Stable Expression of Aspercillus Awamori Glucoamylase in Distiller's Yeast

Cole, G; McCabe, Peter C.; Inlow, Duane; Gelfand, David H.; Ben‐Bassat, Arie; Innis, Michael A.

doi:10.1038/nbt0488-417

Cited by 52 publications

(28 citation statements)

References 14 publications

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“…Many researchers have reported on attempts to resolve this problem by using recombinant glucoamylase-expressing yeasts with the ability to ferment starch to ethanol directly (1,3,7,9,14,16). Recombinant yeasts which coproduce glucoamylase and ␣-amylase have meanwhile been developed to further improve the efficiency of starch fermentation (2,4,6,15,21,22).…”

mentioning

confidence: 99%

Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and α-Amylase

Shigechi¹,

Koh²,

Fujita

et al. 2004

Appl Environ Microbiol

203

106

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Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis ␣-amylase by using the C-terminal-half region of ␣-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.

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mentioning

confidence: 99%

Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and α-Amylase

Shigechi¹,

Koh²,

Fujita

et al. 2004

Appl Environ Microbiol

203

106

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show abstract

“…The plasmid YEpPM18 [16] containing the wild-type GA gene and S. cerevisiae C468 (α leu2-3 leu 2-112 his 3-11 his 3-15 mal -) [4] used as the GA expression organism were gifts from Cetus (Emeryville, CA). S. cerevisiae was grown in YPD (untransformed) (10 mg/mL bacto yeast extract [Difco], 20 mg/mL bacto tryptone [Difco], 20 mg/mL glucose) and SD + His (1.7 mg/mL yeast nitrogen base without amino acids or ammonium sulfate [Difco]), 5 mg/mL ammonium sulfate, 2% glucose, 0.1 mg/mL L-histidine) (transformed) or SD + His + 1% soluble starch (for screening) at 30 °C unless otherwise indicated.…”

Section: Enzymes Strains Plasmids and Mediamentioning

confidence: 99%

“…There are two Nglycosylation sites in the catalytic domain, Asn171 and Asn395, that flank the active site. The A. awamori GA gene has been cloned and can be expressed conveniently in Saccharomyces cerevisiae [4].…”

Section: Introductionmentioning

confidence: 99%

Asp238→Asn Creates a Novel ConsensusN-Glycosylation Site inAspergillus awamori Glucoamylase

2002

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A single mutation, Asp238→Asn (D238N), of Aspergillus awamori glucoamylase (GA) was identified that increases extracellular production of the enzyme in Saccharomyces cerevisiae at 37 °C. The mutant was isolated as a suppressor of Gly396→Ser (G396S), a previously isolated temperature-sensitive mutation that decreases the thermostability and extracellular production of GA expressed in S. cerevisiae. Culture supernatants of the double mutant G396S/D238N contained much more GA than supernatants of G396S at 33.5 and 37 °C but not at 30 °C. Additionally, culture supernatants of the D238N contained 1.5 to 2-fold more GA than supernatants of wild-type when grown at 37 °C but not at 30 or 33.5 °C. The D238N mutation creates a consensus Nglycosylation site in GA. Mass spectrometry showed that the molecular weight of D238N was 2319 Da greater than that of the wild-type GA and that of D238N/G396S was 3094 Da greater than that of G396S, suggesting the presence of an additional Nlinked glycan at residue 238. No difference in thermostability or activity was observed between the G396S and G396S/D238N mutants or between wild-type and D238N GAs, and D238N did not affect intracellular GA levels at 30 or 37 °C.

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“…Some glucoamylase genes have been cloned (Boel et al 1984;Nunberg et al 1984;Yamashita et al 1985) and expressed by yeast (Innis et al 1985). Among them, the glucoamylase gene from Aspergillus awamori has been introduced into distiller's yeast strains (Cole et al 1988;Inlow et al 1988). The resulting transformants have been evaluated for direct fermentation of soluble starch and tested for the possibility of eliminating the saccharification step in alcohol production.…”

Section: Evaluation Of Various Transformantsmentioning

confidence: 99%

Direct fermentation of raw corn to ethanol by yeast transformants containing a modified Rhizopus glucoamylase gene

Ashikari

Kunikasi

Matsumoto

et al. 1989

Appl Microbiol Biotechnol

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Stable Expression of Aspercillus Awamori Glucoamylase in Distiller's Yeast

Cited by 52 publications

References 14 publications

Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and α-Amylase

Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and α-Amylase

Asp238→Asn Creates a Novel ConsensusN-Glycosylation Site inAspergillus awamori Glucoamylase

Direct fermentation of raw corn to ethanol by yeast transformants containing a modified Rhizopus glucoamylase gene

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