Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus

Hiroyuki Inoue, Tatsuya Fujii, Miho Yoshimi, Larry E. Taylor, Stephen R. Decker, Seiichiro Kishishita, Makoto Nakabayashi, Kazuhiko Ishikawa

Research output: Contribution to journalArticle

27 Citations (Scopus)

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 languageEnglish
Pages (from-to)823-830
Number of pages8
JournalJournal of Industrial Microbiology and Biotechnology
Volume40
Issue number8
DOIs
Publication statusPublished - Aug 2013
Externally publishedYes

Fingerprint

Acremonium
Starch
Enzymes
Genes
Cellulose
Plasmids
Genetic Terminator Regions
Cellulose 1,4-beta-Cellobiosidase
Genome
Proteins
Glucan 1,4-alpha-Glucosidase
Uracil
Glycoside Hydrolases
Polymerase chain reaction
Cell culture
Molecular Weight
Molecular weight
Polymerase Chain Reaction
Substrates

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 journalArticle

Inoue, Hiroyuki ; Fujii, Tatsuya ; Yoshimi, Miho ; Taylor, Larry E. ; Decker, Stephen R. ; Kishishita, Seiichiro ; Nakabayashi, Makoto ; Ishikawa, Kazuhiko. / Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus. In: Journal of Industrial Microbiology and Biotechnology. 2013 ; Vol. 40, No. 8. pp. 823-830.
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