Identification of a Genomic Region Containing a Novel Promoter Resistant to Glucose Repression and Over-Expression of [beta]-Glucosidase Gene in Hypocrea orientalis EU7-22

A high concentration of glucose in the medium could greatly inhibit the expression of cellulase in filamentous fungi. The aspartic protease from fungus Hypocrea orientalis EU7-22 could efficiently express under both induction condition and glucose repression condition. Based on the sequence of struc...

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Bibliographic Details
Published in:International journal of molecular sciences 2013-04, Vol.14 (4), p.8479
Main Authors: Long, Chuannan, Cheng, Yijin, Gan, Lihui, Liu, Jian, Long, Minnan
Format: Article
Language:English
Subjects:
Biomass
Carbon
Cellulase
Cellulose
Cloning
Enzymes
Fungi
Genomes
Glucose
Lignocellulose
Polymerase chain reaction
Proteins
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Summary:A high concentration of glucose in the medium could greatly inhibit the expression of cellulase in filamentous fungi. The aspartic protease from fungus Hypocrea orientalis EU7-22 could efficiently express under both induction condition and glucose repression condition. Based on the sequence of structure gene of aspartic protease, the upstream sequence harboring the putative promoter proA for driving the expression of aspartic protease was obtained by genome walking. The upstream sequence contained the typical promoter motifs "TATA" and "CAAT". The β-glucosidase gene (Bgl1) from H. orientalis was cloned and recombined with promoter proA and terminator trpC. The expression cassette was ligated to the binary vector to form pUR5750-Bgl1, and then transferred into the host strain EU7-22 via Agrobacterium tumefaciens mediated transformation (ATMT), using hygromycin B resistance gene as the screening marker. Four transformants Bgl-1, Bgl-2, Bgl-3 and Bgl-4 were screened. Compared with the host strain EU7-22, the enzyme activities of filter paper (FPA) and β-glucosidase (BG) of transformant Bgl-2 increased by 10.6% and 19.1% under induction condition, respectively. The FPA and BG activities were enhanced by 22.2% and 700% under 2% glucose repression condition, respectively, compared with the host strain. The results showed that the putative promoter proA has successfully driven the over-expression of Bgl1 gene in H. orientalis under glucose repression condition.
ISSN:1661-6596
1422-0067
DOI:10.3390/ijms14048479