Discovery of the 2,4’-Dihydroxy-3’-methoxypropiophenone Biosynthesis Genes in Aspergillus oryzae

Significance

Aspergillus oryzae is a filamentous fungus that has long been used by the Japanese in the production of fermented foods such as soybean paste, rice wine and soy sauce. The United States Food and Drug Administration has classified it as a generally regarded as safe (GRAS) organism. Just like any other GRAS organisms, A. oryzae has been found not to produce secondary metabolites that are potentially toxic. Polyketides are a major class of secondary metabolites found in both bacteria and fungi. Polyketide synthases are involved in the synthesis of polyketides. In a previous study, the sequencing of the whole genome of A. oryzae revealed that A. oryzae contained a number of secondary metabolite biosynthetic gene clusters. About 61 secondary metabolite biosynthesis gene clusters were discovered including 27 polyketide synthase gene clusters. All the 27 polyketide synthase gene clusters were found to contain iterative type I polyketide synthase genes. However, it was noticed that secondary metabolites produced potentially by these polyketide synthase gene clusters were mostly unknown.

In a new study conducted by scientists at the University of Tokyo: Dr. Eiichiro Kan, Dr. Hiroya Tomita, Dr. Yohei Katsuyama, Dr. Jun-ichi Maruyama and led by Professor Yasuo Ohnishi, the authors sought to determine previously unidentified polyketides that could be produced by uncharacterized iterative type I polyketide synthase gene clusters in A. oryzae. They discovered a monocyclic polyketide, 2,4’-dihydroxy-3’-methoxypropiophenone (compound 1) and its biosynthetic genes. The work is published in ChemBioChem, a journal combining chemistry and biology. Dr. Yasuji Koyama from the Noda Institute for Scientific Research is a co-author and contributed to the research.

The research team focused their study on eight polyketide synthase gene clusters in A. oryzae. These eight had been earlier reported to be expressed at relatively high levels in a minimal medium. They observed that unlike the A. oryzae strain that lacked all eight polyketide synthase gene clusters, the parent strain of A. oryzae produced three compounds (1, 2, and 3) in a culture condition. These compounds were found to be related polyketides with a propiophenone structure. The research team identified one gene cluster responsible for the biosynthesis of these polyketides and analyzed the function of each gene. The propiophenone synthesis genes A and B were observed to be required for the biosynthesis of compounds 1, 2 and 3 while propiophenone synthesis gene C was not needed for the biosynthesis of compound 1 but was found to be required for improving its yield and also possibly related to the hydroxylation of compound 3. Compound 1 was identified as 2,4’-dihydroxy-3’-methoxypropiophenone, while compound 3 was identified as 4’-hydroxy-3’-methoxypropiophenone, but the team was unable to obtain sufficient quantities of compound 2 for structure elucidation. Compound 3 was also found to be a precursor to compound 1.

2,4’-Dihydroxy-3’-methoxypropiophenone (1) was reported to be a metabolite (evofolin-A) in the heartwood extract of the medicinal plant Tetradium glabrifolium. This compound was also isolated from the herb Sida acuta as a compound to induce quinone reductase in cultured mouse hepatoma cells (Hepa 1c1c7). However, no information on its biosynthetic pathway has been provided. It was surprising to find that A. oryzae also produces this medicinal plant-derived polyketide.

In conclusion, through this landmark study, the authors have identified a rare secondary metabolite of A. oryzae, a monocyclic polyketide, 2,4’-dihydroxy-3’-methoxypropiophenone (1) and proposed its biosynthetic pathway. This study supports previous postulations that A. oryzae seldom produces any endogenous secondary metabolites. Thus the findings from this study has provided new and additional knowledge about the cryptic secondary metabolism in A. oryzae, which will be beneficial for the food industry in using A. oryzae as a safe microorganism, and for future basic research in this field.

About the author

Yasuo Ohnishi received B.S., M.S. and Ph.D. degrees from The University of Tokyo (1991, 1993 and 1996), and carried out postdoctoral research at the same university (1996-1997). He wrote a doctoral thesis on protein secretion in Gram-negative bacteria. Since he received his Ph.D., he has been engaged in research on Streptomyces genetics and secondary metabolite biosynthesis. He was appointed as an assistant professor (1997) and an associate professor (2002) in the Laboratory of Fermentation Microbiology (led by Prof. Sueharu Horinouchi), The University of Tokyo. This laboratory has its origins in the Laboratory of Agricultural Production established in 1900 and was officially established in 1924; it is one of the oldest microbiology laboratories in Japan. Then, he was appointed as a full professor in 2010, succeeding the late Prof. S. Horinouchi. Current research in the Fermentative Microbiology Laboratory focuses on (i) regulation of morphological development in the rare actinomycete Actinoplanes missouriensis, (ii) biosynthesis of secondary metabolites, and (iii) microbial production of industrially useful compounds and enzymes. （See https://www.hakko.bt.a.u-tokyo.ac.jp/en）

About the author

Yohei Katsuyama received B.S., M.S. and Ph.D. degrees from The University of Tokyo (2005, 2007 and 2010), and carried out postdoctoral research at the laboratory organized by Prof. Rolf Müller in Saarland University, Germany (2010-2012). He wrote a doctoral thesis on combinatorial biosynthesis of plant polyketides in Escherichia coli. When he was a postdoctoral researcher, he worked on the biosynthesis of natural products produced by myxobacteria and actinobacteria. He was appointed as a lecturer (2012) and an associate professor (2017) in the Laboratory of Fermentation Microbiology, The University of Tokyo. His main research interests are (1) biosynthesis of secondary metabolites produced by actinomycetes, (2) biochemistry of enzymes involved in secondary metabolism, and (3) microbial production of industrially useful compounds.

About the author

Eiichiro Kan received B.S. and M.S. degrees from Tohoku University (2013 and 2015). After he received his M.S., he joined Kikkoman Corporation, one of the most famous soy sauce companies in the world. He was a visiting researcher in the Laboratory of Fermentation Microbiology, The University of Tokyo, via Noda Institute for Scientific Research from April 2016 to March 2019. He obtained his Ph.D. degree from The University of Tokyo in 2021. He wrote a doctoral thesis on heterologous production of polyketides in Aspergillus oryzae and identification of cryptic polyketides of A. oryzae. Now, he works in the Kikkoman Singapore R & D laboratory in Singapore.

About the author

Jun-ichi Maruyama received B.S., M.S. and Ph.D. degrees from The University of Tokyo (1996, 1998 and 2001), and carried out postdoctoral research at the laboratory organized by Prof. Katsuhiko Kitamoto (2001-2006). He wrote a doctoral thesis on nuclear dynamics in the filamentous fungus Aspergillus oryzae used in Japanese traditional food fermentation. He was appointed as an assistant professor at the same laboratory (2006) and as a project associate professor in Laboratory of Brewing Microbiology, donated by Kikkoman Corporation (2016). He also studied as a guest researcher at the laboratory organized by Prof. Gerhard H Braus in Georg August University, Göttingen, Germany (2010). His current research interests are (1) filamentous fungal multicellularity, cell fusion, heterokaryon incompatibility and sexual development, and (2) molecular breeding for microbial production of heterologous proteins/natural products by genome editing.

Reference

Kan E, Tomita H, Katsuyama Y, Maruyama JI, Koyama Y, Ohnishi Y. Discovery of the 2,4′-Dihydroxy-3′-methoxypropiophenone Biosynthesis Genes in Aspergillus oryzae. Chembiochem. 2021;22(1):203-211.

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