Cloning and Heterologous Expression of Lactate Dehydrogenase Genes from Acid-Tolerant <i>Lactobacillus acetotolerans</i> HT

Abstract

Lactic acid (LA) has been used for wide range of food processing and industrial applications, for example, as a raw material of biodegradable plastics, poly(lactic acid)s (PLAs). Thus, there is a demand to incorporate acid-resistance for effective LA production. Acid-tolerant lactic acid bacteria (LAB), Lactobacillus acetotolerans HT, has been isolated from rice vinegar (pH 2.9, 6 % acetic acid). In this study, genes for the D-and L-lactate dehydrogenases (D-LDH, L-LDH1, and L-LDH2) of Lb. acetotolerans HT, which constitute the key enzymes in cell growth and lactic acid production, were cloned and identified. Through heterologous expression of LDH genes in Escherichia coli DH5α, recombinant E. coli DH5α harboring the D-LDH (ldhD) or L-LDH1 (ldhL1) genes were found to produce D-LA or L-LA, respectively, whereas the strain harboring the L-LDH2 (ldhL2) gene did not produce L-LA. This finding strongly suggests that the translational product of the ldhL2 gene does not exhibit L-LDH activity in vivo.

Recombinant production, purification, and biochemical characterization of a novel L-lactate dehydrogenase from Bacillus cereus NRC1 and inhibition study of mangiferin

“…Additionally, the LDH gene was cloned into the pET-28a (+) vector, yielding the LDH-pET clone, whose expression was triggered by 0.1 mM IPTG. These results corroborate with Goto et al’s (2018) findings of cloning, sequencing, and identifying the LDH gene by heterologous expression in E. coli DH5α™. Additionally, with the help of the plasmid pET28a, the Ruminococcaceae strain CPB6 gene encoding L-LDH was effectively cloned and expressed in E. coli BL21 (DE3) ( Yang et al, 2020 ).…”

“…Additionally, the LDH gene was cloned into the pET-28a (+) vector, yielding the LDH-pET clone, whose expression was triggered by 0.1 mM IPTG. These results corroborate with Goto et al's (2018) findings of cloning, sequencing, and identifying the LDH gene by heterologous expression in E. coli DH5α ™ .…”

Metabolic footprint analysis of volatile metabolites by gas chromatography-ion mobility spectrometry to discriminate between different fermentation temperatures during Streptococcus thermophilus milk fermentation

“…Streptococcus thermophilus is a homofermentative LAB (Radke-Mitchell and Sandine, 1984;Guyot et al, 2015;Cui et al, 2016) and uses the glycolytic pathway for growth and generating lactic acid as a by-product. The glycolysis is driven by the rate-limiting enzymes β-galactosidase (Greenberg and Mahoney, 1982), phosphofructokinase (Viana et al, 2005), lactate dehydrogenase (Goto et al, 2018), and H + -atpase (Liu et al, 2017). Streptococcus thermophilus is significantly associated with β-galactosidase (Greenberg and Mahoney, 1982) and H + -atpase (Liu et al, 2017); a few studies have indicated that S. thermophilus has a gene encoding lactate dehydrogenase with detectable enzyme activity (Bolotin et al, 2001), indirectly indicating favor of glycolysis.…”

Enhancement of lactate fraction in poly(lactate-<i>co</i>-3-hydroxybutyrate) synthesized by <i>Escherichia coli</i> harboring the D-lactate dehydrogenase gene from <i>Lactobacillus acetotolerans</i> HT

“…In our previous study, we cloned and identified the ldhD gene of Lactobacillus acetotolerans HT (Goto et al, 2018). The recombinant E. coli DH5a with the introduced ldhD gene from Lb.…”