Glucansucrase Gtf180-ΔN 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 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-glucan synthesis by mutational engineering of the Gtf180-ΔN enzyme of Lactobacillus reuteri 180 results in the construction of more efficient glycosylation biocatalysts. Gtf180-ΔN mutants (L938F, L981A, and N1029M) with an impaired α-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-α-D-glucoside product, explaining the improved monoglycosylation yields. Analysis of the available high resolution 3D crystal structure of the Gtf180-ΔN protein provided a clear understanding of how mutagenesis of residues L938, L981, and N1029 impaired α-glucan synthesis, thus yielding mutants with an improved glycosylation potential.

Keywords: Acceptor reaction; Catechol; Enzyme engineering; Glucansucrase; Glycosylation; Lactobacillus reuteri.

Fig. 1

a Time-course synthesis of α-D-glucosides of catechol by WT Gtf180-ΔN (400 mM catechol, 1000 mM sucrose, 4 U/mL Gtf180-ΔN). T = 37 °C, pH = 4.7. b Time-course synthesis of catechol-G1 by WT Gtf180-ΔN and mutants derived (400 mM catechol, 1000 mM sucrose, 4 U/mL Gtf180-ΔN). T = 37 °C, pH = 4.7

Fig. 2

Monoglycosylation yields of WT Gtf180-ΔN and the L981A mutant derived (400 mM catechol/resorcinol/hydroquinone/butanol, 58 mM hexanol, 4 mM octanol, 1000 mM sucrose, 4 U/mL Gtf180-ΔN). All monoglycosylation yields represent maximum values (incubation time dependent on acceptor substrate). T = 37 °C, pH = 4.7

Fig. 3

a, b Conversion of the resorcinol acceptor substrate and G1 production by WT Gtf180-ΔN and the L981A mutant derived (400 mM resorcinol, 1000 mM sucrose, 4 U/mL Gtf180-ΔN (mutant)). T = 37 °C, pH = 4.7

Fig. 4

1D ¹H NMR spectra of a butyl glucoside, b hexyl glucoside, c octyl glucoside, d catechol-G1, e resorcinol-G1, f hydroquinone-G1, g catechol-3′G2, and h catechol-6′G2

Fig. 5

Correlation between α-GSP for sucrose as acceptor substrate, K _m for catechol and catechol-G1 as acceptor substrate, and G1 yield of WT Gtf180-ΔN and mutants derived. Data are listed in Tables 1 and 3. Filled circles G1 yield (%), unfilled circles K _m for catechol (mM), unfilled squares K _m for catechol-G1 (mM)

Fig. 6

Stereo view of Gtf180-ΔN with the acceptor maltose (yellow carbon atoms) bound in subsites +1 and +2 (PDB: 3KLL). Residue N1029 from domain A (blue) provides direct and indirect (water-mediated) hydrogen bonds to the non-reducing end glucosyl unit bound at subsite +1. Residues L938 and L981 from domain B (green) are also near subsite +1. This figure has been adapted from Meng et al. (2015)