Development of high cell density Limosilactobacillus reuteri KUB-AC5 for cell factory using oxidative stress reduction approach.

BACKGROUND: Expression systems for lactic acid bacteria have been developed for metabolic engineering applications as well as for food-grade recombinant protein production. But the industrial applications of lactic acid bacteria as cell factories have been limited due to low biomass formation resulted in low efficiency of biomanufacturing process. Limosilactobacillus reuteri KUB-AC5 is a safe probiotic lactic acid bacterium that has been proven as a gut health enhancer, which could be developed as a mucosal delivery vehicle for vaccines or therapeutic proteins, or as expression host for cell factory applications. Similar to many lactic acid bacteria, its oxygen sensitivity is a key factor that limits cell growth and causes low biomass production. The aim of this study is to overcome the oxidative stress in L. reuteri KUB-AC5. Several genes involved in oxidative and anti-oxidative stress were investigated, and strain improvement for higher cell densities despite oxidative stress was performed using genetic engineering. RESULTS: An in-silico study showed that L. reuteri KUB-AC5 genome possesses an incomplete respiratory chain lacking four menaquinone biosynthesis genes as well as a complete biosynthesis pathway for the production of the precursor. The presence of an oxygen consuming enzyme, NADH oxidase (Nox), leads to high ROS formation in aerobic cultivation, resulting in strong growth reduction to approximately 25% compared to anaerobic cultivation. Recombinant strains expressing the ROS scavenging enzymes Mn-catalase and Mn-superoxide dismutase were successfully constructed using the pSIP expression system. The Mn-catalase and Mn-SOD-expressing strains produced activities of 873 U/ml and 1213 U/ml and could minimize the ROS formation in the cell, resulting in fourfold and sevenfold higher biomass formation, respectively. CONCLUSIONS: Expression of Mn-catalase and Mn-SOD in L. reuteri KUB-AC5 successfully reduced oxidative stress and enhanced growth. This finding could be applied for other lactic acid bacteria that are subject to oxidative stress and will be beneficial for applications of lactic acid bacteria for cell factory applications.

Analysis and Reconstitution of the Menaquinone Biosynthesis Pathway in Lactiplantibacillus plantarum and Lentilactibacillus buchneri.

In Lactococcus lactis and some other lactic acid bacteria, respiratory metabolism has been reported upon supplementation with only heme, leading to enhanced biomass formation, reduced acidification, resistance to oxygen, and improved long-term storage. Genes encoding a complete respiratory chain with all components were found in genomes of L. lactis and Leuconostoc mesenteroides, but menaquinone biosynthesis was found to be incomplete in Lactobacillaceae (except L. mesenteroides). Lactiplantibacillus plantarum has only two genes (menA, menG) encoding enzymes in the biosynthetic pathway (out of eight), and Lentilactobacillus buchneri has only four (menA, menB, menE, and menG). We constructed knock-out strains of L. lactis defective in menA, menB, menE, and menG (encoding the last steps in the pathway) and complemented these by expression of the extant genes from Lactipl. plantarum and Lent. buchneri to verify their functionality. Three of the Lactipl. plantarum biosynthesis genes, lpmenA1, lpmenG1, and lpmenG2, as well as lbmenB and lbmenG from Lent. buchneri, reconstituted menaquinone production and respiratory growth in the deficient L. lactis strains when supplemented with heme. We then reconstituted the incomplete menaquinone biosynthesis pathway in Lactipl. plantarum by expressing six genes from L. lactis homologous to the missing genes in a synthetic operon with two inducible promoters. Higher biomass formation was observed in Lactipl. plantarum carrying this operon, with an OD600 increase from 3.0 to 5.0 upon induction.