transcriptomic analysis of temperature responses of aspergillus
TRANSCRIPT
Transcriptomic Analysis of Temperature Responses of Aspergilluskawachii during Barley Koji Production
Taiki Futagami,a* Kazuki Mori,a Shotaro Wada,b Hiroko Ida,b Yasuhiro Kajiwara,b Hideharu Takashita,b Kosuke Tashiro,a
Osamu Yamada,c Toshiro Omori,b Satoru Kuhara,a Masatoshi Gotoa
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japana; Research and Development Laboratory, Sanwa Shurui Co., Ltd.,Usa, Oita, Japanb; National Research Institute of Brewing, Higashi-Hiroshima, Japanc
The koji mold Aspergillus kawachii is used for making the Japanese distilled spirit shochu. During shochu production, A. kawa-chii is grown in solid-state culture (koji) on steamed grains, such as rice or barley, to convert the grain starch to glucose and pro-duce citric acid. During this process, the cultivation temperature of A. kawachii is gradually increased to 40°C and is then low-ered to 30°C. This temperature modulation is important for stimulating amylase activity and the accumulation of citric acid.However, the effects of temperature on A. kawachii at the gene expression level have not been elucidated. In this study, we inves-tigated the effect of solid-state cultivation temperature on gene expression for A. kawachii grown on barley. The results of DNAmicroarray and gene ontology analyses showed that the expression of genes involved in the glycerol, trehalose, and pentosephosphate metabolic pathways, which function downstream of glycolysis, was downregulated by shifting the cultivation temper-ature from 40 to 30°C. In addition, significantly reduced expression of genes related to heat shock responses and increased ex-pression of genes related with amino acid transport were also observed. These results suggest that solid-state cultivation at 40°Cis stressful for A. kawachii and that heat adaptation leads to reduced citric acid accumulation through activation of pathwaysbranching from glycolysis. The gene expression profile of A. kawachii elucidated in this study is expected to contribute to theunderstanding of gene regulation during koji production and optimization of the industrially desirable characteristics of A.kawachii.
Koji molds, such as the yellow koji mold Aspergillus oryzae, areimportant microbes for the production of many traditional
Japanese fermented foods, such as sake, miso, and shoyu (1). Thewhite koji mold Aspergillus kawachii, which was derived from theblack koji mold Aspergillus luchuensis, is used in the production ofshochu, a traditional Japanese distilled spirit (2–4). A. kawachiiproduces large amounts of citric acid in addition to several glyco-side hydrolases, such as �-amylase and glucoamylase, which arealso produced by A. oryzae. Glycoside hydrolases play an impor-tant role in shochu production as they degrade the complex poly-saccharides found in rice and barley into smaller mono- or disac-charides that can be further utilized by the yeast Saccharomycescerevisiae for ethanol fermentation. The citric acid-producingcharacter of A. kawachii is also beneficial for shochu production ascitric acid lowers the pH of the fermentation and thereby helpsprevent microbial contamination, which is particularly problem-atic because shochu is mainly produced in Japan’s southwest is-land of Kyushu, where the climate is relatively warm. Citric acidproduction by A. kawachii is a unique feature because it is notfound in other koji molds, including A. oryzae.
A. kawachii has similar genomic and physiological features asAspergillus niger, which is industrially used for the production ofprotein and citric acid. However, A. kawachii is clearly distinctfrom most A. niger strains with respect to the production of my-cotoxins (5–7). Although several A. niger strains produce the my-cotoxins fumonisin and ochratoxin, A. kawachii does not producethese mycotoxins. For this reason, A. kawachii is considered to bea safe microorganism for industrial use.
For the production of koji used for making shochu, the culti-vation temperature of A. kawachii is strictly controlled. Typically,the temperature is raised to 40°C in the early cultivation stages toincrease amylase activity and lowered to 30°C in the later stages to
promote the production of citric acid (8). The cultivation condi-tions for A. kawachii were developed and refined based on theexperiences of shochu brewers for controlling the production ofoptimal amounts of amylases and citric acid (9). However, thespecific genes involved in these processes that are activated and/orrepressed under the different temperature conditions remain un-clear.
In the present study, we investigated the temperature-medi-ated changes that occur in A. kawachii at the gene expression levelduring the production of shochu koji. For this purpose, the tran-scriptomic profiling of A. kawachii using DNA microarrays wasperformed and compared under two different cultivation condi-tions: normal conditions, in which the temperature was raised to40°C and then lowered to 30°C, and high-temperature conditions,
Received 21 October 2014 Accepted 8 December 2014
Accepted manuscript posted online 12 December 2014
Citation Futagami T, Mori K, Wada S, Ida H, Kajiwara Y, Takashita H, Tashiro K,Yamada O, Omori T, Kuhara S, Goto M. 2015. Transcriptomic analysis oftemperature responses of Aspergillus kawachii during barley koji production. ApplEnviron Microbiol 81:1353–1363. doi:10.1128/AEM.03483-14.
Editor: D. Cullen
Address correspondence to Masatoshi Goto, [email protected].
T.F. and K.M. contributed equally to this article.
* Present address: Taiki Futagami, Education and Research Center forFermentation Studies, Faculty of Agriculture, Kagoshima University, Korimoto,Kagoshima, Japan.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03483-14.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.03483-14
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in which the cultivation temperature was raised from 36 to 40°Cand then maintained at 40°C. In particular, we focused on theeffects of temperature reduction on the gene expression profiles ofA. kawachii during solid-state culture to determine the signifi-cance of temperature control on koji production.
MATERIALS AND METHODSStrain and koji production. The white koji mold A. kawachii SH46, whichwas recently renamed A. luchuensis SH46 (4), was obtained from HiguchiMatsunosuke Shoten Co., Ltd. (Osaka, Japan), and used for koji produc-tion with barley polished to 70%. The barley was steamed (water adsorp-tion rate, 35%) before being used for koji production, as described previ-ously (9). The solid-state cultivation of A. kawachii on the barley wasperformed under normal (N)-temperature conditions, in which the tem-perature was increased from 36 to 40°C over a 25-h period and was thenlowered to 30°C, and under high (H)-temperature conditions, in whichthe temperature was increased from 36 to 40°C over a 25-h period andthen maintained at 40°C for 19 h (Fig. 1A). Barley koji (100 g) was pre-pared in triplicate under both N and H conditions and was used for thefollowing experiments.
Acidity and GlcNAc measurements of barley koji. Acidity was mea-sured to examine and compare citric acid production under the N and Hculture conditions. For the analysis, 40 g of prepared koji was homoge-nized with 200 ml of water, and acidity was then measured by acid-basetitration. GlcNAc levels in koji were also measured to evaluate the growthof A. kawachii on barley according to a previously described method witha slight modification (10). Briefly, approximately 7 g of barley koji wasdried in an oven at 105°C for 1 h and then crushed to a powder. Thecrushed koji (500 mg) was washed three times with 20 ml of 50 mMsodium phosphate buffer (pH 7.0), and the resultant suspension was cen-trifuged at 1,580 � g for 10 min. The obtained pellet was mixed with 10 mlof 50 mM sodium phosphate buffer containing 5 mg/ml yatalase (TaKaRaBio, Shiga, Japan) and incubated at 37°C for 3 h. After further centrifuga-tion at 1,580 � g for 10 min, the amount of GlcNAc in the supernatant wasdetermined colorimetrically as previously described (11).
Quantification of metabolites. Barley koji was collected at varioustime points between 25 and 45 h of cultivation, as indicated in Fig. 1A(sampling points 1, 2, 3, and 5) and frozen immediately at �80°C.Metabolome analysis of the collected samples was performed by HumanMetabolome Technologies, Inc. (Tsuruoka, Japan), as follows. Samples ofkoji (50 mg) were crushed using a hammer, dissolved in 500 �l of meth-anol, and then disrupted by bead beating at 1,500 rpm for 2 min using aShake Master NEO MBS-M10N21 (Biomedical Science, Tokyo, Japan).After the bead beating was repeated five times, the sample was furtherhomogenized by shaking at 4,000 rpm for 1 min using a Micro SmashMS-100R (Tomy Digital Biology, Tokyo, Japan) and was then mixed with500 �l of chloroform and 200 �l of Milli-Q water. After centrifugation ofthe sample at 2,300 � g for 5 min at 4°C, 400 �l of the water phase was
transferred to an ultrafiltration module (Ultrafree MC PLHCC HMT, 5kDa; Millipore) and centrifuged at 9,100 � g for 2 h at 4°C. The obtainedfiltrate was dried and redissolved in 50 �l of Milli-Q water. Metaboliteswere analyzed using a capillary electrophoresis–time-of-flight mass spec-trometry (CE-TOF/MS) system (Agilent Technologies) equipped with afused silica capillary (50 �m [inside diameter] by 80 cm). The data wereanalyzed using MasterHands software (version 2.13.0.8.h). Statistical dif-ferences were calculated from triplicate data of independently producedbarley koji by using Welch’s t test.
Preparation of RNA. Barley koji was collected at various time pointsbetween 25 and 45 h of cultivation, as indicated in Fig. 1A (samplingpoints 2, 3, 4, and 5). The frozen barley koji samples were physicallydisrupted with a hammer in liquid nitrogen to keep the samples frozen.Two grams of the crushed koji was incubated with 5 ml of RNAiso reagent(TaKaRa Bio) at 50°C for 10 min with vigorous vortexing every 2 min. Thesample was then incubated at room temperature for 5 min before beingfrozen at �80°C for over 1 h. The samples were thawed at room temper-ature and then centrifuged at 1,580 � g for 15 min at room temperature.The upper phase was retrieved, and 0.2 volumes of chloroform was addedand vigorously mixed with the remaining sample, which was then furthercentrifuged at 1,580 � g for 15 min at room temperature. The upper phasewas retrieved and purified using an SV total RNA isolation system (Pro-mega, Madison, WI) according to the manufacturer’s protocol. The qual-ity of the RNA sample used for DNA microarray analysis was confirmedby electrophoresis with a BioAnalyzer instrument (Agilent Technologies).
DNA microarray analysis. Probes for DNA microarray analysis weredesigned using a SurePrint G3 8 � 60K eArray (Agilent Technologies,Santa Clara, CA) for the 11,488 predicted coding sequences (CDSs) of A.kawachii IFO 4308 (12). Five different probes were designed for each CDS.Cyanine-3 (Cy3)-labeled cRNA was prepared from 50 ng of RNA using aone-color microarray-based gene expression analysis low-input QuickAmp labeling kit (Agilent Technologies) according to the manufacturer’sinstructions, followed by RNeasy column purification (Qiagen, Valencia,CA). The microarray was hybridized with Cy3-labeled cRNA at 65°C for17 h in a humid chamber and was then washed, dried, and scanned usingan Image analyzer (Agilent Technologies). All microarray experimentswere performed three times with RNA samples obtained from indepen-dently prepared barley koji. The triplicate data of five probes were ana-lyzed by Tukey’s bi-weight method, and the signal intensities were nor-malized by a quantile normalization method using the R packagenormalize.quantiles function (http://cran.r-project.org/index.html) (13).The expression ratio was calculated as log (base 2) ratio. If the q value wasfound to be less than 0.01 using the Limma package (http://www.bioconductor.org/packages/release/bioc/html/limma.html) and the log2
fold change was less than �0.5 or greater than 0.5, it was considered to bea significant change (see Data Set S1 in the supplemental material). Thedifferential gene expression identified in the DNA microarray analysis wasconfirmed by real-time reverse transcription-PCR (RT-PCR), as de-scribed in the following section. The gene expression data were analyzedwith manually constructed metabolic pathways of A. kawachii mainlybased on models in A. niger (14, 15) (see Fig. S1). In addition, the geneswere subjected to gene ontology (GO) analysis (see Data Set S2). The GOannotation of A. kawachii genes was based on InterProScan, and the anal-ysis was performed using a Perl script (16, 17).
Real-time RT-PCR analysis. For the validation of microarray-basedexpression data, real-time RT-PCR was performed. cDNAs were synthe-sized using a PrimeScript RT reagent kit with gDNA Eraser (perfect realtime) (TaKaRa Bio) according to the manufacturer’s protocol using 240ng of total RNA as the template. Real-time RT-PCR was performed usinga thermal cycler Dice real-time system MRQ (TaKaRa Bio) with SYBRpremix Ex Taq II (Tli RNase H Plus) (TaKaRa Bio). Specific primer setsfor the 27 genes listed in Table S1 in the supplemental material were usedfor the amplification reactions. Genomic DNA of A. kawachii IFO 4308(4.5 � 104, 9 � 104, 1.8 � 105, and 4.5 � 105 copies) was used as standardDNA. The actin gene was used to normalize mRNA expression levels, and
FIG 1 Experimental conditions and properties of the barley koji produced inthis study. (A) Cultivation temperature conditions. (B) Acidity of barley koji atthe indicated sampling points (1 to 5) shown in panel A. (C) GlcNAc levels inbarley koji at the sampling points indicated in panel A. Data are presented asmeans � standard deviations. *, P value � 0.01 (Welch’s t test).
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the relative transcriptional levels of the 26 target genes were calculated(Table 1; see also Fig. S2 in the supplemental material).
Microarray data accession number. The microarray data were depos-ited in the Gene Expression Omnibus under accession number GSE58454.
RESULTSGenome comparison. The results of the genome analysis of A.kawachii IFO 4308 performed in this study have been briefly sum-marized in the genome announcements in Eukaryotic Cell (12).Here, differences in the genome structures of A. kawachii, A. niger,and A. oryzae were compared to understand the characteristic pro-ductivities of organic acids and examine the degree of CDS con-servation among these three Aspergillus species (see Fig. S3 in thesupplemental material). The number of predicted CDSs in the ge-nomes of A. kawachii IFO 4308, A. niger CBS 513.88, and A. oryzaeRIB40 were 11,488, 14,056, and 11,902, respectively (12, 18–20)(Aspergillus Genome Database [http://www.aspergillusgenome.org/]). A total of 3,797 CDSs were conserved at a 70% level ofidentity among these three Aspergillus species. The clustering anal-
ysis also identified that 4,852 CDSs were common only between A.kawachii IFO 4308 and A. niger CBS 513.88 and that 309 CDSswere common only between A. kawachii IFO 4308 and A. oryzaeRIB40. As cultures of A. kawachii and A. niger accumulate mark-edly higher levels of citric acid than those of A. oryzae, the presenceof genes involved in the metabolic tricarboxylic acid (TCA) cyclewere compared. Four genes in the A. kawachii genome, encodingmalate dehydrogenase (AKAW_04056), pyruvate carboxylase(AKAW_08633), and two citrate synthases (AKAW_00170 andAKAW_09689), were included in the cluster of genes that werecommon between A. kawachii and A. niger.
An important trait of koji molds used in the fermentation in-dustry is the nonproduction of mycotoxins. Although several A.niger strains are known to produce ochratoxin (5, 7), A. kawachiiIFO 4308 does not produce ochratoxin due to the deletion of apolyketide synthase gene involved in ochratoxin biogenesis (3).Previous genome analysis of A. kawachii IFO 4308 revealed that anapproximately 21-kb genomic region that includes the polyketide
TABLE 1 Genes up- and downregulated in response to lowering the koji cultivation temperature
Reaction no.in Fig. 2 Locus tag Putative functionh
Fold change in expression byg:
Microarray Real-time RT-PCR
N 26.5 h/H 26.5 h
N 44 h/H 44 h
N 26.5 h/H 26.5 h
N 44 h/H 44 h
1 AKAW_05831 Glucokinase 0.43b 1.1 0.44d 0.82 AKAW_01051 Fructose-2,6-bisphosphatase 0.54b 1.7c 0.67e 1.13 AKAW_03207 Glucose-6-phosphate isomerase 0.62b 1.3 0.73e 0.934 AKAW_07001 Fructose bisphosphate aldolase 0.60b 1.2 0.69f 1.25 AKAW_02920 Glyceraldehyde-3-phosphate dehydrogenase 0.61b 1.3 0.9 1.16 AKAW_03245 Phosphoglycerate mutase 2.2a 2.2 1.3e 1.37 AKAW_00804 Pyruvate carboxylase (cytosol) 1.7b 1.6 1.8e 1.28 AKAW_01578 6-Phosphogluconolactonase 0.57b 1.3 0.61e 0.899 AKAW_01810 Phosphogluconate dehydrogenase (decarboxylating) 0.15b 0.18c 0.16f 0.16f
10 AKAW_02256 Ribose-5-phosphate isomerase (A) 0.59a 1.3 0.65e 0.9110 AKAW_00489 Ribose-5-phosphate isomerase (B) 0.39b 2.1c 0.54e 1.311 AKAW_04326 Transaldolase (sedoheptulose-7-phosphate:D-glyceraldehyde-
3-phosphate glyceronetransferase)0.60b 1.5 0.70e 1.1
12 AKAW_00232 Transketolase (Sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycolaldehydetransferase)
0.18b 0.26c 0.26f 0.24f
13 AKAW_03596 �,�-Trehalosephosphate synthase (UDP forming) 0.20b 0.7 0.32e 0.66f
13 AKAW_03597 �,�-Trehalosephosphate synthase (UDP forming) 0.22b 0.9 0.25f 0.70f
13 AKAW_05189 �,�-Trehalosephosphate synthase (UDP forming) 0.24b 0.37 0.31e 0.3414 AKAW_03775 Trehalose phosphatase 0.67b 1.9 0.76f 1.115 AKAW_00120 �,�-Trehalase 0.51b 1.1 0.63f 0.68f
16 AKAW_10295 Glycerol 3-phosphate dehydrogenase (NAD dependent) 0.097b 0.18c 0.14f 0.15f
17 AKAW_05699 Glycerol 3-phosphate dehydrogenase (FAD dependent) (FAD-dependent sn-glycerol-3-phosphate dehydrogenase)
0.56a 0.78 0.74f 0.57e
18 AKAW_01027 Glycerol kinase 0.54b 0.95 0.70f 0.7919 AKAW_09531 Glycerone kinase 0.45b 1.1 0.63f 0.7320 AKAW_01925 PDH (acetyl-transferring) (PDH component E1-�) 0.26b 0.20c 0.21e 0.17f
21 AKAW_08633 Pyruvate carboxylase (mitochondrial) 0.64b 1 0.4e 0.3422 AKAW_08158 Fumarate hydratase (S)-malate hydrolyase (fumarate forming) 0.59b 1.2 0.69f 0.7423 AKAW_08716 Malate synthase 2.8b 2.4 2.5d 1.4a q value of �0.001.b q value of �0.01.c q value of �0.05.d P value of �0.001.e P value of �0.01.f P value of �0.05.g Ratios were determined for the indicated conditions and time points, identified such that N 26.5 h, for example, indicates the value for the N condition at 26.5 h.h FAD, flavin adenine dinucleotide; PDH, pyruvate dehydrogenase.
Temperature Responses of A. kawachii
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synthase gene is deleted in this strain (12). A. kawachii also doesnot produce fumonisin (7), which is consistent with the findingthat A. kawachii IFO 4308 lacks orthologs of nearly all fum genesinvolved in fumonisin biogenesis in A. niger (see Table S2 in thesupplemental material) (19, 21).
Properties of barley koji. Acid production by A. kawachii IFO4308 in solid-state culture for barley koji production was mea-sured for evaluating the accumulation of citric acid under twodifferent culture conditions (Fig. 1A). The acidity of koji preparedunder the normal (N) temperature condition was significantlyhigher than that of koji produced under sustained high-tempera-ture conditions (Fig. 1B).
The growth of A. kawachii at each temperature condition wasevaluated by measuring the amount of GlcNAc in the koji samplesover time (Fig. 1C). The measurement of GlcNAc, which is pres-ent in the fungus-specific cell wall component chitin, is generallyused for evaluating the growth of koji molds because it is difficult toaccurately determine the weight of cells grown in solid-state culture(10). The amount of cell growth that was determined based on theGlcNAc content per gram of koji was not significantly different be-tween the two cultivation conditions, indicating that the temperatureshift did not markedly affect the cell yield of A. kawachii.
Expression of amylolytic enzymes. The major component ofkoji is starch from the polished grains, such as barley and rice.Thus, one of the important features of A. kawachii is the produc-tion of amylolytic enzymes, particularly �-amylase, glucoamylase,and �-glucosidase, which belong to the glycoside hydrolase (GH)GH13, GH15, and GH31 protein families in the CAZy (Carbohy-drate-Active enZYmes) database (http://www.cazy.org/Welcome-to-the-Carbohydrate-Active.html). A total of 17 GH13, 2 GH15,and 5 GH31 family proteins were identified in the A. kawachii IFO4308 genome (see Table S3 and Fig. S4 in the supplemental mate-rial).
During koji production, A. kawachii produces two types of�-amylases, acid-labile �-amylase (alAA) (AmyA, AKAW_11452)and acid-stable �-amylase (asAA) (AamA, AKAW_02026) (22,23). asAA is critical for shochu koji production due to largeamounts of citric acid produced by A. kawachii. In addition toalAA and asAA, the putative �-amylases AmyC (AKAW_09852),AmyD (AKAW_04889), and AmyE (AKAW_09723) (24) werefound in the present genomic analysis (see Table S3 in the supple-mental material). In the case of glucoamylase and �-glucosidase,the A. kawachii genome possesses two glucoamylases, GlaA(AKAW_08979) and GlaB (AKAW_07267), and four �-glucosi-dases, AgdA (AKAW_09853), AgdB (AKAW_05480), AgdC(AKAW_02436), and AgdD (AKAW_10689). As AmyD, AmyE,AgdC, and AgdD do not possess N-terminal signal sequences,these GHs do not appear to be directly involved in the saccharifi-cation of extracellular starch. The amyE gene of A. kawachii islocated in a gene cluster with agtA and agsE, which are putative cellwall 4-�-glucanotransferase and �-1,3-glucan synthase genes, re-spectively, an organization that is similar to that of other Aspergil-lus species (25).
The starch-binding domain (family CBM_20) of amylases isrequired for the degradation of raw, but not gelatinized, starch(26). Among the GH13, GH15, and GH31 family proteins identi-fied in the A. kawachii genome, only asAA and GlaA possess astarch-binding domain. The number and domain structure ofthese GH enzymes was highly similar to that of A. niger CBS 513.88(24). Notably, the glucoamylase GlaB that was found in A. kawa-
chii IFO 4308 is phylogenetically distant from A. oryzae GlaB,which is specifically expressed during solid-state culture and has acrucial role in the digestion of starch during sake production (27,28) (see Fig. S4 in the supplemental material).
The conditions used in the traditional shochu koji-makingprocess, which involve raising the temperature from 36 to 40°Cand subsequently lowering the temperature to 30°C, have beenoptimized for the effective saccharification of starch and produc-tion of citric acid (9, 29). However, among the examined amylo-lytic enzymes, only the expression of the amyC, agdC, and glaBgenes of A. kawachii IFO 4308 were changed significantly (log2
fold change greater than 0.5; q � 0.05) between conditions N andH (see Table S3 in the supplemental material). No marked differ-ences in gene expression of the major amylolytic enzymes, includ-ing alAA, asAA, and GlaA, were observed. These results indicatedthat lowering the temperature in the later stages of koji productionis important for producing large amounts of citric acid but doesnot affect the expression levels of these amylases.
Organic acid production. A. kawachii produces larger amountsof citric acid under the normal temperature conditions used forshochu koji production than at high temperature (9). The levels ofnearly all other examined organic acids of the TCA (tricarboxylicacid) cycle, including pyruvate, citrate, cis-aconitate, isocitrate,�-ketoglutarate, succinate, and malate, did not change signifi-cantly under N temperature conditions at 25 and 26.5 h, whichrepresented short-term responses to temperature reduction (Ta-ble 2). However, after a further 19 h of cultivation at a lowertemperature (N condition), citric acid and isocitrate had accumu-lated in koji at levels 1.8- and 2.7-fold, respectively, of those de-tected at higher temperature (H condition). In particular, isoci-trate significantly increased under condition N, indicating thatthe isocitrate dehydrogenation step in the TCA cycle might bethe rate-limiting step for the citric acid production under con-dition H.
Among the other identified metabolites, the levels of glycerol3-phosphate and glycerol were significantly higher (1.6- and 2.2-fold, respectively), and those of dihydroxyacetone phosphate(DHAP) and trehalose 6-phosphate were significantly lower (0.6-and 0.11-fold, respectively) under condition N at 26.5 h than thosedetected at 25 h under condition H (Tables 2 and 3). After 44 h ofcultivation, the amounts of glycerol 3-phosphate and glycerol un-der condition N remained significantly higher (1.4- and 1.7-fold,respectively), and those of DHAP and trehalose 6-phosphate re-mained significantly lower (0.5- and 0.3-fold, respectively) thanthose under condition H. In addition, the amount of ribulose5-phosphate was significantly higher (1.5-fold) and that of UDPwas significantly lower (0.6-fold) under condition N than thoseunder condition H after 44 h of cultivation.
Gene expression related to citric acid production. The ex-pression profiles of A. kawachii during solid-state cultivation at 40and 30°C after 26.5 h were compared to identify temperature-sensitive genes related to citric acid production. Changes in geneexpression were considered to be statistically significant if the qvalue was less than 0.01 and the log2-fold change was less than�0.5 or greater than 0.5. Using these criteria, a total of 1,114differentially expressed genes, which included 566 upregulatedgenes and 548 downregulated genes, were identified in the DNAmicroarray analysis (see Data Set S1 in the supplemental mate-rial). Among the differentially expressed genes, 26 genes weremapped to metabolic pathways related to organic acid production
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(see Fig. S1). The trends of up- and downregulation of these genes,with the exception of one gene (AKAW_02920), were confirmedby real-time RT-PCR analysis (P value � 0.05) (Table 1). Both themicroarray and real-time RT-PCR results suggested that the 22genes related to the Embden-Meyerhof-Parnas (EMP) pathway,pentose phosphate pathway, glycerol pathway, trehalose pathway,and TCA cycle were downregulated after the temperature reduc-tion from 40 to 30°C, whereas 3 genes, phosphoglycerate mutase(AKAW_03245), cytosolic pyruvate carboxylase, (AKAW_00804),and malate synthase (AKAW_08716), were upregulated (Table 1and Fig. 2).
To better understand the mechanisms underlying the accumu-lation of citric acid in solid-state cultures of A. kawachii, wesearched for genes related to citric acid production among the1,114 differentially expressed genes. In A. niger, the export of ci-trate from the mitochondria into the cytoplasm is proposed to beone of the major steps controlling citric acid accumulation(30–32). However, the mitochondrial tricarboxylate transporterfor citric acid has not been functionally identified in Aspergillus
species. The present microarray analysis identified a significantgene expression change (log2 fold change less than �0.5 or greaterthan 0.5) for 10 putative mitochondrial carrier genes: a putativemitochondrial tricarboxylate transporter (AKAW_03754), whichis a homolog of the mitochondrial citrate transporter Ctp1 of Sac-charomyces cerevisiae (33); two putative C4-dicarboxylate trans-porter/malic acid transport proteins (AKAW_02799 and AKAW_05361); a putative mitochondrial dicarboxylate carrier protein(AKAW_02096); and six putative mitochondrial carrier proteins(AKAW_00314, AKAW_04269, AKAW_05213, AKAW_04250,AKAW_08131, and AKAW_09097) (see Table S4 in the supple-mental material).
Transcriptional regulation also plays an important role inglobal metabolic processes in living organisms. In the present geneexpression analyses, seven putative transcriptional factors, includ-ing several C6 transcription factors (AKAW_03658, AKAW_09811,AKAW_02766, and AKAW_01957), a Zn(II)2Cys6 transcriptionfactor (AKAW_02903), and two bZIP transcription factors(AKAW_09548 and AKAW_06968), were identified among the
TABLE 2 Metabolites identified in barley koji prepared with A. kawachii
Compound namea
Concn (nmol/mg koji)b Concn ratiob
N 25 h N 26.5 h N 44 h H 44 h N 26.5 h/N 25 h N 44 h/H 44 h
Glucose 6-phosphate 27 19 20 18 0.7 1.1Fructose 6-phosphate 6.6 5.5 4.8 4.4 0.8 1.1Fructose 1,6-diphosphate 4.7 2.4 ND 1.8 0.5 NADHAP 4.8 2.7 2.1 4.2 0.6e 0.5e
Glycerol 3-phosphate 12 19 36 25 1.6e 1.4e
3-Phosphoglycerate 2.8 3.5 3.3 3.8 1.3 0.96-Phosphogluconate 1.9 1.8 2.2 1.7 1 1.3Ribulose 5-phosphate 3.7 3.1 2.7 2 0.8 1.4Ribose 5-phosphate 3.5 2.8 3.7 2.4 0.8 1.5c
Sedoheptulose 7-phosphate 3.1 4 4.9 2.5 1.3 2d
Pyruvate 78 55 18 16 0.7 1.1Citrate 32,444 39,597 139,094 78,370 1.2 1.8d
cis-Aconitate 509 503 769 826 1 0.9Isocitrate 371 383 2,644 967 1 2.7c
�-Ketoglutarte 885 871 689 813 1 0.8Succinate 175 163 105 105 0.9 1Fumarate 408 269 259 238 0.7e 1.1Malate 7,865 7,271 4,272 5,041 0.9 0.8GABA 32 27 26 44 0.8 0.6UDP 1.3 1.1 1.2 2 0.8 0.6d
a DHAP, dihydroxyacetone phosphate; GABA, �-aminobutyric acid.b Values were determined for N and H conditions at the indicated time points, identified such that N 25 h, for example, indicates the value for the N condition at 25 h. ND, notdetected; NA, not available.c P value of �0.001.d P value of �0.01.e P value of �0.05.
TABLE 3 Signal intensity of metabolites in barley koji prepared with A. kawachii
Compound
Signal intensitya Signal ratioa
N 25 h N 26.5 h N 44 h H 44 h N 26.5 h/N 25 h N 44 h/H 44 h
ADP-ribose 5.90E�06 5.00E�06 9.40E�06 4.70E�06 0.8 2Glycerol 5.50E�03 1.20 E�02 1.70E�02 1.00E�02 2.2d 1.7b
Trehalose 6-phosphate 1.80E�04 1.90E�05 2.50E�05 8.50E�05 0.11c 0.3d
a Values were determined for N and H conditions at the indicated time points, identified such that N 25 h, for example, indicates the value for the N condition at 25 h.b P value of �0.001.c P value of �0.01.d P value of �0.05.
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FIG 2 Differentially expressed genes mapped on the proposed metabolic pathways of A. kawachii. Bold and dashed arrows indicate downregulated andupregulated reactions, respectively, 26.5 h after the cultivation temperature lowered from 40 to 30°C during koji production. The numbers (1 to 23) next to thearrows correspond to the numbering of locus tags in Table 1. Abbreviations: CoA, coenzyme A; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate;FAD, flavin adenine dinucleotide; Gln, glutamine; PRPP, 5-phospho-alpha-D-ribose 1-diphosphate; TP, transporter protein.
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genes with significant changes in expression between conditions Nand H (see Table S5 in the supplemental material). AKAW_03658,AKAW_09811, AKAW_06968, and AKAW_09548 are predicted tobe orthologs of rosA, nosA, atfA, and atfB, respectively, of Asper-gillus nidulans and A. oryzae (34–37).
Gene ontology analysis of the 1,114 genes that responded to adecreasing koji cultivation temperature. To examine the signif-icance of reducing the cultivation temperature during shochu kojiproduction on global gene expression, GO analysis was performedfor the 1,114 differentially expressed genes. GO enrichment anal-ysis focusing on biological processes identified 42 enriched GOterms (Fig. 3) (see Data Set S2 in the supplemental material).Because many categories shared the same gene transcripts, over-lapping genes were manually removed. Finally, 26 genes related tothe EMP pathway, pentose phosphate pathway, glycerol pathway,trehalose pathway, and TCA cycle were included in the GO termsglucose metabolic process (GO:0006006), trehalose metabolicprocess (GO:0005991), glycerol-3-phosphate metabolic process(GO:0006072), carbohydrate metabolic process (GO:0005975),and metabolic process (GO:0008152). In addition, the phospho-glycerate mutase gene (AKAW_03245) and fumarate hydratase(S)-malate hydrolyase (fumarate-forming) gene (AKAW_08158)were also statistically significantly changed between the two culti-vation conditions although these two genes were not included inthe GO analysis.
With the exception of the genes directly involved in the pro-duction of citric acid, we focused on differentially expressed genesthat were categorized in the GO terms marked with asterisks inFig. 3, and the representatives of these were protein folding (GO:0006457), amino acid transport (GO:0006865), glutamine familyamino acid metabolic process (GO:0009064), and potassium iontransport (GO:0006813) (Table 4). The protein folding (GO:0006457) functional group consisted of 12 downregulated genesin response to lowered temperature and included genes encodingchaperones, such as mitochondrial DnaJ (DnaJ/Hsp40 family)and calnexin, an endoplasmic reticulum-associated chaperone in-volved in glycoprotein quality control. This result indicated thatcultivation at 40°C is a stressful environment for A. kawachii andthat chaperones may be involved in adaptation to heat stress con-ditions. The amino acid transport (GO:0006865) functionalgroup consisted of 17 upregulated and 1 downregulated aminoacid transporter, and the glutamine family amino acid metabolicprocess (GO:0009064) functional group contained 6 upregulatedand 3 downregulated genes involved in glutamine and prolinemetabolism. The observed gene expression changes indicated thatamino acid transport and metabolism were activated after the re-moval of heat stress. Furthermore, the potassium ion transport(GO:0006813) functional group contained one upregulated andthree downregulated genes, indicating that potassium ion trans-port is regulated in response to lowered temperature.
DISCUSSION
We performed gene expression profiling analysis at conventionaland elevated temperatures for barley koji production for shochubrewing to investigate how cultivation temperature influencesgene expression in A. kawachii during solid-state cultivation.DNA microarray analysis identified a total of 1,114 differentiallyexpressed genes between the two examined temperature condi-tions. The observed profile of these genes showed that cultivationat 40°C is stressful for A. kawachii and that heat adaptation leads to
reduced citric acid accumulation through activation of pathwaysbranching from glycolysis, as discussed in detail below.
The gene expression profile of A. kawachii when cultivated at40°C in solid-state culture for an extended period (24 h) indi-cates that the pentose phosphate, trehalose, and glycerol pathwaysare upregulated. Thus, the reduction in citric acid accumulation inbarley koji produced under high-temperature conditions may beassociated with the enhanced activity of these pathways. Glyceroland trehalose function as stress protectants in microorganisms,including aspergilli. For A. kawachii, cultivation at 40°C appearsto induce a stress response because several heat shock proteins andchaperones are significantly upregulated at this temperature,based on the results of GO analysis (Table 4). This finding is inagreement with a previous report that found that heat shock stressinduces oxidative stress and an antioxidant response in A. niger,resulting in increased accumulation of the storage carbohydratestrehalose and glycogen (38). It was suggested that trehalose mobi-lization is required to facilitate cell recovery by A. niger after heat-induced damage (39). In A. niger, cultivation temperature is alsoan important factor for the production of citric acid, which isoptimally produced in liquid culture between 24 and 30°C (40).Here, citrate accumulation by A. kawachii was not observed 1.5 hafter the shift to the lower temperature during solid-state cultiva-tion (compare the values for N 26.5 h and N 25 h in Table 2);however, the concentration of metabolites related to glycerol andtrehalose metabolism (DHAP, glycerol 3-phosphate, glycerol, andtrehalose 6-phosphate) significantly changed between N 26.5 hand N 25 h and between N 44 h and H 44 h (Tables 2 and 3),indicating that changes in the accumulation of these metabolitesoccur more rapidly than those involved in citrate accumulation.The glycerol pathway is regulated by the high-osmolarity glycerol(HOG) signal transduction pathway. Here, the gene expression ofypdA (AKAW_05530), hogA (sakA) (AKAW_06644), and atfA(AKAW_06968) of the HOG pathway were downregulated signif-icantly in A. kawachii (q � 0.01) upon lowering the cultivationtemperature from 40 to 30°C (see Data Set S1 in the supplementalmaterial). AtfA is required for the expression of the NAD-depen-dent glycerol 3-phosphate dehydrogenase gene (gfdB) and is in-volved in glycerol accumulation in A. nidulans and A. oryzae (36,37). Consistent with these functions, the transcriptional level of aputative NAD-dependent glycerol 3-phosphate dehydrogenase-encoding gene (AKAW_10295) of A. kawachii was significantlyreduced at the lower cultivation temperature (Table 1). However,the glycerol concentration of koji produced at 30°C (condition Nat 44 h) was 1.7-fold higher than the koji made at 40°C (conditionH at 44 h) (q � 0.001) (Table 3). This difference might be due tocompetition for substrates between the glycerol pathway and theenhanced trehalose and/or pentose phosphate pathways when A.kawachii is cultivated at 40°C.
GO analysis also indicated that increasing the cultivation tem-perature to 40°C is stressful for A. kawachii. The 12 genes related toprotein folding were found to be downregulated after the temper-ature shift from 40°C to 30°C (Table 4). The induction of genesrelated to protein folding during heat stress response under liquidcultivation condition have also been observed in the other fila-mentous fungi such as Aspergillus fumigatus, Blastocladiella emer-sonii, and Curvularia protuberata (41–44). In the case of A. fu-migatus known as a human-pathogenic fungus, a total of 323genes were more highly expressed at 48°C than at 37°C (41). Theyinclude seven homologs of the 12 differentially expressed protein
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folding-related genes (AKAW_00421, AKAW_03188, AKAW_03383,AKAW_04621, AKAW_06285, AKAW_06769, and AKAW_07910;BLASTP E values, 0) in A. kawachii, indicating that culture condi-tions such as liquid or solid-state are not determinant for the
expression of these genes. The GO analysis has also shown that theexpression of genes related to amino acid transport and biosyn-thesis of glutamine family amino acids was upregulated upon low-ering the cultivation temperature (Table 4), indicating that the
FIG 3 Biological process GO terms identified by GO enrichment analysis among significantly regulated genes in A. kawachii as determined by lowering thecultivation temperature. The 1,114 differentially expressed genes identified in A. kawachii by lowering the cultivation temperature for 26.5 h were used for theanalysis. The GO terms are shown in order of P value (from lowest to highest; all P values are �0.01). *, select genes in this group are listed in Table 4.
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TABLE 4 Differentially expressed genes related to protein folding, amino acid transport, glutamine family amino acid biosynthetic process, andpotassium ion transport in response to lowered cultivation temperature
Locus tag Putative function
Fold changee
GO term(s) (functions)N 26.5 h/H 26.5 h
N 44 h/H 44 h
AKAW_00421 Mitochondrial DnaJ chaperone 0.44b 0.58c Protein folding, temperature stimulus, response to heat, response toabiotic stimulus
AKAW_06769 Mitochondrial protein import proteinMas5
0.41b 0.78 Protein folding, temperature stimulus, response to heat, response toabiotic stimulus
AKAW_00043 Calnexin precursor 0.29a 0.46 Protein foldingAKAW_00798 Peptidyl-prolyl cis-trans isomerase B 0.56b 0.69 Protein foldingAKAW_01492 DnaJ and TPR domain protein 0.47b 0.66 Protein foldingAKAW_02250 10-kDa heat shock protein, mitochondrial 0.40b 0.42c Protein foldingAKAW_03188 Heat shock protein SSC1, mitochondrial
precursor0.41b 0.36b Protein folding
AKAW_03383 Peptidyl-prolyl cis-trans isomerase Cpr7 0.17a 0.58 Protein foldingAKAW_04505 DnaJ domain protein 0.51a 0.65 Protein foldingAKAW_04621 Heat shock protein 60, mitochondrial
precursor0.44b 0.51 Protein folding
AKAW_06285 Heat shock protein (SspB) 0.21b 0.54 Protein foldingAKAW_07910 DnaJ domain protein Psi 0.29b 0.63 Protein foldingAKAW_00265 Amino acid permease 2.8b 2 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_01162 High-affinity methionine permease 1.5b 0.94 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_01807 Proline-specific permease 3.4b 0.52c Amino acid transport, organic acid transport, carboxylic acid transportAKAW_02935 Amino acid permease 2.5b 1.9c Amino acid transport, organic acid transport, carboxylic acid transportAKAW_03046 Amino acid permease 1.8b 1.4 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_03122 Amino acid transporter 2.0b 1.7c Amino acid transport, organic acid transport, carboxylic acid transportAKAW_03197 GABA permease 2.9b 0.56c Amino acid transport, organic acid transport, carboxylic acid transportAKAW_04302 Amino acid permease 0.6b 0.82 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_05043 Amino acid transporter 7.3b 1.3 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_05161 Amino acid permease family protein 3.0b 2.6c Amino acid transport, organic acid transport, carboxylic acid transportAKAW_06587 GABA permease 2.4b 1.1 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_06915 GABA permease GabA 1.9b 2.1c Amino acid transport, organic acid transport, carboxylic acid transportAKAW_07190 Arginine permease 2.8b 1.1 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_07406 Amino acid permease 2.6b 1.9b Amino acid transport, organic acid transport, carboxylic acid transportAKAW_07981 GABA permease 4.7b 0.51 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_09139 Proline permease 4.9b 0.79 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_09349 GABA permease 2.2b 1.4 Amino acid transport, organic acid transport, carboxylic acid transportAKAW_00538 Delta-1-pyrroline-5-carboxylate
dehydrogenase, mitochondrialprecursor
2.5b 1.1 Glutamine family amino acid metabolic process, glutamine familyamino acid biosynthetic process, proline metabolic process
AKAW_03315 Pyrroline-5-carboxylate dehydrogenase 2.8b 0.84 Glutamine family amino acid metabolic process, glutamine familyamino acid biosynthetic process, proline metabolic process
AKAW_03319 Proline oxidase Put1 5.0a 1.4 Glutamine family amino acid metabolic process, glutamine familyamino acid biosynthetic process, proline metabolic process
AKAW_09924 Proline oxidase Put1 5.9a 2.1c Glutamine family amino acid metabolic process, glutamine familyamino acid biosynthetic process, proline metabolic process
AKAW_09925 Pyrroline-5-carboxylate reductase 3.3b 1.4 Glutamine family amino acid metabolic process, glutamine familyamino acid biosynthetic process, proline metabolic process
AKAW_10512 Acetylglutamate synthase 0.66b 1.5 Glutamine family amino acid metabolic process, glutamine familyamino acid biosynthetic process, proline metabolic process
AKAW_00077 Glutamine synthetase 1.6b 1.3 Glutamine family amino acid metabolic process, glutamine familyamino acid biosynthetic process
AKAW_02324 NAD-specific glutamate dehydrogenase 0.4b 0.5c Glutamine family amino acid metabolic processAKAW_05543 bifunctional pyrimidine biosynthesis
protein (PyrABCN)0.7b 0.6c Glutamine family amino acid metabolic process
AKAW_00143 Voltage-gated K channel beta subunit 0.69b 1.2 Potassium ion transportAKAW_00287 Potassium transporter 5 1.9a 3.4c Potassium ion transportAKAW_03290d Ankyrin repeat-containing protein 0.62b 1.5 Potassium ion transportAKAW_06471 Ion channel 0.49b 0.92 Potassium ion transporta q value of �0.001.b q value of �0.01.c q value of �0.05 statistical significance.d AKAW_03290 shows a best BLASTP hit to potassium transporter Trk2 of Saccharomyces cerevisiae although it was annotated just as ankyrin repeat-contaning protein by InterProScan.e Ratios were determined for the indicated conditions and time points, identified such that N 26.5 h, for example, indicates the value for the N condition at 26.5 h.
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growth of A. kawachii appears to be activated by the relief of heatstress. We observed that colony formation by A. kawachii wasmarkedly reduced by heat stress when cells were cultured on yeast-glucose, Czapek-Dox, and minimal agar media (data not shown).However, this finding is inconsistent with the result of GlcNAcanalysis, which indicated that high-temperature solid-state culti-vation does not significantly affect the growth of A. kawachii. Be-cause the analytical method for GlcNAc surveyed both living anddead cells, the cells carried over after the temperature shift maycontribute to higher GlcNAc levels. In addition, the regulation ofchitin biogenesis might be affected by cultivation temperature, asevidenced by the fact that the expression of mpkA (AKAW_00136)of the cell wall integrity (CWI) signal transduction pathway wasincreased 1.8-fold (q � 0.01) by reducing the cultivation temper-ature to 30°C (see Data Set S1 in the supplemental material).
The enzymatic activity of the prepared barley koji cannot beexplained based on gene expression profiles alone. For example,phosphofructokinase is inhibited by citrate, but this inhibition isrelieved by the NH4
ion, AMP, and fructose 2,6-bisphosphate inA. niger (32, 45, 46). In addition, pyruvate kinase activity in A.niger is controlled by various physiological effectors, includingH, Mn2, K, Mg2, and NH4 (47, 48). The present GO anal-ysis showed that expression of the genes related to potassium iontransport was significantly changed between conditions N and H,a response that might affect the activity of pyruvate kinase in A.kawachii (Table 4). Moreover, A. niger hexokinase is strongly in-hibited by physiological concentrations of trehalose 6-phosphate(49). Because the trehalose 6-phosphate concentration in shochukoji was significantly reduced at lower temperature (Table 3), itmight have adversely affected the glucose phosphorylation activityin the EMP pathway, leading to downregulation of glycolytic car-bon flow.
In conclusion, the present study has determined the gene ex-pression profile of A. kawachii during solid-state culture condi-tions. The results suggest that the high-temperature solid-statecultivation of A. kawachii adversely affects the central pathways ofglucose metabolism, leading to depressed citric acid production.Koji is typically produced using traditional solid-state culturetechniques and is widely used in the fermentation industry in Ja-pan. Thus, the gene expression information obtained in this studyis expected to improve the understanding of gene regulation dur-ing the koji-making process and to allow optimization of the in-dustrially desirable characteristics of A. kawachii. In addition, thepresent findings also provide insight into the mechanisms under-lying the accumulation of citric acid by A. kawachii during solid-state culture.
ACKNOWLEDGMENTS
This work was supported in part by Grants-in-Aid for Scientific Research(no. 24580116 to M.G. and no. 25450106 to T.F.) from the Japan Societyfor the Promotion of Science.
REFERENCES1. Machida M, Yamada O, Gomi K. 2008. Genomics of Aspergillus oryzae:
learning from the history of Koji mold and exploration of its future. DNARes 15:173–183. http://dx.doi.org/10.1093/dnares/dsn020.
2. Kitahara K, Yoshida M. 1949. On the so-called Awamori white mold partIII. (1) Morphological and several physiological characteristics. J FermentTechnol 27:162–166.
3. Yamada O, Takara R, Hamada R, Hayashi R, Tsukahara M, Mikami S.2011. Molecular biological researches of Kuro-Koji molds, their classifi-
cation and safety. J Biosci Bioeng 112:233–237. http://dx.doi.org/10.1016/j.jbiosc.2011.05.005.
4. Hong SB, Yamada O, Samson RA. 2014. Taxonomic re-evaluation ofblack koji molds. Appl Microbiol Biotechnol 98:555–561. http://dx.doi.org/10.1007/s00253-013-5332-9.
5. Frisvad JC, Larsen TO, Thrane U, Meijer M, Varga J, Samson RA,Nielsen KF. 2011. Fumonisin and ochratoxin production in industrialAspergillus niger strains. PLoS One 6:e23496. http://dx.doi.org/10.1371/journal.pone.0023496.
6. Gibbons JG, Rokas A. 2013. The function and evolution of the Aspergillusgenome. Trends Microbiol 21:14 –22. http://dx.doi.org/10.1016/j.tim.2012.09.005.
7. Hashimoto R, Asano K, Tokashiki T, Onji Y, Hirose-Yasumoto M,Takara R, Toyosato T, Yoshino A, Ikehata M, Ying L, Kumeda Y,Yokoyama K, Takahashi H. 2013. Mycotoxin production and geneticanalysis of Aspergillus niger and related species including the kuro-kojimold. JSM Mycotoxins 63:179 –186. http://dx.doi.org/10.2520/myco.63.179.
8. Iwano K, Mikami S, Fukuda K, Nose A, Shiinoki S. 1987. Influence ofcultural conditions on various enzyme activities of shochu koji. J Brew SocJpn 82:200 –204. http://dx.doi.org/10.6013/jbrewsocjapan1915.82.200.
9. Omori T, Takeshima N, Shimoda M. 1994. Formation of acid-labile�-amylase during barley-koji production. J Ferment Bioeng 78:27–30.http://dx.doi.org/10.1016/0922-338X(94)90173-2.
10. Fujii F, Ozeki K, Kanda A, Hamachi Y, Nunokawa Y. 1992. A simplemethod for the determination of grown mycelial content in rice-koji usingcommercial cell wall lytic enzyme, yatalase. J Brew Soc Jpn 87:757–759.http://dx.doi.org/10.6013/jbrewsocjapan1988.87.757.
11. Reissig JL, Storminger JL, Leloir LF. 1955. A modified colorimetricmethod for the estimation of N-acetylamino sugars. J Biol Chem 217:959 –966.
12. Futagami T, Mori K, Yamashita A, Wada S, Kajiwara Y, Takashita H,Omori T, Takegawa K, Tashiro K, Kuhara S, Goto M. 2011. Genomesequence of the white koji mold Aspergillus kawachii IFO 4308, used forbrewing the Japanese distilled spirit shochu. Eukaryot Cell 10:1586 –1587.http://dx.doi.org/10.1128/EC.05224-11.
13. Bolstad BM, Irizarry RA, Astrand M, Speed TP. 2003. A comparison ofnormalization methods for high density oligonucleotide array data basedon variance and bias. Bioinformatics 19:185–193. http://dx.doi.org/10.1093/bioinformatics/19.2.185.
14. Andersen MR, Nielsen ML, Nielsen J. 2008. Metabolic model integrationof the bibliome, genome, metabolome and reactome of Aspergillus niger.Mol Syst Biol 4:178. http://dx.doi.org/10.1038/msb.2008.12.
15. Flipphi M, Sun J, Robellet X, Karaffa L, Fekete E, Zeng AP, Kubicek CP.2009. Biodiversity and evolution of primary carbon metabolism in Aspergillusnidulans and other Aspergillus spp. Fungal Genet Biol 46:S19–S44. http://dx.doi.org/10.1016/j.fgb.2008.07.018.
16. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, DavisAP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L,Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM,Sherlock G. 2000. Gene ontology: tool for the unification of biology. NatGenet 25:25–29. http://dx.doi.org/10.1038/75556.
17. Harris MA, Clark J, Ireland A, Lomax J, Ashburner M, Foulger R,Eilbeck K, Lewis S, Marshall B, Mungall C, Richter J, Rubin GM, BlakeJA, Bult C, Dolan M, Drabkin H, Eppig JT, Hill DP, Ni L, Ringwald M,Balakrishnan R, Cherry JM, Christie KR, Costanzo MC, Dwight SS,Engel S, Fisk DG, Hirschman JE, Hong EL, Nash RS, Sethuraman A,Theesfeld CL, Botstein D, Dolinski K, Feierbach B, Berardini T,Mundodi S, Rhee SY, Apweiler R, Barrell D, Camon E, Dimmer E, LeeV, Chisholm R, Gaudet P, Kibbe W, Kishore R, Schwarz EM, SternbergP, Gwinn M, et al. 2004. The Gene Ontology (GO) database and infor-matics resource. Nucleic Acids Res 32:D258 –D261. http://dx.doi.org/10.1093/nar/gkh036.
18. Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, KusumotoK-I, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H,Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE,Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, ClevelandTE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M,Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, KokubunA, Maeda H, Maeyama N, Maruyama J, Nagasaki H, Nakajima T, OdaK, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K,Terabayashi Y, Wortman JR, et al. 2005. Genome sequencing and anal-
Futagami et al.
1362 aem.asm.org February 2015 Volume 81 Number 4Applied and Environmental Microbiology
on February 8, 2018 by guest
http://aem.asm
.org/D
ownloaded from
ysis of Aspergillus oryzae. Nature 438:1157–1161. http://dx.doi.org/10.1038/nature04300.
19. Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ,Turner G, de Vries RP, Albang R, Albermann K, Andersen MR,Bendtsen JD, Benen JAE, van den Berg M, Breestraat S, Caddick MX,Contreras R, Cornell M, Coutinho PM, Danchin EGJ, Debets AJM,Dekker P, van Dijck PWM, van Dijk A, Dijkhuizen L, Driessen AJM, d’Enfert C, Geysens S, Goosen C, Groot GSP, de Groot PWJ, GuillemetteT, Henrissat B, Herweijer M, van den Hombergh JPTW, van denHondel CAMJJ, van der Heijden RTJM, van der Kaaij RM, Klis FM,Kools HJ, Kubicek CP, van Kuyk PA, Lauber J, Lu X, van der MaarelMJEC, Meulenberg R, Menke H, Mortimer MA, Nielsen J, Oliver SG, etal. 2007. Genome sequencing and analysis of the versatile cell factoryAspergillus niger CBS 513.88. Nat Biotechnol 25:221–231. http://dx.doi.org/10.1038/nbt1282.
20. Cerqueira GC, Arnaud MB, Inglis DO, Skrzypek MS, Binkley G, Simi-son M, Miyasato SR, Binkley J, Orvis J, Shah P, Wymore F, Sherlock G,Wortman JR. 2014. The Aspergillus Genome Database: multispecies cu-ration and incorporation of RNA-Seq data to improve structural geneannotations. Nucleic Acids Res 42:D705–D710. http://dx.doi.org/10.1093/nar/gkt1029.
21. Baker SE. 2006. Aspergillus niger genomics: past, present and into thefuture. Med Mycol 44(Suppl 1):S17–S21. http://dx.doi.org/10.1080/13693780600921037.
22. Kaneko A, Sudo S, Takayasu-Sakamoto Y, Tamura G, Ishikawa T, Oba T.1996. Molecular cloning and determination of the nucleotide sequence of agene encoding an acid-stable �-amylase from Aspergillus kawachii. J FermentBioeng 81:292–298. http://dx.doi.org/10.1016/0922-338X(96)80579-4.
23. Suganuma T, Fujita K, Kitahara K. 2007. Some distinguishable proper-ties between acid-stable and neutral types of alpha-amylases from acid-producing koji. J Biosci Bioeng 104:353–362. http://dx.doi.org/10.1263/jbb.104.353.
24. Yuan XL, van der Kaaij RM, van den Hondel CA, Punt PJ, van derMaarel MJ, Dijkhuizen L, Ram AF. 2008. Aspergillus niger genome-wideanalysis reveals a large number of novel alpha-glucan acting enzymes withunexpected expression profiles. Mol Genet Genomics 279:545–561. http://dx.doi.org/10.1007/s00438-008-0332-7.
25. de Groot PW, Brandt BW, Horiuchi H, Ram AF, de Koster CG, KlisFM. 2009. Comprehensive genomic analysis of cell wall genes in Aspergil-lus nidulans. Fungal Genet Biol 46(Suppl 1):S72–S81. http://dx.doi.org/10.1016/j.fgb.2008.07.022.
26. Goto M, Semimaru T, Furukawa K, Hayashida S. 1994. Analysis of theraw starch-binding domain by mutation of a glucoamylase from Aspergil-lus awamori var. kawachi expressed in Saccharomyces cerevisiae. Appl En-viron Microbiol 60:3926 –3930.
27. Hata Y, Ishida H, Ichikawa E, Kawato A, Suginami K, Imayasu S. 1998.Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB)expressed in solid-state culture of Aspergillus oryzae. Gene 207:127–134.http://dx.doi.org/10.1016/S0378-1119(97)00612-4.
28. Hata Y, Ishida H, Kojima Y, Ichikawa E, Kawato A, Suginami K, ImayasuS. 1997. Comparison of two glucoamylases produced by Aspergillus oryzae insolid-state culture (koji) and in submerged culture. J Ferment Bioeng84:532–537. http://dx.doi.org/10.1016/S0922-338X(97)81907-1.
29. Kajiwara Y, Takeshima N, Ohba H, Omori T, Shimoda M, Wada H.1997. Production of acid-stable �-amylase by Aspergillus kawachii duringbarley Shochu-Koji production. J Ferment Bioeng 84:224 –227. http://dx.doi.org/10.1016/S0922-338X(97)82058-2.
30. Torres N. 1994. Modelling approach to control of carbohydrate metabo-lism during citric acid accumulation by Aspergillus niger. I. Model defini-tion and stability of the steady state. Biotechnol Bioeng 44:104 –111.
31. Torres N. 1994. Modelling approach to control of carbohydrate metabo-lism during citric acid accumulation by Aspergillus niger. II. Sensitivityanalysis. Biotechnol Bioeng 44:112–118. http://dx.doi.org/10.1002/bit.260440116.
32. Karaffa L, Kubicek CP. 2003. Aspergillus niger citric acid accumulation:do we understand this well working black box? Appl Microbiol Biotechnol61:189 –196. http://dx.doi.org/10.1007/s00253-002-1201-7.
33. Kaplan RS, Mayor JA, Gremse DA, Wood DO. 1995. High level expres-
sion and characterization of the mitochondrial citrate transport proteinfrom the yeast Saccharomyces cerevisiae. J Biol Chem 270:4108 – 4114. http://dx.doi.org/10.1074/jbc.270.8.4108.
34. Vienken K, Scherer M, Fischer R. 2005. The Zn(II)2Cys6 putative Aspergillusnidulans transcription factor repressor of sexual development inhibits sexualdevelopment under low-carbon conditions and in submersed culture. Genet-ics 169:619–630. http://dx.doi.org/10.1534/genetics.104.030767.
35. Vienken K, Fischer R. 2006. The Zn(II)2Cys6 putative transcription factorNosA controls fruiting body formation in Aspergillus nidulans. Mol Micro-biol 61:544–554. http://dx.doi.org/10.1111/j.1365-2958.2006.05257.x.
36. Hagiwara D, Asano Y, Yamashino T, Mizuno T. 2008. Characterizationof bZip-type transcription factor AtfA with reference to stress responses ofconidia of Aspergillus nidulans. Biosci Biotechnol Biochem 72:2756 –2760.http://dx.doi.org/10.1271/bbb.80001.
37. Sakamoto K, Iwashita K, Yamada O, Kobayashi K, Mizuno A, Akita O,Mikami S, Shimoi H, Gomi K. 2009. Aspergillus oryzae atfA controlsconidial germination and stress tolerance. Fungal Genet Biol 46:887– 897.http://dx.doi.org/10.1016/j.fgb.2009.09.004.
38. Abrashev RI, Pashova SB, Stefanova LN, Vassilev SV, Dolashka-Angelova PA, Angelova MB. 2008. Heat-shock-induced oxidative stressand antioxidant response in Aspergillus niger 26. Can J Microbiol 54:977–983. http://dx.doi.org/10.1139/W08-091.
39. Svanström A, Melin P. 2013. Intracellular trehalase activity is required fordevelopment, germination and heat-stress resistance of Aspergillus nigerconidia. Res Microbiol 164:91–99. http://dx.doi.org/10.1016/j.resmic.2012.10.018.
40. Kitos PA, Campbell JJ, Tomlinson N. 1953. Influence of temperature onthe trace element requirements for citric acid production by Aspergillusniger. Appl Microbiol 1:156 –159.
41. Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, Arroyo J,Berriman M, Abe K, Archer DB, Bermejo C, Bennett J, Bowyer P, Chen D,Collins M, Coulsen R, Davies R, Dyer PS, Farman M, Fedorova N,Fedorova N, Feldblyum TV, Fischer R, Fosker N, Fraser A, García JL,García MJ, Goble A, Goldman GH, Gomi K, Griffith-Jones S, Gwilliam R,Haas B, Haas H, Harris D, Horiuchi H, Huang J, Humphray S, Jiménez J,Keller N, Khouri H, Kitamoto K, Kobayashi T, Konzack S, Kulkarni R,Kumagai T, Lafton A, Latgé JP, Li W, Lord A, Lu C, et al. 2005. Genomicsequence of the pathogenic and allergenic filamentous fungus Aspergillus fu-migatus. Nature 438:1151–1156. http://dx.doi.org/10.1038/nature04332.
42. Georg RC, Gomes SL. 2007. Transcriptome analysis in response to heatshock and cadmium in the aquatic fungus Blastocladiella emersonii. Eu-karyot Cell 6:1053–1062. http://dx.doi.org/10.1128/EC.00053-07.
43. Albrecht D, Guthke R, Brakhage AA, Kniemeyer O. 2010. Integrativeanalysis of the heat shock response in Aspergillus fumigatus. BMC Genom-ics 11:32. http://dx.doi.org/10.1186/1471-2164-11-32.
44. Morsy MR, Oswald J, He J, Tang Y, Roossinck MJ. 2010. Teasing aparta three-way symbiosis: transcriptome analyses of Curvularia protuberatain response to viral infection and heat stress. Biochem Biophys Res Com-mun 401:225–230. http://dx.doi.org/10.1016/j.bbrc.2010.09.034.
45. Arts E, Kubicek CP, Röhr M. 1987. Regulation of phosphofructokinasefrom Aspergillus niger: effect of fructose-2,6-bisphosphate on the action ofcitrate, ammonium ions and AMP. Microbiology 133:1195–1199. http://dx.doi.org/10.1099/00221287-133-5-1195.
46. Habison A, Kubicek CP, Röhr M. 1983. Partial purification and regula-tory properties of phosphofructokinase from Aspergillus niger. Biochem J209:669 – 676.
47. Mesecar AD, Nowak T. 1997. Metal-ion-mediated allosteric triggering ofyeast pyruvate kinase. 2. A multidimensional thermodynamic linked-function analysis. Biochemistry 36:6803– 6813.
48. Meixner-Monori B, Kubicek CP, Röhr M. 1984. Pyruvate kinase fromAspergillus niger: a regulatory enzyme in glycolysis? Can J Microbiol 30:16 –22. http://dx.doi.org/10.1139/m84-003.
49. Panneman H, Ruijter GJ, van den Broeck HC, Visser J. 1998. Cloningand biochemical characterisation of Aspergillus niger hexokinase: the en-zyme is strongly inhibited by physiological concentrations of trehalose6-phosphate. Eur J Biochem 258:223–232. http://dx.doi.org/10.1046/j.1432-1327.1998.2580223.x.
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