Production of Lactate by Metabolically Engineered Scheffersomyces stipitis

Lactate is a valuable compound used in food, chemical, and pharmaceutical industries. High-value, optically pure L- or D-lactate can be synthesized microbially via specific dehydrogenases. The non-conventional yeast <i>Scheffersomyces stipitis</i>, which is known for fermenting both hexoses and pentoses, is a promising host for biochemical production from lignocellulosic biomass but does not naturally produce lactate. In this study, we engineered <i>S. stipitis</i> to produce lactate by expressing two codon-optimized bacterial L-lactate dehydrogenase genes under the control of strong native promoters. The engineered strain produced 7.42 g/L (0.46 g/g yield) and 11.67 g/L (0.58 g/g yield) lactate from glucose and xylose, respectively. The highest titer, 19.27 g/L (0.52 g/g yield), was achieved from 50 g/L xylose after 74 h. Increasing the fermentation temperature from 28 °C to 32 °C improved yield by 30%, while a neutralizing agent further enhanced yield by 25% and prevented lactate degradation following carbon depletion. Although the wildtype strain produced a significant amount of ethanol on both glucose and xylose, the engineered strain produced ethanol as a side product exclusively on glucose and not on xylose. This phenomenon could be advantageous for biotechnological applications and may reflect shifts in gene expression depending on the carbon source or even on the presence of lactate.