Biomanufacturing of L‐Lactic Acid from Inulin Exploiting Kluyveromyces marxianus and Engineered Saccharomyces cerevisiae Cell Factories

The transition to sustainable lactic acid (LA) biomanufacturing necessitates a shift from first-generation feedstocks toward residual biomass and low-pH fermentation, eliminating the costly, waste-intensive salt purification and acidification steps. This study presents a novel, two-yeast strategy designed to valorize inulin, a fructose polymer also found in cardoon roots, for high-efficiency LA production at a pH below its pKa. The key novelty of this work lies in identifying and pairing the unique physiological strengths of each organism: Kluyveromyces marxianus as the optimal yeast for inulin hydrolysis, and Saccharomyces cerevisiae as the superior host for robust LA production at low pH and high titers. We discovered that if ammonium sulphate is substituted with urea as a nitrogen source for K. marxianus cultivation, the inulinase activity increased by nearly 20-fold, significantly enhancing the initial breakdown of the substrate. Inulinase-enriched K. marxianus supernatant was then used to cultivate S. cerevisiae m850, an engineered homolactic strain, and convert inulin into L-LA. Comparing separate hydrolysis and fermentation (SHF) with simultaneous saccharification and fermentation (SSF), we achieved a maximal yield > 0.77 g/g with a final pH of 2.5. While SSF was limited by pH-induced inulinase inactivation, the SHF process achieved complete substrate conversion. These results demonstrate a promising platform for producing LA from a residual feedstock, drastically reducing impacts and downstream processing costs.