Secretion of Bacillus α‐amylase from yeast directed by glucoamylase I signal sequence of Saccharomyces diastaticus

Abstract

For the secretion of Badllus stearothermophilus a-amylase from yeast, a recombinant plasmid pGAT17 was constructed by fusing B. stearothermophilus a -amylase structural gene in frame to the promoter and signal sequence of Saccharomyces diastaticus glucoamytase I gene (STAJ). The secretion of the heterologous a -amylase from S. diastaticus transformed with pGAT17 was confirmed by the halo formation around colonies on selective starch agar medium. About 80% of the total ~-amylase activity was detected in the extraceUular culture medium. The secreted a-amylase was glycosylated and its molecular weight increased from 61 kDa to 75 kDa. The thermostability of the the glycosylated a -amylase was markedly enhanced, compared with that of the non-glycosytated enzyme from E. coli. ~TRODUCTIONThe yeast S. diastaticus which is closely related to S. cerevisiae genetically and physiologically, produces extracellular glucoamylase isozyme I, II or III encoded by STA1, STA2 or STA3 gene, respectively, and can ferment starch (1,2). We previously cloned STAI gene (3) and identified the N-terminal signal sequence targeting nascent polypeptide to the endoplasmic reticulum of the secretory apparatus (4). The signal sequence of the a-mating factor (5), invertase (6), acid phosphatase (7) or killer toxin (8) were most extensively used for the secretion of heterologous proteins from yeast. However, few studies on glucoamylase I signal sequence have been reported so far (9-11).The abbreviations used are: DNS, dinitrosalicylic acd; SDS-PAGE, sodium dodesyl sulfate-polyacrylamide gel electrophoresis

A re-evaluation of diastatic Saccharomyces cerevisiae strains and their role in brewing

“…Accordingly, most attempts to engineer S. cerevisiae strains for glucoamylase production have involved introduction of genes from fungal species other than S. cerevisiae (Görgens et al 2015). The STA1 signal peptide that mediates the extracellular expression of the enzyme has however been exploited for the expression of other hydrolytic enzymes such as α-amylase, cellobiohydrolase, and β-glucosidase (Nam et al 1994;Kang et al 1996;Marín-Navarro et al 2011;Gurgu et al 2011;Gurgu et al 2013).…”

Mutagenesis of the glucoamylase signal peptide ofSaccharomyces diastaticusand functional analysis inSaccharomyces cerevisiae

“…Each complementary DNA strand was phosphorylated, annealed and ligated to pBluescript and the PGK promoter. Each 1.7 kb Bam HI– Sal I DNA fragment was isolated from the resultant plasmid pBPGKs (pBPGKwt, m1, m2, m3, m4) and cloned into the Bam HI and Sal I site of pAA7 containing yeast GAL7 transcription terminator, generating plasmid pAPSs [10]. The CMCase structural gene isolated from pUEC19 [12] was ligated in frame with the GSPs of pAPSs treated with Sal I and alkaline phosphatase.…”

Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae

“…Each complementary DNA strand was phosphorylated, annealed and ligated to pBluescript and the PGK promoter. Each 1.7 kb BamHI^SalI DNA fragment was isolated from the resultant plasmid pBPGKs (pBPGKwt, m1, m2, m3, m4) and cloned into the BamHI and SalI site of pAA7 containing yeast GAL7 transcription terminator, generating plasmid pAPSs [10]. The CMCase structural gene isolated from pUEC19 [12] was ligated in frame with the GSPs of pAPSs treated with SalI and alkaline phosphatase.…”

“…The glucoamylase I gene (STA1) of Saccharomyces diastaticus is translated as a large precursor composed of three characteristic regions: an N-terminal signal peptide (21 amino acids) required for targeting glucoamylase to the endoplasmic reticulum, a threonine-and serine-rich region, and a C-terminal functional domain [7]. A few studies on heterologous protein secretion using the glucoamylase signal peptide (GSP) have been reported, but the secretion e¤ciency was not high [8,9,10,11].…”