40NISR Research GRANT
2008 Research Grant

Establishment of marker-recycling method in filamentous
fungi and development of an Aspergillus aculeatus multi-auxotrophic hostTakashi KAWAGUCHI
Graduate School of Life and Environmental Sciences, Osaka Prefecture University

Research aims
Bioprocess for converting cellulosic substances to liquidfuels, so-called ‘bioethanol’, is becoming more importantand getting closer to the stage for industrial use, eventhough saccharification of cellulosic substances is still
mostly achieved by sulfate treatment. Since enzymatic saccharification is more desirable than the acid degradation
procedure from the viewpoint of environmental advantage,
it is expected that enzymatic saccharification at industrial
scale will be put into practice in future. However, the economical cost for cellulase production prevents the industrialization of enzymatic saccharification because their activity is not sufficiently high yet. To overcome this problem,
we need to establish a high-production system for cellulases, as well as to improve properties of cellulases throughprotein engineering and to elucidate the synergistic effects
among cellulases with different properties.
A. aculeatus, a filamentous fungus isolated from soil,
produces a number of extracellular cellulose- and hemicellulose-degrading enzymes which shows synergism with Trichoderma cellulases. It was shown that alkali-treated ricestraw was hydrolyzed almost completely into monosaccharide by the mixed culture filtrate of these two strains1,2).
Meanwhile, we constructed a host-vector system of A. aculeatus, and the A. aculeatus b -mannosidase gene was expressed by the system. The A. aculeatus expression systemproduced a 10-fold higher level of the enzyme than the A.
oryzae expression system, indicating the high potential ofA. aculeatus as a factory for production of useful proteins3,4). On the other hand, the concerted action of several
cellulases with different properties is considered to be essential for the effective hydrolysis of cellulose into monosugar. Thus, we aim to breed a ‘Super Mold’ that simultaneously produces many types of cellulases, which should
be useful for cost reduction of enzyme production. For that
purpose, a host strain carrying multiple selection markers
for transformation must be constructed by gene disruption.
However, knock-out of a target gene is not so easy in filamentous fungi because DNA repair by nonhomologous
end-joining (NHEJ) occurs prior to homologous recombination. Recently, it was demonstrated that gene disruptionof Ku70/80 or Lig4 gene, which are involved in NHEJ
event, resulted in the elevated frequency of homologous re-combination5–9). In this context, the aims of this study are:i) gene disruption of Ku70/80 or Lig4 in A. aculeatus, ii)
development of marker-recycling method in filamentous
fungi, and iii) construction of a multi-auxotrophic A. aculeatus host strain.
Methods
A pyrG-deficient mutant (pyrG13, uridine auxotroph),
spontaneously isolated from the A. aculeatus wild type by
selection for resistance to 5-fluoroorotic acid (5-FOA)3),
was transformed with a DNA fragment designed for disruption of ku80 homolog gene (Aaku80), and transformants
grown on the uridine-free plate were obtained (Fig. 1-a).

Fig. 1.

Disruption of Aaku gene and marker-recyling method.

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Out of these transformants, a strain of which the Aaku80
locus was replaced by a single copy of the disruption cassette via double crossover recombination was identified bySouthern blot analysis (R1). Then, conidia of the strain
were spread on a plate containing 5-FOA, and one of 5FOA resistant colonies obtained was confirmed as theAaku80 disruptant (MR12), in which the pyrG gene was excised by intramolecular homologous recombination between direct repeat (DR) (Fig. 1-b). The effect of the
Aaku80 disruption on the gene-targeting efficiency was assessed by performing the disruption of the argB locus.
An additional mutant gene (argB –) was successfully introduced into the argB locus of the MR12 strain (Aaku80 –,pyrG–) by the marker-recycling method and an A. aculeatus
host strain carrying double selection marker for transformation was established. Also, effects of the DR length on theexcision frequency were examined by comparing the occurrence rates of 5-FOA resistant derivatives from each transformant into which one of the three disruption cassettes
with the different length of DR (20, 98, and 495 bp) was introduced.
Results
Disruption of Aaku80 gene and recycling of pyrG
marker: The pyrG13 was transformed with the Aaku80disruption cassette and 40 transformants were obtained by
the selection on a uridine/uracil-free selection medium. Outof these transformants, one strain (R1) in which one copyof the disruption cassette was integrated via double homologous recombination at the Aaku80 locus was identified bySouthern blot analysis. Then, we tried to excise the pyrGcontaining fragment sandwiched by a pair of DR in order toreuse pyrG as a selection marker according to the methodcommonly used for Saccharomyces cerevisiae10). Conidia(about 104) were spread onto a 5-FOA-containing plate and
20 to 30 colonies, which should be 5-FOA resistant, appeared on the plate after 10 days cultivation. Southern blot
analysis showed that the expected excision event throughintramolecular homologous recombination occurred in themore than 90% of clones examined. Also, it was confirmedthat the 5-FOA resistant clones were uridine/uracil-auxotrophs. One of these 5-FOA resistant clones (MR12) wasused for further experiments.
Characteristic of Aaku80 disruptant: Growth andsporulation of MR12 (Aaku80 –, pyrG +) were indistinguishable from those of pyrG13 (Aaku80 +, pyrG +). Meanwhile,
growth of MR12 on 0.5% methyl methanesulfonate (MMS)
was poorer than that of pyrG13. This finding was in goodagreement with the previous observations that ku disruption
increased sensitivity against the DNA-damaging reagent in
N. crassa5) and A. fumigatus9).
Gene-targeting efficiency at the argB locus in Aaku80
disruptant: The strains pyrG13 and MR12 were transformed with the argB disruption cassette carrying complete
pyrG gene as a selection marker within argB coding region
and the first selection was done on the uridine/uracil-freeplate. One strain out of 32 transformants from pyrG13
found to be an arginine auxotroph (targeting frequency;3%), while 29 out of 31 transformants from MR12 wereshown to be arginine auxotrophs (94%). Southern blot

analysis showed that all the arginine auxotrophic clones derived from MR12 had a single copy of the disruption cassette at the target locus, suggesting that integration of thecassette took place by homologous recombination via double crossover.
Consturuction of pyrG, argB double-auxotrophic hostand effect of the DR length on intramolecular homologous recombination: Three types of argB disruption cassettes with different length of DR (20, 98, and 495 bp) andthe recycling-marker of pyrG were constructed and introduced into MR12 to yield 15, 10, and 20 uridine/uracil-prototrophic transformants, respectively. Out of these transoformants, 11, 10, and 17 clones were arginine auxotrophs,respectively. Then, three clones carrying a single copy ofeach disruption cassette were chosen and named mrTA1-14,
which carries 20-bp DR, mrTA2-1, which carries 98-bp
DR, and mrTA3-6, which carries 495-bp DR, respectively.
Firstly, 106 conidia of mrTA3-6 were spread on a plate containing 5-FOA, uridine, and arginine and ten colonies wereobtained. All of the ten clones were found to beuridine/uracil-auxotrophs. Thus, it was concluded that an A.
aculeatus host strain with double selection markers fortransformation was successfully constructed. Moreover, theoccurrence frequencies of the 5-FOA resistant clones from
mrTA1-14, mrTA2-1, and mrTA3-6 were estimated to be
5.7106, 1.4105, and 1.2104, respectively. These results indicate that the frequency of precise excision event
increases as the length of DR increases.
Conclusion
In this study, it was shown that the gene targeting efficiency was drastically elevated by the ku disruption in A.
aculeatus, as shown for many other filamentous fungi.
Also, it was demonstrated that the marker-recycling technique commonly used in yeast can be applied to filamentous fungi. Once a mutant with a two-way (positive andnegative) selectable marker like pyrG is isolated, marker-recycling enables consecutive gene disruption. Though it wasreported that 20-bp is the sufficient length of DR for intramolecular homologous recombination in yeast, it has
been supposed that longer DR is required for homologous
recombination in filamentous fungi. However, this study indicated that correct excision events via homologous recombination can occur even with 20-bp DR in A. aculeatus, although the frequency is relatively low. Our findings thatDRs as short as 20 bp were functional for promoting correct
excision via homologous recombination should be useful indesigning disruption cassettes for marker-recycling in filamentous fungi. We are now constructing an A. aculeatus
host strain with multi-selectable markers by the method developed in this study and breeding the ‘Super Mold’ producing a number of different-type cellulases to hydrolyze
cellulosic substances with high efficiency.
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