The SPT15 gene encodes a Saccharomyces cerevisiae TATA-binding protein, which is able to globally control the transcription levels of various metabolic and regulatory genes. In this study, a SPT15 gene mutant (S42N, S78R, S163P, and I212N) was expressed in S. cerevisiae BY4741 (BSPT15-M3), of which effects on fermentative yeast properties were evaluated in a series of culture types. By applying different nitrogen sources and air supply conditions in batch culture, organic nitrogen sources and microaerobic condition were decided to be more favorable for both cell growth and ethanol production of the BSPT15-M3 strain than the control S. cerevisiae BY4741 strain expressing the SPT15 gene (BSPT15wt). Microaerobic fed-batch cultures of BSPT15-M3 with glucose shock in the presence of high ethanol content resulted in a 9.5-13.4% higher glucose consumption rate and ethanol productivity than those for the BSPT15wt strain. In addition, BSPT15-M3 showed 4.5 and 3.9% increases in ethanol productivity from cassava hydrolysates and corn starch in simultaneous saccharification and fermentation processes, respectively. It was concluded that overexpression of the mutated SPT15 gene would be a potent strategy to develop robust S. cerevisiae strains with enhanced cell growth and ethanol production abilities.

Yeast with multiple tolerance onto harsh conditions has a number of advantages for bioethanol production. In this study, an alcohol yeast of Issatchenkia orientalis MTY1 is isolated in a Korean winery and its multiple tolerance against high temperature and acidic conditions is characterized in microaerobic batch cultures and by metabolomic analysis. In a series of batch cultures using 100 g L glucose, I. orientalis MTY1 possesses wider growth ranges at pH 2-8 and 30-45 °C than a conventional yeast of Saccharomyces cerevisiae D452-2. Moreover, I. orientalis MTY1 showes higher cell growth and ethanol productivity in the presence of acetic acid or furfural than S. cerevisiae D452-2. I. orientalis MTY1 produces 41.4 g L ethanol with 1.5 g L h productivity at 42 °C and pH 4.2 in the presence of 4 g L acetic acid, whereas a thermo-tolerant yeast of Kluyvermyces marxianus ATCC36907 does not grow. By metabolomics by GC-TOF MS and statistical analysis of 125 metabolite peaks, it is revealed that the thermo-tolerance of I. orientalis MTY1 might be ascribed to higher contents of unsaturated fatty acids, purines and pyrimidines than S. cerevisiae D452-2. Conclusively, I. orientalis MTY1 could be a potent workhorse with multiple tolerance against harsh conditions considered in cellulosic bioethanol production.

Carboxypeptidase Y (CPY) is a yeast vacuolar protease with useful applications including C-terminal sequencing of peptides and terminal modification of target proteins. To overexpress CPY with the pro-sequence (proCPY) encoded by the Saccharomyces cerevisiae PRC1 gene in recombinant S. cerevisiae, the proCPY gene was combined with the gene coding for a signal sequence of S. cerevisiae mating factor α (MFα), invertase (SUC2), or Kluyveromyces marxianus inulinase (INU1). Among the three constructs, the MFα signal sequence gave the best specific activity of extracellular CPY. To enhance the CPY expression level, folding accessory proteins of Kar2p, Pdi1p and Ero1p located in the S. cerevisiae endoplasmic reticulum were expressed individually and combinatorially. A single expression of Kar2p led to a 28 % enhancement in extracellular CPY activity, relative to the control strain of S. cerevisiae CEN.PK2-1D/p426Gal1-MFαCPY. Coexpression of Kar2p, Pdi1p and Ero1p gave a synergistic effect on CPY expression, of which activity was 1.7 times higher than that of the control strain. This work showed that engineering of signal sequences and protein-folding proteins would be helpful to overexpress yeast proteins of interest.