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Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes, which can catalyze the oxidative cleavage of polysaccharide β-1,4 glycosidic bonds to improve the hydrolysis efficiency of the substrate by other glycoside hydrolases. To improve the conversion efficiency of cellulose and chitin, a strain was screened from the soil of Yuelu Mountain in Hunan province, China. The gene sequence of a novel AA10 LPMO (Ba LPMO10) was successfully cloned from the genome of the strain and heterologously expressed in E. coli BL21 (DE3). The optimal enzyme activity of Ba LPMO10 was observed at pH 6.0 and 70 °C using 2,6-dimethoxyphenol as substrate, and its maximum specific activity was 91.4 U/g. When Ba LPMO10 synergized with glycoside hydrolase to degrade microcrystalline cellulose and colloidal chitin, the reducing sugar content increased by 7% and 23%, respectively, compared to glycoside hydrolase alone. Moreover, the results of molecular docking and molecular dynamics simulation showed that the distance between Ba LPMO10 and cellohexaose were further than that of Ba LPMO10 and chitohexaose, and the number of hydrogen bonds between Ba LPMO10 and cellohexaose were lower than that of Ba LPMO10 and chitohexaose. Finally, the hot spot residues of Ba LPMO10 interacting with chitohexaose/cellohexaose were identified. • A novel strain manifesting cellulolytic activity was isolated and screened from soil. • A novel AA10 LPMO (Ba LPMO10) was cloned and expressed in E. coli BL21. • Ba LPMO10 has a synergistic degradation effect on both chitin and cellulose, but it is more effective on chitin. • Molecular mechanism of interactions between Ba LPMO10 and substrates was clarified. • It offers new ideas for industrial applications of cellulose and chitin. [ABSTRACT FROM AUTHOR]

Uricase (or Urate oxidase), a key enzyme involved in purine metabolism, is commonly used in treating conditions such as gout, hyperuricemia, and tumor lysis syndrome. In this study, a uricase-producing strain (named CSAJ-16) was isolated from the soil sample of Cangshan Mountain, Yunnan Province, China. This strain was identified as Arthrobacter sp. CSAJ-16. Based on the gene sequence alignment, the uricase gene (named aruox ) of Arthrobacter sp. CSAJ-16 was amplified and heterologously expressed. The recombinant uricase (ArUOX) was about 32 kDa. The optimal pH and temperature of ArUOX were pH 7 and 20°C, respectively. The ArUOX remained above 50% relative activity after incubation at 37°C for 100 min or at pH 6.0-8.6 for 24 h. Moreover, metal ions such as K + , Mg 2+ , Ca 2+ , Ba 2+ and Pb 2+ can significantly enhance the activity of ArUOX (> 200%). These enzymatic properties indicate that ArUOX has potential applications in pharmaceutical enzymes and uric acid detection kits., (© 2023 Xin Yan et al., published by Sciendo.)

The CBL-interacting protein kinase (CIPK) protein plays a vital role in response to abotic stresses. Dendrobium catenatum has been listed as a rare and endangered plant in China because it is difficult for it to adapt suboptimal environmental stress. However, studies on its responses to abiotic stresses are rare. Herein, the CIPK24 gene was cloned from D. catenatum. Expression pattern analysis showed that it displayed a tissue-specificity with a relatively high expression level in sepal and petal, while its expression was up-regulated by polyethylene glycol (PEG) treatment in stems and leaves, and also by salt stress in roots. Localization analysis indicated that DcCIPK24 is located to the nucleus, plasma membrane and cytoplasm. DcCIPK24 overexpression increased the tolerance of transgenic Arabidopsis to drought and salt stresses when compare with wild-type (WT) plants. Transgenic plants had lower malondialdehyde (MDA) content, while had a higher proline content and superoxide dismutase (SOD) activity than WT plants under drought or salinity stress. Furthermore, drought treatment induced faster influxes of Ca2+ and H+ in transgenic roots compared with those in WT roots. Upon treatment with NaCl, Na+ efflux of the transgenic roots was faster, but K+ efflux rate was lower compared with those of WT roots, resulting in less Na+ accumulation in transgenic plants. RNA-seq analysis showed that 114 and 497 differentially expressed genes (DEGs) were found in DcCIPK24 transgenic plants under drought and salt stress, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses found that DEGs enriched in starch and sucrose metabolism, cutin and wax biosynthesis, and peroxisome pathway under drought stress, and enriched in phenylpropanoid biosynthesis pathway and mitogen-activated protein kinase (MAPK) signaling pathway under salt stress. These findings indicate that DcCIPK24 is a positive regulator of D. catenatum 's response to drought and salt stresses, suggesting it has potential application in genetic modification of D. catenatum to enhance drought and salt tolerance. [Display omitted] • DcCIPK24 was induced by salt and drought stress. • DcCIPK24 improved both salt and drought tolerance of transgenic Arabidopsis. • DcCIPK24 enhanced salt and drought tolerance by scavenging reactive oxygen species. [ABSTRACT FROM AUTHOR]

Pyropia haitanensis is an important laver species in China. Its quality traits are closely related to the content of glutamic acid. Glutamate dehydrogenase (GDH) is a crucial enzyme in the glutamic acid metabolism. In this study, two GDH genes from P. haitanensis, PhGDH1 and PhGDH2, were cloned and successfully expressed in Escherichia coli. The in vitro enzyme activity assay demonstrated that the catalytic activity of PhGDHs is mainly in the direction of ammonium assimilation. The measured Km values of PhGDH1 for NADH, (NH4)2SO4, and α-oxoglutarate were 0.12, 4.99, and 0.16 mM, respectively, while the corresponding Km values of PhGDH2 were 0.02, 3.98, and 0.104 mM, respectively. Site-directed mutagenesis results showed that Gly193 and Thr361 were important catalytic residues for PhGDH2. Moreover, expression levels of both PhGDHs were significantly increased under abiotic stresses. These results suggest that PhGDHs can convert α-oxoglutarate to glutamic acid, and enhance the flavor and stress resistance of P. haitanensis. [ABSTRACT FROM AUTHOR]

Verrucosispora sp. SCSIO 07399, a rare marine-derived actinomycete, produces a set of ansamycin-like polyketides kendomycin B-D ( 1 - 3 ) which possess potent antibacterial activities and moderate tumor cytotoxicity. Structurally, kendomycin B-D contain a unique aliphatic macrocyclic ansa scaffold in which the highly substituted pyran ring is connected to the quinone moiety. In this work, a type I/type III polyketide synthase (PKS) hybrid biosynthetic gene cluster coding for assembly of kendomycin B ( kmy ), and covering 33 open reading frames, was identified from Verrucosispora sp. SCSIO 07399. The kmy cluster was found to be essential for kendomycin B biosynthesis as verified by gene disruption and heterologous expression. Correspondingly, a biosynthetic pathway was proposed based on bioinformatics, cluster alignments, and previous research. Additionally, the role of type III PKS for generating the precursor unit 3,5-dihydroxybenzoic acid (3,5-DHBA) was demonstrated by chemical complementation, and type I PKS executed the polyketide chain elongation. The kmy cluster was found to contain a positive regulatory gene kmy4 whose regulatory effect was identified using real-time quantitative PCR (RT-qPCR). These advances shed important new insights into kendomycin B biosynthesis and help to set the foundation for further research aimed at understanding and exploiting the carbacylic ansa scaffold.

• Nosema bombycis polar tube protein 1 (NbPTP1) was expressed and secreted in Sf9 cells. • The heterologous expression protein was glycosylated. • Nosema bombycis proliferation in NbPTP1-expressing Sf9 cells was inhibited. Nosema bombycis , the first identified microsporidium, causes heavy losses to the sericulture industry in China. During infection, microsporidia discharge a long and hollow polar tube, which delivers the sporoplasm into host cells. Polar tube protein 1 was the major component on the polar tube. Previously, we expressed the polar tube protein 1 from Nosema bombycis (NbPTP1) intercellularly in Drosophila S2 cells. Here, the microsporidian protein was expressed in Lepidopteran Sf9 cells. During heterologous expression, NbPTP1 protein was secreted and glycosylated. Microsporidian proliferation decreased in NbPTP1-expressing Sf9 cells. This confirms that NbPTP1 protein can interact with the host cell membrane receptor protein to facilitate microsporidian invasion. [ABSTRACT FROM AUTHOR]

Main Conclusion: Two sesquiterpene synthases were identified through comparative transcriptome analysis of Leonurus sibiricus. LsSqTPS2 could produce high titer of δ-cadinene in vivo which suggests the terpene specificity of L. sibiricus. Leonurus sibiricus L., a medicinal herb, is widely used in China due to its pharmacological activities. Cadinene type sesquiterpenes, one of major bioactive components mainly present in aerial parts of L. sibiricus, showed antibacterial, anti-inflammatory, antioxidant and antiproliferative properties. However, there is no report about the sesquiterpene biosynthesis in L. sibiricus. This study identified L. sibiricus sesquiterpene synthases (LsSqTPSs) through comparative transcriptome analysis of L. sibiricus leaf and root samples using the BGISEQ-500 sequencing technique. A total of 83,244 unigenes were obtained with an average length of 1025 bp. Among them, 50,356 unigenes (60.49%) acquired annotations according to the BLAST searching results. A total of 68 differentially expressed genes (DEGs) were potentially involved in the sesquiterpene biosynthesis. Furthermore, four candidate DEGs encoding LsSqTPSs were characterized. The enzymatic characterization in engineered yeast showed that LsSqTPS1 produced α-farnesene as the single product and LsSqTPS2 mainly produced 76.23 mg/L of δ-cadinene, which constituted the major component of L. sibiricus leaf essential oil. This work contributes to the investigation of sesquiterpene biosynthesis in L. sibiricus.

2-O-α-D-glu-copyranosyl-sn-glycerol is a high value-added product with prospective application in food, cosmetics, health products and pharmaceutical industries. However, industrial scale of 2-O-α-D-glu-copyranosyl-sn-glycerol has not yet been applied in China, and there are few related reports on 2-O-α-D-glu-copyranosyl-sn-glycerol synthesis. The purpose of this experiment is to develop a method for catalyzing the synthesis of food-grade 2-O-α-D-glu-copyranosyl-sn-glycerol using whole cells of "Generally Recognized as Safe" (GRAS) recombinant Bacillus subtilis. In our work, a recombinant B. subtilis 168/pMA5-gtfA that heterologously expressing Leuconostoc mesenteroides sucrose phosphorylase was constructed and used as a whole-cell catalyst to synthesize 2-O-α-D-glu-copyranosyl-sn-glycerol. Optimizing the culture temperature, time and whole cell transformation conditions has increased the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol. The results showed that 1.43 U/mL of sucrose phosphorylase was achieved in B. subtilis 168/pMA5-gtfA after culturing for 20 h at 30 °C in fermentation medium. The highest conversion rate reached 75.1%, and the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol was 189.3 g/L with an average transformation rate of 15.6 mmol/(L·h) after 48 hours whole-cell transformation with the sucrose concentration of 1 mol/L and the glycerol concentration of 2.5 mol/L at 30 °C, OD₆₀₀ 40 and pH 7.0. This is the highest yield of 2-O-α-D-glu-copyranosyl-sn-glycerol synthesized catalytically by recombinant B. subtilis that was ever reported, and this study provides the theoretical and experimental basis for the industrial production and application of 2-O-α-D-glucopyranosyl-sn-glycerol.