Abstract:With the development of sequencing and in vitro culture technique, more and more unknown methanogens have been identified. In recent years, the 7^th order of methanogen (Methanomassiliicoccales, Mmc) has been gradually discovered and established. Mmc is close to Thermoplasmatales in evolutionary ralationship, but it exists at a relatively long distance and forms an independent cluster. Mmc is widely distributed in the digestive tract of mammalian and insects, paddy fields, wetlands and other environments, but strains from different environments exhibit habitat preference. Mmc lacks the complete metabolic pathway to reduce CO₂ to methyl coenzyme M, which leads to their strict use of hydrogen to reduce methyl substrates to genetrate methane. A comprehensive and in-depth understanding of the functions of Mmc in rumen of ruminants will be conducive to the proposal of a new and efficient strategy for methane emission reduction in ruminants. Therefore, we review here the isolation culture, physiochemical and genomic properties of Mmc as well as its role in the rumen methane production.

Abstract:Mangrove forest is a special coastal wetland ecosystem and formed by the periodic mixing of freshwater and seawater. The sediment of mangrove wetland is rich in organic matters, accelerating nutrient cycling driven by resident microbes. Due to high concentrations of sulfate and a variety of sulfide in mangrove sediment, mangrove wetland has been considered as an ideal ecosystem to explore sulfur cycling. This review aims to understand the microbially-driven sulfur cycling, especially sulfur oxidation and sulfate reduction processes in the sediment of mangrove wetland. The major environmental factors affecting sulfur oxidation and sulfate reduction are also discussed. Moreover, future research expectations for microbially-driven sulfur cycling in mangrove ecosystem are indicated.

Abstract:[Objective] To investigate the inhibitory activity of trans-cinnamaldehyde (TC) and cinnamaldehyde (CD) on spoilage and biofilm formation of Pseudomonas isolated from spoiled beef. [Methods] The minimal inhibitory concentration (MIC) on Pseudomonas lundensis was measured by plate counting. Effects of sub-MIC two cinnamaldehyde on the biofilm formation and extracellular enzymes were evaluated by crystal violet staining, bead vortexing, confocal laser scanning microscope, folin method. The effects of sub-MIC TC and CD on the gene expressions of lapA, fliC, aprX and lip were detected by real-time quantitative PCR. [Results] MIC of TC and CD was 200 μg/mL and 225 μg/mL, respectively. Cinnamaldehyde at sub-inhibitory concentration of 1/8 MIC, 1/4 MIC, 1/2 MIC decreased significantly the biofilm biomass and adhesion of P. lundensis. Compared to the control, the biofilm of P. lundensis decreased by 60.27% and 52.05%, when supplemented with exogenous 1/2 MIC of TC or CD, respectively. The 1/2 MIC of TC and CD decreased the bacterial adhesion by 56.35% and 61.10%, respectively. The treatment by TC or CD resulted in the thinner biofilm thickness of P. lundensi, even partial biofilm cell was killed by TC. Two cinnamaldehyde strongly inhibited swimming motility, and the inhibitory rates were 58.08% and 42.29% by 1/2 MIC of TC and CD, respectively. Furthermore, protease of P. lundensis decreased by 61.90% and 76.19%, while lipolytic activity inhibited by 40.17% and 47.01% in the presence of 1/2 MIC of TC and CD, respectively, indicating that sub-MIC TC or CD markedly repressed the two extracellular enzymes activity. Cinnamaldehyde caused greatly down-regulated gene expressions of lapA, fliC, aprX and lip (P<0.05), except no significant difference in lapA gene treated by 1/8 MIC of CD. The expressions of lapA, fliC, aprX and lip decreased to 0.05-0.16 and 0.02-0.12 fold in P. lundensis treated with 1/2 MIC of TC and CD (P<0.05), respectively. [Conclusion] Two cinnamaldehyde isomers at sub-MIC significantly repressed biofilm formation and spoilage of P. lundensis. TC had a stronger inhibitory effect on biofilm formation, while CD was more effective in decrease of extracellular enzymes, which was closely associated with down-regulation of expression of related genes by cinnamaldehyde.

Abstract:[Objective] The aim of the study was to analyze changes of pre-formed Vibrio parahaemolyticus biofilm under cold shock. The source of persistent infections can be biofilms that occur naturally on food surfaces and medical biomaterials. The change of pre-formed Vibrio parahaemolyticus biofilm under cold shock was studied.[Methods] The changes of biofilm biomass were checked by crystal violet staining, exopolysaccharides and proteins were quantified by modified sonication method and Lowry method, and genes related to the expression of biofilm were extracted and purified by RNA extraction kit. The Confocal laser scanning microscopy (CLSM) images reveal biofilm morphological structure, the correlation analysis shows inner relationship among biofilm structure parameters and biofilm related genes.[Results] V. parahaemolyticus biofilm biomass increased significantly during this cold shock period. Polysaccharides and proteins increased gradually in the extracellular polymeric matrix (EPS). In addition, the expression of flagella and virulence-related genes were differentially regulated. The architecture of the biofilm, quantified using mean thickness, average diffusion distance, porosity, biofilm roughness and homogeneity also changed during the cold shock and these parameters were correlated (P < 0.01). However, the correlation between biofilm architecture and biofilm-related gene expression was relatively weak (P<0.05). [Conclusion] Cold shock at 4℃ and 10℃ is not sufficient to reduce V. parahaemolyticus biofilm formation.

Abstract:[Objective] The objective of this research is to develop a new approach for labelling the cell wall peptidoglycan (PG) of living bacteria and provide a simple and convenient tool for the study of PG biosynthesis and metabolism as well as its role in the infection and pathogenesis. [Methods] N-Acetylglucosamine 1-phosphate (GlcNAc-1-P) is a general and essential precursor of PG biosynthesis in eubacteria. We designed and synthesized an analogue of GlcNAc-1-P (designated as Ac₃GlcNAz-1-P) that carries an azido group for the fluorescence labelling of PG in bacteria. Escherichia coli BL21 were used as the model bacteria and were cultured in the presence of Ac₃GlcNAz-1-P to enable the metabolic incorporation of this compound into the PG biosynthetic pathway. Subsequently the PG component was isolated from these pretreated bacteria and characterized by transform infrared spectroscopy (FTIR) and liquid chromatography-high resolution mass spectroscopy (LC-MS). The labelling of bacterial PG was performed by the incubation of the pretreated bacteria with an alkyne-functionalized fluorescent dye (DBCO-Cy3), which could directly couple to azido groups through click reaction. Fluorescence was probed by confocal fluorescence microscopy (CFM). [Results] Ac₃GlcNAz-1-P was successfully synthesized in four steps from D-glucosamine hydrochloride with a total yield of 79%. LC-MS and FTIR analysis of the PG extracts from E. coli pretreated with Ac₃GlcNAz-1-P revealed the presence of azide-labelled sugar units in the PG structure. CFM observation confirmed that the pretreated bacteria could be stained by DBCO-Cy3 selectively and effectively. [Conclusion] The azido-containing sugar unit could be incorporated into the structure of bacterial cell wall PG by the preincubation of bacteria with Ac₃GlcNAz-1-P, allowing the PG of living bacterial to be easily labelled by alkyne-bearing fluorescent reagents (bioorthogonal fluorescent probes). This method would be useful for the study of biological and pathological functions of bacterial PG.

Abstract:[Objective] We classified the antifungal strain Streptomyces sp. IMS002 that was isolated from the rhizosphere soil. We identified its bioactive compound in organic phase. [Methods] Through the concatenation analysis of 16S rDNA sequence and 5 concatenated sequences (atpD, gyrB, recA, rpoB and trpB), and physiological and biochemical detection, we classified Streptomyces IMS002. Besides, we observed the hyphae and spore morphology of sp. IMS002 by scanning electron microscope sp.. Using silica gel column chromatography, gel column chromatography and HPLC, we isolated and purified the active compound against Fusarium oxysporum. Furthermore, its chemical structure was determined by a liquid mass coupled high resolution mass spectrometer, a 500 MHz NMR spectrometer, and a circular dichroism spectrometer. [Results] Streptomyces sp. IMS002 had a close relationship with Streptomyces ambofaciens, and its fermentation broth had good antifungal activity against Fusarium oxysporum. With the separation and purification and modern spectrum identification, we identified this compound as Borrelidin. [Conclusion] Streptomyces sp. IMS002 produced Borrelidin that has bioactivity against Fusarium oxysporum.

Abstract:β-N-acetylglucosaminidases (NAGases) participate in the removal of N-acetylglucosamine (GlcNAc) from the non-reducing end of peptidoglycan or chitin, and are important for many biological functions and industrial applications. [Objective] A new NAGase encoding gene NagZ703 was cloned from a facultative alkaliphilic Bacillus pseudofirmus and expressed in Escherichia coli, for the enzymatic production of GlcNAc. [Methods] The gene NagZ703 was cloned into the expression vector pET28a to get the recombinant plasmid pET28a-NagZ703. The recombinant NagZ703 was induced for expression in E. coli BL21 (DE3) and purified through His-Trap column. Then the purified NagZ703 was characterized. [Results] The multiple sequence alignments showed that NagZ703 belonged to GH 3 NAGase with 2 domains, and the catalytic active sites were composed of 3 conserved residues (Arg232, His234 and Arg318) at the N-terminal domain. NagZ703 shared only 37% identity with the most studied BsNagZ from B. subtilis. The purified NagZ703 exhibited optimal activity at 60℃ and pH 6.5, the specific activity was 10.79 U/mg, and the K_m and V_max of NagZ703 were 0.276 mmol/L and 0.612 mmol/(mg·min) toward p-nitrophenyl β-N-acetylglucosaminide, respectively. NagZ703 retained more than 80% residual activity after pre-incubation at 50℃ for 30 min, or at pH 6.0-10.5 for 12 h. NagZ703 was a non-metalloenzyme because EDTA could not affect its activity, whereas Hg²⁺ completely inhibited its activity. NagZ703 hydrolyzed colloidal chitin to produce GlcNAc in vitro. [Conclusion] A NAGaseNagZ703 from facultative alkaliphilic Bacillus pseudofirmus was characterized carefully. The ability of NagZ703 to produce GlcNAc from colloidal chitin provided a promising approach for the production of GlcNAc by enzymatic hydrolysis.

Abstract:[Objective] Wheat leaf rust caused by Puccinia triticina (Pt) is one of the most serious wheat diseases worldwide. Urediniospores germination is the premise to ensure the normal infection of leaf rust. Therefore, we studied differential expression characteristics of germinating urediniospores, to provide a basis for revealing the mechanism of germination and its relationship with pathogenesis. [Methods] We used RNA-seq to analyze the differentially expressed genes at resting and germinated urediniospores of THTT pathotype. [Results] We found 4400 up-regulated unigenes and 5325 down-regulated unigenes at germination stage compared with resting urediniospores. GO enrichment analysis showed that the up-regulated differentially unigenes mainly referred to cellular process, the metabolic process of organic matter, signal transduction, single-organism process and catalytic enzyme activity etc. The down-regulated unigenes mainly referred to single-organism process, single-organism cellular process, the metabolic process of organic matter, catalytic activity etc. All differentially expressed unigenes referred to 109 pathways. We choose Mitogen-activated protein kinase (MAPK) signaling pathway and Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) interactions in vesicular transport, which are related with germinating. The results of quantitative analysis of 10 unigenes were consistent with the results of gene digital expression profiling. [Conclusion] The result discovered the significantly differential unigenes in germinated urediniospores and this lay a base for understanding the pathogenic mechanism of Pt.

Abstract:[Objective] In order to provide high-efficient strains for bioremediation of Cr(VI) pollution, growth and reduction characteristics of a strain of highly efficient Cr(VI) reducing bacteria separated from sewer sludge of electroplating plant were studied. [Methods] Cr(VI) reducing strains were isolated and screened from sludge by enrichment culture method. The primary identification was carried out by physiological and biochemical analysis and 16S rRNA gene sequence analysis. Single factor experiments were used to determine the optimal culture conditions and the resistance characteristics to stress environment. In addition, the best electronic donor was screened out by investigating the effect of electronic donor on reduction ability of strain. [Results] An efficient Cr(VI) reducing strain was isolated and preliminarily identified as Microbacterium sp., named as BD6. Strain BD6 is suitable to grow in medium temperature and alkaline environment. Meanwhile, it can tolerate high-salt environment with NaCl concentration up to 50.0 g/L. The test showed that growth of the strain is strongly inhibited by Mn²⁺, slightly inhibited by Ni²⁺, Zn²⁺ and Cd²⁺ and promoted by Cu²⁺. The minimum inhibitory concentration of Cr(VI) on BD6 was 1700 mg/L. The reduction of Cr(VI) by BD6 is significantly improved by electronic donors, including glycerol, fructose, lactose, glucose and sodium pyruvate. Glycerol is considered as the best electron donor for the reduction of Cr(VI) by BD6. The reduction rate of 100 mg/L Cr(VI) is only 69.63% in 96 h without adding electron donor, however, it could reach 100% in 36 h after adding 2 g/L glycerol. The reduction of the strain to higher initial concentration of Cr(VI) can be enhanced by adding the amount of glycerol, but restrained by the toxicity of Cr(VI). Reduction test shows that the optimum reduction conditions and growth conditions of the strain are consistent. In the high-salt environment with NaCl concentration of 50.0 g/L and toxic environment with Cd²⁺ concentration of 50 mg/L, the reduction rate of the strain to 100 mg/L Cr(VI) are 96.79% within 72 h and 99.86% within 54 h respectively with presence of 2 g/L glycerol. [Conclusion] Microbacterium sp. BD6 is a potential candidate strain for bioremediation of Cr(VI) contamination.

Abstract:[Objective] Based on genome mining, we analyzed the secondary metabolites from mangrove-derived Streptomyces avicenniae 9-9, targeting on polycyclic tetramate macrolactams (PTMs). [Methods] Bioinformatics analysis was carried out to analyze the genome sequence of Streptomyces avicenniae 9-9. We searched PTMs by the method of high-performance liquid chromatography and mass spectroscopy. [Results] We found four genes, named aviA-D involved in PTMs biosynthetic gene clusters in Streptomyces avicenniae 9-9. Two PTMs, 1 and 2, were identified as ikaruganmycin and capsimycin B, respectively. Compound 3 through 5 also belonged to PTMs family, and their structures were proposed based on MS/MS fragmentation pattern. [Conclusion] Mangrove-derived Streptomyces avicenniae 9-9 can produce PTMs compounds with the 5-6-5 fused tricyclic skeleton.

Abstract:[Objective] Elucidation of the biosynthetic mechanism of the deoxyaminosugar moiety in acarbose from Actinoplanes sp. SE50/110. [Methods] On the basis of BlastP analysis, AcbA, AcbB and AcbV were proposed to be associated with the biosynthesis of deoxyaminosugar moiety. Firstly, the in-frame deletion and trans-complementation of acbA, acbB and acbV were performed in SE50/110 to investigate their involvement in acarbose biosynthesis. Then, AcbA, AcbB and AcbV were heterologously expressed in E. coli BL21(DE3)/pGro7 and purified by Ni affinity chromatography. Finally, using D-glucose-1-phosphate as the starting substrate, the biosynthetic process of deoxyaminosugar moiety was elucidated by enzymatic assay. In addition, the properties of these involved enzymes were characterized. [Results] Deletion mutants of acbA, acbB and acbV in SE50/110 were named as ZD03, ZD04 and ZD05, respectively, all of which lost the productivity of acarbose. And then, the production of acarbose was recovered through trans-complementation of acbA, acbB and acbV in ZD03, ZD04 and ZD05, respectively. In vitro enzymatic analysis suggested that AcbA, a D-glucose-1-phosphate thymidylyltransferase, is responsible for the biosynthesis of dTDP-D-glucose from D-glucose-1-phosphate and dTTP. It showed a K_m of (0.185±0.053) mmol/L and a V_max of (2.366±0.217) μmol/(min·mg) with D-glucose-1-phosphate, as well as a K_m of (4.964±1.089) mmol/L and a V_max of (60.310±5.419) μmol/(min·mg) with dTTP. AcbB, a TDP-D-glucose-4,6-dehydratase, catalyzed the dehydration of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose. The K_m and V_max of AcbB are (0.353±0.089) mmol/L and (306.401±28.740) μmol/(min·mg), respectively. AcbV is a dTDP-4-keto-6-deoxy-D-glucose-aminotransferase and catalyzes the transamination of dTDP-4-keto-6-deoxy-D-glucose to dTDP-4-amino-4,6-dideoxy-D-glucose using glutamic acid as amino donor. The K_m and V_max of AcbV are (1.411±0.293) mmol/L and (3.447±0.279) μmol/(min·mg), respectively. [Conclusion] This study elucidated the biosynthetic pathway of deoxyaminosugar moiety in acarbose, which paved a solide way for a full elucidation of acarbose biosynthesis. Meanwhile, the characterization of the involved enzymes provided important information for the metabolic engineering of SE50/110 to improve acarbose production.

Abstract:Epiphytic microorganisms on the bark surface play an important role in host plants' health and adaptability. The maintenance mechanism of microbial community structure and diversity in bark microhabitats need attention. [Objective] We studied the distribution characteristics and fitness mechanisms of the bacterial community on the bark surface of Larix principis-rupprechtii along elevation gradients and light in Pangquangou Natural Reserve.[Methods] By using PCR-DGGE and high-throughput sequencing technology, we studied the structure of bacterial community. By using nonmetric multidimensional scaling (NMDS) ordination, we analyzed the spatial distribution characteristic of bacterial community. By using redundancy analysis, we analyzed the relationship between bacterial community and environmental factors and by using One-way ANOVA, we compared bacterial community composition between sunny and shady surface. Based on zero-deviation analysis, the driving factors of bacterial community assembly on the shady and sunny surface were studied. [Results] Bacterial community structure was significantly different among elevation gradients (ANOSIM; P<0.05). The redundancy analysis showed that pH and total carbon (TC) on bark surface were significantly correlated with the community structure (P<0.05). The relative abundances of photosynthetic autotrophic bacteria (norank_Cyanobacteria) on the sunny surface were significantly higher than those on shady one, while the relative abundance of Rhizobiales showed an opposite trend. We suggested that light might be a driving factor in determining the structure of bark bacterial community. The results of zero-deviation analysis showed that the diversity pattern of the bacterial community on the bark surface was mainly affected by environmental filtration. [Conclusion] The determination processes were dominant factors in shaping the structure and diversity pattern of bacterial community on the bark surface of Larix principis-rupprechtii in this area.

Abstract:[Objective] To explore the diversity and differentiation of endophytic nitrogen-fixing bacteria community in Pennisetum sp. in different habitats. [Methods] High-throughput sequencing of nitrogen-fixing enzyme nifH target gene method was used to study the endophytic nitrogen-fixing bacteria in Pennisetum sp. in 6 typical areas of China, including Fujian Minhou County, Xinjiang Moyu County, Inner Mongolia Alashan Zuoqi, Qinghai Guide County, Gansu Anding District, and Hainan Nada Town. The environmental driving mechanism of nitrogen-fixing bacteria diversity was analyzed by combining geographical and climatic factor statistics. [Results] A total of 64122 valid sequences of nifH genes were obtained, 640 OTUs belonging to 6 phyla, 10 classes, 17 orders, 24 families, 33 genera and 39 species. The species and abundance of predominant endophytic nitrogen-fixing bacteria in Pennisetum sp. from different regions were quite different. At the phylum level, Proteobacteria were the dominant bacteria in the five areas of Fuzhou Minhou County, Gansu Anding District, Xinjiang Moyu County, Inner Mongolia Alashan Zuoqi and Qinghai Guide County, whereas Proteobacteria and Cyanobacteria were the dominant phylum in Hainan Nada town. At the genus level, the most dominant endogphytic nitrogen-fixing bacteria groups in Pennisetum sp. in different regions as below:Minhou County of Fuzhou province (undetermined genus of Proteobacteria, 80.56%), Moyu County of Xinjiang province (undetermined genus of Proteobacteria, 33.14%), Alashan Zuoqi of Inner Mongolia (undetermined genus of Proteobacteria, 76.23%), Anding District of Gansu Province (undetermined genus of Alphaproteobacteria, 53.78%), Nada Town of Hainan (undetermined genus of Proteobacteria, 38.79%), Guide County of Qinghai (undetermined genus of Proteobacteria, 46.12%). The analysis of alpha-diversity and beta-diversity showed that the diversity of endophytic nitrogen-fixing bacteria communities in Pennisetum sp. in different regions was different. The diversity and richness of endophytic nitrogen-fixing bacteria in Pennisetum sp. in Nada Town of Hainan Province were the highest, while those in Minhou County of Fujian province were the lowest. The canonical correspondence analysis (CCA) showed that annual average rainfall and annual average temperature were the main factors affecting the change of endophytic nitrogen-fixing bacteria in Pennisetum sp., followed by soil organic matter, soil total nitrogen and soil pH. [Conclusion] The composition and abundance of endophytic nitrogen-fixing bacteria community in Pennisetum sp. in different areas were quite different. The species and relative abundance of endophytic nitrogen-fixing bacteria in Pennisetum sp. in Nada town of Hainan province were relatively high. This research can provide theoretical supports for the development of endophytic nitrogen-fixing bacteria in Pennisetum sp. and the breeding and production of nitrogen-fixing microbial fertilizers.

Abstract:[Objective] To analyze the dominant methanogenic communities and methanogenic pathways in the different saline-alkaline lakes at the Qinghai-Tibet Plateau. [Methods] Using the sediments from three saline-alkaline lakes, Gongzhu Co, Kunzhong Co and Zigetang Co, that have different salinities and plantation types, as objects, we analyzed archaeal 16S rRNA diversity via high-throughput sequencing, and quantified the dominant methanogenic groups using quantitative PCR. The dominant methanogenic groups were then confirmed by comparing methane producing rates of the sediments from all known methanogenic substrates (methanol, trimethylamine, acetate and H₂/CO₂). Upon inhibition of methane production by a methanogenesis-specific inhibitor (2-bromoethanesulfonate), accumulated methanogenic precursors were determined so as to infer methanogenic pathways in the lake sediments. [Results] Methanosarcinaceae (11%), Methanosaetaceae (7.9%) and Methanomicrobiales (7.4%) were determined to be dominant in Kunzhong Co; Methanosaetaceae was accounted for 15% and 15.3% in Gongzhu Co and Zigetang Co, respectively, including the genera of Methanobacterium and Methanolobus. The highest methane production rates were determined from acetate and methanol in Gongzhu Co and Kunzhong Co respectively, but there was no difference in methane production rate from different methanogenic substrates in Zigetang Co. Methanol and acetate were respectively accumulated in methanogensis inhibited Kunzhong Co and Gongzhu Co, but no substance was obviously accumulated in Zigetang Co. [Conclusion] Methylotrophic methanogens and their implemented methanogenic pathway could be dominant in Kunzhong Co; and acetoclastic methanogens were prevalent in Gongzhu Co. However, not much methane production and substrate accumulation were determined in Zigetang Co. Based on the analysis, the dominant methanogenic pathways and methanogenic communities were predicted to be associated more with vegetation type but less with salinities of the saline-alkaline lakes on the Tibetan Plateau.

Abstract:[Objective] We aimed to construct recombinant Kluyveromyces lactis (K. lactis) strains to produce cold-adapted chitinases (chitinase A, chiA; chitinase C, chiC) of Pseudoalteromonas sp. DL-6. We purified and characterized the recombinant proteins. [Methods] We synthesized optimized-codons chitinase genes. The recombinant K. lactis expression plasmids (pKLAC1-chiA, pKLAC1-chiC) were constructed successfully and transformed into K. lactis by electric field pulses to achieve soluble expression of cold-adapted chitinases. The recombinant proteins were further purified using Ni-NTA chromatography. [Results] The recombinant K. lactis producing cold-adapted chitinases was successfully constructed and obtained high purity. The purified recombinant proteins, ChiA and ChiC, appeared as a single band of approximately 110 kDa and 90 kDa by SDS-PAGE, respectively. The activities of ChiA and ChiC were 51.45 U/mg and 108.56 U/mg measured by Schales' method. The optimum temperature was 20℃ and 30℃, and the optimum pH was 8.0 and 9.0, respectively. ChiA and ChiC were stable at pH 8.0-12.0 and below 40℃. ChiA and ChiC have obvious degradation activity to colloidal chitin and powdered chitin including α-chitin and β-chitin. Besides, they acted synergistically to efficiently degrade chitin substrates. [Conclusion] The expression, purification, enzymatic properties and degradation products of cold-adapted chitinases in K. lactis were achieved for the first time. These research results provide reference for the study of other cold-adapted chitinases.

Abstract:[Objective] Lytic polysaccharide monooxygenases (LPMOs) are new lignocellulose-degrading enzymes that break glycosidic bonds of polysaccharides via oxidation. This study aims to explore and characterize novel LPMOs. [Methods] LPMO genes were cloned from Aspergillus oryzae and expressed in Pichia pastoris. [Results] Sequence analysis indicated that both AoLPMO2 and AoLPMO5 belong to auxiliary activity 9 family. AoLPMO5 was transformed into P. pastoris GS115 to obtain a 2-copy insertion yeast GS/AO5-4. After induction by 1% methanol for 4 d, protein yield in culture supernatant reached 0.19±0.01 g/L. The molecular mass of recombinant protein was approximate 34 kDa, which was higher that the theorical calculation, indicating probably post-translation modification proceeded in yeast host. The optimal temperature and pH of AoLPMO5 to catalyze locust bean gum were 60℃ and 5.0. Its K_m and V_max were 8.72±1.99 mg/mL and 109.4±12.8 μmol/(s·mg), respectively. Addition of 0.1 mmol/L Cu²⁺, 0.5, 2.0 and 2.5 mmol/L H₂O₂ enhanced its catalytic activity by (7.10±1.32)% (P<0.05), (21.11±6.17)% (P<0.01), (20.22±1.13)% (P<0.01) and (18.40±2.86)% (P<0.01), respectively, while gallic acid or vitamin C did not show promotion. The AoLPMO5 was found to bind with locust bean gum substrate at the early stage of reaction, resulting in temporary inhibition of mannanase (BsMAN3) activity. However, these two enzymes showed synergic effects on substrate deconstruction at the later period. [Conclusion] AoLPMO5 is a novel biomass-degrading enzyme. Insights into its enzymatic properties and substrate-degrading patterns will contribute to natural lignocellulosic biomass conversion and biorefinery, especially second-generation bioethanol and functional oligosaccharides production.

Abstract:[Objective] Terpenoids are widely present in various organisms and play key roles in all forms of life. Two pathways have been found for the biosynthesis of terpenoids:the mevalonate (MVA) pathway and 2-methyl-D-erythritol-4-phosphate (MEP) pathway. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a key enzyme in the MEP pathway that catalyzes 1-deoxy-D-xylulose-5-phosphate to generate MEP. DXR is encoded by a dxr gene in Bacillus subtilis, while two dxr genes (dxr1 and dxr2) encode DXR in Bacillus thuringiensis (Bt). To determine the mechanism of transcriptional regulation and the function of dxr gene, we analyzed the transcriptional regulation of dxr1 gene and phenotype of dxr1 and dxr2 mutants of Bt HD73. [Methods] Transcriptional start site was analyzed by 5'RACE. Transcriptional activity of dxr1 promoter (Pdxr1) was analyzed by promoter fusions with lacZ gene. dxr1 and dxr2 mutants of Bt HD73 strain were constructed by homologous recombination. Comparison of the Cry1Ac protein production was determined by protein quantitation. DXR activity was determined by DXR detection kit. [Results] A G residue located 39 bp upstream from the dxr1 start codon was identified. The transcriptional activity of Pdxr1 was sharply decreased in sigH mutant compared with that in HD73 wild-type. Mutation of dxr1 or dxr2 has no significant differences on growth, sporulation efficiency and Cry1Ac protein production. However, the DXR activity was decreased in the mutants. [Conclusion] Transcription of dxr1 is controlled by SigH. Deletion of dxr genes influenced the activity of DXR.