74NISR RESEARCH GRANT
2002 Research Grant

Structural and Functional Analyses of Novel Transposons of aKoji-mold, Aspergillus oryzae, and Their Application for Efficient Insertional Mutagenesis
Katsuya GOMI
Graduate School of Agricultural Science, Tohoku University
Aspergillus oryzae is an important filamentous fungus
in the Japanese fermentation industry, such as sake, soysauce, and miso manufacturing, as well as commercial enzyme production. Since no sexual life cycle has been foundin A. oryzae, it is difficult to identify the genes involved inindustrially important pathways by classical genetic analysis. Thus, there is a need to develop general methods toidentify and clone genes from A. oryzae and related fungi.
For this purpose, isolation of transposable elements fromfilamentous fungi could give a useful tool for gene tagging.
The aim of the research is to isolate transposable elementswith potential for gene tagging from A. oryzae.
Isolation and characterization of DNA transposons fromA. oryzae
We found an EST clone highly homologous to A. nigertransposable element, Ant1, in the EST database. A genomic library of A. oryzae RIB40 was screened using thisEST clone as a probe. Nucleotide sequencing revealed thatthe gene contained an ORF of 981-bp encoding 326 aminoacid residues. It showed extensive sequence homology(70% amino acid identity) to that of Ant1. Additionally, theencoded protein included the D35E motif common to thetransposases of the IS630-Tc1 superfamily. As the ORF wasfound in EST database, the gene most likely encodes afunctional transposase and thus we designated it tnpA(transposase of A. oryzae). In general, transposase gene islocated within the IS (insertion sequence) element that has
TIRs at the both ends. However, the tnpA gene was not surrounded with typical TIRs, but such an IS element wasfound about 1-kb downstream of the tnpA. IS element is approximately 1.9-kb in length, and had 39-bp imperfectTIRs, which showed 72–82% nucleotide homology to thatof Ant1. In addition, there was no ORF within the 1.9-kb ISelement. To our knowledge, this is the first example among
fungal DNA transposons that the IS element larger than 1kb contains no ORF and transposase gene is located out ofthe element. Although it remains to be elucidated whether
the element could transpose, we designated the element as
Aot1 (Aspergillus oryzae transposon 1). Southern blot
analysis of A. oryzae RIB40 showed that tnpA gene andAot1 exist as three copies in the chromosome, two of which
are located immediately downstream of the amyB and
amyC genes. Northern blot hybridization analysis revealed

that the tnpA gene was expressed in the submerged culturesof rich media, especially in the medium containing wheatbran extract.
In order to test the transposition activity of Aot1, weconstructed niaD disruptants by insertion of the Aot1 into
the intron sequence of the resident niaD gene. Conidiospores of the resultant disruptants were inoculated ontoa minimal medium and then putative revertants that could
grow on the medium were isolated. To examine whether therevertants are generated by transposition of Aot1 from theniaD gene is underway.
Isolation and characterization of retrotransposons fromA. oryzae
We have also found an EST clone (JZ1636) highly homologous to the gene encoding reverse transcriptase (RT)of the LINE-like DNA element in a fungus, Ascobolus immersus. LINEs (long interspersed nuclear elements) andLINE-like elements are the most abundant classes of transposable elements with non-LTR (long terminal repeat).
This EST clone seemed to be transcribed from the gene in
an A. oryzae LINE-like element, and to be involved in its
transposition through an RNA intermediate. Screening ofthe genomic library with the EST clone as a probe resulted
in isolation of two positive phage clones, which are derived
from different chromosome loci. One phage clone contained
wholeputative
retrotransposon,Aoret1(Aspergillus oryzae retrotransposon), and the other had
Aoret2 about 6-kb downstream of Aoret1. Both putative
retrotransposons contained two long ORFs encoded on thestrand of the same direction.
The first ORF (ORF1) of Aoret1 and Aoret2 encoded aputative protein of 411 and 444 amino acid residues, respectively. These putative proteins contained cysteine-richmotifs near the C-terminal region, which are probably involved in binding DNA. The cysteine-rich motifs are commonly found in gag-like proteins encoded in retrotransposons of other organisms. Amino acid homology between
the proteins of ORF1s of Aoret1 and Aoret2 was 42%. The
second ORF (ORF2) of Aoret1 encoded a protein of 1301
amino acids, and ORF2 of Aoret2 was 1255 amino acids inlength, both of which showed homology to the RT domainsof various retrotransposons. Eight highly conserved regions
are found in the RT domain, including a YXDD motif that75

is considered to be a significant sequence for RT activity.
The YXDD motif is FIDD and FVDD in the Aoret1 ORF2
and the Aoret2 ORF2, respectively. There was no recognizable protease motif, but a region homologous to endonuclease was found at N-terminus in the both proteins. In addition, a motif homologous to RNase H was also found at theC-terminal region in the protein encoded by ORF2 ofAoret1. The proteins of both ORF2s showed 37% identity
each other.
Distribution of the retrotransposons in the A. oryzae
strains and its related fungi
In order to determine the copy number of the retrotransposons in various strains of A. oryzae and its closely
related fungi, Southern blot analysis was done with Aoret1or Aoret2 as a probe. While most A. oryzae strains including RIB40 have only single or two copies but some strains
such as RIB143, RIB326, RIB647, and RIB67 have multiple (approximately 10) copies of Aoret1, all strains examined have at least 10 copies of Aoret2. Interestingly, A.
flavus that is a putative ancestor of A. oryzae have no detectable signals for Aoret1 and Aoret2, and all A. sojae
strains examined have multiple copies of both retrotransposons. These observations suggest that those retroelements might have transposed between A. oryzae and A.
sojae in the koji-making processes, where both species
were possibly cultured, in ancient era.
Expression analysis of the retrotransposons in A. oryzae
Northern blot analysis using a respective ORF2 ofboth retrotransposons as a probe revealed that major transcripts of approximately 5.5-kb in length were transcribedfrom both retrotransposons. The transcripts were somewhat
larger than the expected size for ORF2, but coincided withthe full-length transcripts used as a LINEs RNA transposition intermediate. This was proved by the observation thatORF1 also hybridized to the same size of the transcript.
Thus if two proteins, gag-like protein and RT protein,would be produced, they may be translated from a single
full-length transcript of the both retrotransposons. In addition, both retrotranspsons are transcribed extensively in
YPD medium. To compare the expression level of the retrotransposon among the strains with low and high copy number of Aoret1, Northern blot analysis was done using thestrains, RIB40 (2 copies) and RIB67 (approx. 10 copies).
No difference in expression level of Aoret1 was observed,
suggesting that transcriptional gene silencing may occur ormost of Aoret1 may be defective in RIB67. Since varioustransposons have been activated under stress conditions, A.
oryzae RIB40 was cultured at 42°C or in the presence ofCuSO4. Whereas heat shock treatment did not affect the expression level of Aoret1, treatment with CuSO4 led to a significant increase in the Aoret1 transcript. It should be investigated whether the transposition of Aoret1 could be enhanced under such a stress condition.
Addendum – The Aspergillus oryzae genome sequencing
project revealed the existence of another LINE-like retrotransposon, Aoret3, and two novel LTR retrotransposons,
AoLTR1 and AoLTR2 in the genome of A. oryzae.