Exploration of cryptic organic photosensitive compound as Zincphyrin IV in Streptomyces venezuelae ATCC 15439.

Zincphyrin IV is a potential organic photosensitizer which is of significant interest for applications in biomedicine, materials science, agriculture (as insecticide), and chemistry. Most studies on Zincphyrin are focused on Zincphyrin III while biosynthesis and application of Zincphyrin IV is comparatively less explored. In this study, we explored Zincphyrin IV production in Streptomyces venezuelae ATCC 15439 through combination of morphology engineering and "One strain many compounds" approach. The morphology engineering followed by change in culture medium led to activation of cryptic Zincphyrin IV biosynthetic pathway in S. venezuelae with subsequent detection of Zincphyrin IV. Morphology engineering applied in S. venezuelae increased the biomass from 7.17 to 10.5 mg/mL after 48 h of culture. Moreover, morphology of engineered strain examined by SEM showed reduced branching and fragmentation of mycelia. The distinct change in color of culture broth visually demonstrated the activation of the cryptic biosynthetic pathway in S. venezuelae. The production of Zincphyrin IV was found to be initiated after overexpression ssgA, resulting in the increase in titer from 4.21 to 7.54 µg/mL. Furthermore, Zincphyrin IV demonstrated photodynamic antibacterial activity against Bacillus subtilis and photodynamic anticancer activity against human ovarian carcinoma cell lines.

Functional analysis of the GlcP promoter in Streptomyces peucetius var. caesius.

In Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. Glucose is mainly transported by GlcP, a membrane protein encoded by glcp. In Streptomyces coelicolor, this protein is encoded by sco5578. However, there is little information about the physiology of the GlcP promoter in Streptomyces. The aim of the present work was to clone and perform a functional analysis of the sp7066 promoter (ortholog of sco5578) from Streptomyces peucetius var. caesius. Hydrophobicity and cellular location analysis of the putative amino acid sequence of the cloned gene predicted SP7066 would be a membrane protein with a topology of six plus six transmembrane segments interrupted by a large cytoplasmic loop. In silico analysis of the upstream region of the sp7066 transcription initiation site predicted the sequences 5'-AGGAATAGT-3' and 5'-TTGACT-3' for regions -10 and -35 of sp7066 promoter. To reflect sp7066 expression, the promoter sequence was amplified, subcloned, and fused to the egfp reporter gene. Immunoblot analysis revealed that D-glucose and its analog 2-deoxyglucose were able to induce sp7066 expression. This effect was not modified by the presence of equimolar concentrations of D-galactose or N-acetylglucosamine. No expression of egfp was detected with the use of other carbon sources such as L-arabinose, D-fructose, and glycerol. Based on these analyses, we conclude that D-glucose is a preferred carbon source in S. peucetius var. caesius and that the sp7066 expression product, a putative non-PTS glucose permease, likely is a H+/symporter, localized to the membrane, and shows a strong specificity for D-glucose for inducing expression.

Heterologous production of spectinomycin in Streptomyces venezuelae by exploiting the dTDP-D-desosamine pathway.

Spectinomycin is an aminoglycoside antibiotic composed of actinamine and actinospectose, which are fused together by a putative glycosyltransferase, SpcG, during spectinomycin biosynthesis. Although previous studies have revealed the involvement of SpcA (myo-inositol monophosphatase), SpcB (dehydrogenase), SpcS2 (aminotransferase), and SpcM (methyltransferase) in the biosynthesis of actinamine, heterologous biosynthesis of spectinomycin via actinospectose has not been clearly elucidated. In this study, Streptomyces venezuelae was utilized as a source of dTDP-actinospectose from the pikromycin biosynthetic desosamine sugar pathway, and a recombinant vector, pSM5, carrying spcA, spcB, spcS2, spcM, and spcG was inserted into S. venezuelae. The formation of dTDP-spectinose was suspected through the use of dehydrogenase in the S. venezuelae chromosome. Herewith, the genetically engineered strain, S. venezuelae SM5, effectively produced up to 89.2mg/L in optimized medium. However, pSM5 in S. venezuelae YJ003, a dTDP-actinospectose-deficient strain, did not produce spectinomycin. This result demonstrates the use of a dTDP-actinospectose precursor produced in the desosamine pathway for heterologous production of spectinomycin in S. venezuelae.

Development of a Streptomyces venezuelae-based combinatorial biosynthetic system for the production of glycosylated derivatives of doxorubicin and its biosynthetic intermediates.

Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and l-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4'-epi-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone ε-rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of Streptomyces venezuelae mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from ε-rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-epi-l-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the S. venezuelae-based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.

Metabolic engineering of Streptomyces venezuelae 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.

Identification and functional characterization of an afsR homolog regulatory gene from Streptomyces venezuelae ATCC 15439.

Sequencing analysis of a 5-kb DNA fragment from Streptomyces venezuelae ATCC 15439 revealed the presence of one 3.1-kb open reading frame (ORF), designated afsRsv. The deduced product of afsR-sv (1,056 aa) was found to have high homology with the global regulatory protein AfsR. Homology-based analysis showed that afsR-sv represents a transcriptional activator belonging to the Streptomyces antibiotic regulatory protein (SARP) family that includes an Nterminal SARP domain containing a bacterial transcriptional activation domain (BTAD), an NB-ARC domain, and a Cterminal tetratricopeptide repeat domain. Gene expression analysis by reverse transcriptase PCR (RT-PCR) demonstrated the activation of transcription of genes belonging to pikromycin production, when afsR-sv was overexpressed in S. venezuelae. Heterologous expression of the afsR-sv in different Streptomyces strains resulted in increased production of the respective antibiotics, suggesting that afsR-sv is a positive regulator of antibiotics biosynthesis.

Heterologous production of epothilones B and D in Streptomyces venezuelae.

Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 microg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 microg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.

Heterologous production and detection of recombinant directing 2-deoxystreptamine (DOS) in the non-aminoglycoside-producing host Streptomyces venezuelae YJ003.

2-Deoxystreptamine is a core aglycon that is vital to backbone formation in various aminoglycosides. This core structure can be modified to develop hybrid types of aminoglycoside antibiotics. We obtained three genes responsible for 2-deoxystreptamine production, neo7, neo6, and neo5, which encode 2-deoxy-scyllo-inosose synthase, L-glutamine: 2-deoxy-scyllo-inosose aminotransferase, and dehydrogenase, respectively, from the neomycin gene cluster. These genes were cloned into pIBR25, a Streptomyces expression vector, resulting in pNDOS. The recombinant pNDOS was transformed into a non-aminoglycoside-producing host, Streptomyces venezuelae YJ003, for heterologous expression. Based on comparisons of the retention time on LC-ESI/MS and ESIMS data with those of the 2-deoxystreptamine standard, a compound produced by S. venezuelae YJ003/pNDOS was found to be 2-deoxystreptamine.

Heterologous expression of metK1-sp and afsR-sp in Streptomyces venezuelae for the production of pikromycin.

Two regulator genes, metK1-sp and afsR-sp, from Streptomyces peucetius ATCC 27952 were heterologously expressed in S. venezuelae ATCC 15439, to produce 14-membered pikromycin antibiotics. The production of pikromycin was increased by 1.6-fold and 2.6-fold by the expression of metK1-sp and afsR-sp, respectively. The overexpression of metK1-sp and afsR-sp in S. venezuelae stimulated the expression of the pathway-specific regulatory genes, pikD and ketosynthase, as demonstrated by RT-PCR. The elevated transcripts of the pikD and ketosynthase genes were consistent with the enhanced production of pikromycin.

Metabolic engineering of noviose: heterologous expression of novWUS and generation of a new hybrid antibiotic, noviosylated 10-deoxymethynolide/narbonolide, from Streptomyces venezuelae YJ003-OTBP1.

NovW, novU and novS genes have been characterized as dTDP-4-keto-6-deoxy-D-glucose 3-epimerase, C-5 methyltransferase and dTDP-glucose 4-ketoreductase, respectively involved in noviose biosynthetic pathway. We have cloned and expressed the Streptomyces spheroids novWUS genes in S. venezuelae YJ003-OTBP1. This established the function of novWUS and, at the same time, it also proved that the noviosyl derivative of 10-deoxymethynolide(2)/narbonolide(4) obtained from S. venezuelae YJ003-OTBP1 is a novel hybrid antibiotic.

Genetically engineered biosynthesis of macrolide derivatives including 4-amino-4,6-dideoxy-L-glucose from Streptomyces venezuelae YJ003-OTBP3.

Two sugar biosynthetic cassette plasmids were used to direct the biosynthesis of a deoxyaminosugar. The pOTBP1 plasmid containing TDP-glucose synthase (desIII), TDP-glucose-4,6-dehydratase (desIV), and glycosyltransferase (desVII/desVIII) was constructed and transformed into S. venezuelae YJ003, a strain in which the entire gene cluster of desosamine biosynthesis is deleted. The expression plasmid pOTBP3 containing 4-aminotransferase (gerB) and 3,5-epimerase (orf9) was transformed again into S. venezuelae YJ003- OTBP1 to obtain S. venezuelae YJ003-OTBP3 for the production of 4-amino-4,6-dideoxy-L-glucose derivatives. The crude extracts obtained from S. venezuelae ATCC 15439, S. venezuelae YJ003, and S. venezuelae YJ003-OTBP3 were further analyzed by TLC, bioassay, HPLC, ESI/MS, LC/MS, and MS/MS. The results of our study clearly shows that S. venezuelae YJ003-OTBP3 constructs other new hybrid macrolide derivatives including 4-amino-4,6-dideoxy-L-glycosylated YC-17 (3, [M+ Na+] m/z=464.5), methymycin (4, m/z=480.5), novamethymycin (6, m/z=496.5), and pikromycin (5, m/z=536.5) from a 12- membered ring aglycon (10-deoxymethynolide, 1) and 14-membered ring aglycon (narbonolide, 2). These results suggest a successful engineering of a deoxysugar pathway to generate novel hybrid macrolide derivatives, including deoxyaminosugar.

Heterologous expression of the kanamycin biosynthetic gene cluster (pSKC2) in Streptomyces venezuelae YJ003.

The pSKC2 cosmid, which has 32 kb and 28 open-reading frames, was isolated from Streptomyces kanamyceticus ATCC12853 as the gene cluster of kanamycin. This gene cluster includes the minimal biosynthetic genes of kanamycin with the resistance and regulatory genes. It was heterologously expressed in Streptomyces venezuelae YJ003, which has the advantage of fast growth, good efficiency of the transformation host, and rapid production of the aminoglycosides antibiotic. The isolated compound was analyzed by electrospray ionization-mass spectrometry, liquid chromatography-mass spectrometry, high-performance liquid chromatography, and tandem mass spectrometry and shows a molecular weight of 485 as kanamycin A.

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.

New olivosyl derivatives of methymycin/pikromycin from an engineered strain of Streptomyces venezuelae.

A mutant strain of Streptomyces venezuelae was engineered by deletion of the entire gene cluster related to biosynthesis of the endogenous deoxysugar (TDP-D-desosamine) and replacement with genes required for biosynthesis of an intermediate sugar (TDP-4-keto-6-deoxy-D-glucose) or an exogenous sugar (TDP-D-olivose), from the oleandomycin and urdamycin deoxysugar pathways. The 'sugar-flexible' glycosyltransferase (DesVII) was able to attach the intermediate sugar and the new sugar to both 12- and 14-membered macrolactones thus producing quinovose or olivose glycosylated 10-deoxymethynolide and narbonolide, respectively. In addition, hydroxylated analogs of the new metabolites were detected. These results demonstrate a successful attempt of engineering the deoxysugar pathway for generation of novel hybrid macrolide antibiotics.