Potent L-lactic acid assimilation of the fermentative and heterothallic haploid yeast Saccharomyces cerevisiae NAM34-4C.

We screened an industrial thermotolerant Saccharomyces cerevisiae strain, KF7, as a potent lactic-acid-assimilating yeast. Heterothallic haploid strains KF7-5C and KF7-4B were obtained from the tetrads of the homothallic yeast strain KF7. The inefficient sporulation and poor spore viability of the haploid strains were improved by two strategies. The first strategy was as follows: (i) the KF7-5C was crossed with the laboratory strain SH6710; (ii) the progenies were backcrossed with KF7-5C three times; and (iii) the progenies were inbred three times to maintain a genetic background close to that of KF7. The NAM12 diploid between the cross of the resultant two strains, NAM11-9C and NAM11-13A, showed efficient sporulation and exhibited excellent growth in YPD medium (pH 3.5) at 35°C with 1.4-h generation time, indicating thermotolerance and acid tolerance. The second strategy was successive intrastrain crosses. The resultant two strains, KFG4-6B and KFG4-4B, showed excellent mating capacity. A spontaneous mutant of KFG4-6B, KFG4-6BD, showed a high growth rate with a generation time of 1.1 h in YPD medium (pH 3.0) at 35°C. The KFG4-6BD strain produced ascospores, which were crossed with NAM11-2C and its progeny to produce tetrads. These tetrads were crossed with KFG4-4B to produce NAM26-14A and NAM26-15A. The latter strain had a generation time of 1.6 h at 35°C in pH 2.5, thus exhibiting further thermotolerance and acid tolerance. A progeny from a cross of NAM26-14A and NAM26-15A yielded the strain NAM34-4C, which showed potent lactic acid assimilation and high transformation efficiency, better than those of a standard laboratory strain.

Isolation and characterization of a mutant recombinant Saccharomyces cerevisiae strain with high efficiency xylose utilization.

A recombinant xylose-utilizing Saccharomyces cerevisiae strain carrying one copy of heterologous XYL1 and XYL2 from Pichia stipitis and endogenous XKS1 under the control of the TDH3 promoter in the chromosomal DNA was constructed from the industrial haploid yeast strain NAM34-4C, which showed thermotolerance and acid tolerance. The recombinant S. cerevisiae strain SCB7 grew in minimal medium containing xylose as the sole carbon source, and its shortest generation time (G(short)) was 5 h. From this strain, four mutants showing rapid growth (G(short) = 2.5 h) in the minimal medium were isolated. The mutants carried four mutations that were classified into three linkage groups. Three mutations were dominant and one mutation was recessive to the wild type allele. The recessive mutation was in the PHO13 gene encoding para-nitrophenyl phosphatase. The other mutant genes were not linked to TAL1 gene encoding transaldolase. When the mutants and their parental strain were used for the batch fermentation in a complex medium at pH 4.0 containing 30 g/L xylose at 35 °C with shaking (60 rpm) and an initial cell density (Absorbance at 660 nm) of 1.0, all mutants showed efficient ethanol production and xylose consumption from the early stage of the fermentation culture. In two mutants, within 24 h, 4.8 g/L ethanol was produced, and the ethanol yield was 47%, which was 1.4 times higher than that achieved with the parental strain. The xylose concentration in the medium containing the mutant decreased linearly at a rate of 1 g/L/h until 24 h.

Improvement of ethanol production from D-lactic acid by constitutive expression of lactate transporter Jen1p in Saccharomyces cerevisiae.

To improve ethanol production from D-lactate, Jen1p, a monocarboxylate-proton symporter, was constitutively expressed in Saccharomyces cerevisiae NAM34-4C. The mutant produced 2.4 g/L of ethanol, approximately 2.4 times higher than that of the wild-type strain. A monocarboxylate/proton symporter gene (JEN1) null mutant was also constructed. It produced 0.19 g/L of ethanol, 5 times lower than that of the wild-type strain.