Satoshi Saitoh

Efficient production of L-lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene

ABSTRACT We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the l-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2μm plasmid-based vectors and two genome-integrated strains.

Genetically engineered wine yeast produces a high concentration of L-lactic acid of extremely high optical purity.

ABSTRACT For mass production of lactic acid, we newly constructed a transgenic wine yeast strain that included six copies of the bovine l-lactate dehydrogenase gene on the genome. On fermentation in inexpensive cane juice-based medium, l-lactate production of this recombinant reached 122 g/liter and the optical purity was 99.9% or higher.

The Effect of Pyruvate Decarboxylase Gene Knockout in Saccharomyces cerevisiae on L-Lactic Acid Production

A plant- and crop-based renewable plastic, poly-lactic acid (PLA), is receiving attention as a new material for a sustainable society in place of petroleum-based plastics. We constructed a metabolically engineered Saccharomyces cerevisiae that has both pyruvate decarboxylase genes (PDC1 and PDC5) disrupted in the genetic background to express two copies of the bovine L-lactate dehydrogenase (LDH) gene. With this recombinant, the yield of lactate was 82.3 g/liter, up to 81.5% of the glucose being transformed into lactic acid on neutralizing cultivation, although pdc1 pdc5 double disruption led to ineffective decreases in cell growth and fermentation speed. This strain showed lactate productivity improvement as much as 1.5 times higher than the previous strain. This production yield is the highest value for a lactic acid-producing yeast yet reported.

Metabolic engineering of Saccharomyces cerevisiae for efficient production of pure l−(+)−lactic acid

We developed a metabolically engineered Saccharomyces cerevisiae, which produces optically pure L-lactic acid efficiently using cane juice-based medium. In this recombinant, the coding region of pyruvate decarboxylase (PDC)1 was completely deleted, and six copies of the bovine L-lactate dehydrogenase (L-LDH) genes were introduced on the genome under the control of the PDC1 promoter. To confirm optically pure lactate production in low-cost medium, cane juice-based medium was used in fermentation with neutralizing conditions. L-lactate production reached 122 g/L, with 61% of sugar being transformed into L-lactate finally. The optical purity of this L-lactate, that affects the physical characteristics of poly-L-lactic acid, was extremely high, 99.9% or over.We developed a metabolically engineered Saccharomyces cerevisiae, which produces optically pure l-lactic acid efficiently using cane juice-based medium. In this recombinant, the coding region of pyruvate decarboxylase (PDC)1 was completely deleted, and six copies of the bovine l-lactate dehydrogenase (l-LDH) genes were introduced on the genome under the control of the PDC1 promoter. To confirm optically pure lactate production in lowcost medium, cane juice-based medium was used in fermentation with neutralizing conditions. l-lactate production reached 122 g/L, with 61% of sugar being transformed into l-lactate finally. The optical purity of this l-lactate, that affects the physical characteristics of poly-l-lactic acid, was extremely high, 99.9% or over.

Breeding of Industrial Diploid Yeast Strain with Chromosomal Integration of Multiple β-Glucosidase Genes

We constructed a double auxotrophic OC-2 industrial diploid strain of Saccharomyces cerevisiae and introduced 4 copies of cell surface displaying beta-glucosidase (BGL) genes into the chromosome. The engineered OC-2 strain showed 5-fold higher BGL activity compared with the yeast carrying 2 copies of BGL gene and directly produced ethanol from cellobiose.