Introduction

The filamentous hemiascomycete Ashbya gossypii is a natural producer of riboflavin (Demain 1972). Riboflavin is an important growth factor in higher eukaryotes because it is the precursor of flavocoenzymes such as flavin mononucleotide and flavin adenine dinucleotide. A. gossypii has been utilized for industrial riboflavin production, and recently, its entire genome has been completely sequenced and annotated (Dietrich et al. 2004; Hemida et al. 2005). Currently, A. gossypii is used in the biorefining of waste vegetable oil. However, when waste oily resources are used as the carbon source, increased riboflavin productivity is required for the process to be economically viable. Therefore, several research groups (Schmidt et al. 1996a; Park et al. 2007) have applied classical mutagenesis and mutant selection techniques using antimetabolites such as itaconate and oxalate for this purpose. Schmidt et al. (1996b) found that itaconate is inhibitory to isocitrate lyase and itaconate-resistant strain is useful to improve riboflavin yield. Thus, metabolic engineering has been currently practiced for improving the riboflavin yield by overexpression and modification of key enzymes, e.g., threonine aldolase (Monschau et al. 1998) and phosphoribosyl pyrophosphate synthase (Jiménez et al. 2005, 2008).

The glyoxylate cycle is a C4-dicarboxylic acid interconversion pathway, which has been characterized as a “glyoxylate bypass of tricarboxylic acid (TCA) cycle” because the malate dehydrogenase, citrate synthase, and aconitase activities are shared by both cycles (Kornberg and Madsen 1957). The glyoxylate cycle plays an essential role in cell growth on nonfermentable carbon sources such as acetate, ethanol, and fatty acids and in fungal virulence in microorganisms. Dysfunctional mutants of Candida albicans that lacked isocitrate lyase (ICL1, E.C. 4.1.3.1) or malate synthase (MLS1, E.C. 2.3.3.9) in the glyoxylate cycle lost their ability to form pseudohypha and their fungal virulence in mice (Lorenz and Fink 2001). In Saccharomyces cerevisiae, disruptants of these genes were unable to utilize carbon sources such as ethanol, acetate, or oleic acid (Fernandez et al. 1992; Hartig et al. 1992; Kunze et al. 2002). The metabolic importance of ICL1 has been well studied as a key enzyme in riboflavin biosynthesis from oils in A. gossypii since its activity was positively correlated to the riboflavin yield (Kanamasa et al. 2007; Maeting et al. 1999; Schmidt et al. 1996a).

Malate synthase is an acyltransferase that converts glyoxylate and acetyl-CoA to malate. In this reaction, the acetyl residue from acetyl-CoA is transferred to glyoxylate. The malate that is generated is either converted to oxaloacetate for continuous glyoxylate cycle or is used as the initial substrate in gluconeogenesis for conversion to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (PCK1, E.C. 4.1.1.49). Although MLS1 activity is believed to be necessary for mycelial growth on nonfermentable carbon sources, A. gossypii MLS1 has not been functionally identified and characterized in riboflavin biosynthesis from vegetable oils.

In this study, the MLS1 homolog was disrupted and overexpressed in A. gossypii to facilitate the identification and characterization of the gene product. We could demonstrate that MLS1 is one of the important key enzymes for the improved production of riboflavin from rapeseed oil. Moreover, supplementation malate into the A. gossypii culture was effective in improving riboflavin productivity.

Materials and methods

Strains and growth conditions

A. gossypii ATCC 10895 (AgWT) and Escherichia coli DH5α were used as the A. gossypii wild-type and DNA manipulation host strain, respectively. E. coli DH5α was grown in LB medium (pH 7.5) consisting of 1% (w/v) polypeptone-S (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan), 0.5% (w/v) bacto yeast extract (Becton, Dickinson and Company, NJ, USA), and 0.5% (w/v) sodium chloride (Wako Pure Chem. Ind., Ltd., Osaka, Japan).

The media used for A. gossypii culture were as follows: YD medium (pH 6.8) containing 1% (w/v) yeast extract (Oriental Yeast Co., Ltd., Tokyo, Japan) and 1% (w/v) glucose; YR medium (pH 6.8) containing 1% (w/v) yeast extract and 1% (w/v) rapeseed oil; seed medium for riboflavin production (per liter) consisting of 30 g corn steep liquor (Wako), 9 g yeast extract, and 15 g rapeseed oil (pH 6.8); production medium (per liter) containing 60 g corn steep liquor, 30 g gelatin (Wako), 1.5 g KH2PO4, 1.5 g glycine, 4.4 mg CoCl2, 17.9 mg MnCl2⋅4H2O, 44.2 mg ZnSO4⋅7H2O, 10.3 mg MgSO4⋅7H2O, and 50 g rapeseed oil (pH 6.8). Cultures were performed in 500-ml shaker flasks with a working volume of 50 ml of each medium. The cultures were incubated on a rotary shaker (Bio Shaker; Takasaki Scientific Instrument Co.) at 220 rpm and 28°C. For selective growth of the transformants, Geneticin (Wako) was added to the cultures to a final concentration of 200 μg/ml.

Homology search of A. gossypii malate synthase using BLAST

The amino acid sequence of A. gossypii malate synthase was obtained from the Ashbya Genome Database (http://agd.vital-it.ch/index.html) described by Hemida et al. (2005). The amino acid sequence of the malate synthase from S. cerevisiae that was identified by Hartig et al. (1992) was used as the query sequence for cross-species BLAST homology searching in the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/). The acquired FASTA format amino acid sequences of several species, including A. gossypii, were multialigned by ClustalX (Larkin et al. 2007) and modified by GeneDog.

Plasmid constructions

DNA was manipulated using standard procedures (Sambrook and Russell 2001). The control plasmid (pARK) and expression plasmid with the ACR268C gene in A. gossypii (pAMK) were constructed using pAUR123 (TaKaRa Bio Inc., Shiga, Japan), as shown in Scheme 1A. The ACR268C gene was polymerase chain reaction (PCR)-amplified with 100 ng of AgWT chromosomal DNA as the template using the KOD-Plus DNA polymerase (Toyobo, Co., Ltd., Osaka, Japan). PCR was carried out using the AgMLS1-Ex-F and AgMLS1-Ex-R primers (Table 1) under the following conditions: 1 cycle at 95°C for 2 min followed by 35 cycles at 95°C for 30 s, 55°C for 1 min, and 68°C for 2 min. The amplified fragment was purified by GFX PCR DNA and the Gel Band Purification Kit (GE Healthcare UK Ltd., Buckinghamshire, England, UK). The ACR268C gene was inserted between the KpnI and XbaI sites located downstream of the alcohol dehydrogenase promoter (ADH pro) in pAUR123, using a DNA ligation kit (Mighty Mix, TaKaRa Bio Inc.). The resulting plasmid was designated pARM.

Scheme 1
scheme 1

A Flow diagram of plasmid construction. Black arrows indicate TEF1 promoter–kanamycin-resistance gene–TEF1 terminator (TEF1pro-Kanr). Diagonal squares denote the ACR268C (MLS1) gene, which was amplified using the AgMLS1-Ex-F and AgMLS1-Ex-R primers. B Disruption of the ACR268C gene in the A. gossypii wild-type strain. The asterisk and diagonal box represent the 527th codon encoding the aspartate residue, which acts as a proton donor, and a 60-bp homologous region of the ACR268C gene, respectively

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Table 1 Primers list
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The kanamycin-resistance gene cassette (TEF1pro-Kanr), which contains the kanamycin-resistance gene used as a dominant marker for Geneticin resistance in eukaryotes (Jiménez and Davies 1980) under the control of the A. gossypii translation elongation factor 1α promoter (TEF1pro) and terminator (TEF1ter), was amplified using pPKT as the template (Kato and Park 2004) and the AgTEFproH3-F and AgTEFterH3-R primers (Table 1). PCR was carried out using LA-Taq Hot Start Version (TaKaRa Bio Inc.) under the following conditions: 1 cycle at 95°C for 2 min followed by 35 cycles at 95°C for 30 s, 55°C for 1 min, and 72°C for 1.5 min. Final extension was carried out with 1 cycle at 72°C for 10 min. The amplified fragment was purified as described above and inserted into the HindIII sites of pARM and pAUR123. The resulting plasmids were designated pAMK and pARK, respectively. pAMK harbored both TEF1pro-Kanr and ACR268C, while pARK contained only TEF1pro-Kanr.

PCR-based gene targeting disruption

The knockout mutant of the ACR268C gene was constructed by PCR-based gene targeting disruption, as described by Wendland et al. (2000). Technical protocol of transformation in A. gossypii was performed according to the method described by Monschau et al. (1998) and Kanamasa et al. (2007) with some modifications. The 60-bp homologous sequence of the ACR268C gene on both ends of TEF1pro-Kanr was amplified using pPKT as the template, as shown in Scheme 1A, and the Agmls901-F and Agmls1600-R primers (Table 1). PCR amplification was carried out using LA-Taq Hot Start Version (TaKaRa Bio Inc.) under the following conditions: 1 cycle at 95°C for 2 min followed by 35 cycles at 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min 42 s. Final extension was carried out with 1 cycle at 72°C for 10 min. The PCR product was purified as described above.

Spores of AgWT (approximately 1.0 × 106) were grown on YD medium for 27 h. The grown mycelia were harvested by filtration, washed with distilled water, and suspended in 50 mM potassium phosphate buffer (pH 6.8) containing 25 mM 2-mercaptoethanol. The suspension was incubated at 30°C for 30 min with gentle agitation, and the mycelia were collected by filtration and washed with transformation buffer consisting of 270 mM sucrose, 10 mM Tris–HCl (pH 7.5), and 1 mM MgCl2. The mycelia were finally resuspended in cooled transformation buffer, and 350 μl of the mycelial suspension was mixed with 300 ng of the above-purified gene disruption cassette (Scheme 1B). The cassette was introduced into the mycelium by electroporation in a Gene Pulser Xcell system (Bio-Rad Lab. Inc., Hercules, CA, USA) at 1.5 kV/cm, 400 Ω, and 25 μF using 2-mm pre-chilled electrocuvettes (Bio-Rad). The postelectroporated mycelia were incubated on a YD plate to regenerate the mycelia at 30°C for 6 h. Subsequently, the mycelia were covered with 20 ml YD medium containing 0.6% agar and 300 μg/ml Geneticin for isolating the transformants.

Confirmation of transformants

The disruption of the ACR268C gene was confirmed by PCR using Ex-Taq Hot Start Version (TaKaRa Bio Inc.) with the AgMLS1-V1 and AgMLS1-V2 primers (Table 1) and 200 ng chromosomal DNA from the transformant. The PCR conditions were as follows: 1 cycle at 95°C for 2 min followed by 35 cycles at 95°C for 30 s, 55°C for 1 min, and 72°C for 3 min. Final extension was carried out by 1 cycle at 72°C for 10 min. For Southern blotting analysis of the mutant, 100 μg of the chromosomal DNA was simultaneously digested with BamHI and XhoI, and the products were separated by 0.9% (w/v) agarose gel electrophoresis. The DNA fragments were transferred onto Hybond-N+ (GE Healthcare). A DNA fragment of the kanamycin-resistance gene was used as the hybridization probe. Preparation of the labeled probe and chemiluminescent detection were carried out with the AlkPhos Direct Labeling and Detection system (GE Healthcare), according to the manufacturer’s protocol. Positive signals were detected by Fluor-S/MAX (Bio-Rad).

To confirm the transformants carrying pARK or pAMK, 10 μg of total DNA from each transformant was introduced into E. coli cells, and the rescued plasmid was confirmed by restriction enzyme mapping.

Quantification of malate synthase messenger RNA by real-time quantitative reverse transcriptase-polymerase chain reaction

A. gossypii mycelia grown in production medium in flask for 26 h were harvested by filtration. Resulting mycelia were mixed with 0.3 g of acid washed glass beads (Sigma) and 1 ml of ISOGEN (NIPPON GENE Co., Ltd. Tokyo, Japan), and then fractured by vigorous agitation. Complementary DNA was obtained by reverse transcription-PCR using PrimeScript RT-PCR Kit (TaKaRa Bio Inc.) with the extracted total RNA as the template with random 6-mers primer in the elongation condition at 40°C for 1 h. The template RNA was degraded by 10 mg of RNase (Sigma) at 37°C for 30 min. Messenger RNA levels of malate synthase and actin were quantified by using FullVelocity SYBR Green QPCR Master Mix (Agilent Technologies Inc., CA, USA) under the condition of 1 cycle at 95°C for 5 min, 60 cycles at 95°C for 10 s, and 60°C for 30 s for the amplification plot, and 1 cycle at 95°C for 1 min, 55°C for 30 s, and 95°C for 5 min for the dissociation plot. QMLS1-F, QMLS1-R and QACT1-F, QACT1-R primers (Table 1) were used for malate synthase and actin, respectively. Actin was used as an internal standard because of its constitutive expression.

Enzyme assay

The malate synthase activity was determined according to the method of Dixon and Kornberg (1959). Ten microliters of the enzyme solution was added to 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 2 mM sodium glyoxylate, and 50 μM acetyl-CoA (Wako), and the final volume was made up to 1 ml. The specific absorbance of acetyl-CoA was measured at 232 nm. One unit of malate synthase activity was defined as the amount of enzyme required to deacetylate 1 μmol of acetyl-CoA per minute. Nonspecific deacetylated acetyl-CoA was measured in the absence of MgCl2 and sodium glyoxylate. The protein concentration was determined by the Bradford method using a protein assay kit (Bio-Rad) with bovine serum albumin as the standard.

Analytical methods

The riboflavin and residual oil concentrations were measured according to the method previously described by Park and Ming (2004). The dry cell weight was measured as follows. The mycelia from the culture broth were filtered using filter paper No. 5A (Advantec, Tokyo, Japan). The mycelia paste was dried overnight in an oven at 100°C, and the dry cell weight was measured.

Results

Multiple alignment of amino acid sequences of A. gossypii ACR268Cp

When multiple alignment analysis of amino acid sequences of the proteins of yeasts and fungi was carried out, several conserved regions and similar peroxisomal-targeting sequences were observed in the C-terminal region; these were designated SRL and SKL. The malate synthase of S. cerevisiae has a signal sequence and is believed to be transported into the peroxisome (Kunze et al. 2002). The amino acid sequence of ACR268Cp showed 73% identity to the malate synthase from S. cerevisiae (ScMLS1), 60% identity to the enzyme from Aspergillus niger (AnMLS1), and 57% identity to the enzymes from C. albicans (CaMLS1) and Neurospora crassa (NcMLS1). Characterization of the features based on the ScMLS1 amino acid sequence obtained by UniProt (http://www.pir.uniprot.org/) suggested that ACR268Cp had two active sites, i.e., a proton acceptor and donor on the 247th arginine residue and 527th aspartate residue, respectively.

ACR268C gene disruption and its phenotype

The ACR268C gene was disrupted, and six Geneticin-resistant colonies of A. gossypii were isolated with an efficiency of approximately 20 colony-forming units (cfu)/μg of DNA. The ACR268C gene disruption was confirmed by genomic PCR using the AgMLS1-V1 and AgMLS1-V2 primers. The presence of the 2.7-kb fragment (lane 2 in Fig. 1a) indicated the introduction of TEF1pro-Kanr into the ACR268C gene. This also led to the identification of the Geneticin-resistant colony as an ACR268C gene disruptant. Southern blot analysis was carried out to confirm the disruption of the ACR268C gene. The 2.6-kb DNA fragment was detected using a chemiluminescent kanamycin-resistance gene probe from the chromosomal DNA that had been double-digested with BamHI and XhoI at both ends of the ACR268C gene (Fig. 1b). These results demonstrated that the TEF1pro-Kanr gene cassette was integrated into the ACR268C gene, and the gene disruptant was designated AgΔmls1.

Fig. 1
figure 1

Confirmation of the ACR268C gene-targeted disruptant by PCR and Southern blot analysis. a The PCR products were amplified from chromosomal DNA using the AgMLS1-V1 and AgMLS1-V2 primers (Scheme 1B). The up and down arrows indicate fragments of sizes 2.7 and 1.6 kb, respectively. b In the Southern blot analysis, chromosomal DNA was digested simultaneously with BamHI and XhoI. The kanamycin-resistance gene, which is absent in the AgWT chromosomal DNA, was used as the probe. Lanes 1 and 2 in a and b denote AgWT and AgΔmls1 (ACR268C gene-disruptant), respectively

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To investigate the phenotypic variations between the AgWT and AgΔmls1 strains, both strains were cultured in the production medium. The specific malate synthase activity of AgΔmls1 was less than 5 mU/mg protein, which was one-seventh that of the AgWT strain (Fig. 2a). This indicated that replacement of the +961 to +1599 region of the ACR268C gene, including the 527th aspirate residue, with TEF1pro-Kanr leads to complete loss of enzyme activity. This resulted in a significant decrease in riboflavin production by the AgΔmls1 strain—approximately 10-fold less than that by the AgWT strain (Fig. 2b). The oil consumption and dry cell weight of AgΔmls1 were half or less than half that of the AgWT strain (Fig. 2c, d).

Fig. 2
figure 2

Time course of malate synthase activity (a), riboflavin concentration (b), residual oil concentration (c), and dry cell weight (d) in cultures of the AgWT (closed rhombus) and AgΔmls1 (open triangle) strains. Both cultures were carried out in triplicates, and the average data and standard deviations are shown

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The mycelial morphology of AgΔmls1 differed from that of AgWT. In the AgWT strain, as the culture progressed, the mycelia transformed into hypertrophic cells (HM in Fig. 3). In contrast, the AgΔmls1 cells maintained their morphology as thin filamentous mycelia (FM in Fig. 3) from the beginning, i.e., when the culture was initiated. Oil droplets in the AgΔmls1 strain remained even after a culture time of 6 days, indicating the presence of residual oil (O in Fig. 3). Fewer riboflavin-accumulating yellowish mycelia (R in Fig. 3) were observed in AgΔmls1 than in AgWT. Crystallized riboflavin (CR in Fig. 3) was observed in hypertrophic mycelia of AgWT but not in those of the AgΔmls1 strain.

Fig. 3
figure 3

Morphological changes in the ACR268C (MLS1) gene disruptant and AgWT (A–C) and AgΔmls1 (a–c) strains. The mycelia are shown at 3 days (A and a), 5 days (B and b), and 7 days (C and c) of culture. HM hypertrophic mycelia, O residual oil droplet, FM filiform mycelia, R riboflavin, CR crystallized riboflavin, S spore. Bars indicate a 10-μm scale

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Riboflavin production, malate synthase activity, and the transcriptional level in the malate synthase-overexpressing transformant

The presence of the pARK and pAMK plasmids in the transformant was verified by digesting the extracted plasmid DNA with KpnI and XbaI. Electrophoretic analysis demonstrated the presence of bands of size approximately 850 and 1.9 kb, which represent the kanamycin-resistance gene and ACR268C gene, respectively (lane 3 in Fig. 4a). The transformants with AgWT/pARK and AgWT/pAMK were designated AgWP and AgMLS1, respectively.

Fig. 4
figure 4

Confirmation of constructed plasmids (a) and time course of malate synthase activity (b) and riboflavin concentration (c) in cultures of the AgWP (open square) and AgMLS1 (closed circle) strains. a The plasmids were digested with KpnI and XbaI. M, 1-kb ladder; lane 1, pAUR123; lane 2, pARK; and lane 3, pAMK. Each of the constructed plasmids pARK and pAMK was prepared from the total DNA extracted from the AgWP and AgMLS1 strains, respectively. Each culture was carried out in triplicates, and the average data and standard deviation are shown

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The specific malate synthase activity of AgMLS1 was significantly higher than the activities of the AgWP (Fig. 4b) and AgΔmls1 strains (Fig. 2a), indicating that the ACR268C gene encodes malate synthase. When the AgWP and AgMLS1 transformants were cultured in the production medium, the riboflavin production by AgMLS1 was 1.7-fold higher than that by AgWP (Fig. 4c), even though the values of oil consumption and dry cell weight were similar to those of AgWP (data not shown).

Real-time quantitative RT-PCR revealed that MLS1 messenger RNA (mRNA) level of AgMLS1 strain was approximately 1.6- and 8-fold higher than those of AgWP and AgΔmls1, respectively (Figs. 4b and 5). This indicates that the plasmid pAMK expressing MLS1 functions appropriately.

Fig. 5
figure 5

Analysis of intracellular transcriptional level using quantitative real-time PCR. Quantification of MLS1 mRNA was represented by ratio of MLS1 and ACT1. Total RNA was extracted from 26-h grown mycelia (n = 4)

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Additive effect of glyoxylate and malate on riboflavin production in the ACR268C gene disruptant

Malate synthase gene disruption led to a significant decrease in riboflavin production, suggesting that continuous glyoxylate cycling and/or gluconeogenesis bypass from malate might be blocked. Glyoxylate and malate are the substrate and product of malate synthase, respectively. Therefore, to investigate the effects of glyoxylate and malate on metabolic flux, the AgWT and AgΔmls1 strains were cultured in glyoxylate- or malate-supplemented production medium. Remarkable phenotypic differences were not observed between the strains upon culture in the 50-mM glyoxylate-supplemented culture (data not shown). However, the 50-mM malate-supplemented cultures of AgWT and AgΔmls1 strains showed higher riboflavin production, oil consumption, and dry cell weight in comparison with the values of the control culture. The riboflavin concentrations of the AgΔmls1 and AgWT strains were more than 2-fold higher than those of the strains cultured without the malate additive (Fig. 6a). In particular, in the culture of the AgΔmls1 strain, 50 g/l of oil was completely consumed (Fig. 6b), which differed drastically from the result obtained in the absence of the malate additive (Fig. 2c).

Fig. 6
figure 6

Riboflavin production (a) and residual oil concentration (b) in cultures of AgWT (rhombus) and AgΔmls1 (triangle) strains with (closed symbols) or without (open symbols) 50 mM malate. Each culture was carried out in triplicate, and the average data and standard deviations are shown

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Discussion

In this study, the ACR268C gene was identified to encode malate synthase, and its role in riboflavin biosynthesis in A. gossypii was investigated using rapeseed oil as the sole carbon source. We analyzed the amino acid sequence of ACR268Cp and found sequences that were similar to two active sites and the C-terminal peroxisome-targeting signal (−SRL; Gould et al 1988) of S. cerevisiae malate synthase. In the ACR268C gene-disruptant, the 527th aspartate, which functions as a proton donor, was replaced with the kanamycin-resistance gene, and the disruptant did not exhibit any malate synthase activity (Fig. 2a). This strain also showed decreases in the riboflavin concentration, oil consumption, and dry cell weight. Therefore, malate synthase is important for riboflavin biosynthesis and the assimilation of vegetable oils. This is similar to the functioning of isocitrate lyase (Schmidt et al. 1996b). However, Kanamasa et al. reported that an isocitrate lyase gene-disrupted mutant lost the ability to produce riboflavin but grew well in the production medium (Kanamasa et al. 2007). This suggests that malate synthase may have an important role not only in riboflavin production but also in the maintenance of hyphal growth and turnover of carbon assimilated from nonfermentable carbon sources under aerobic conditions. With regard to mycelial morphology, hypertrophic mycelia were hardly observed in the disruptant in comparison with the wild-type strain, even during the late culture period. Empirically, hypertrophic mycelia are predominant in riboflavin producing A. gossypii throughout the late culture period. This suggests that the stagnation of the glyoxylate cycle results in retardation of both cell growth and riboflavin biosynthesis because the supply of both malate and oxaloacetate may be limited. Malate and oxaloacetate are substrates for glyoxylate cycle turnover, malate/aspartate shuttle (Schmitt and Edwards 1983), and gluconeogenesis (Fig. 7). These processes are necessary for the biosynthesis of the sugar phosphate (such as ribulose-5-phosphate) and purine nucleotide (such as guanosine triphosphate) for the riboflavin scaffold (Stahmann et al. 2000).

Fig. 7
figure 7

Putative metabolic pathway for riboflavin biosynthesis in A. gossypii. The pathways in which malate synthase and isocitrate lyase are involved are indicated by the thick solid line and dotted line, respectively

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As for malate synthase-overexpressing strain, even though the expression promoter and/or replication origin was adopted relatively at low mRNA level, as compared to those of TEF1 promoter and 2-μm origin, the riboflavin concentration of the MLS1-overexpressing strain was 1.7-fold higher than that of the wild-type. This is due to an increase in the malate synthase activity and the mRNA level (Figs. 4b, c and 5). Therefore, we expect and suggest that adoption of strong expression promoters may increase the riboflavin yield to a better extent in this experiment.

MLS1 disruptant strain did not respond to a malate-supplemented culture. Although malate addition led to an increase in oil consumption, riboflavin production was not restored significantly in the disruptant, unlike the wild-type strain. It is probable that ACR268Cp may have another function distinct from malate synthase activity. On the other hand, a glyoxylate (substrate of malate synthase)-supplemented culture did not have any effect on riboflavin production, oil consumption, and cell growth in both the A. gossypii wild-type and disruptant strains, in spite of the presence of excess substrate (data not shown). This suggests that the efficiency of acetyl-CoA turnover is a limiting factor for malate synthase activity in glyoxylate additive culture, and that malate can be a driving force for effective turnover of glyoxylate cycle, gluconeogenesis, and TCA cycle (Fig. 7). Therefore, efficient turnover and/or excess supplementation of key metabolites such as malate around log-phase on mycelial growth may be important for effective riboflavin production of A. gossypii. An effective oil consumption due to improved mycelia lipase activity (Stahmann et al. 1997) or fortification of metabolic activity on β-oxidation may be necessary for improving riboflavin productivity.

Using an MLS1 disruptant and MLS1-overexpressing transformant, we demonstrated that malate synthase is one of the important key enzymes for improving riboflavin production in A. gossypii. However, riboflavin production of AgWP and AgMLS1 strains was half or less than that of the wild-type. It may be due to addition of antibiotics for maintaining subnuclear plasmid, resulting in lower mycelial growth of AgWP strain than that of AgWT strain (data not shown). Kato and Park (2004) showed similar phenomenon with 2-μm origin plasmid transformants as well as yeast autonomously replicating sequence (ARS1) contained in pARK and pAMK, both of which were functioned in A. gossypii as replication origins (Wright and Philippsen 1991). Therefore, to further improve riboflavin production, it is necessary to overexpress important key genes in the A. gossypii genome. Jiménez et al. (2008) have succeeded in chromosomal integration by gene-targeting recombination using a specific gene cassette with tandem placement of a drug-resistant gene and riboflavin production-positive gene expression cassettes. In the near future, chromosomal integration by malate synthase recombination will be useful for improving the riboflavin yield of A. gossypii.