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 <i>Saccharomyces cerevisiae</i> strains by incorporating a codon-optimized <i>D-LDH</i> gene from <i>Leuconostoc pseudomesenteroides</i> (LpDLDH) into the specific genomic loci of the <i>CYB2</i>, <i>PDC1</i>, <i>MPC1</i>, <i>PDC6</i>, <i>ADH1</i>, and <i>PDC5</i> genes to redirect pyruvate toward lactic acid. Strains with the <i>LpDLDH</i> gene integrated at the <i>PDC1</i> locus achieved the highest D-LA titers (51 g/L) with minimal ethanol byproduct, followed by strains with integrations into the <i>CYB2</i> locus at 31.92 g/L, the <i>MPC1</i> locus at 10 g/L, and the <i>PDC6</i> locus at 0.026 g/L. In contrast, strains with <i>LpDLDH</i> integrated at the <i>ADH1</i> and <i>PDC5</i> 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 (<i>FLO5</i>), stress adaptation (<i>HSP150</i>), and cell wall integrity (<i>YGP1</i>, <i>SED1</i>, and <i>SCW11</i>). The <i>CYB2</i>-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 <i>S. cerevisiae</i> yeast.