Project description:The bacterial pathogen Pseudomonas tolaasii severely damages white button mushrooms by secretion of the pore-forming toxin tolaasin, the main virulence factor of brown blotch disease. Yet, fungus-associated helper bacteria of the genus Mycetocola (Mycetocola tolaasinivorans and Mycetocola lacteus) may protect their host by an unknown detoxification mechanism. By a combination of metabolic profiling, imaging mass spectrometry, structure elucidation, and bioassays, we found that the helper bacteria inactivate tolaasin by linearizing the lipocyclopeptide. Furthermore, we found that Mycetocola spp. impair the dissemination of the pathogen by cleavage of the lactone ring of pseudodesmin. The role of pseudodesmin as a major swarming factor was corroborated by identification and inactivation of the corresponding biosynthetic gene cluster. Activity-guided fractionation of the Mycetocola proteome, matrix-assisted laser desorption/ionization (MALDI) analyses, and heterologous enzyme production identified the lactonase responsible for toxin cleavage. We revealed an antivirulence strategy in the context of a tripartite interaction that has high ecological and agricultural relevance.
Project description:Bacillus subtilis CGMCC 12426 is an efficient producer of poly-?-glutamate with regular stereochemistry. Here, the complete genome sequence of B. subtilis CGMCC 12426 is presented, which may facilitate the design of rational strategies for further strain improvements with industrial potential.
Project description:Here we report the complete genome sequence of Pseudomonas stutzeri strain CGMCC 1.1803 (equivalent to ATCC 17588), the type strain of P. stutzeri, which encodes 4,138 open reading frames on a 4,547,930-bp circular chromosome. The CGMCC 1.1803 genome contains genes involved in denitrification, benzoate/catechol degradation, chemotaxis, and other functions.
Project description:Here, we report the 4.1-Mb draft genome sequence of Bacillus subtilis subsp. natto strain CGMCC 2108, a high producer of poly-?-glutamic acid (?-PGA). This sequence will provide further help for the biosynthesis of ?-PGA and will greatly facilitate research efforts in metabolic engineering of B. subtilis subsp. natto strain CGMCC 2108.
Project description:Variovorax is a metabolically diverse genus of plant growth-promoting rhizobacteria (PGPR) that engages in mutually beneficial interactions between plants and microbes. Unlike most PGPR, Variovorax cannot synthesize the phytohormone indole-3-acetic acid (IAA) via tryptophan. However, we found that Variovorax boronicumulans strain CGMCC 4969 can produce IAA using indole-3-acetonitrile (IAN) as the precursor. Thus, in the present study, the IAA synthesis mechanism of V. boronicumulans CGMCC 4969 was investigated. V. boronicumulans CGMCC 4969 metabolized IAN to IAA through both a nitrilase-dependent pathway and a nitrile hydratase (NHase) and amidase-dependent pathway. Cobalt enhanced the metabolic flux via the NHase/amidase, by which IAN was rapidly converted to indole-3-acetamide (IAM) and in turn to IAA. IAN stimulated metabolic flux via the nitrilase, by which IAN was rapidly converted to IAA. Subsequently, the IAA was degraded. V. boronicumulans CGMCC 4969 can use IAN as the sole carbon and nitrogen source for growth. Genome sequencing confirmed the IAA synthesis pathways. Gene cloning and overexpression in Escherichia coli indicated that NitA has nitrilase activity and IamA has amidase activity to respectively transform IAN and IAM to IAA. Interestingly, NitA showed a close genetic relationship with the nitrilase of the phytopathogen Pseudomonas syringae Quantitative PCR analysis indicated that the NHase/amidase system is constitutively expressed, whereas the nitrilase is inducible. The present study helps our understanding of the versatile functions of Variovorax nitrile-converting enzymes that mediate IAA synthesis and the interactions between plants and these bacteria.IMPORTANCE We demonstrated that Variovorax boronicumulans CGMCC 4969 has two enzymatic systems-nitrilase and nitrile hydratase/amidase-that convert indole-3-acetonitrile (IAN) to the important plant hormone indole-3-acetic acid (IAA). The two IAA synthesis systems have very different regulatory mechanisms, affecting the IAA synthesis rate and duration. The nitrilase was induced by IAN, which was rapidly converted to IAA; subsequently, IAA was rapidly consumed for cell growth. The nitrile hydratase (NHase) and amidase system was constitutively expressed and slowly but continuously synthesized IAA. In addition to synthesizing IAA from IAN, CGMCC 4969 has a rapid IAA degradation system, which would be helpful for a host plant to eliminate redundant IAA. This study indicates that the plant growth-promoting rhizobacterium V. boronicumulans CGMCC 4969 has the potential to be used by host plants to regulate the IAA level.
Project description:Laccases are a class of multi-copper oxidases with important industrial values. A thermotolerant laccase produced by a basidiomycete fungal strain Cerrena unicolor CGMCC 5.1011 was studied. With glycerin and peptone as the carbon and nitrogen sources, respectively, a maximal laccase activity of 121.7 U/mL was attained after cultivation in the shaking flask for 15 days. Transcriptomics analysis revealed an expressed laccase gene family of 12 members in C. unicolor strain CGMCC 5.1011, and the gene and cDNA sequences were cloned. A glycosylated laccase was purified from the fermentation broth of Cerrena unicolor CGMCC 5.1011 and corresponded to Lac2 based on MALDI-TOF MS/MS identification. Lac2 was stable at pH 5.0 and above, and was resistant to organic solvents. Lac2 displayed remarkable thermostability, with half-life time of 1.67 h at 70 ºC. Consistently, Lac2 was able to completely decolorize malachite green (MG) at high temperatures, whereas Lac7 from Cerrena sp. HYB07 resulted in accumulation of colored MG transformation intermediates. Molecular dynamics simulation of Lac2 was conducted, and possible mechanisms underlying Lac2 thermostability were discussed. The robustness of C. unicolor CGMCC 5.1011 laccase would not only be useful for industrial applications, but also provide a template for future work to develop thermostable laccases.
Project description:Genome sequences of marine streptomycetes are valuable for the discovery of useful enzymes and bioactive compounds by genome mining. However, publicly available complete genome sequences of marine streptomycetes are still limited. Here, we present the complete genome sequence of a marine streptomycete Streptomyces sp. S063 CGMCC 14582. Species delineation based on the pairwise digital DNA-DNA hybridization and genome comparison ANI (average nucleotide identity) value showed that Streptomyces sp. S063 CGMCC 14582 possesses a unique genome that is clearly different from all of the other available genomes. Bioactivity tests showed that Streptomyces sp. S063 CGMCC 14582 produces metabolites with anti-complement activities, which are useful for treatment of numerous diseases that arise from inappropriate activation of the human complement system. Analysis of the genome reveals no biosynthetic gene cluster (BGC) which shows even low similarity to that of the known anti-complement agents was detected in the genome, indicating that Streptomyces sp. S063 CGMCC 14582 may produce novel anti-complement agents of microbial origin. Four BGCs which are potentially involved in biosynthesis of non-ribosomal peptides were disrupted, but no decrease of anti-complement activities was observed, suggesting that these four BGCs are not involved in biosynthesis of the anti-complement agents. In addition, LC-MS/MS analysis and subsequent alignment through the Global Natural Products Social Molecular Networking (GNPS) platform led to the detection of novel peptides produced by the strain. Streptomyces sp. S063 CGMCC 14582 grows rapidly and is salt tolerant, which benefits efficient secondary metabolite production via seawater-based fermentation. Our results indicate that Streptomyces sp. S063 has great potential to produce novel bioactive compounds, and also is a good host for heterologous production of useful secondary metabolites for drug discovery.
Project description:Background:2-Phenylethanol (2-PE) is a higher aromatic alcohol that is widely used in the perfumery, cosmetics, and food industries and is also a potentially valuable next-generation biofuel. In our previous study, a new strain Enterobacter sp. CGMCC 5087 was isolated to produce 2-PE from glucose through the phenylpyruvate pathway. Results:In this study, candidate genes for 2-PE biosynthesis were identified from Enterobacter sp. CGMCC 5087 by draft whole-genome sequence, metabolic engineering, and shake flask fermentation. Subsequently, the identified genes encoding the 2-keto acid decarboxylase (Kdc) and alcohol dehydrogenase (Adh) enzymes from Enterobacter sp. CGMCC 5087 were introduced into E. coli BL21(DE3) to construct a high-efficiency microbial cell factory for 2-PE production using the prokaryotic phenylpyruvate pathway. The enzymes Kdc4427 and Adh4428 from Enterobacter sp. CGMCC 5087 showed higher performances than did the corresponding enzymes ARO10 and ADH2 from Saccharomyces cerevisiae, respectively. The E. coli cell factory was further improved by overexpressing two upstream shikimate pathway genes, aroF/aroG/aroH and pheA, to enhance the metabolic flux of the phenylpyruvate pathway, which resulted in 2-PE production of 260 mg/L. The combined overexpression of tktA and ppsA increased the precursor supply of erythrose-4-phosphate and phosphoenolpyruvate, which resulted in 2-PE production of 320 mg/L, with a productivity of 13.3 mg/L/h. Conclusions:The present study achieved the highest titer of de novo 2-PE production of in a recombinant E. coli system. This study describes a new, efficient 2-PE producer that lays foundation for the industrial-scale production of 2-PE and its derivatives in the future.
Project description:Complete genome sequence of Gluconacetobacter xylinus CGMCC 2955 for fine control of bacterial cellulose (BC) synthesis is presented here. The genome, at 3,563,314?bp, was found to contain 3,193 predicted genes without gaps. There are four BC synthase operons (bcs), among which only bcsI is structurally complete, comprising bcsA, bcsB, bcsC, and bcsD. Genes encoding key enzymes in glycolytic, pentose phosphate, and BC biosynthetic pathways and in the tricarboxylic acid cycle were identified. G. xylinus CGMCC 2955 has a complete glycolytic pathway because sequence data analysis revealed that this strain possesses a phosphofructokinase (pfk)-encoding gene, which is absent in most BC-producing strains. Furthermore, combined with our previous results, the data on metabolism of various carbon sources (monosaccharide, ethanol, and acetate) and their regulatory mechanism of action on BC production were explained. Regulation of BC synthase (Bcs) is another effective method for precise control of BC biosynthesis, and cyclic diguanylate (c-di-GMP) is the key activator of BcsA-BcsB subunit of Bcs. The quorum sensing (QS) system was found to positively regulate phosphodiesterase, which decomposed c-di-GMP. Thus, in this study, we demonstrated the presence of QS in G. xylinus CGMCC 2955 and proposed a possible regulatory mechanism of QS action on BC production.