Objective: The deletion of pyruvate decarboxylase-like enzyme gene (YDL080C gene) in industrial Saccharomyces cerevisiae haploids a-8 and alpha-22 were respectively constructed, and the effect of YDL080C gene deletion on the production of higher alcohols, especially isoamyl alcohol, was investigated in high gravity ethanol fermentation.
Methods: Upper and down stream fragments YA (460 bp) and YB (630 bp) of YDL080C gene were amplified by PCR using the genomic DNA of S. cerevisiae haploids a-8 and alpha-22, respectively; KanMX marker for G418 resistance from plasmid pUG6 was cloned by PCR. Fragments YA, YB and KanMX were respectively inserted into the same plasmid pUC19 using EcoRI and KpnI, KpnI and BamHI, and KpnI sites in the order of YA-KanMX-YB to construct the recombined vector pUC-YABK. The recombinant cassette YA-KanMX-YB was cloned from plasmid pUC-YABK by PCR and respectively transformed into industrial yeast haploid a-8 and alpha-22. By the double homologous recombination, YDL080C gene deletion mutants were constructed and selected by the growth extent on YEPD agar plates containing 600 microg/mL G418. At the end of high gravity ethanol fermentation, fermentation performance and higher alcohols production of parental haploids and their mutants were determined.
Results: YDL080C deletion mutants were respectively selected from their corresponding parental haploid a-8 and alpha-22. The alcohol fermentation results showed that higher alcohols, especially isoamyl alcohol, were almost invariable among the mutants and their corresponding parental haploids. However, mutants yielded more amount of ethanol of 0.6 (%, v/v) and 0.4 (%, v/v) over its parental haploid, respectively.
Conclusion: Deletion of YDL080C gene in S. cerevisiae haploids has no noticeable effect on decreasing the production of higher alcohols, especially isoamyl alcohol, but it seems to somehow increase the production of ethanol.