Temperature stress improved exopolysaccharide yield from Tetragenococcus halophilus: Structural differences and underlying mechanisms revealed by transcriptomic analysis.

This study aimed to enhance exopolysaccharide production by Tetragenococcus halophilus, and results showed that low temperature (20 °C) significantly improved exopolysaccharide production. Based on the analysis of batch fermentation kinetic parameters, a temperature-shift strategy was proposed, and the exopolysaccharide yield was increased by 28 %. Analysis of the structure of exopolysaccharide suggested that low temperature changed the molecular weight and monosaccharide composition. Transcriptomic analysis was performed to reveal mechanisms of low temperature improving exopolysaccharide production. Results suggested that T. halophilus regulated utilization of carbon sources through phosphotransferase system and increased the expression of key genes in exopolysaccharide biosynthesis to improve exopolysaccharide production. Meanwhile, metabolic pathways involved in glycolysis, amino acids synthesis, two-component system and ATP-binding cassette transporters were affected at low temperature. Results presented in this paper provided a theoretical basis for biosynthetic pathway of exopolysaccharide in T. halophilus and aided to strengthen its production and application in many areas.

Function of a hydrophobin in growth and development, nitrogen regulation, and abiotic stress resistance of Ganoderma lucidum.

Fungal hydrophobins have many important physiological functions, such as maintaining hydrophobicity and affecting virulence, growth, and development. In Ganoderma lucidum, the molecular regulation mechanisms of hydrophobins in mushroom are unclear. In this study, we investigated a hydrophobin protein 1 (Hyd1) in G. lucidum, which belongs to the fungal Class I hydrophobins. The hyd1 gene was highly expressed during the formation of primordia, and expression was the lowest in fruiting bodies. Through the construction of hyd1 silenced strains, we found that primordia formation was not initiated in these strains. This finding indicated that Hyd1 played an important role in the development of G. lucidum. Second, AreA, a key transcription factor in nitrogen metabolism, negatively regulated the expression of hyd1. In an areA-silenced strain, the expression of hyd1 increased by ∼14-fold compared with that of the wild-type (WT) strain. Electrophoretic mobility shift assays (EMSA) indicated binding of AreA to the promoter of hyd1. Additionally, expression of hyd1 was determined in the presence of different nitrogen sources. Compared with that in the ammonia nitrogen source, the expression of hyd1 in nitrate nitrogen source significantly increased. Finally, we found that hyd1 plays important roles not only in nitrogen regulation but also in the resistance to other abiotic stresses. After silencing of hyd1, the resistance to heat, cell wall, and salt stresses decreased. Our findings reveal the important roles of Hyd1 in the development and resistance to abiotic stresses in G. lucidum and provide insights into the nitrogen regulation mechanism of hydrophobins in higher basidiomycetes.

Citric Acid Induces the Increase in Lenthionine Content in Shiitake Mushroom, Lentinula edodes.

Shiitake mushroom, Lentinula edodes, is the second largest edible fungus in the world, with a characteristic aroma. 1,2,3,5,6-pentathioheterocycloheptane, commonly known as lenthionine, is the main source of this aroma. Lenthionine has high commercial value, and if we explore the possible induction mechanism of citric acid in lenthionine synthesis, we can provide a reference for the effective application of citric acid as an inducer. In this paper, the single-factor treatment of Lentinula edodes with variable citric acid concentration and treatment duration showed that the best citric acid concentration for L. edodes was 300 µM, and the best treatment duration was 15 days. Additionally, the optimal design conditions were obtained using the response surface method (RSM); the treatment concentration was 406 µM/L, the treatment duration was 15.6 days, and the lenthionine content was 130 µg/g. γ-Glutamyl transpeptidase (LEGGT) and cystine sulfoxide lyase (LECSL) are the key enzymes involved in the biosynthesis of lanthionine. The expression levels of LEGGT and LECSL genes increased significantly under citric acid treatment. Additionally, the lenthionine content of the silenced strains of LEGGT and LECSL was significantly decreased.

Hydrolase CehA and a Novel Two-Component 1-Naphthol Hydroxylase CehC1C2 are Responsible for the Two Initial Steps of Carbaryl Degradation in Rhizobium sp. X9.

Carbaryl is a widely used carbamate pesticide in agriculture. The strain Rhizobium sp. X9 possesses the typical carbaryl degradation pathway in which carbaryl is mineralized via 1-naphthol, salicylate, and gentisate. In this study, we cloned a carbaryl hydrolase gene cehA and a novel two-component 1-naphthol hydroxylase gene cehC1C2. CehA mediates carbaryl hydrolysis to 1-naphthol and CehC1, an FMNH2 or FADH2-dependent monooxygenase belonging to the HpaB superfamily, and hydroxylates 1-naphthol in the presence of reduced nicotinamide-adenine dinucleotide (FMN)/flavin adenine dinucleotide (FAD), and the reductase CehC2. CehC1 has the highest amino acid similarity (58%) with the oxygenase component of a two-component 4-nitrophenol 2-monooxygenase, while CehC2 has the highest amino acid similarity (46%) with its reductase component. CehC1C2 could utilize both FAD and FMN as the cofactor during the hydroxylation, although higher catalytic activity was observed with FAD as the cofactor. The optimal molar ratio of CehC1 to CehC2 was 2:1. The Km and Kcat/Km values of CehC1 for 1-naphthol were 74.71 ± 16.07 µM and (8.29 ± 2.44) × 10-4 s-1·µM-1, respectively. Moreover, the enzyme activities and substrate spectrum between CehC1C2 and previously reported 1-naphthol hydroxylase McbC were compared. The results suggested that McbC had a higher 1-naphthol hydroxylation activity, while CehC1C2 had a broader substrate spectrum.