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Use of the kojA promoter, involved in kojic acid biosynthesis, for polyketide production in Aspergillus oryzae: implications for long-term production.

ABSTRACT: BACKGROUND:Aspergillus 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. RESULTS:A 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. CONCLUSIONS:The 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.

SUBMITTER: Tamano K

PROVIDER: S-EPMC6814975 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Obesity and its related diseases are increasing worldwide. One of the best therapeutic strategies for obesity management is through the inhibition of pancreatic lipase (PL) enzyme. So far orlistat is the only FDA approved PL inhibitor, but with unpleasant side effects. New efficacious anti-obesity drugs are needed to achieve a successful reduction in the incidence and prevalence of obesity. Many microbial metabolites have PL inhibitory activity. Screening soil inhabitants for PL inhibitors could help in increasing the available anti-obesity drugs. We aimed to isolate and identify alternative PL inhibitors from soil flora. RESULTS:We screened the crude mycelial methanolic extracts of 39 soil samples for PL inhibitory activity by the quantitative lipase colorimetric assay, using the substrate p-nitrophenyl palmitate and orlistat as positive control. AspsarO, a PL inhibitor producer, was isolated from an agricultural field soil in Giza, Egypt. It was identified as Aspergillus oryzae using colony morphology, microscopical characteristics, 18S rDNA sequencing, and molecular phylogeny. Increasing the PL inhibitor activity, in AspsarO cultures, from 25.9?±?2% to 61.4?±?1.8% was achieved by optimizing the fermentation process using a Placket-Burman design. The dried 100% methanolic fraction of the AspsarO culture had an IC50 of 7.48??g/ml compared to 3.72??g/ml for orlistat. It decreased the percent weight gain, significantly reduced the food intake and serum triglycerides levels in high-fat diet-fed Sprague-Dawley rats. Kojic acid, the active metabolite, was identified using several biological guided chromatographic and 1H and 13C NMR techniques and had an IC50 of 6.62??g/ml. Docking pattern attributed this effect to the interaction of kojic acid with the key amino acids (Lys80, Trp252, and Asn84) in PL enzyme binding site. CONCLUSION:Combining the results of the induced obesity animal model, in silico molecular docking and the lipase inhibitory assay, suggests that kojic acid can be a new therapeutic option for obesity management. Besides, it can lower serum triglycerides in obese patients.

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Use of the kojA promoter, involved in kojic acid biosynthesis, for polyketide production in Aspergillus oryzae: implications for long-term production.

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