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.

FIG. 1.

Maps of the plasmid vectors and breeding of transgenic S. cerevisiae. (A) T157 (LDH gene, four copies) strain. The constructed DNA fragment, which was obtained by digesting the pBBLE-LDHKCB vector with SacI and ApaI, was integrated into the PDC5 downstream locus of chromosome XII in the YIBO-7A (LDH gene, two copies) strain, and PDC5 and SLX4 were not disrupted through an integration step. The method used to modify the bovine l-LDH gene and the construction of the YIBO-7A strain were described in detail in the previous report (10). (B) T165 (LDH gene, six copies) strain. The constructed DNA fragment, which was obtained by digesting the pBG418-LDHKCB vector with SacI and KpnI, was integrated into the PDC6 upstream locus of chromosome VII in the T157 (LDH gene, four copies) strain. PDC6 and CTT1 were not disrupted.

FIG. 2.

Comparison of LDH specific activity with increasing gene copy number. Shown are the control strain OC-2T with no LDH gene (◊), YIBO-7A strain with two copies of the LDH gene (▪), T157 strain with four copies of the LDH gene (▴), and T165R strain with six copies of the LDH gene (•). The average and deviation for three independent experiments are presented.

FIG. 3.

Time courses of l-lactate production of four transgenic strains on a flask scale. Shown are the control strain OC-2T with no LDH gene (◊), YIBO-7A strain with two copies of the LDH gene (▪), T157 strain with four copies of the LDH gene (▴), and T165R strain with six copies of the LDH gene (•). The average and deviation for three independent experiments are presented.

FIG. 4.

Fermentation analysis with the S. cerevisiae T165R strain in the cane juice-based medium (sugar concentration, approximately 20%). ○, l-lactate; ▪, ethanol. The jar conditions were kept at 32°C and pH 5.2, with aeration at 0.15 liters/min. The agitation rate was controlled at 60 rpm, and 1 N NaOH was used for neutralization.