Effects of Nitrogen and Phosphorus Limitation on Fatty Acid Contents in Aspergillus oryzae

Abstract

Aspergillus oryzae, commonly known as koji mold, has been widely used for the large-scale production of food products (sake, makgeolli, and soy sauce) and can accumulate a high level of lipids. In the present study, we showed the dynamic changes in A. oryzae mycelium growth and conidia formation under nitrogen and phosphorus nutrient stress. The fatty acid profile of A. oryzae was determined and the content of unsaturated fatty acid was found increased under nitrogen and phosphorus limitation. Oleic acid (C_18:1), linoleic acid (C_18:2), and γ-linolenic acid (C_18:3) production were increased on five nitrogen and phosphorus limitation media, especially on nitrogen deep limitation and phosphorus limitation group, showing a 1. 2-, 1. 6-, and 2.4-fold increment, respectively, compared with the control. Transcriptomic analysis showed the expression profile of genes related to nitrogen metabolism, citrate cycle, and linoleic acid synthesis, resulting in the accumulation of unsaturated fatty acid. qRT-PCR results further confirmed the reliability and availability of the differentially expressed genes obtained from the transcriptome analysis. Our study provides a global transcriptome characterization of the nitrogen and phosphorus nutrient stress adaptation process in A. oryzae. It also revealed that the molecular mechanisms of A. oryzae respond to nitrogen and phosphorus stress. Our finding facilitates the construction of industrial strains with a nutrient-limited tolerance.

Keywords: Aspergillus oryzae; differentially expressed genes; fatty acid; nitrogen and phosphorus limitation; transcriptome.

FIGURE 1

The dry biomass and density of spores as affected by nitrogen and phosphorus. (A) The dry biomass under nitrogen and phosphorus limitation and the control. The mycelia were collected by peeling them off from the plates and dried overnight for the determination of biomass. (B) The density of spores under nitrogen and phosphorus limitation and the control. (C–H) The phenotype of A. oryzae strain under the condition of control and distinct N/P limitation. CK, N-, P-, N- -, N- P-, and N- -P- represent the control and distinct N/P-limited groups, respectively. The bars represent the average (±SE) of biological repeats. Asterisks indicate statistically significant differences between groups (Student’s t-test): *p < 0.05, **p < 0.01, ns, no significant difference.

FIGURE 2

Distribution of differentially expressed genes (DEGs) in the six samples. (A) DEG distribution between control and nitrogen and phosphorus limitation. (B) Venn diagram exhibiting DEG distribution in three samples. CK, the control; P1, the single P limitation (P-); N1, the single N limitation (N-); NP, N deep limitation and P limitation (N- -P-).

FIGURE 3

Key enzymes encoded by the DEGs involved in the nitrogen metabolism are enriched by KEGG analysis. CK, the control; P1, the single P limitation (P-); N1, the single N limitation (N-); NP, N deep limitation and P limitation (N- -P-).

FIGURE 4

Key enzymes encoded by the DEGs involved in the carbohydrate metabolism are enriched by KEGG analysis.

FIGURE 5

DEGs are involved in the linoleic acid biosynthesis pathways and the changes of C18 fatty acid in response to different nitrogen and phosphorus limitation. (A) The expression pattern of DEGs involved in the linoleic acid biosynthesis pathways. (B) The relative expression levels of four genes in response to different nitrogen and phosphorus limitation. The bars represent the average (±SE) of biological repeats. Asterisks indicate statistically significant differences between groups (Student’s t-test): **p < 0.01, ns: no significant difference. CK: the control; P1: the single P limitation (P-); N1: the single N limitation (N-); NP: N deep limitation and P limitation (N- -P-).