Abstract
A starch-inducible homologous expression system in Acremonium cellulolyticus was constructed to successfully produce recombinant cellulolytic enzymes. A. cellulolyticus Y-94 produced amylolytic enzymes and cellulolytic enzymes as major proteins in the culture supernatant when grown with soluble starch (SS) and Solka-Flock cellulose (SF), respectively. To isolate a strong starch-inducible promoter, glucoamylase (GlaA), which belongs to glycoside hydrolase family 15, was purified from the SS culture of Y-94, and its gene was identified in the genome sequence. The 1.4-kb promoter and 0.4-kb terminator regions of glaA were amplified by polymerase chain reaction (PCR) and used in the construction of a plasmid that drives the expression of the cellobiohydrolase I (Cel7A) gene from A. cellulolyticus. The resultant expression plasmid, containing pyrF as a selection marker, was randomly integrated into the genome of the A. cellulolyticus Y-94 uracil auxotroph. The prototrophic transformant, Y203, produced recombinant Cel7A as an extracellular protein under control of the glaA promoter in the SS culture. Recombinant and wild-type Cel7A were purified from the SS culture of Y203 and the SF culture of A. cellulolyticus CF-2612, respectively. Both enzymes were found to have the same apparent molecular weight (60 kDa), thermostability (T m 67.0 C), and optimum pH (pH 4.5), and showed similar catalytic properties for soluble and insoluble substrates. These results suggest that the A. cellulolyticus starch-inducible expression system will be helpful for characterization and improvement of fungal cellulolytic enzymes.
| Original language | English |
|---|---|
| Pages (from-to) | 823-830 |
| Number of pages | 8 |
| Journal | Journal of Industrial Microbiology and Biotechnology |
| Volume | 40 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - Aug 2013 |
| Externally published | Yes |
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Keywords
- Acremonium cellulolyticus
- Cellobiohydrolase I
- Cellulase production
- Glucoamylase
- Homologous expression
- Protein expression
ASJC Scopus subject areas
- Biotechnology
- Applied Microbiology and Biotechnology
Cite this
Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus. / Inoue, Hiroyuki; Fujii, Tatsuya; Yoshimi, Miho; Taylor, Larry E.; Decker, Stephen R.; Kishishita, Seiichiro; Nakabayashi, Makoto; Ishikawa, Kazuhiko.
In: Journal of Industrial Microbiology and Biotechnology, Vol. 40, No. 8, 08.2013, p. 823-830.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus
AU - Inoue, Hiroyuki
AU - Fujii, Tatsuya
AU - Yoshimi, Miho
AU - Taylor, Larry E.
AU - Decker, Stephen R.
AU - Kishishita, Seiichiro
AU - Nakabayashi, Makoto
AU - Ishikawa, Kazuhiko
PY - 2013/8
Y1 - 2013/8
N2 - A starch-inducible homologous expression system in Acremonium cellulolyticus was constructed to successfully produce recombinant cellulolytic enzymes. A. cellulolyticus Y-94 produced amylolytic enzymes and cellulolytic enzymes as major proteins in the culture supernatant when grown with soluble starch (SS) and Solka-Flock cellulose (SF), respectively. To isolate a strong starch-inducible promoter, glucoamylase (GlaA), which belongs to glycoside hydrolase family 15, was purified from the SS culture of Y-94, and its gene was identified in the genome sequence. The 1.4-kb promoter and 0.4-kb terminator regions of glaA were amplified by polymerase chain reaction (PCR) and used in the construction of a plasmid that drives the expression of the cellobiohydrolase I (Cel7A) gene from A. cellulolyticus. The resultant expression plasmid, containing pyrF as a selection marker, was randomly integrated into the genome of the A. cellulolyticus Y-94 uracil auxotroph. The prototrophic transformant, Y203, produced recombinant Cel7A as an extracellular protein under control of the glaA promoter in the SS culture. Recombinant and wild-type Cel7A were purified from the SS culture of Y203 and the SF culture of A. cellulolyticus CF-2612, respectively. Both enzymes were found to have the same apparent molecular weight (60 kDa), thermostability (T m 67.0 C), and optimum pH (pH 4.5), and showed similar catalytic properties for soluble and insoluble substrates. These results suggest that the A. cellulolyticus starch-inducible expression system will be helpful for characterization and improvement of fungal cellulolytic enzymes.
AB - A starch-inducible homologous expression system in Acremonium cellulolyticus was constructed to successfully produce recombinant cellulolytic enzymes. A. cellulolyticus Y-94 produced amylolytic enzymes and cellulolytic enzymes as major proteins in the culture supernatant when grown with soluble starch (SS) and Solka-Flock cellulose (SF), respectively. To isolate a strong starch-inducible promoter, glucoamylase (GlaA), which belongs to glycoside hydrolase family 15, was purified from the SS culture of Y-94, and its gene was identified in the genome sequence. The 1.4-kb promoter and 0.4-kb terminator regions of glaA were amplified by polymerase chain reaction (PCR) and used in the construction of a plasmid that drives the expression of the cellobiohydrolase I (Cel7A) gene from A. cellulolyticus. The resultant expression plasmid, containing pyrF as a selection marker, was randomly integrated into the genome of the A. cellulolyticus Y-94 uracil auxotroph. The prototrophic transformant, Y203, produced recombinant Cel7A as an extracellular protein under control of the glaA promoter in the SS culture. Recombinant and wild-type Cel7A were purified from the SS culture of Y203 and the SF culture of A. cellulolyticus CF-2612, respectively. Both enzymes were found to have the same apparent molecular weight (60 kDa), thermostability (T m 67.0 C), and optimum pH (pH 4.5), and showed similar catalytic properties for soluble and insoluble substrates. These results suggest that the A. cellulolyticus starch-inducible expression system will be helpful for characterization and improvement of fungal cellulolytic enzymes.
KW - Acremonium cellulolyticus
KW - Cellobiohydrolase I
KW - Cellulase production
KW - Glucoamylase
KW - Homologous expression
KW - Protein expression
UR - http://www.scopus.com/inward/record.url?scp=84880773351&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84880773351&partnerID=8YFLogxK
U2 - 10.1007/s10295-013-1286-2
DO - 10.1007/s10295-013-1286-2
M3 - Article
VL - 40
SP - 823
EP - 830
JO - Journal of Industrial Microbiology and Biotechnology
JF - Journal of Industrial Microbiology and Biotechnology
SN - 1367-5435
IS - 8
ER -