CRISPR/Cas9-mediated gene editing of L. bulgaricus d-lactate dehydrogenase (ldh) gene to monitor yogurt characteristics

Abstract

Yogurt is a coagulated dairy product that results from the fermentation of milk and milk products by lactic acid bacteria, specifically Lactobacillus bulgaricus and Streptococcus thermophilus. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is an emerging tool in molecular biology research which can be used extensively in prokaryotes and eukaryotes. Lactate dehydrogenase (LDH) is an important enzyme of glycolytic pathway. In this research, we have edited L. bulgaricus d-lactate dehydrogenase (ldh) gene using CRISPR/Cas9 system. Genome editing was accomplished through synthesizing gDNA on the basis of PAM region of ldh gene and ligation of synthesized gDNA with pCRISPR-SacB plasmid which was later co-transformed with mutagenic donor DNA into competent cells containing pCasRed. This method combined CRISPR/Cas9 with γ Red machinery to achieve editing of ldh gene. The PCR screening and sequencing results confirmed that, base deletion has been occurred in the target region of ldh gene. Differences in several characteristics including texture, pH were observed on the yogurt samples from newly developed L. bulgaricus strains compared to normally produced yogurt. Our results suggest that, other genes of lactobacillus can be edited through CRISPR/Cas9 technology which can be extensively used for metabolic engineering.