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Synthesis of Isomaltooligosaccharides by Saccharomyces cerevisiae Cells Expressing Aspergillus niger ?-Glucosidase.

ABSTRACT: The ?-glucosidase encoded by the aglA gene of Aspergillus niger is a secreted enzyme belonging to family 31 of glycoside hydrolases. This enzyme has a retaining mechanism of action and displays transglycosylating activity that makes it amenable to be used for the synthesis of isomaltooligosaccharides (IMOs). We have expressed the aglA gene in Saccharomyces cerevisiae under control of a galactose-inducible promoter. Recombinant yeast cells expressing the aglA gene produced extracellular ?-glucosidase activity about half of which appeared cell bound whereas the other half was released into the culture medium. With maltose as the substrate, panose is the main transglycosylation product after 8 h of incubation, whereas isomaltose is predominant after 24 h. Isomaltose also becomes predominant at shorter times if a mixture of maltose and glucose is used instead of maltose. To facilitate IMO production, we have designed a procedure by which yeast cells can be used directly as the catalytic agent. For this purpose, we expressed in S. cerevisiae gene constructs in which the aglA gene is fused to glycosylphosphatidylinositol anchor sequences, from the yeast SED1 gene, that determine the covalent binding of the hybrid protein to the cell membrane. The resulting hybrid enzymes were stably attached to the cell surface. The cells from cultures of recombinant yeast strains expressing aglA-SED1 constructions can be used to produce IMOs in successive batches.

SUBMITTER: Casa-Villegas M

PROVIDER: S-EPMC6045415 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Synthesis of Isomaltooligosaccharides by Saccharomyces cerevisiae Cells Expressing Aspergillus niger α-Glucosidase.

Casa-Villegas Mary M Marín-Navarro Julia J Polaina Julio J

ACS omega 20171116 11

The α-glucosidase encoded by the aglA gene of Aspergillus niger is a secreted enzyme belonging to family 31 of glycoside hydrolases. This enzyme has a retaining mechanism of action and displays transglycosylating activity that makes it amenable to be used for the synthesis of isomaltooligosaccharides (IMOs). We have expressed the aglA gene in Saccharomyces cerevisiae under control of a galactose-inducible promoter. Recombinant yeast cells expressing the aglA ge ...[more]

PMID: 30023572

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Project description:Backgroundβ-glucosidases (BGLs) catalyze the hydrolysis of soluble cellodextrins to glucose and are a critical component of cellulase systems. In order to engineer Saccharomyces cerevisiae for the production of ethanol from cellulosic biomass, a BGL tailored to industrial bioconversions is needed.ResultsWe applied a directed evolution strategy to a glycosyl hydrolase family 3 (GH3) BGL from Aspergillus niger (BGL1) by expressing a library of mutated bgl1 genes in S. cerevisiae and used a two-step functional screen to identify improved enzymes. Twelve BGL variants that supported growth of S. cerevisiae on cellobiose and showed increased activity on the synthetic substrate p-nitrophenyl-β-D-glucopyranoside were identified and characterized. By performing kinetic experiments, we found that a Tyr → Cys substitution at position 305 of BGL1 dramatically reduced transglycosidation activity that causes inhibition of the hydrolytic reaction at high substrate concentrations. Targeted mutagenesis demonstrated that the position 305 residue is critical in GH3 BGLs and likely determines the extent to which transglycosidation reactions occur. We also found that a substitution at Gln(140) reduced the inhibitory effect of glucose and could be combined with the Y305C substitution to produce a BGL with decreased sensitivity to both the product and substrate. Using the crystal structure of a GH3 BGL from A. aculeatus, we mapped a group of beneficial mutations to the β/α domain of the molecule and postulate that this region modulates activity through subunit interactions. Six BGL variants were identified with substitutions in the MFα pre-sequence that was used to mediate secretion of the protein. Substitutions at Pro(21) or Val(22) of the MFα pre-sequence could produce up to a twofold increase in supernatant hydrolase activity and provides evidence that expression and/or secretion was an additional factor limiting hydrolytic activity.ConclusionsUsing directed evolution on BGL1, we identified a key residue that controls hydrolytic and transglycosidation reactions in GH3 BGLs. We also found that several beneficial mutations could be combined and increased the hydrolytic activity for both synthetic and natural substrates.

Dataset Information

Synthesis of Isomaltooligosaccharides by Saccharomyces cerevisiae Cells Expressing Aspergillus niger ?-Glucosidase.

Publications

Synthesis of Isomaltooligosaccharides by <i>Saccharomyces cerevisiae</i> Cells Expressing <i>Aspergillus niger</i> α-Glucosidase.

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