Abstract
BackgroundAspergillus oryzae, a useful industrial filamentous fungus, produces limited varieties of secondary metabolites, such as kojic acid. Thus, for the production of valuable secondary metabolites by genetic engineering, the species is considered a clean host, enabling easy purification from cultured cells. A. oryzae has been evaluated for secondary metabolite production utilizing strong constitutive promoters of genes responsible for primary metabolism. However, secondary metabolites are typically produced by residual nutrition after microbial cells grow to the stationary phase and primary metabolism slows. We focused on a promoter of the secondary metabolism gene kojA, a component of the kojic acid biosynthetic gene cluster, for the production of other secondary metabolites by A. oryzae.ResultsA kojA disruptant that does not produce kojic acid was utilized as a host strain for production. Using this host strain, a mutant that expressed a polyketide synthase gene involved in polyketide secondary metabolite production under the kojA gene promoter was constructed. Then, polyketide production and polyketide synthase gene expression were observed every 24 h in liquid culture. From days 0 to 10 of culture, the polyketide was continuously produced, and the synthase gene expression was maintained. Therefore, the kojA promoter was activated, and it enabled the continuous production of polyketide for 10 days.ConclusionsThe combined use of the kojA gene promoter and a kojA disruptant proved useful for the continuous production of a polyketide secondary metabolite in A. oryzae. These findings suggest that this combination can be applied to other secondary metabolites for long-term production.
Highlights
Aspergillus oryzae, a useful industrial filamentous fungus, produces limited varieties of secondary metabolites, such as kojic acid
Heterologous production of secondary metabolites of polyketides, terpenes, and non-ribosomal peptides has ever been attempted in A. oryzae, and from the data presented here, it is indicated that none or only one peak of compounds with similar physical properties exist in the wild-type strain [11,12,13,14]
Production of a polyketide YWA1 synthesized by A. oryzae wA under the kojA promoter in the kojA disruptant To begin with, we validated our assumption that the expression of primary metabolism genes attenuates toward the end of culture
Summary
Aspergillus oryzae, a useful industrial filamentous fungus, produces limited varieties of secondary metabolites, such as kojic acid. For the production of valuable secondary metabolites by genetic engineering, the species is considered a clean host, enabling easy purification from cultured cells. A. oryzae has been evaluated for secondary metabolite production utilizing strong constitutive promoters of genes responsible for primary metabolism. It has a strong ability to produce hydrolases, such as amylase, A. oryzae produces a limited array of secondary metabolites, such as kojic acid [7, 8], and does not produce. Knockout mutants of kojic acid biosynthesis genes are considered more effective hosts for secondary metabolite production than the wild-type strain because they do not produce any kojic acid
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