Exploring differential traits of lipid-producing stages of the wild type and morphologically engineered strain of Aspergillus oryzae by comparative kinetic modeling.

Comparative profilings of cell growth and lipid production in the morphologically engineered strain (Δags1) and the wild type (WT) of Aspergillus oryzae BCC7051 were implemented. Using various nitrogen sources, a discrimination in cell morphology between the two strains was found, of which the Δags1 culture exhibited mycelial growth as small pellets in contrast to the WT. Of them, sodium nitrate and potassium nitrate were optimal for lipid production of the WT and Δags1 strains, respectively, which the highest lipid concentrations of 7.2 and 7.9 g L-1 were obtained in the respective cultures. The mathematical models of the growth kinetics and lipid phenotypes of both fungal strains were developed, enabling to distinguish three lipid-producing stages, including low lipid-producing, lipid accumulation, and lipid turnover stages. The model validation showed good performances in all nitrogen sources tested for the WT, but only NaNO3 and mixed yeast extract/NH4Cl were fitted well for the Δags1. The difference in the period of lipid-producing stages between the WT and Δags1 indicated the metabolic alterations of A. oryzae by the defect of a gene involved in the cell wall biosynthesis, which exhibited benefits for bioprocessing practices in addition to the high productivities of biomass and lipid. These findings would further permit the manipulation in the metabolic hub of the fungal production platform for other industrial purposes.

Metabolic Regulation of Sugar Assimilation for Lipid Production in Aspergillus oryzae BCC7051 through Comparative Transcriptome Perspective.

Microbial lipid production with cost effectiveness is a prerequisite for the oleochemical sector. In this work, genome-wide transcriptional responses on the utilization of xylose and glucose in oleaginous Aspergillus oryzae were studied with relation to growth and lipid phenotypic traits. Comparative analysis of the active growth (t1) and lipid-accumulating (t2) stages showed that the C5 cultures efficiently consumed carbon sources for biomass and lipid production comparable to the C6 cultures. By pairwise comparison, 599 and 917 differentially expressed genes (DEGs) were identified in the t1 and t2 groups, respectively, in which the consensus DEGs were categorized into polysaccharide-degrading enzymes, membrane transports, and cellular processes. A discrimination in transcriptional responses of DEGs set was also found in various metabolic genes, mostly in carbohydrate, amino acid, lipid, cofactors, and vitamin metabolisms. Although central carbohydrate metabolism was shared among the C5 and C6 cultures, the metabolic functions in acetyl-CoA and NADPH generation, and biosynthesis of terpenoid backbone, fatty acid, sterol, and amino acids were allocated for leveraging biomass and lipid production through at least transcriptional control. This study revealed robust metabolic networks in the oleaginicity of A. oryzae governing glucose/xylose flux toward lipid biosynthesis that provides meaningful hints for further process developments of microbial lipid production using cellulosic sugar feedstocks.

Fermentation process development of recombinant Hansenula polymorpha for gamma-linolenic acid production.

Development in the strain and the fermentation process of Hansenula polymorpha was implemented for the production of gamma-linolenic acid (GLA, C18:3 delta 6,9,12), which is an n-6 polyunsaturated fatty acid (PUFA) and has been reported to possess a number of health benefits. The mutated delta 6-desaturase (S213A) gene of Mucor rouxii was expressed in H. polymorpha under the control of the methanol oxidase (MOX) promoter. Without utilization of methanol a high cell-density culture of the yeast recombinant carrying the delta 6-desaturase gene was achieved by fed-batch fermentation using glycerol-limited conditions. The delta 6-desaturated products, octadecadienoic acid (C18:2 delta6,9), GLA and stearidonic acid (C18:4 delta6,9,12,15), accumulated at high levels under the derepression condition. The GLA production was also optimized by adjusting specific growth rates. The results show that the specific growth rate affected both lipid content and fatty acid composition of the GLA-producing recombinant. Among the various specific growth rates studied, the highest GLA concentration, which was at of 697 mg/l, was obtained in the culture with the specific growth rate of 0.08 /h. Interestingly, the fatty acid profile of the yeast recombinant bearing the Mucor delta 6-desaturase gene was similar to that of blackcurrant oil with both containing similar proportions of n-3 and n-6 essential fatty acids.

Physiological Traits of Dihomo-γ-Linolenic Acid Production of the Engineered Aspergillus oryzae by Comparing Mathematical Models.

Dihomo-γ-linolenic acid (DGLA; C20:3 n-6) is expected to dominate the functional ingredients market for its role in anti-inflammation and anti-proliferation. The DGLA production by the engineered strain of Aspergillus oryzae with overexpressing Pythium Δ6-desaturase and Δ6-elongase genes was investigated by manipulating the nutrient and fermentation regimes. Of the nitrogen sources tested, the maximum biomass and DGLA titers were obtained in the cultures using NaNO3 grown at pH 6.0. For establishing economically feasible process of DGLA production, the cost-effective medium was developed by using cassava starch hydrolysate (CSH) and NaNO3 as carbon and nitrogen sources, respectively. The supplementation with 1% (v/v) mother liquor (ML) into the CSH medium promoted the specific yield of DGLA production (Y DGLA / X ) comparable with the culture grown in the defined NaNO3 medium, and the DGLA proportion was over 22% in total fatty acid (TFA). Besides, the GLA was also generated at a similar proportion (about 25% in TFA). The mathematical models of the cultures grown in the defined NaNO3 and CSH/ML media were generated, describing that the lipid and DGLA were growth-associated metabolites corresponding to the relevant kinetic parameters of fermentations. The controlled mode of submerged fermentation of the engineered strain was explored for governing the PUFA biosynthesis and lipid-accumulating process in relation to the biomass production. This study provides an informative perspective in the n-6 fatty acid production through physiological manipulation, thus leading to a prospect in viable production of the DGLA-enriched oil by the engineered strain.