title: Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus.
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.
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-0046, Japan. inoue-h@aist.go.jp
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Inoue
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fujii
authors.forename: Miho
authors.initials: M
authors.lastname: Yoshimi
authors.forename: Larry E
authors.initials: LE
authors.lastname: Taylor
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Decker
authors.forename: Seiichiro
authors.initials: S
authors.lastname: Kishishita
authors.forename: Makoto
authors.initials: M
authors.lastname: Nakabayashi
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishikawa
mesh_terms: D000164:Acremonium / Q000201:enzymology* / Q000235:genetics*; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D013213:Starch / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D002482:Cellulose; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase; D043366:Cellulose 1,4-beta-Cellobiosidase