Metabolic analysis reveals the amino acid responses of Streptomyces lydicus to pitching ratios during improving streptolydigin production

Pitching ratio has been reported to impact not only on the primary metabolism, but also the secondary metabolism. Comparative metabolomics was used to explore the metabolic responses of Streptomyces lydicus E9 to pitching ratios (1, 10, and 30 %, v/v). We identified more than 120 metabolites involved in glycolysis, tricarboxylic acid cycle, and amino acid and secondary metabolism, of which there are significant differences in the quantified 32 metabolites under different pitching ratios by gas chromatography coupled to time-of-flight mass spectrometry. The intracellular levels of most amino acids (e.g., valine, alanine, and isoleucine) declined with the increases of pitching ratios. Especially, the relative abundances of glutamate and proline were not only decreased with the increases of pitching rations, but also had much low level at stages II and III, which might be related to the significant enhancement in streptolydigin of S. lydicus E9 under 30 % high pitching ratio. Moreover, principal component analysis revealed that eight metabolites, including glucopyranoside, maltose, cAMP, glycine, proline, lysine, isoleucine, and valine, were considered as potential biomarkers to distinguish the influences of pitching ratios on streptolydigin production. Further investigations demonstrated that the additions of exogenous glutamate and proline (100 mg?L^?1) enhanced significantly the accumulation of streptolydigin, indicating that glutamate was the synthetic precursor of streptolydigin, while proline in S. lydicus E9 was converted into glutamate and consequently improved streptolydigin biosynthesis. Therefore, these findings provide new insights into the amino acid responses of S. lydicus E9 to pitching ratios and provide potential strategies to improve streptolydigin production.     

Insights into the roles of exogenous glutamate and proline in improving streptolydigin production of Streptomyces lydicus with metabolomic analysis

The addition of precursors was one strategy to improve antibiotic production. The exogenous proline and glutamate, as precursors of streptolydigin, could significantly improve the streptolydigin production, but their underlying molecular mechanisms remain unknown. Herein, metabolomic analysis was carried out to explore the metabolic responses of Streptomyces lydicus to the additions of proline and glutamine. The significant differences in the quantified 53 metabolites after adding the exogenous proline and glutamate were enunciated by gas chromatography coupled to time-of-flight mass spectrometry. Among them, the levels of some fatty acids (e.g., dodecanoic acid, octadecanoic acid, hexadecanoic acid) were significantly decreased after adding glutamate and proline, indicating that the inhibition of fatty acid synthesis might be benefit for the accumulation of streptolydigin. Particularly, the dramatic changes of the identified metabolites, which are involved in glycolysis, the tricarboxylic acid cycle, and the amino acid and fatty acid metabolism, revealed that the additions of glutamate and proline possibly caused the metabolic cross-talk in S. lydicus. Additionally, the level of intracellular glutamate dramatically enhanced at 12 h after adding proline, showing that exogenous proline may be firstly convert into glutamate and consequently result in crease of the streptolydigin production. The high levels of streptolydigin at 12 and 24 h after adding glutamate unveiled that part glutamate were rapidly used to synthesize the streptolydigin. Furthermore, there is the significant difference in metabolomic characteristics of S. lydicus after adding glutamate and proline, uncovering that multiple regulatory pathways are involved in responses to the additions of exogenous glutamate and proline. Taken together, exogenous glutamate and proline not only directly provided the precursors of streptolydigin biosynthesis, but also might alter the metabolic homeostasis of S. lydicus E9 during improving the production of streptolydigin.     

Amino acid precursor supply in the biosynthesis of the RNA polymerase inhibitor streptolydigin by Streptomyces lydicus

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ～2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin.     

Biosynthesis of the RNA polymerase inhibitor streptolydigin in Streptomyces lydicus: tailoring modification of 3-methyl-aspartate

The asparaginyl-tRNA synthetase-like SlgZ and methyltransferase SlgM enzymes are involved in the biosynthesis of the tetramic acid streptolydigin in Streptomyces lydicus. Inactivation of slgZ led to a novel streptolydigin derivative. Overexpression of slgZ, slgM, or both in S. lydicus led to a considerable increase in streptolydigin production.     

Purification and partial characterization of polygalacturonase from Streptomyces lydicus

Polygalacturonase produced by Streptomyces lydicus was purified to homogeneity by ultrafiltration and a combination of ion exchange and gel filtration chromatographic procedures. The purified enzyme was an exo-polygalacturonase with a molecular weight of 43 kDa. It was optimally active at 50 degrees C and pH 6.0. The enzyme was stable from pH 4.0 to 7.0 and at or below 45 degrees C for 90 min. K(m) value for polygalacturonic acid was 1.63 mg/mL and the corresponding V(max) was 677.8 microM min(-1) mg(-1). The inhibition constant (K(i)) for gluconic acid d-lactone was 20.75 mM. Purified enzyme had been inhibited by N-bromosuccinimide, while l-tryptophan could induce enzyme activity, indicating the involvement of tryptophan at the active site.     

基因组重排与传统育种相结合选育大观霉素高产菌株

以壮观链霉菌(Streptomyces spectabilis)为研究对象,采用基因组重排技术与传统诱变育种相结合的方法选育大观霉素的高产菌株.通过原生质体紫外诱变获得壮观链霉菌突变体群体,高产突变菌株间进行两轮的基因组重排,筛选的高产菌株用NTG诱变得新霉素和链霉素的抗性突变菌株,抗性突变菌株间进行两轮基因组重排,从...     

Cardiac proteomic responses to ischemia-reperfusion injury and ischemic preconditioning

Cardiac ischemia and ischemia-reperfusion (I/R) injury are major contributors to morbidity and mortality worldwide. Pathological mechanisms of I/R and the physiological mechanisms of ischemic preconditioning (IPC), which is an effective cardiac protective response, have been widely investigated in the last decade to search for means to prevent or treat this disease. Proteomics is a powerful analytical tool that has provided important information to identify target proteins and understand the underlying mechanisms of I/R and IPC. Here, we review the application of proteomics to I/R injury and IPC to discover target proteins. We analyze the functional meaning of the accumulated data on hundreds of proteins using various bioinformatics applications. In addition, we review exercise-induced proteomic alterations in the heart to understand the potential cardioprotective role of exercise against I/R injury. Further developments in the proteomic field that target specialized proteins will yield new insights for optimizing therapeutic targets and developing a wide range of therapeutic agents against ischemic heart disease.     

Cloning Streptomyces genes for antibiotic production

The cloning and recombination of the genes of Streptomyces bacteria offer a method of increasing antibiotic yields and generating new antibiotics. Novel vectors, both plasmids and phages, have been developed for use with Streptomyces. This article describes some of these vectors and relevant cloning and screening techniques.     

A proteomic analysis of Streptomyces coelicolor programmed cell death

Programmed cell death (PCD) is an active cellular suicide that occurs in eukaryotes and bacteria in response to both abiotic and biotic stresses. In contrast to eukaryotic apoptosis, little is known about the molecular machinery that regulates bacterial PCD. In a previous work, we described the existence of PCD phenomena in Streptomyces (Manteca et al., Res. Microbiol. 2006, 157, 143-152). In the present study, we performed a proteomic analysis of PCD in Streptomyces coelicolor, for which we developed a system to obtain dead and live cell-enriched samples. PCD in this filamentous bacterium is accompanied by the appearance of enzymes involved in the degradation of cellular macromolecules, regulatory proteins, and stress-induced proteins. We argue that some of these proteins have specific functions in the PCD pathway and putative roles for the identified proteins have been proposed. The increased amounts of several antioxidant proteins suggest oxidative stress as either the cause or consequence of the cell death.     

Efficient transformation and expression of the glucanase gene from Bacillus megaterium in the biocontrol strain Streptomyces lydicus A02

Streptomyces have been used extensively as the biocontrol agents due to their ability to produce various antimicrobial compounds, such as antibiotics and hydrolytic enzymes. Streptomyces lydicus strain A02, which was isolated from the soil of suburban forest field in Beijing (China), is capable of producing natamycin and has proved to be a potential biocontrol agent to several plant fungal diseases, including wilts caused by Fusarium oxysporum f. spp. However, hydrolytic enzymes like glucanase have not been detected in S. lydicus A02 on CMC-Na plates by congo red staining. Glucanase, a pathogenesis-related (PR) protein, degrades fungal cell walls and has been widely used as antifungal agent in plant protection. Therefore, a recombinant S. lydicus expressing a glucanase gene, which was cloned from the biocontrol strain Bacillus megaterium L103 and driven by the Streptomyces erythraea ermE* promoter, was constructed in this study. The engineered S. lydicus AG02 shared a similar yield of natamycin with the wild-type A02 strain. Compared to the wild-type strain A02, the engineered S. lydicus AG02 had a remarkably higher glucanase activity, as well as antifungal activity to F. oxysporum f. sp. conglutinans, F. oxysporum f. sp. niveum and Rhizoctonia cerealis. This demonstrated the improved biocontrol effect of S. lydicus AG02 attributed to transforming the exogenous glucanase from B. megaterium, which acted synergistically with natamycin to increase the antifungal activity of the strain.     

常压室温等离子体(ARTP)诱变及高通量筛选那西肽高产菌株

采用新型常压室温等离子体(ARTP)诱变活跃链霉菌(Streptomyces actuosu),并应用抑菌圈和48孔板培养方法高通量筛选高产那西肽菌株。研究表明抑菌圈径的大小与48孔板效价之间以及48孔板效价与摇瓶效价之间均有较好的相关性,系数R分别达到0.534和0.896。通过多轮ARTP诱变及高通量筛选最终获得了3株相对效价提高50%以上的遗传性能稳定的突变株。ARTP诱变技术作为获得那西肽高产菌株的有效途径,与传统摇瓶发酵筛选相比,48孔板及抑菌圈法能显著提高那西肽高产菌株的筛选效率。     

Evaluation of the possible proteomic application of trypsin from Streptomyces griseus

Trypsin (EC 3.4.21.4) is the protease of choice for proteome analysis using mass spectrometry of peptides in sample digests. In this work, trypsin from Streptomyces griseus (SGT) was purified to homogeneity from pronase. The enzyme was evaluated in in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analyses of the digests. We recognized a remarkable cleavage performance of SGT. The number of produced and matching tryptic peptides was higher than in the case of commonly used bovine trypsin (BT) and allowed us to obtain higher identification scores in database searches. Interestingly, SGT was found to also generate nonspecific peptides whose sequencing by MALDI-TOF/TOF tandem mass spectrometry (MS/MS) revealed a partial F-X, Y-X, and W-X cleavage specificity. To suppress autolysis, either arginine or arginine plus lysine residues in SGT were modified by chemical reagents. In consequence, the autolytic pattern of SGT was reduced significantly, but specific activity dropped dramatically. As demonstrated by relative quantification of peptides at different times, SGT is more stable at 37 °C than is its bovine counterpart. We conclude that SGT represents a convenient alternative for proteomic applications involving protein digestion. Moreover, parallel digestions of sample aliquots by SGT and BT provide the possibility of combining partially different results (unique matching peptides) to improve protein identification.     

Sensitivity of bacteriophage Mu-1 development to rifampicin and streptolydigin

Summary Mu-1 development is blocked by addition of rifampicin in a rif ⁸ but not in rif ^r strain. This suggests that Mu does not synthesize any stable protein which can replace the host RNA polymerase.The fact that Mu development is only partially blocked by streptolydigin is discussed.Aspirants from the Fonds National de la Recherche Scientifique.     

Streptomyces lividans as host for heterologous protein production

Abstract Streptomycetes are Gram-positive soil bacteria with a differentiated morphology. They are considered interesting candidates for the production of heterologous proteins for several reasons, including their efficient secretion mechanism by which the secreted proteins are localized into the culture supernatant. In view of this potential, this review article describes different aspects of gene expression and regulation in Streptomyces , and summarizes and discusses results obtained using Streptomyces lividans as host for secretion of heterologus proteins of prokaryotic and eukaryotic origin.