Identification and characterization of genes responsible for biosynthesis of kojic acid, an industrially important compound from Aspergillus oryzae
Introduction
The products of filamentous fungi, Aspergilli, have wide applications in a variety of industrial fields, including the food, chemical, and pharmaceutical industries. Aspergillus oryzae has been utilized for the production of indigenous Japanese foods such as sake, shoyu, miso, and vinegar. Other Aspergilli have been used in the production of citric acid by fermentation (e.g., Aspergillus niger) and the anti-hypercholesterolemic agent lovastatin (e.g., Aspergillus terreus). Utilization of Aspergilli in industry is largely based on their ability to produce useful enzymes and metabolites.
Kojic acid (5-hydroxy-2-(hydroxymethyl)-4-pyrone; KA) is a major secondary metabolite produced by a limited range of microorganisms, including A. oryzae, Aspergillus flavus, and Aspergillus tamarii, as well as Penicillium species and certain bacteria in the stationary phase of growth (Bentley, 2006, Wilson, 1971). KA was originally isolated from a koji-culture, a solid-state culture of steamed rice inoculated with A. oryzae (Saito, 1907), and conditions affecting production have been intensively studied (Futamura et al., 2001a, Futamura et al., 2001b, Wan et al., 2005, Wan et al., 2004). KA is now used in a variety of applications, including use as an antibiotic (Bentley, 2006, Beélik, 1956), as an additive to prevent browning of food materials, and as an antioxidant (Bentley, 2006, Nohynek et al., 2004). Recently, there has been interest in KA as an inhibitor of tyrosinase (Saruno et al., 1979, Cabanes et al., 1994) due to chelation of copper ions required in the active site of the enzyme, as is observed in the crystal structure of copper-dependent quercetin 2,3-dioxygenase with KA (Steiner et al., 2002). As a result, this compound is utilized as a skin-lightning agent in the cosmetic industry and in medicine for the treatment of chloasma.
KA can be effectively produced by conversion from glucose with greater than 65% molar productivity (Kluyver and Perquin, 1933). Effective conversion and established methods for analyzing KA production have made KA a good model for studying the regulation of secondary metabolism in filamentous fungi. In particular, Aspergilli have a highly diversified metabolism that is used for industrial production of useful metabolites. However, it is said that approximately 25% of the genes in the genome of lower eukaryotes are unexpressed or are uninduced under culture conditions ordinarily used in laboratories (Naitou et al., 1997). This means that lower eukaryotes, such as Aspergilli, may have various unknown metabolic functions that might produce various novel substances. Thus, information on the biosynthesis of KA, such as identifying genes and their regulator(s), could aid in studying other secondary metabolites.
Despite extensive studies, neither a biosynthetic pathway nor the genes responsible for KA production have been discovered during the more than 100 years since its first isolation (Saito, 1907). Arnstein and Bentley analyzed the KA biosynthesis pathway by the isotope tracer technique in the beginning of the 1950s (e.g., Arnstein and Bentley, 1953a, Arnstein and Bentley, 1953b, Arnstein and Bentley, 1953c). Bentley suggested that, at most, two or three enzymes are needed for the conversion of glucose to KA (2006) and noted that it was surprising that no enzyme system for the formation of KA had been obtained.
The identification of specific genes for KA formation is important for further study, but no genes have been identified. One reason might be the difficulty of purifying enzymes to measure their catalytic activity. Conversion of glucose to KA could not be detected in vitro after the A. flavus mycelium was broken (Bajpai et al., 1982). Moreover, no possible biosynthetic intermediate has been definitively identified (Bentley, 2006).
The complete genome sequence of A. oryzae has been published (Machida et al., 2005) together with the genomes of two other Aspergillus species, A. nidulans (Galagan et al., 2005) and A. fumigatus (Nierman et al., 2005). Subsequently, sequencing of other species, such as A. niger and A. flavus, have been completed (Andersen and Nielsen, 2009). This has enabled analysis of these important organisms at the genome level, facilitating the discovery of novel industrial uses for the fungi in biotechnology (Tamano et al., 2007, Machida et al., 2008). Here, we report for the first time the identification and characterization of genes involved in KA biosynthesis.
Section snippets
Strains and media
A. oryzae RIB40 was isolated in 1950 from cereal (raw material of shoyu) in Kyoto, Japan. This strain is distributed from the National Research Institute of Brewing, Japan (http://www.nrib.go.jp/ken/asp/strain.html). For genetic engineering, an A. oryzae strain having a genotype of ligD::ptrA (pyrG-, niaD-) was used, which had been derived from a ligD::ptrA mutant of NS4 (Mizutani et al., 2008). This ligD::ptrA mutant was derived from NS4 having a genotype of (sC-, niaD-) (Yamada et al., 1997).
KA production by A. oryzae
KA production by the A. oryzae strain RIB40 was detected by three independent methods, the colorimetric method measuring intensity of the red color formed by chelating ferric ions (Bentley, 1957) (Fig. 1), the TLC method, and mass spectrometry (data not shown). The fungus produced KA both in the submerged culture and on the agar plate. When mycelia were grown in the standard liquid medium, the production of KA was detected within 3–4 days after inoculation and the production continued for 2 weeks
Discussion
This is the first report identifying and characterizing the genes responsible for KA biosynthesis employing a reverse genetic method coupled with a DNA microarray technique. An essential step was to find pairs of conditions with significant differences in the production of the metabolite of interest, affecting other physiological factors in the cell as little as possible. The amount and type of the nitrogen source affects the production of some metabolites such as penicillin (Thykaer and
Acknowledgments
This work was partly supported by a Grant-in-Aid from the High-Tech Research Center Project for Private Universities; a matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), 2004–2008; and the Industrial Technology Research Grant Program in 2007 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
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2022, Journal of Hazardous MaterialsCitation Excerpt :The isolates from the northern region had the lowest levels of CPA, which were significantly different from those in the central and southern regions (p = 0.041 and 0.021, respectively) (Fig. 2c3). No clear regional differences in the level of kojic acid (KA) (Terabayashi et al., 2010) produced among the isolates were observed (Fig. 2c4). We also found that the A. flavus population possesses rich secondary metabolic diversity within the species.
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These authors contributed equally to the study.