The lldD lactate dehydrogenase is a determinant of lactic acid tolerance in Pichia kudriavzevii by pyruvate metabolism pathway.

Lactic acid stress is common in traditional fermented foods. Pichia kudriavzevii owes its industrial prevalence to its superior acid tolerance, making deciphering its response mechanism imperative for sustainable fermentation processes. This work explored the remarkable acid tolerance of P. kudriavzevii, which can tolerate 80 g/L lactic acid. The key lactic acid tolerance gene lldD of P. kudriavzevii was determined through transcriptomic analysis. Importantly, this work knocked out the key gene lldD related to lactic acid tolerance for the first time by using the CRISPR-Cas9 technology. Meanwhile, the lldD knockout strain (pk-ΔlldD) was successfully constructed. There were significant differences between pk-ΔlldD and P. kudriavzevii in terms of tolerance to lactic acid, metabolism of lactic acid, utilization of glucose and ethanol production. In addition, the deletion of the gene lldD has a significant impact on the ABC transporter and metabolites of the amino acid metabolic pathway in P. kudriavzevii. In conclusion, this work provides a theoretical basis for engineering high acid-tolerant industrial yeast strains through targeted genetic modification. It helps enhance the stability of fermentation processes under lactic acid stress and ultimately lays a foundation for promoting efficient and low-loss production in the fermentation industry.