J. Biol. Macromol., 16 (1), 9-15 (2016)

Note

Chara cterization of the g atA g ene f ro m Asp ergillus o r yza eMotoaki Sano, Mitsuko Dohmoto, Shinichi Ohashi
G e n o m e B i o t e c h n o l o g y L a b o r a t o r y, K a n a z a w a I n s t i t u t e o f Te c h n o l o g y,
Ya t s u k a h o 3 - 1 , H a k u s a n , I s h i k a w a 9 2 4 - 0 8 3 8 , J a p a n

Received April 12, 2016; Accepted May 10, 2016

Weanalyzed

theroleof

thegeneencoding

GABA

transaminase (gatA) in Aspergillus oryzae by gene disruption.
The gatA -disrup ted strain could no t g row on mini ma l mediu mw ith GAB A as the sole ni trog en sou rce. Th es e resul ts indicatethat gatA is essential for GABA metabolism in A. oryzae.

K eywor ds: Aspergi l lus oryzae , gat A, GA BA t r ansa mi nase , G ABA

γ-Aminobutyric acid (GABA) is aGABA regulates pain and anxiety andubiquitous non-protein amino acid foundreduces blood pressure (2); furthermore,in plants, animals, and microorganisms.

daily ingestion of GABA, which is

It acts as a neuronal transmission

approved as a dietary supplement, has ainhibitor at inhibitory synapses in thebeneficial

brain

hypertension (3).

bybindingtoaspecifictherapeuticeffectfortransmembrane receptor in the plasma

GABA is produced from glutamate bymembrane (1). Clinical administration of

the action of glutamate decarboxylase in

To whom correspondence should bethe GABA shunt pathway or fromaddressed:

putrescine degradation; it is converted toE-mail:msano@neptune.kanazawa-it.ac.jp

succinate semialdehyde (SSA) through10

Characterization of the gatA gene from Aspergillus oryzae

transamination by GABA transaminase

accumulated within A. oryzae cells. In(gatA). SSA dehydrogenase converts

this study, we investigated the role ofSSA

gatA in A. oryzae by gene disruption.

tosuccinate(SUCC),

which

subsequently feeds into the TCA cycle.

gatA (XM_001823026) from A. oryzae

GABA transaminase activity has been

was homologous to gatA from otherdetected in several fungal and yeastorganisms, showing 87%, 98%, andstrains. Moreover, the GABA shunt

100% homology to gatA from A.

pathway is conserved from bacteria tonidulans, A. fumigates, and A. flavus,

fungi, plants, and vertebrates (4).

respectively.

TheGABA is a metabolic by-product ofsimilarity

indicated

plants and microorganisms. Although it

conserved in Aspergillus species (7).

is not found in fresh food, it is produced

To analyze the expression of gatA using

by microbial fermentation. In Japan,

real-time PCR, we isolated total RNA

GABA-rich dietary components such as

and mRNA from A. oryzae RIB40. The

sake and beni-koji (5) are marketed asstrain was precultured at 30 °C for 24 hsupplements.

contains

in YPD medium. The main culture wasapproximately 20 ppm GABA, andthen established for 6 h at 30 °C inrice-koji contains approximately 200 mg

minimal medium (2% glucose, 0.3%GABA/kg dry koji (1). The filamentous

KH2PO4, 0.1% KCl, and 0.1% MgSO 4)

fungus Aspergillus oryzae is widely used

containing 100 mM GABA or 100 mM

in the preparation of traditional Japanese(NH4)2SO4. Total RNA was extracted

fermented foods such as sake, soy sauce,using RNAiso Plus (TaKaRa, Shiga,miso, and mirin (6). GABA levels in thisJapan) according to the manufacturer’s

fungal strain must be enhanced toinstructions. mRNA was purified using

improve the health benefits of theseanfoods.

Purification Kit (TaKaRa) following theSake

Therefore,

gatA-disrupted

wemutant.

producedThisamutant

could not degrade GABA, so GABA

highdegreeofgatAisthat

Oligotex-dT30<super>

manufacturer’s

protocol.

mRNAReverse

transcription and real-time PCR were

Sano, Dohmoto, and Ohashi

11

performed using the SuperScript III
Platinum Two-Step qRT-PCR Kit withSYBR Green (Invitrogen, Carlsbad, CA)(8).

Relativequantificationwasperformed using the 2 -ΔΔCt technique(ABI User Bulletin 2). The sequences(5′→3′) of the gatA primers were
GCAACTTCCCCTCTGCTGACT(forward)

andFig.

1.

Relative

gatA

expression

GCGGTGAAGACCTGGTTCA

analyzed by real-time PCR.

(reverse). Histone H1 was used as angatA expression in cells grown inendogenous control. gatA expression wasminimal medium containing 100 mM

strongly induced by addition of 100 mM(NH4)2SO4 was set to 1.0. Error bar

GABA and was 39-fold higher than withindicates standard deviation (n = 3).

addition of 100 mM (NH4)2SO4 (Fig. 1).
In Saccharomyces cerevisiae, GABA

System (Roche Diagnostics, Mannheim,transaminase is encoded by UGA1.

Germany). The 5′- and 3′-arms of gatA

When S. cerevisiae was grown with

were amplified from genomic DNA withGABA as the sole nitrogen source, thethe primers LU/LL (5′-arm) and RU/RL

expression of UGA1 was similar to the(3′-arm). pyrG was amplified with theexpression of gatA. Thus, the real-timeprimer pair PU/PL. Amplified fragments

PCR assays indicated that gatA waswere purified using the Wizard Gel

inducible by GABA (9).

extraction kit (Promega, Madison, WI).

∆gatA was created as described byThe

5′-flanking:pyrG:3′-flanking

Tamano et al. (10). The gatA gene

amplicon (1:3:1 molar ratio) was used as

disruption cassette was generated bya template. The PCR products were usedfusion polymerase chain reaction (PCR)

for a second round of PCR with theusing an Expand High Fidelity PCRprimer pair LU/RL to fuse the 5′ and 3′

12Characterization of the gatA gene from Aspergillus oryzae

regions of the target gene at each end ofPurification Kit (Promega, Madison, WI)

the pyrG gene. The primer sequences

according(5′→3′)

instructions. Disruption of gatA was

were

as

follows:

CCATGATCTCGTAGAGAGG(LU),

toconfirmedbythemanufacturer’s

Southernblotting.

CATCACAGGGTACGTCTGTTGTGG

Southern blotting of genomic DNA wasTGTACAGAGGTGTGG

performed(LL),

followingdigestion

with

AATCGCCACACCTCTGTACACCAC

HindIII. The DNA fragment containing

AACAGACGTACCCTGTGA(PU),sequence upstream of gatA gene was

AACCAATGAACGCCGGACTCTGGC

used as a probe. The DIG-DNA labeling

AGATACAACTGCACCTC

and detection kit (Roche) was used for(PL),

TCTTCTGAGGTGCAGTTGTATCTGC

signaldetection.

Weobserved

CAGAGTCCGGCGTTCATT (RU), andexpected hybridization signals at 1.3 kb

AGGTCCGTCGTCGCAATT (RL). The

and 4.6 kb for digested genomic DNA

amplified fragment was purified with theisolated from the control and disrupted

Wizard Gel extraction kit and used forstrains,respectively(Fig.

transformation.

kb!

A. oryzae strain ∆ligD (pyrG-, niaD-)
was used as a host for transformation(11).

Theuridine2).
1! 2!

HindIII! HindIII!
Probe!
Control!
gatA!

prototrophic

transformants were subjected to twothe

1.3 kb!

4.6!

" gatA!
pyrG!

consecutive rounds of single sporing.

4.6 kb!

1.3!
HindIII!

The control strain for these experiments
was transformed with only pyrG. The
gatA-disruptedstrains

mutantwereandcontrolFig. 2.

Southern blot

grownongenomicDNA(20analysis ofµg/lane)fromyeast-peptone-dextrose (YPD) medium

gatA-disrupted (lane 1) and control (laneat 30 °C for 24 h. DNA was extracted2) strains.

using

theWizard

GenomicDNA

Sano, Dohmoto, and Ohashi

Theseresults

homologous

indicatedsuccessfulrecombination

attheencoding

13

aputativeGABA

aminotransferase, is essential for GABA

resident gatA locus.

metabolism in A. oryzae. Furthermore,

We observed that A. oryzae could

the function of gatA in A. oryzae isefficiently utilize GABA as a nitrogensimilar to that of UGA1 in S. cerevisiae

source. However, the gatA-disrupted

but differs from that of ugatA in U.

strain did not grow on GABA plates (Fig.

maydis.

3). These data suggest that gatA isessential for GABA metabolism in A.
oryzae. An S. cerevisiae mutant strain
lacking UGA1 was incapable of growthon minimal medium with GABA as thesolenitrogensource.

However,aUstilago maydis strain with a GABA
transaminase gene disruption (ugatA)Fig.

was able to grow with GABA as the sole

gatA-disrupted

nitrogenresultgatA-disrupted strains were grown atindicated the presence of an alternative

30 °C for 5 days on minimal medium

pathway for the utilization of GABA incontaining 100 mM GABA.

source(12).

This3.

Growthstrains.

phenotypeofControlandU. maydis. These results revealed thatthe pathway for GABA metabolism isdiverse among microorganisms.

Acknowledgement
This work was supported by JPSPIn conclusion, we analyzed the function

KAKENHI Grand Number 26450506.

of gatA in A. oryzae. We found that gatAWe thank Harue Kitagawa for technical

was inducible by GABA and that aassistance.

gatA-disrupted strain could not be grownwith GABA as the sole nitrogen source.
These results indicate that gatA, the gene

14

Characterization of the gatA gene from Aspergillus oryzae

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Communicated by Ishijima Sumio

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