Identification of a genomic region containing a novel promoter resistant to glucose repression and over-expression of β-glucosidase gene in Hypocrea orientalis EU7-22

Abstract

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.

Figure 1

Gel electrophoresis of amplified products after the three nested genome walking PCRs. Lanes 1–4 were the first nested PCR products with the genome DNA as template using primer SP1 and AP1, SP1 andAP2, SP1 and AP3, SP1 and AP4, respectively; Lanes 5–8 were the second nested PCR products with the firstly nested PCR products as template using primer SP2 and AP1, SP2 and AP2, SP2 and AP3, SP2 and AP4, respectively; Lanes 9–12 were the third nested PCR products with the secondly nested PCR products as template using primer SP3 and AP1, SP3 and AP2, SP3 and AP3, SP3 and AP4, respectively. The arrow indicates the target amplified fragment of the unknown region. Lane M represents the DNA size standard markers (200 bp DNA Ladder Marker). The numbers on the right represent the DNA fragment length in bp.

Figure 2

The nucleotide sequence of upstream of proA gene. The putative TATA (Indicated by “——”) and CAAT (Indicated by “– –”) boxes are indicated. The consensus binding sequences for the fungal transcription factors CRE I (5′-SYGGRG-3′, indicated by “······”), AREA (5′-HGATAR-3′, indicated by “——”), and PACC (5′-GCCARG-3′, indicated by “ ”) involved in carbon, nitrogen, and pH regulations, respectively. Arrow underline the sequences indicated the reverse primer SP3. M = A or C; S = G or C; Y = C or T; R = A or G; H = A, C or T.

Figure 3

Molecular analysis of integration of T-DNA into H.orientalis. (A) PCR analysis of the phosphotransferase gene (hph) using specific primers hph-F&R to amplify an 811 bp fragment of transformants Lanes 1-4: transformants Bgl-1, Bgl-2, Bgl-3 and Bgl-4, respectively; Ma: 200 bp DNA Marker; (B) PCR analysis of the gene expression cassette (PproA-Bgl1-TtrpC) using specific primers PproA-F&TtrpC-R to amplify a 4194 bp fragment of transformants. Lane 1: host strain EU7-22; Lanes 2–5: transformants Bgl-1, Bgl-2, Bgl-3 and Bgl-4. Mb: DL10 000 DNA Marker.

Figure 4

Analysis of FPA and BG activities and bgl1 gene relative expression amount of host strain EU7-22 and transformant Bgl-2. (A) FPA and BG activities under induction condition; (B) FPA and BG activities under repression condition; (C) relative expression amount of Bgl1 gene under induction condition; (D) relative expression amount of Bgl1 gene under repression condition.

Figure 5

Sketch map of designed reverse primers sites for SP1, SP2 and SP3. The reverse primers (SP1, SP2 and SP3) were designed according to the interest gene (proA) sequence from H. orientalis EU7-22.

Figure 6

Schematic illustration of the binary expression vectors pUR5750-Bgl1 construction.