Improvement of Free Fatty Acid Secretory Productivity in Aspergillus oryzae by Comprehensive Analysis on Time-Series Gene Expression.

Pui Shan Wong,Koichi Tamano,Sachiyo Aburatani

doi:10.3389/fmicb.2021.605095

Pui Shan Wong, Koichi Tamano + Show 1 more

Open Access

https://doi.org/10.3389/fmicb.2021.605095

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Abstract

Aspergillus oryzae is a filamentous fungus that has historically been utilized in the fermentation of food products. In recent times, it has also been introduced as a component in the industrial biosynthesis of consumable compounds, including free fatty acids (FFAs), which are valuable and versatile products that can be utilized as feedstocks in the production of other commodities, such as pharmaceuticals and dietary supplements. To improve the FFA secretory productivity of A. oryzae in the presence of Triton X-100, we analyzed the gene expression of a wild-type control strain and a disruptant strain of an acyl-CoA synthetase gene, faaA, in a time-series experiment. We employed a comprehensive analysis strategy using the baySeq, DESeq2, and edgeR algorithms to clarify the vital pathways for FFA secretory productivity and select genes for gene modification. We found that the transport and metabolism of inorganic ions are crucial in the initial stages of FFA production and revealed 16 candidate genes to be modified in conjunction with the faaA disruption. These genes were verified through the construction of overexpression strains, and showed that the manipulation of reactions closer to the FFA biosynthesis step led to a higher increase in FFA secretory productivity. This resulted in the most successful overexpression strains to have an FFA secretory productivity more than two folds higher than that of the original faaA disruptant. Our study provides guidance for further gene modification for FFA biosynthesis in A. oryzae and for enhancing the productivity of other metabolites in other microorganisms through metabolic engineering.

Highlights

Free fatty acids (FFAs) are lipid compounds that have a carboxyl group in a free form within the molecule
RNA-Seq expression data were obtained from the A. oryzae RIB40 control strain and the faaA strain cultured in the presence of 1% Triton X-100 and sampled at 48, 72, 96, 120, 144, and 168 h, as described in section “Materials and Methods.”
Three differential expression algorithms were used to identify the genes affected by the faaA disruption so that free fatty acids (FFAs) secretory productivity could be increased by gene modification: baySeq (Hardcastle and Kelly, 2010), DESeq2 (Love et al, 2014), and edgeR (Robinson et al, 2009; McCarthy et al, 2012; Figure 1)

Summary

Introduction

Free fatty acids (FFAs) are lipid compounds that have a carboxyl group in a free form within the molecule. FFAs are valuable, because their derivatives can be used as source materials for biofuels, various pharmaceuticals, and dietary supplements (Lennen and Pfleger, 2013; Tamano, 2014). RNA-Seq-Driven Improvement of Metabolite Productivity fatty acid methyl esters (FAMEs) that are generally used as biodiesel fuel (Ichihara and Fukubayashi, 2010). FFAs can act as feedstocks for the production of pharmaceutical compounds [e.g., prostaglandin E1 (PGE1)] and functional lipids [e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] (Lennen and Pfleger, 2013; Zhou et al, 2016). EPA and DHA are possibly beneficial in the normal aging processes of the brain and are used as dietary supplements (Dyall, 2015)

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