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.

Characterization of a feruloyl esterase B from Talaromyces cellulolyticus

“…18) The results that the purified TcFaeB exhibits a smeared single band by SDS-PAGE (Fig. S1) and its molecular mass is slightly larger than that calculated from the sequence suggest the glycosylation modification of the Ser-/Thr-rich linker region in the enzyme.…”

“…The T. cellulolyticus YP-4 uracil autotroph from Y-94 strain (CBS136886) was maintained on potato dextrose agar plates containing uracil and uridine at final concentrations of 1 g/L each. 18) Transformants of T. cellulolyticus YP-4 were maintained on minimum medium agar plates. 19) Construction of the recombinant enzyme.…”

Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus)

“…We successfully overexpressed the cellulase and hemicellulase genes in this organism and constructed a starchinducible homologous expression system. 5,6) The expression of cellulase and hemicellulase genes is regulated by various transcriptional factors in filamentous fungi. [7][8][9][10] The transcriptional factor XlnR/ Xyr1 was isolated as an inducer of xylanase genes in Aspergillus niger.…”

“…cellulolyticus YP-4, 5) which is a uracil auxotrophic strain derived from T. cellulolyticus Y-94 1) (CBS 136886, FERM BP-5826), was maintained on potato dextrose agar (Difco, Detroit, MI) plates containing 1 g/L uracil and 1 g/L uridine. The transformants were maintained on MM plates (1% glucose, 10 mM NH 4 Cl, 10 mM potassium phosphate (pH 6.5), 7 mM KCl, and 2 mM MgSO 4 ).…”

Gene Targeting by RNAi-Mediated Knockdown of Potent DNA Ligase IV Homologue in the Cellulase-Producing Fungus Talaromyces cellulolyticus

“…cellulolyticus strain Y-94 (FERM BP-5826, CBS136886) and its uracil auxotrophic mutant YP-4 strain were maintained in potato dextrose agar plates (Difco, USA) in the presence or absence of uracil and uridine [22]. Genome sequences of the T. cellulolyticus strain Y-94 were combed to identify the potent DNA ligase IV homologous gene (ligD) by performing a DNA-BLAST search using the ligD sequence from Talaromyces marneffei as a probe sequence [23].…”