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Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans

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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.

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Acknowledgments

We thank Dr. David Mills for graciously providing plasmid pMSP3535H3 used in this study. We also thank Dr. Karl Vermillion for obtaining the NMR spectra on these samples and Suzanne Unser and Kristina Glenzinski for their expert technical support. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.

Conflict of interest

The authors declare that they have no competing interests.

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  1. 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

    Christopher D. Skory & Gregory L. Côté

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  1. Christopher D. Skory
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  2. Gregory L. Côté
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Correspondence to Christopher D. Skory.

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Skory, C.D., Côté, G.L. Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans. Appl Microbiol Biotechnol 99, 10001–10010 (2015). https://doi.org/10.1007/s00253-015-6854-0

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