Improving the productivity of malic acid by alleviating oxidative stress during Aspergillus niger fermentation

Abstract

Background: As an attractive platform chemical, malic acid has been commonly used in the food, feed and pharmaceutical field. Microbial fermentation of biobased sources to produce malic acid has attracted great attention because it is sustainable and environment-friendly. However, most studies mainly focus on improving yield and ignore shortening fermentation time. A long fermentation period means high cost, and hinders the industrial applications of microbial fermentation. Stresses, especially oxidative stress generated during fermentation, inhibit microbial growth and production, and prolong fermentation period. Previous studies have shown that polypeptides could effectively relieve stresses, but the underlying mechanisms were poorly understood.

Results: In this study, polypeptides (especially elastin peptide) addition improves the productivity of malic acid in A. niger, resulting in shortening of fermentation time from 120 to 108 h. Transcriptome and biochemical analyses demonstrated that both antioxidant enzyme-mediated oxidative stress defense system, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), and nonenzymatic antioxidant system, such as glutathione, were enhanced in the presence of elastin peptide, suggesting elastin peptide relieving oxidative stresses is involved in many pathways. In order to further investigate the relationship between oxidative stress defense and malic acid productivity, we overexpressed three enzymes (Sod1, CAT, Tps1) related to oxidation resistance in A. niger, respectively, and these resulting strains display varying degree of improvement in malic acid productivity. Especially, the strain overexpressing the Sod1 gene achieved a malate titer of 91.85 ± 2.58 g/L in 96 h, corresponding to a productivity of 0.96 g/L/h, which performs better than elastin peptide addition.

Conclusions: Our investigation provides an excellent reference for alleviating the stress of the fungal fermentation process and improving fermentation efficiency.

Keywords: Aspergillus niger; Elastin peptide; L-Malic acid; Metabolic engineering; Oxidative stress tolerance.

Fig. 1

Schematic illustrating protein peptides relieving stress and enhancing the fluxes of glycolysis, r-tricarboxylic acid (TCA) and the glyoxylic acid bypass pathway in A. niger during malic acid bioproduction. a Fermentation assessment with peptides in A. niger; b transcriptomic analysis combined with biochemical analysis of elastin peptide supplementation in the fermentation period; c Overexpression of the gene for the stress-defense system and enhancement of the productivity of A. niger

Fig. 2

Fermentation kinetics of different peptides supplemented in A. niger RG0095 fermentation culture medium. a glucose consumption; b malic acid production; c malic acid productivity; d cell dry weight. Data represent the mean ± standard deviation (SD) of three independent replicates. Statistical significance was determined by Student’s t-test (n = 3). *p < 0.05, **p < 0.01

Fig. 3

Transcriptomic profiling and annotation of differentially expressed genes between the fermentation medium with elastin peptide-supplemented group and the nonsupplemented group. a Score plots of the transcriptome of the elastin peptide-supplemented group compared to the nonsupplemented group; b volcano plots of the transcriptome of the elastin peptide-supplemented group compared to the nonsupplemented group; c up- and downregulated DEGs in the elastin peptide-supplemented group compared to the nonsupplemented group; d GO annotation of DEGs in the elastin peptide-supplemented group compared to the nonsupplemented group; e KEGG annotation of the DEGs in the elastin peptide-supplemented group compared to the nonsupplemented group

Fig. 4

Proposed cell signal transduction pathway regulating malic acid production in A. niger during elastin peptide supplementation

Fig. 5

Validation of RNA-seq data using qRT-PCR analysis of the selected key genes. Error bars represent three technical replicates

Fig. 6

Biochemical analysis of the stress defense system in A. niger. a H₂O₂ assay; b ROS assay; c SOD assay; d CAT assay; e GST assay; f GR assay; g GSSH assay; h GSSG assay; i TS assay; j THL assay; k TOC assay; l HK assay; m Pfk assay; n PK assay; o Pyc assay; p MS assay. Statistical significance was determined by Student’s t-test (n = 3). **p < 0.01

Fig. 7

Time course of fermentation of A. niger strains. a Growth profiles of RG0095, RG0580, RG0581 and RG0582 on potato dextrose agar (PDA) plates at 28 °C for 48 h; b expression analysis of the indicated genes by qRT-PCR in RG0095 and RG0580, RG0581 and RG0582; c glucose consumption; d malic acid production. e Malic acid productivity. Data represent the mean ± SD of three independent replicates. Statistical significance was determined by Student’s t-test (n = 3). *p < 0.05, **p < 0.01