title: Regulation of EPS production in Lactobacillus casei LC2W through metabolic engineering.
abstract: Lactobacillus casei LC2W is an exopolysaccharide(EPS)-producing strain with probiotic effects. The low efficiency and unclear regulation mechanism of EPS biosynthesis have become main constraints for its application in food industry. To investigate the major rate-limiting factors of EPS biosynthesis and to improve its yield, metabolic engineering was applied to this strain. Eight relevant genes related to central metabolism, sugar-nucleotides supply, glycosyltransferase and cofactor engineering were cloned and overexpressed. The results suggested that nox, pfk, rfbB and galT genes were the largest contributors to EPS biosynthesis in this study, which elevated EPS yield by 46·0, 20, 17·4 and 19·6% respectively. Notably, under aerobic condition which was not a suitable condition for lactobacilli to grow in, recombinant strain LC-nox achieved the highest EPS yield of 263·7 mg l(-1) , which was increased by 75% compared to that of the starting strain. The oxygen stress was excluded since the phenomenon was not observed in the control strain under the same condition. Therefore, it was probably that higher NADH oxidase activity led to a decreased NADH availability and reduced lactate concentration, which resulted in the elevation of EPS yield.
  
  
  CONCLUSIONS
  This study contributed to the understanding of EPS biosynthesis in Lact. casei through metabolic engineering and provided a starting point for introducing cofactor engineering into this strain. Overexpression of NADH oxidase was found to have a most significant effect on the EPS production. It is the first report that EPS could be accumulated to such a high level under aerobic condition in lactobacilli. Our results provided a novel strategy for the improvement of EPS production in lactic acid bacteria.
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd., Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China.
authors.forename: N
authors.initials: N
authors.lastname: Li
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd., Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd., Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd., Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China.
authors.forename: C
authors.initials: C
authors.lastname: You
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Dairy Research Institute, Bright Dairy & Food Co., Ltd., Synergetic Innovation Center of Food Safety and Nutrition, Shanghai, China.
authors.forename: B
authors.initials: B
authors.lastname: Guo
mesh_terms: D005285:Fermentation; D019344:Lactic Acid / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000235:genetics* / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D046928:Peptidoglycan Glycosyltransferase / Q000235:genetics / Q000378:metabolism; D025221:Phosphofructokinases / Q000096:biosynthesis / Q000235:genetics; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D019936:Probiotics / Q000378:metabolism; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D009097:Multienzyme Complexes; D011135:Polysaccharides, Bacterial; D009243:NAD; D019344:Lactic Acid; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D046928:Peptidoglycan Glycosyltransferase; D025221:Phosphofructokinases; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase
keywords: Cofactor engineering; Lactobacillus casei; NADH oxidase; exopolysaccharide; metabolic engineering