title: Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans.
abstract: We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.
authors.affiliation: Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., Peoria, IL, 61604, USA. chris.skory@ars.usda.gov.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Skory
authors.affiliation: Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., Peoria, IL, 61604, USA.
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Côté
mesh_terms: D019149:Bioreactors / Q000382:microbiology; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression; D005822:Genetic Vectors; D005936:Glucans / Q000378:metabolism*; D016695:Glycosyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000302:isolation & purification; D013294:Lactococcus lactis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics; D009561:Nisin / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D003470:Culture Media; D005936:Glucans; D011994:Recombinant Proteins; D009561:Nisin; D019344:Lactic Acid; D016695:Glycosyltransferases; C033837:alternansucrase
keywords: Dextran; Dextransucrase; Glucansucrase; Insoluble polysaccharide; Mutan