced quorum-sensing mechanism associated with the flocculation of cells, making them potentially more suitable for very high gravity (VHG) culture and fermentation with VHG substrate to increase product titers and reduce the discharge of waste water. The fusant SPSC01derived from an industrial Saccharomyces cerevisiae with excellent ethanol fermentation performance and another yeast Schizosaccharomyces pombe with flocculating phenotype combines the merits of the parent strains, which has been patented and applied in fuel ethanol production. In this project, molecular mechanism underlying the flocculation of yeast cells was investigated, with the identification and cloning of FLO genes from the yeast strain, with an objective to developing a platform for engineering regular non-flocculating yeast strains with the flocculating phenotype for more efficient production of ethanol and other fermentation products.The SPSC01genomic library consisting of4663clones was constructed with the copy-control fosmid vector to isolate the intact SPSC01FLO1. The library covered14.6-fold genome equivalent, and the average size of the insert DNA fragments was approximately38kb. Four primers for the PCR screening of the fosmid library were synthesized based on the sequence of S. cerevisiae S288C FLO1from the Genome Database. Five fosmid clones containing the intact SPSC01FLO1sequence were subsequently obtained. The sequence analysis revealed a8049bp open reading frame that encoded a2682-amino acid protein, which was much longer than that detected with S288C. The N-terminal and C-terminal regions of the putative SPSC01Flolp sequence exhibited93%and92%identity with the corresponding regions of the sequence of S288C Flolp. but the middle part of the SPSC01Flolp contained43repeat units of45-amino acid residues, while only18repeat units were identified in S288C Flolp.On the basis of homologous recombination, the PCR-mediated gene disruption was performed using the KanMX4selectable marker Hanking about45bp of homology to the SPSC01FLO1sequence. The gene disruption cassette was generated by PCR amplification. The PCR products were purified and directly transformed into SPSC01by electroporation, and resistant colonies were selected on the YPD plates containing G418. Transformants were then picked and incubated in the YPD medium containing G418. As a result, no flocculation was observed for the cultures, indicated that the disruption of SPSC01FLO1led to a complete abolishment of the flocculation phenotype. Furthermore, the HO locus that involves in the conversion of the mating-type of the yeast, and has no effect on yeast growth if it is replaced with a different gene was chosen as the integration site for the integrative expression vector pHO1-1developed from the cloning vector pHO, which contained FLO1from SPSC01under the control of the strong constitutive PGK1promoter from S. cerevisiae S288C, and the CYC1terminator from the plasmid p424GPD for efficient transcription of the SPSC01FLO1. The pHO1-1vector linearized with Not I was transformed into the regular non-flocculating industrial S. cerevisiae6525. The transformants exhibited strong flocculation ability when grown in the YPD medium, while the integrated expression of the empty vector pHO containing the KunMX4selectable marker flanking by the HO fragments in S. cerevisiae6525had no impact on the phenotype of the host strain.The flocculating genes FLO9, FLO10and FLO11were further isolated from SPSC01,. and transformed into S. cerevisiae6525. The sequence analysis of SPSC01FLO9revealed a2904bp open reading frame,831bp N-terminal region,1395bp C-terminal region, and675bp middle part with5repeats compared to13repeats detected with S288C FLO9. No flocculation was observed for the transformant6525FLO9. The sequence analysis of SPSC010FLO10revealed a4221bp open reading frame,906bp N-terminal region,1686bp C-terminal region, and1629bp middle part with18repeats (A) and5repeats (B) compared to10and4detected with S288C FLO10. The transformant6525FLO10exhibited weak flocculation ability. The sequence analysis of SPSC01FLO11revealed a3315bp open reading frame,789bp N-terminal region,1392bp C-terminal region, and1134bp middle part with18repeats (A) and9repeats (B), compared to25and16detected with S288C FLO11. The transformant6525FLO11didn’t exhibit pseudohyphal formation and flocculation phenotype.In order to evaluate the genomic stability, the transformants were grown in the YPD medium without G418and sub-cultured daily. After two weeks, the G418resistance and flocculation of transformant were not changed significantly. The recombinant6525FLO1displayed strong flocculation. with a flocculation index of approximately95%, which was inhibited by mannose, indicating that the flocculation of the transformant is the Flo1type. The stable flocculation was observed for the transformant6525FLO1when temperature was in20-50℃, pH from3.0to7.0, and Ca2+concentration exceeded10mM. The flocculation index of the transformant6525FLO10was about85%, and the flocculation was inhibited by mannose and glucose for the NewFlo type. The flocculation of6525FLO10was stable when temperature was below50℃, pH from4.0to6.0, and Ca2+concentration was above10mM. The results of VHG fermentation experiments with medium containing250g/L glucose illustrated that glucose was consumed within48h, and about110g/L ethanol was produced. When yeast cells form the flocs in millimeter scale, the rates of glucose utilization and ethanol production decreased slightly, but the flocculation didn’t affect the yeast growth, since no significant difference was observed in the biomass between the transformants and their wild-type.In summary, four FLO genes were isolated from SPSC01, their sequence analysis and functional verification were performed, and platform for engineering regular non-flocculating yeast strain with the flocculating phenotype for more efficient production of ethanol and other fermentation products was developed.