The effect of lactate dehydrogenase expression from different sources on lactate utilization in Saccharomyces cerevisiae

Abstract

The excessive accumulation of lactic acid in corn pulp, a by-product of wet processing of corn starch, seriously restricts its resource utilization, especially in the field of feed, which is prone to cause digestive dysfunction in animals. This study aims to explore the expression characteristics of lactate dehydrogenase (LDH) from different sources in yeast and its influence on lactate metabolism, in order to optimize the biological fermentation process of corn slurry. This study aimed at the problem of insufficient natural lactic acid metabolism efficiency of Saccharomyces cerevisiae, systematically analyzed the microbial lactic acid catabolic network, and constructed highly efficient degradation strains through genetic engineering means. The heterologous expression strategy of multi-source lactate dehydrogenase (LDH) was adopted. Through genetic engineering technology, the yeast endogenous gene FCB2, LctO derived from Lactococcus lactis and LctD derived from Escherichia coli were introduced into Saccharomyces cerevisiae to construct three groups of engineered strains. Combined with CRISPR-Cas9 gene editing, yeast transformation and high performance liquid chromatography (HPLC) analysis, Systematically evaluate the effects of different LDH on the lactic acid utilization efficiency of Saccharomyces cerevisiae. Through fermentation tests on seven well-performing industrial strains of Saccharomyces cerevisiae preserved in the laboratory, a strain Y02 with the best fermentation effect of corn slurry was screened out. To further increase the lactic acid metabolism rate of strain Y02, lactate dehydrogenase genes from different sources were overexpressed in the form of chromosomal integration expression. In the YPL medium with lactic acid as the sole carbon source, the FCB2 gene (cytotin c REDOX enzyme) derived from yeast itself significantly increased the utilization rate of lactic acid. It is nearly twice as high as the original strain. However, continuing to increase the expression copy number of FCB2 instead led to a decrease in the lactic acid metabolism rate. It was found through the detection of intracellular pyruvate content that the growth of the strain was inhibited due to excessive accumulation of pyruvate. Therefore, moderate expression of the FCB2 gene can effectively enhance the lactic acid catabolism capacity of Saccharomyces cerevisiae.