Factors Affecting D-Lactic Acid Production by Flocculant Saccharomyces cerevisiae Under Non-Neutralizing Conditions

Integrating heterogeneous genes is widely used in metabolic engineering to produce D-lactic acid (D-LA), an essential compound in bioplastics and pharmaceuticals. However, research on the effects of integrating various loci on gene expression, especially regarding flocculation behavior, remains limited. This study constructed Saccharomyces cerevisiae strains by incorporating a codon-optimized D-LDH gene from Leuconostoc pseudomesenteroides (LpDLDH) into the specific genomic loci of the CYB2, PDC1, MPC1, PDC6, ADH1, and PDC5 genes to redirect pyruvate toward lactic acid. Strains with the LpDLDH gene integrated at the PDC1 locus achieved the highest D-LA titers (51 g/L) with minimal ethanol byproduct, followed by strains with integrations into the CYB2 locus at 31.92 g/L, the MPC1 locus at 10 g/L, and the PDC6 locus at 0.026 g/L. In contrast, strains with LpDLDH integrated at the ADH1 and PDC5 loci failed to produce detectable levels of D-LA and exhibited a complete loss of flocculation. Gene expression analysis revealed a significant expression of genes related to flocculation (FLO5), stress adaptation (HSP150), and cell wall integrity (YGP1, SED1, and SCW11). The CYB2-integrating strain showed strong flocculant properties, contributing to its robustness. These findings highlight the influence of genomic locus selection on metabolic flux and stress adaptation, offering insights into optimizing D-LA production in flocculant S. cerevisiae yeast.