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

Abstract

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 gene produced extracellular α-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.

Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans

“…α-Glucosidases of filamentous fungi have been suggested as feasible catalysts for transglycosylation. Thus, an Aspergillus enzyme with high transglycosylating activity was reported to produce panose and isomaltose from maltose [18,59,60]. When an A. nidulans α-glucosidase with strong transglycosylating activity was reacted with 5 g/L maltose during 6 h, approximately 50% of maltose was converted to transglycosylation products, 60% of which was isomaltose [60].…”

Identification and characterization of novel transglycosylating α‐glucosidase from Aspergillus neoniger

“…(2017) and Casa‐Villegas et al . (2017) when homology models of α‐glucosidase from A. niger strains were used. Docking studies also showed Phe693, Thr694, Thr228, Trp343, Met491 and Trp453 as the adjacent sites to catalytic site (cf.…”

Expression and function of an HAC1-regulated multi-copy xylanase gene in Saccharomyces cerevisiae

“…S3). SphI-treated pYES2-PαXC-rDNA, linearized at the rDNA loci, was transformed into S. cerevisiae INVSc1 using the optimized LiAC/ssDNA chemical method [19] .…”

“…Because HAC1 contains a 250-bp intron, it is not expressed in normal S. cerevisiae cells. However, when improperly folded proteins accumulate in the ER, HAC1 is spliced, the intron is removed, and active HAC1 protein is expressed [19] . Therefore, in the current study, improper protein folding was induced in wild-type S. cerevisiae using DTT, a protein folding inhibitor, allowing amplification of activated HAC1.…”

“…In addition, further investigation showed The promoter is one of the most crucial elements of an expression vector because it influences the mRNA synthesis rate. Accordingly, a strong promoter contributes to higher expression of a heterologous protein [19] . To improve the expression of cellobiohydrolase in A. niger, Qin et al used the glaA promoter sequence, which promotes the expression of glucoamylase, to drive the expression of the CBH gene.…”