Dataset Information

Glucansucrase Gtf180-?N of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules.

ABSTRACT: Glucansucrases have a broad acceptor substrate specificity and receive increased attention as biocatalysts for the glycosylation of small non-carbohydrate molecules using sucrose as donor substrate. However, the main glucansucrase-catalyzed reaction results in synthesis of ?-glucan polysaccharides from sucrose, and this strongly impedes the efficient glycosylation of non-carbohydrate molecules and complicates downstream processing of glucosylated products. This paper reports that suppressing ?-glucan synthesis by mutational engineering of the Gtf180-?N enzyme of Lactobacillus reuteri 180 results in the construction of more efficient glycosylation biocatalysts. Gtf180-?N mutants (L938F, L981A, and N1029M) with an impaired ?-glucan synthesis displayed a substantial increase in monoglycosylation yields for several phenolic and alcoholic compounds. Kinetic analysis revealed that these mutants possess a higher affinity for the model acceptor substrate catechol but a lower affinity for its mono-?-D-glucoside product, explaining the improved monoglycosylation yields. Analysis of the available high resolution 3D crystal structure of the Gtf180-?N protein provided a clear understanding of how mutagenesis of residues L938, L981, and N1029 impaired ?-glucan synthesis, thus yielding mutants with an improved glycosylation potential.

SUBMITTER: Devlamynck T

PROVIDER: S-EPMC4980424 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Glucansucrase Gtf180-ΔN of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules.

Devlamynck Tim T Te Poele Evelien M EM Meng Xiangfeng X van Leeuwen Sander S SS Dijkhuizen Lubbert L

Applied microbiology and biotechnology 20160406 17

Glucansucrases have a broad acceptor substrate specificity and receive increased attention as biocatalysts for the glycosylation of small non-carbohydrate molecules using sucrose as donor substrate. However, the main glucansucrase-catalyzed reaction results in synthesis of α-glucan polysaccharides from sucrose, and this strongly impedes the efficient glycosylation of non-carbohydrate molecules and complicates downstream processing of glucosylated products. This paper reports that suppressing α-g ...[more]

PMID: 27052379

Similar Datasets

Project description:BackgroundLactic acid bacteria (LAB) are one of the microorganisms of choice for the development of protein delivery systems for therapeutic purposes. Although there are numerous tools to facilitate genome engineering of lactobacilli; transformation efficiency still limits the ability to engineer their genomes. While genetically manipulating Lactobacillus reuteri ATCC PTA 6475 (LR 6475), we noticed that after an initial transformation, several LR 6475 strains significantly improved their ability to take up plasmid DNA via electroporation. Our goal was to understand the molecular basis for how these strains acquired the ability to increase transformation efficiency.ResultsStrains generated after transformation of plasmids pJP067 and pJP042 increased their ability to transform plasmid DNA about one million fold for pJP067, 100-fold for pSIP411 and tenfold for pNZ8048. Upon sequencing of the whole genome from these strains, we identified several genomic mutations and rearrangements, with all strains containing mutations in the transformation related gene A (trgA). To evaluate the role of trgA in transformation of DNA, we generated a trgA null that improved the transformation efficiency of LR 6475 to transform pSIP411 and pJP067 by at least 100-fold, demonstrating that trgA significantly impairs the ability of LR 6475 to take-up plasmid DNA. We also identified genomic rearrangements located in and around two prophages inserted in the LR 6475 genome that included deletions, insertions and an inversion of 336 Kb. A second group of rearrangements was observed in a Type I restriction modification system, in which the specificity subunits underwent several rearrangements in the target recognition domain. Despite the magnitude of these rearrangements in the prophage genomes and restriction modification systems, none of these genomic changes impacted transformation efficiency to the level induced by trgA.ConclusionsOur findings demonstrate how genetic manipulation of LR 6475 with plasmid DNA leads to genomic changes that improve their ability to transform plasmid DNA; highlighting trgA as the primary driver of this phenotype. Additionally, this study also underlines the importance of characterizing genetic changes that take place after genome engineering of strains for therapeutic purposes.

Dataset Information

Glucansucrase Gtf180-?N of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules.

Publications

Glucansucrase Gtf180-ΔN of Lactobacillus reuteri 180: enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure