Jingwen Yu,Yuxiang Fu,Zeyuan Deng,Yawei Fan,Hongyan Li
In this study, soluble dietary fiber (SDF, including oligosaccharides and polysaccharides) of soybean residue (SR) fermented by Neurospora crassa was used as a research object. In vitro fermentation technology was used to analyze the fermentation properties of SDF from fermented soybean residue (FSR). Moreover, the effects of SDF from FSR on the composition and diversity of intestinal microflora of rats were studied by high-throughput sequencing technology. Results showed that the SDF content of fermented soybean residue was 27.21%. The addition of SDF in the range 2 to 10 g L−1 could increase the levels of gas production and short-chain fatty acids (SCFAs), as well as decrease the pH and ammonia N concentration after 24 h fermentation in the fermentation broth compared with the control group (p < 0.05). The animal-based experiments showed that Bacteroidetes and Firmicutes were the major dominant phyla in all the groups. Compared with the control group, oligosaccharides and polysaccharides of FSR changed the relative abundance and diversity of the bacterial community, and increased the numbers of beneficial flora, such as Prevotellaceae and Lactobacillales. It was shown that SDF of SR fermented by Neurospora crassa had great effects on the intestinal environment and the composition of intestinal flora in rats.
Li Yuanjing,Yan Pengfei,Lu Xiaojie,Qiu Yanling,Liang Shang,Liu Gang,Li Shuangfei,Mou Lin,Xie Ning
The sucrose non-fermenting 1/AMP-activated protein kinase (SNF1/AMPK) is a central regulator of carbon metabolism and energy production in the eukaryotes. In this study, the functions of the Podospora anserina SNF1 (PaSNF1) ortholog were investigated. The ΔPaSNF1 mutant displays a delayed development of mycelium and fruiting bodies and fails to form ascospores. The expression of the PaSNF1 gene in the strain providing female organs in a cross is sufficient to ensure fertility, indicating a maternal effect. Results of environmental stress showed that ΔPaSNF1 was hypersensitive to stress, such as osmotic pressure and heat shock, and resistant to fluconazole. Interestingly, the knockout of PaSNF1 significantly promoted sterigmatocystin (ST) synthesis but suppressed cellulase [filter paperase (FPA), endoglucanase (EG), and β-glucosidase (BG)] activity. Further, transcriptome analysis indicated that PaSNF1 made positive regulatory effects on the expression of genes encoding cellulolytic enzymes. These results suggested that PaSNF1 may function in balancing the operation of primary and secondary metabolism. This study suggested that SNF1 was a key regulator concerting vegetative growth, sexual development, and stress tolerance. Our study provided the first genetic evidence that SNF1 was involved in the ST biosynthesis and that it may also be a major actor of lignocellulose degradation in P. anserina.
Ergothioneine (ERG) is an unusual sulfur-containing amino acid with antioxidant activity that can be synthesized by certain bacteria and fungi. Microbial fermentation is a promising method for ERG production. In this study, the bifunctional enzyme methyltransferase–sulfoxide synthase NcEgt1 from Neurospora crassa was truncated to obtain sulfoxide synthase TNcEgt1, which showed a higher expression level in Escherichia coli BL21(DE3). Then, the genes egtD encoding methyltransferase EgtD and egtE encoding C–S lyase EgtE from Mycobacterium smegmatis were cloned with TncEgt1 into E. coli BL21(DE3) to produce 70 mg/L ERG. To improve ERG production, TNcEgt1 and EgtD were modified, and the resulting mutants were screened with an established high-throughput method which could directly analyze the ERG content in culture broths. After several rounds of mutation and screening, the optimal mutant MD4 was obtained and produced 290 mg/L ERG. Furthermore, a fed-batch culture was conducted in a 5 L bioreactor. After optimizing the fermentation process, the ERG yield reached 5.4 g/L after 94 h of cultivation supplemented with amino acids and glycerol, which is the highest ERG yield reported to date. The results showed that ERG production was significantly improved by modifying the key enzymes, and the engineered strains constructed in this study have potential industrial application prospects.
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