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.

Synthesis of glucooligosaccharides with prebiotic potential by glucansucrase URE 13–300 acceptor reactions with maltose, raffinose and lactose

“…Glucansucrases of the glycoside hydrolase family 70 (GH70) produced by LAB of the genera Lactobacillus, Leuconostoc, Weissella and Streptococcus are of particular interest as they are capable of synthesizing short-chain GOSs with specific and defined linkages depending on the type of the used enzyme [6,11,12]. This strategy makes possible to extend significantly the range of enzymes used for synthesis of oligosaccharides with specific properties [19,[21][22][23][24]. Currently, it is considered that GOSs containing more α-1,2 or α-1,3-linked glucose residues, i.e.…”

Production of isomelezitose from sucrose by engineered glucansucrases

“…Transformation into L. lactis LM0230 and enzyme production methods were similar to that previously described [10].…”

“…The resultant plasmid pDsrS(L459).3535. usp45 relied on the Usp45 peptidase cleavage site fragment that was designed such that the DsrS(L459P) processed by L. lactis should be identical to that secreted by L. mesenteroides NRRL B-1118 with the exception of L459P. Transformation into L. lactis LM0230 and enzyme production methods were similar to that previously described [10].…”

“…The resultant plasmid, pAsr.3535.usp45, was further altered by modifying the codon for L544 ( Fig. 1) with oligonucleotides Asr.L544 P1/ P2 (Table 1) for amino acid substitutions P, S, E, and R. examined the effect of analogous modifications to other heterologously expressed glucansucrases, and expressed several of these enzymes extracellularly in Lactococcus lactis [10].…”

“…It has been produced as a minor acceptor product in reactions of alternansucrase [2] and pDsrI.3535.usp45 [10] PCR amplified with p3535.Gib P1/ P2 (Table 1). The resultant plasmid pDsrS(L459).3535. usp45 relied on the Usp45 peptidase cleavage site fragment that was designed such that the DsrS(L459P) processed by L. lactis should be identical to that secreted by L. mesenteroides NRRL B-1118 with the exception of L459P.…”