Isolation and Characterization of the mheA (Most Highly Expressed) Gene of Aspergillus oryzae

Abstract

The amino acid sequence of the mheA gene of Aspergillus oryzae encodes a putative metallothionein-like protein 1. The size of the mheA transcript was 497 nt and the mheA promoter was induced by glucose, consistent with results of analysis by Northern hybridization and with the pdcA promoter, respectively.

Various strong promoters in Aspergillus oryzae have been identified [1]. These strong promoters can be divided into two major groups: one group includes promoters of genes encoding various amylases, and the other group includes promoters of genes encoding enzymes involved in glycolysis. The promoter, which is not included in these two groups, is that of the pdcA gene coding for pyruvate decarboxylase. The pdcA gene appears to be one of the most highly expressed genes in A. oryzae, and induction of expression by the pdcA promoter has been suggested to occur in the presence of glucose [1, 2].

Sequence analysis of randomly selected clones in a 3'-directed expressed sequence tag (EST) library of a tissue or a cell type can identify highly expressed genes in a given tissue or a cell type [3]. Analysis of 345 randomly selected clones from a 3'-directed EST library of A. oryzae has been conducted; according to the results, the pdc gene and the mheA gene appear to show the highest level of expression [2]. The pdcA gene has been isolated previously [4]. For isolation of the mheA gene, the restriction map around a 3'-directed EST of the mheA gene (the EST No. AO07B12 corresponding to the nucleotide No. 4,966 to 5,165 in the sequence, whose GenBank accession No. is DQ004254) was constructed by Southern hybridization of chromosomal DNA, which demonstrated that the chromosomal DNA having the entire mheA gene can be obtained by combination of two genomic clones isolated from two genomic libraries (result not shown). One is a HindIII-digested genomic library and the other is a PstI-digested genomic library. A 1.5 kb clone was isolated by colony hybridization, using the 3'-directed EST DNA as a probe, from a HindIII-digested library, which has been described previously [4]. The nucleotide sequence of the 1.5 kb fragment indicated that the 1.5 kb fragment may include a part of the mheA promoter therefore, another DNA fragment, a 4.8 kb PstI-digested fragment, was isolated from a PstI-digested genomic library using a 600 bp SacI/PstI-digested fragment of the 1.5 kb clone as a probe. The PstI-digested genomic library was constructed by insertion of PstI-digested chromosomal DNA fragments into pUC19. In total, a 5.5 kb genomic DNA fragment containing the mheA gene was isolated from two genomic libraries. According to the results of a GenBank Blast search, the nucleotide sequence and the amino acid sequence of the 5.5 kb DNA suggested that the mheA gene encodes a putative metallothionein-like protein 1 (MT1-1, GenBank accession No. AAS48540.1) of Leptosphaeria maculans (Fig. 1). Fourteen amino acids out of 21 in the MheA open reading frame (ORF) were exactly matched to those of the MT1-1.

Fig. 1.

Alignment of the total amino acids of the MheA ORF and the MT1-1 ORF. mheA, the MheA open reading frame (ORF) of Aspergillus oryzae; MT1-1, metallothionein-like protein 1 of Leptosphaeria maculans (GenBank accession No. AAS48540.1). Asterisks indicate the identical amino acids between the two.

For identification of the mheA promoter region, examination of the 5' end of the mheA transcript was performed using 5'-rapid amplification of cDNA end (5'-RACE) according to the procedure described previously [5]. The total RNA was isolated from 48 hr-cultured mycelia of a wild type strain, A. oryzae Fungal Genetics Stock Center (FGSC, Kansas, MO, USA) A815, in liquid yeast extract-peptone-dextrose (YEPD) medium. Isolation of mRNA from total RNA was performed using an oligo-dT cellulose column. The 5' part of the mheA transcript was synthesized using a primer, 5'-AAG CCC TCA TCA TCA CCA-3' (5,119~5,102), followed by inverse PCR using a pair of primers whose sequences were 5'-TCA TCC CAT CCT CAC TGG-3' (5,004~5,021) and 5'-TAC TTA TCT TCC TCG GGG-3' (5,051~5,068). Results of 5'-RACE showed that transcription of the mheA gene starts at the 4,668th nucleotide "T" and that the full-length of the mheA transcript is 497 nt without poly(A). This result was consistent with that of analysis by Northern hybridization, showing that the size of the mheA transcript was approximately 500 nt (result not shown).

Because the principles for isolation of the pdcA gene and the mheA gene were identical, the question of whether expression of the mheA gene is induced in the presence of glucose was examined. Total RNA was isolated from 48 hr-cultured mycelia of the wild type strain A. oryzae A815 in liquid YEPD medium and hybridized using a mheA-specific probe. When another carbon source was supplemented, 2% starch or sucrose was added instead of glucose. The mheA-specific probe was the AO07B12 EST DNA, which was labeled with [α-P³²]-dCTP using the Prime-a-Gene Labeling System (Promega, Medison, WI, USA). As shown in Fig. 2, the mheA transcript level was higher in the presence of glucose than in the presence of starch or sucrose, indicating that expression by the mheA promoter is induced by glucose.

Fig. 2.

Comparison of the promoter activity of the mheA promoter in the presence of different carbon sources. Total RNA was isolated from 48 hr-cultured mycelia of a wild type strain of Aspergillus oryzae Fungal Genetics Stock Center (FGSC, Kansas, MO, USA) A815 in liquid yeast extract-peptone-dextrose (YEPD) medium supplemented with 2% glucose, starch, or sucrose, as shown on each lane, and hybridized with a mheA-specific probe.