Heterologous expression of Streptomyces coelicolor trehalose synthase and whole-cell biocatalyst production of trehalose in Escherichia coli / 生物工程学报

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.

Optimized sample preparation for metabolome studies on Streptomyces coelicolor / 生物工程学报

Streptomycetes produce many antibiotics and are important model microorgansims for scientific research and antibiotic production. Metabolomics is an emerging technological platform to analyze low molecular weight metabolites in a given organism qualitatively and quantitatively. Compared to other Omics platform, metabolomics has greater advantage in monitoring metabolic flux distribution and thus identifying key metabolites related to target metabolic pathway. The present work aims at establishing a rapid, accurate sample preparation protocol for metabolomics analysis in streptomycetes. In the present work, several sample preparation steps, including cell quenching time, cell separation method, conditions for metabolite extraction and metabolite derivatization were optimized. Then, the metabolic profiles of Streptomyces coelicolor during different growth stages were analyzed by GC-MS. The optimal sample preparation conditions were as follows: time of low-temperature quenching 4 min, cell separation by fast filtration, time of freeze-thaw 45 s/3 min and the conditions of metabolite derivatization at 40 degrees C for 90 min. By using this optimized protocol, 103 metabolites were finally identified from a sample of S. coelicolor, which distribute in central metabolic pathways (glycolysis, pentose phosphate pathway and citrate cycle), amino acid, fatty acid, nucleotide metabolic pathways, etc. By comparing the temporal profiles of these metabolites, the amino acid and fatty acid metabolic pathways were found to stay at a high level during stationary phase, therefore, these pathways may play an important role during the transition between the primary and secondary metabolism. An optimized protocol of sample preparation was established and applied for metabolomics analysis of S. coelicolor, 103 metabolites were identified. The temporal profiles of metabolites reveal amino acid and fatty acid metabolic pathways may play an important role in the transition from primary to secondary metabolism in S. coelicolor.

Advances in actinobacterial proteomics / 生物工程学报

Protein is the executor of physiological function, and direct embodiment of the life phenomena. Proteomics aims to systematically clarify all or parts of proteins' role and function in life movement. In post genome era, proteomics began to play more important role in life science field. Actinobacteria are closely linked to human production and life, which have produced many clinically important secondary metabolites, including antibiotics, antitumorals and enzymes. Actinobacterial systematics and its model organism Streptomyces coelicolor in 2001 genome sequence laid the foundation for further functional genomic studies. Actinobacterial proteomics was more directly and exactly to interpret the activity of life than genomics and transcriptomics, which grew much faster and received so much attention from scientists in the near years. Complex morphological differention, stronge environment adaptiveness, nitrogen-fixing capacity, metabolic mechanism, pathogenicity and natural produces' discovery were systematically reviewed in this study, which was expected to be the basis for promoting Actinobacterial proteomics study in the near future.

Biodegradation of a blended starch/natural rubber foam biopolymer and rubber gloves by Streptomyces coelicolor CH13

Background: The growing problem of environmental pollution caused by synthetic plastics has led to the search for alternative materials such as biodegradable plastics. Of the biopolymers presently under development, starch/natural rubber is one promising alternative. Several species of bacteria and fungi are capable of degrading natural rubber and many can degrade starch. Results: Streptomyces coelicolor CH13 was isolated from soil according to its ability to produce translucent halos on a mineral salts medium, MSM, supplemented with natural rubber and to degrade starch. Scanning electron microscope studies showed that it colonized the surfaces of strips of a new starch/natural rubber biopolymer and rubber gloves and caused degradation by forming holes, and surface degradation. Starch was completely removed and polyisoprene chains were broken down to produce aldehyde and/or carbonyl groups. After 6 weeks of cultivation with strips of the polymers in MSM, S. coelicolor CH13 reduced the weight of the starch/NR biopolymer by 92 percent and that of the rubber gloves by 14.3 percent. Conclusions: This study indicated that this bacterium causes the biodegradation of the new biopolymer and natural rubber and confirms that this new biopolymer can be degraded in the environment and would be suitable as a ‘green plastic’ derived from natural sources.

Structural basis for prokaryotic calcium-mediated regulation by a Streptomyces coelicolor calcium binding protein

The important and diverse regulatory roles of Ca(2+) in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.

Sequence Analysis and Potential Action of Eukaryotic Type Protein Kinase from Streptomyces coelicolor A3(2)

Protein kinase C (PKC) is a family of kinases involved in the transduction of cellular signals that promote lipid hydrolysis. PKC plays a pivotal role in mediating cellular responses to extracellular stimuli involved in proliferation, differentiation and apoptosis. Comparative analysis of the PKC-alpha, beta, epsilon isozymes of 200 recently sequenced microbial genomes was carried out using variety of bioinformatics tools. Diversity and evolution of PKC was determined by sequence alignment. The ser/thr protein kinases of Streptomyces coelicolor A3 (2), is the only bacteria to show sequence alignment score greater than 30% with all the three PKC isotypes in the sequence alignment. S.coelicolor is the subject of our interest because it is notable for the production of pharmaceutically useful compounds including anti-tumor agents, immunosupressants and over two-thirds of all natural antibiotics currently available. The comparative analysis of three human isotypes of PKC and Serine/threonine protein kinase of S.coelicolor was carried out and possible mechanism of action of PKC was derived. Our analysis indicates that Serine/ threonine protein kinase from S. coelicolor can be a good candidate for potent anti-tumor agent. The presence of three representative isotypes of the PKC super family in this organism helps us to understand the mechanism of PKC from evolutionary perspective.

Sequence Analysis and Potential Action of Eukaryotic Type Protein Kinase from Streptomyces coelicolor A3(2)

Protein kinase C (PKC) is a family of kinases involved in the transduction of cellular signals that promote lipid hydrolysis. PKC plays a pivotal role in mediating cellular responses to extracellular stimuli involved in proliferation, differentiation and apoptosis. Comparative analysis of the PKC-alpha, beta, epsilon isozymes of 200 recently sequenced microbial genomes was carried out using variety of bioinformatics tools. Diversity and evolution of PKC was determined by sequence alignment. The ser/thr protein kinases of Streptomyces coelicolor A3 (2), is the only bacteria to show sequence alignment score greater than 30% with all the three PKC isotypes in the sequence alignment. S.coelicolor is the subject of our interest because it is notable for the production of pharmaceutically useful compounds including anti-tumor agents, immunosupressants and over two-thirds of all natural antibiotics currently available. The comparative analysis of three human isotypes of PKC and Serine/threonine protein kinase of S.coelicolor was carried out and possible mechanism of action of PKC was derived. Our analysis indicates that Serine/ threonine protein kinase from S. coelicolor can be a good candidate for potent anti-tumor agent. The presence of three representative isotypes of the PKC super family in this organism helps us to understand the mechanism of PKC from evolutionary perspective.

Spatio-temporal expression of the pathway-specific regulatory gene redD in S. coelicolor / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Confocal laser scanning microscopy was used to observe the spatio-temporal expression of the pathway-specific gene redD during S. coelicolor cell cultivation. The corresponding mutant S. coelicolor lyqRY1522 carrying redD::eyfp in the chromosome was constructed. The temporal expression results of the fusion protein during submerged cultivation demonstrated that expression of redD began in the transition phase, continuing through the exponential growth phase to the stationary phase, and reached maximum in the stationary phase. On the other hand, redD was expressed only in substrate mycelia during solid-state culture, while aerial mycelia remained essentially non-fluorescent throughout culture. Results demonstrated that the expression pattern of redD coincides with that of the biosynthesis of the antibiotics during culture, revealing a direct correlation between the spatio-temporal distribution of regulatory gene expression and second metabolism.

Analyses of Streptomyces coelicolor inner membrane proteome by multidimentional protein identification technology / 生物工程学报

Streptomyces coelicolor is the model species among streptomycetes. Until now, proteomic analyses of S. coelicolor have been conducted using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, few integral membrane proteins were identified due to the hydrophobic and low-abundance nature of these proteins. In this work, 154 possible inner membrane proteins from S. coelicolor were identified using high pH-proteinase K sample preparation method and multidimensional protein identification technology, among them 44 are integral membrane proteins containing at least one transmembrane domain, most peptides and their corresponding proteins were identified experimentally for the first time.