Ethanol production using nuclear petite yeast mutants

Abstract

Two respiratory-deficient nuclear petites, FY23 delta pet191 and FY23 delta cox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23 rho(0). Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant, Ki, of 2.3% (w/v) and a specific rate of ethanol production, qp, of 0.90 g ethanol g dry cells-1 h-1. FY23 rho(0) was the most sensitive to ethanol, exhibiting a Ki of 1.71% (w/v) and qp of 0.87 ethanol g dry cells-1 h-1. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23 delta pet191, having a Ki of 2.14% (w/v) and the 85% respiratory-deficient FY23 delta cox5a, having a Ki of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23 rho(0) is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation.

Evidence that the antiproliferative effects of auranofin in Saccharomyces cerevisiae arise from inhibition of mitochondrial respiration

“…However, it is also reported that functional mitochondria are essential to maintain the tolerance to ethanol and, hence, a high growth rate (Hutter and OliverS.G., 1998). Also in our case, despite the increased ATP production, AF treated cells slowdown the growth rate after 6 h. All these data strengthen the idea of mitochondria as AF target.…”

“…damages enable the achievement of a fermentation rate higher than in wild-type strains (Hutter and OliverS.G., 1998). However, it is also reported that functional mitochondria are essential to maintain the tolerance to ethanol and, hence, a high growth rate (Hutter and OliverS.G., 1998).…”

Fluconazole resistance in Candida glabrata is associated with increased bud formation and metallothionein production

“…The respiratory status of the two FL-resistant mutant isolates was investigated on yeast extractpeptone-glycerol medium containing 1 % (w/v) yeast extract, 2 % (w/ v) peptone and 3 % (w/v) glycerol as the sole carbon source (Hutter & Oliver, 1998). Thus, cells unable to respire were negatively selected on this medium.…”

Production of Ethanol from Starch by Respiration-Deficient Recombinant Saccharomyces cerevisiae

“…Several studies have pointed out the potential of utilizing respiration-deficient nuclear petites for the commercial production of ethanol (8,13). Despite the vast number of strategies adopted for the construction of amylolytic strains of S. cerevisiae, there have been no reports about the application of respiration-deficient nuclear petites for the production of ethanol from starch.…”

“…The 100%-respiration-deficient nuclear petite FY23⌬pet191 mutant of the parental haploid S. cerevisiae FY23 strain (MATa ura3-52 trp⌬63 leu2⌬1) (19) was generated using PCR-mediated disruption of the pet191 gene with a kanMX4 disruption cassette that determines G418 sulfate (Geneticin) resistance in yeast (8). The S. cerevisiae NPB-G strain was generated by transforming (17) the FY23⌬pet191 strain with the pPB-G plasmid (5), which contains the B. subtilis ␣-amylase and the A. awamori glucoamylase genes expressed under the control of the PGK1 promoter as an excreted fusion protein.…”