Bioconversion of 12-, 14-, and 16-membered ring aglycones to glycosylated macrolides in an engineered strain of <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis>

To develop a system for combinatorial biosynthesis of glycosylated macrolides, Streptomyces venezuelae was genetically manipulated to be deficient in the production of its macrolide antibiotics by deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of thymidine diphosphate (TDP)-d-quinovose or TDP-d-olivose in conjunction with the glycosyltransferase–auxiliary protein pair DesVII/DesVIII derived from S. venezuelae were expressed in the mutant strain. Feeding the representative 12-, 14-, and 16-membered ring macrolactones including 10-deoxymethynolide, narbonolide, and tylactone, respectively, to each mutant strain capable of producing TDP-d-quinovose or TDP-d-olivose resulted in the successful production of the corresponding quinovose- and olivose-glycosylated macrolides. In mutant strains where the DesVII/DesVIII glycosyltransferase–auxiliary protein pair was replaced by TylMII/TylMIII derived from Streptomyces fradiae, quinovosyl and olivosyl tylactone were produced; however, neither glycosylated 10-deoxymethynolide nor narbonolide were generated, suggesting that the glycosyltransferase TylMII has more stringent substrate specificity toward its aglycones than DesVII. These results demonstrate successful generation of structurally diverse hybrid macrolides using a S. venezuelae in vivo system and provide further insight into the substrate flexibility of glycosyltransferases. Won Seok Jung and Ah Reum Han contributed equally to this work.     

Identification of glucose kinase‐dependent and ‐independent pathways for carbon control of primary metabolism,development and antibiotic production in Streptomyces coelicolor by quantitative proteomics

Members of the soil‐dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient availability is a major determinant for the switch to development and antibiotic production in streptomycetes. Carbon catabolite repression (CCR), a main signalling pathway underlying this phenomenon, was so far considered fully dependent on the glycolytic enzyme glucose kinase (Glk). Here we provide evidence of a novel Glk‐independent pathway in Streptomyces coelicolor, using advanced proteomics that allowed the comparison of the expression of some 2000 proteins, including virtually all enzymes for central metabolism. While CCR and inducer exclusion of enzymes for primary and secondary metabolism and precursor supply for natural products is mostly mediated via Glk, enzymes for the urea cycle, as well as for biosynthesis of the γ‐butyrolactone Scb1 and the responsive cryptic polyketide Cpk are subject to Glk‐independent CCR. Deletion of glkA led to strong downregulation of biosynthetic proteins for prodigionins and calcium‐dependent antibiotic (CDA) in mannitol‐grown cultures. Repression of bldB, bldN, and its target bldM may explain the poor development of S. coelicolor on solid‐grown cultures containing glucose. A new model for carbon catabolite repression in streptomycetes is presented.     

Heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces venezuelae

Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. venezuelae and introduce S. venezuelae as a rapid heterologous expression system for the production of secondary metabolites.     

Recombinant production of <Emphasis Type="Italic">Streptococcus equisimilis</Emphasis> streptokinase by <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Background  

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.     

In Streptomyces lividans,acetyl‐CoA synthetase activity is controlled by O‐serine and Nɛ‐lysine acetylation

Protein acetylation is a rapid mechanism for control of protein function. Acetyl‐CoA synthetase (AMP‐forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK⁶¹⁰). S608 acetylation rendered SlAcs inactive and non‐acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD⁺‐dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin‐dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O‐Ser and N^?‐Lys acetylation.     

Development,Antibiotic Production,and Ribosome Assembly in Streptomyces venezuelae Are Impacted by RNase J and RNase III Deletion

RNA metabolism is a critical but frequently overlooked control element affecting virtually every cellular process in bacteria. RNA processing and degradation is mediated by a suite of ribonucleases having distinct cleavage and substrate specificity. Here, we probe the role of two ribonucleases (RNase III and RNase J) in the emerging model system Streptomyces venezuelae. We show that each enzyme makes a unique contribution to the growth and development of S. venezuelae and further affects the secondary metabolism and antibiotic production of this bacterium. We demonstrate a connection between the action of these ribonucleases and translation, with both enzymes being required for the formation of functional ribosomes. RNase III mutants in particular fail to properly process 23S rRNA, form fewer 70S ribosomes, and show reduced translational processivity. The loss of either RNase III or RNase J additionally led to the appearance of a new ribosomal species (the 100S ribosome dimer) during exponential growth and dramatically sensitized these mutants to a range of antibiotics.     

Transcript profiling of jasmonate‐elicited Taxus cells reveals a β‐phenylalanine‐CoA ligase

Plant cell cultures constitute eco‐friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate‐elicited Taxus baccata cell cultures by complementary DNA‐amplified fragment length polymorphism (cDNA‐AFLP) indicated a correlation between an extensive elicitor‐induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate‐induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl‐CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl‐CoA ligase that localizes to the cytoplasm and is able to convert β‐phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β‐phenylalanyl‐CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.     

The structure of DesR from Streptomyces venezuelae,a β‐glucosidase involved in macrolide activation

Antibiotics have, indeed, altered the course of human history as is evidenced by the increase in human life expectancy since the 1940s. Many of these natural compounds are produced by bacteria that, by necessity, must have efficient self‐resistance mechanisms. The methymycin/pikromycin producing species Streptomyces venezuelae, for example, utilizes β‐glucosylation of its macrolide products to neutralize their effects within the confines of the cell. Once released into the environment, these compounds are activated by the removal of the glucose moiety. In S. venezuelae, the enzyme responsible for removal of the sugar from the parent compound is encoded by the desR gene and referred to as DesR. It is a secreted enzyme containing 828 amino acid residues, and it is known to be a retaining glycosidase. Here, we describe the structure of the DesR/D ‐glucose complex determined to 1.4‐Å resolution. The overall architecture of the enzyme can be envisioned in terms of three regions: a catalytic core and two auxiliary domains. The catalytic core harbors the binding platform for the glucose ligand. The first auxiliary domain adopts a “PA14 fold,” whereas the second auxiliary domain contains an immunoglobulin‐like fold. Asp 273 and Glu 578 are in the proper orientation to function as the catalytic base and proton donor, respectively, required for catalysis. The overall fold of the core region places DesR into the GH3 glycoside hydrolase family of enzymes. Comparison of the DesR structure with the β‐glucosidase from Kluyveromyces marxianus shows that their PA14 domains assume remarkably different orientations.     

Heterologous production of clavulanic acid intermediates in <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis>

Heterologous expression can enhance production of diverse secondary metabolites by redirecting precursor pools towards compound of interest. In this study, Streptomyces venezuelae YJ028 was utilized as the heterologous host for the expression of four structural clavulanic acid biosynthesis genes, which encode carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2), and proclavaminate amidinohydrolase (pah2). These genes were cloned into pIBR25 expression vector containing ermE* promoter to generate pBS4. The cas2 gene was also cloned into pSET152 to generate pCas2. It was then integrated into the genome of S. venezuelae YJ028. Upon metabolite profiling of recombinant strains by ultra-pressure liquid chromatography-photodiode array (UPLC-PDA) and high resolution liquid chromatography quadruple time-offlight electrospray ionization mass spectrometry (HR-LC-QTOF-ESI/MS), the production of following clavulanic acid intermediates in S. venezuelae recombinant were confirmed: deoxygaunidinoproclavaminic acid, guanidinoproclavaminic acid, and dihydroclavaminic acid. This work demonstrates the production of β-lactam intermediates of the clavulanic acid pathway by heterologous expression in S. venezuelae YJ028.     

Metabolic engineering of <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> for malonyl-CoA biosynthesis to enhance heterologous production of polyketides

Using metabolic engineering, we developed Streptomyces venezuelae YJ028 as an efficient heterologous host to increase the malonyl-CoA pool to be directed towards enhanced production of various polyketides. To probe the applicability of newly developed hosts in the heterologous production of polyketides, we expressed type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase, in these hosts. Flaviolin production was doubled by expression of acetyl-CoA carboxylase (ACCase) and 4-fold by combined expression of ACCase, metK1-sp and afsR-sp. Thus, the newly developed Streptomyces venezuelae YJ028 hosts produce heterologous polyketides more efficiently than the parent strain.     

Characterization and engineering of the ethylmalonyl-CoA pathway towards the improved heterologous production of polyketides in Streptomyces venezuelae

Streptomyces venezuelae has an inherent advantage as a heterologous host for polyketide production due to its fast rate of growth that cannot be endowed easily through metabolic engineering. However, the utility of S. venezuelae as a host has been limited thus far due to its inadequate intracellular reserves of the (2S)-ethylmalonyl-CoA building block needed to support the biosynthesis of polyketides preventing the efficient production of the desired metabolite, such as tylactone. Here, via precursor supply engineering, we demonstrated that S. venezuelae can be developed into a more efficient general heterologous host for the quick production of polyketides. We first identified and functionally characterized the ethylmalonyl-CoA pathway which plays a major role in supplying the (2S)-ethylmalonyl-CoA extender unit in S. venezuelae. Next, S. venezuelae was successfully engineered to increase the intracellular ethylmalonyl-CoA concentration by the deletion of the meaA gene encoding coenzyme B₁₂-dependent ethylmalonyl-CoA mutase in combination with ethylmalonate supplementation and was engineered to upregulate the expression of the heterologous tylosin PKS by overexpression of the pathway specific regulatory gene pikD. Thus, a dramatic increase (～10-fold) in tylactone production was achieved. In addition, the detailed insights into the role of the ethylmalonyl-CoA pathway, which is present in most streptomycetes, provides a general strategy to increase the ethylmalonyl-CoA supply for polyketide biosynthesis in the most prolific family of polyketide-producing bacteria.     

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α‐position and peptides on N‐terminal position

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N‐α or ε‐acetyl‐L‐lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε‐position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N‐acylation on the α‐position of the lysine or of the amino‐acid in N‐terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N‐α‐acetyl‐L‐lysine could be attributed to the product of SAM23877_1734 gene.     

Stereoselective synthesis of fully protected (2S,4S,6S)‐2‐amino‐6‐hydroxy‐4‐methyl‐8‐oxodecanoic acid (AHMOD)

The stereocontrolled synthesis of fully protected (2S,4S,6S)‐2‐amino‐6‐hydroxy‐4‐methyl‐8‐oxodecanoic acid was accomplished using a glutamate derivative as starting material. The key steps of this stereochemical synthetic pathway involved an Evans asymmetric alkylation, a Sharpless asymmetric epoxidation, and a Grignard reaction. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Effects and statistical optimization of fermentation conditions on growth and poly (vinyl alcohol)-degrading enzyme production of Streptomyces venezuelae GY1

The effects of environmental conditions, including temperature, pH and dissolved oxygen, on growth and production of polyvinyl alcohol (PVA)-degrading enzymes of the newly-isolated strain Streptomyces venezuelae GY1 were investigated. The medium composition for strain GY1 was studied first by single factorial design and then optimized using a central composite design. PVA with high saponification is better for growth of, and PVA-degrading enzyme production by S. venezuelae GY1 compared with PVA with low saponification, in contrast with the characteristics of other bacteria producing PVA-degrading enzymes. The optimal temperature and initial pH for production of PVA-degrading enzyme by strain GY1 was 30°C and 7.0, respectively. The optimal medium composition for PVA-degrading enzyme production is: 1.01 g L^?1 of PVA1799, 0.307 g L^?1 of NaNO₃ and 0.512 g L^?1 of MgSO₄?7H₂O.     

Interactions of actinomycetes with Macrophomina phaseoli (Maubl.) Ashby; The cause of root rot of cotton

Macrophomina phaseoli, the cause of root rot of cotton, was inhibited byStreptomyces albus, S. griseus andS. noursei in agar culture.S. aureofaciens, S. flaveolus, S. rimosus, S. scabies andS. venezuelae were non-antagonistic. Only the antagonisticStreptomyces were found to reduceMacrophomina infection on cotton seedlings in soil without any deleterious effect on cotton growth.     

Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> ISP5230

Consensus amino acid sequences of FADH₂-dependent bacterial halogenases were used to design PCR primers amplifying a halogenase gene fragment from the chloramphenicol producer Streptomyces venezuelae ISP5230. The sequence-specific degenerate primers (MPF1 and MPR2) were used with a touchdown PCR procedure in the first PCR-assisted cloning of a halogenase gene fragment. In the region of the 290-bp PCR product containing the reverse primer, the deduced amino acid sequence exhibited characteristics of a β–α–β fold present in FAD-binding sites of certain monooxygenases. When used to probe Southern blots of restriction-enzyme-digested DNA, the [α-³²P]dCTP-labeled PCR product hybridized specifically with DNA fragments from genomic DNA of S. venezuelae ISP5230. Primers MPF1 and MPR2 also allowed amplification by PCR of approximately 290-bp DNA fragments from several other streptomycetes. The fragments from Streptomyces aureofaciens NRRL2209 and Streptomyces coelicolor A3(2) showed sequence identity with halogenase genes from these species. Thus, the PCR primers are of potential value for amplification and subsequent isolation of actinomycete halogenase genes. Journal of Industrial Microbiology & Biotechnology (2002) 29, 1–5 doi:10.1038/sj.jim.7000263 Received 25 June 2001/ Accepted in revised form 02 April 2002     

Are you talking to me? A possible role for γ‐butyrolactones in interspecies signalling

The secreted γ‐butyrolactone signalling molecule SVB1 regulates the biosynthesis of jadomycin in Streptomyces venezuelae. Interestingly, this molecule is identical to SCB3, a secreted regulator of secondary metabolism in Streptomyces coelicolor. This is a departure for this class of signalling molecules as there are no previous reports of identical signalling molecules produced in different species. One implication of this work is that different species of bacteria could use shared extracellular signals to co‐ordinate secondary metabolism when and if it is advantageous to do so.     

Specific amino acid substitutions in β strand S2 of FtsZ cause spiraling septation and impair assembly cooperativity in Streptomyces spp.

Bacterial cell division is orchestrated by the Z ring, which is formed by single‐stranded treadmilling protofilaments of FtsZ. In Streptomyces, during sporulation, multiple Z rings are assembled and lead to formation of septa that divide a filamentous hyphal cell into tens of prespore compartments. We describe here mutant alleles of ftsZ in Streptomyces coelicolor and Streptomyces venezuelae that perturb cell division in such a way that constriction is initiated along irregular spiral‐shaped paths rather than as regular septa perpendicular to the cell length axis. This conspicuous phenotype is caused by amino acid substitutions F37I and F37R in β strand S2 of FtsZ. The F37I mutation leads, instead of regular Z rings, to formation of relatively stable spiral‐shaped FtsZ structures that are capable of initiating cell constriction. Further, we show that the F37 mutations affect the polymerization properties and impair the cooperativity of FtsZ assembly in vitro. The results suggest that specific residues in β strand S2 of FtsZ affect the conformational switch in FtsZ that underlies assembly cooperativity and enable treadmilling of protofilaments, and that these features are required for formation of regular Z rings. However, the data also indicate FtsZ‐directed cell constriction is not dependent on assembly cooperativity.