Metabolic engineering of Nocardia sp. CS682 for enhanced production of nargenicin A1.

A number of secondary metabolites having therapeutic importance have been reported from the genus Nocardia. One of the polyketide antibiotic compounds isolated from Nocardia is nargenicin A(1). Recently, nargenicin A(1) has been isolated from Nocardia sp. CS682, a new Nocardia strain isolated from soil in Jeonnam, South Korea. It possesses strong antibacterial activity against methicillin-resistant Staphylococcus aureus. In this study, we applied a metabolic engineering approach based on recombinant DNA technology in order to boost the production of nargenicin A(1) from Nocardia sp. CS682. Initially, we optimized the transformation of this new strain by electroporation method. Heterologous expression of S-adenosylmethionine synthetase (MetK1-sp) in Nocardia sp. CS682 enhanced the production of nargenicin A(1) by about 2.8 times due to transcriptional activation of biosynthetic genes as revealed by reverse transcription polymerase chain reaction analysis. Similarly, expression of acetyl-CoA carboxylase genes improved nargenicin A(1) production by about 3.8 times in Nocardia sp. ACC18 compared to that in Nocardia sp. CS682 and Nocardia sp. NV18 by increasing precursor pool. Thus, enhanced production of nargenicin A(1) from Nocardia sp. CS682 can be achieved by expression of transcriptional activator genes and precursor genes from Streptomyces strains.

Enhancement of pradimicin production in Actinomadura hibisca P157-2 by metabolic engineering.

Actinomadura hibisca P157-2 produces potent antifungal antibiotic pradimicins. To enhance pradimicin production, ACCase from Streptomyces coelicolor and two regulatory genes metK1-sp and afsR-sp from Streptomyces peuticus were overexpressed into A. hibisca using an integration vector pSET152 under the control of the strong ermE* promoter. The constructed plasmids pACC152, pSAM152, pAFS152, pSA152 and pASA152 were transformed into A. hibisca by the conjugal method. The recombinant strains A. hibisca ACC, A. hibisca SAM, A. hibisca AFS, A. hibisca SA and A. hibisca ASA produced greater amounts of pradimicin than the parental strain with an increment of 3-fold, 2.1-fold, 2.8-fold, 3.4-fold, and 4.5-fold respectively. To increase the acetyl-coA pool, the strains were fed methyl oleate and acetate as carbon sources. The production was increased in wild-type A. hibisca, A. hibisca ACC and A. hibisca ASA by 2.2-fold, 4.12-fold and 5.98-fold respectively, with oleate and by 1.12-fold, 3.8-fold and 5.38-fold respectively with acetate. The strain A. hibisca ASA remained the best strain for the production of pradimicin. The higher transcriptional levels of structural genes in the strains harboring metK1-sp and afsR-sp compared to the wild-type strain were consistent with the enhanced production.

Proteomic approach to enhance doxorubicin production in panK-integrated Streptomyces peucetius ATCC 27952.

Biosynthesis of polyketide compounds depends upon the starter and extender units of coenzyme A derivatives of carboxylic acids present in the host organism. To increase the coenzyme A (CoA) pool, pantothenate kinase (panK) gene from Escherichia coli was integrated into S. peucetius ATCC 27952 (panK-integrated strain, BG200), which resulted in increase in aglycone polyketide ε-rhodomycinone (RHO), but decrease in the desired product, i.e., doxorubicin (DXR). To reduce RHO accumulation by synthesizing daunorubicin (DNR) from RHO more efficiently, glycosyltransferase (dnrQS) was overexpressed (pIBR25::dnrQS in panK-integrated strain, BG201). However, DnrQS overexpression still resulted in less production of DXR compared with the parental strain. To understand the results in detail by investigating the proteome changes in the panK-integrated strain, two-dimensional (2D) gel electrophoresis was performed. Among the several proteins that are up- or downregulated in BG200, efflux protein DrrA was our main target of interest, because it is directly related to DXR/DNR production in S. peucetius. DXR transporter DrrAB was additionally introduced in BG200 to enhance secretion of toxic DXR. Compared with S. peucetius ATCC 27952, BG204 (pIBR25::drrAB in panK-integrated strain), produced two times higher amount of DXR, which is 9.4-fold higher than that of panK-integrated strain BG200. The results show that the proteomic approach is quite useful in host development of Streptomyces and understanding cell physiology for antibiotic production.

Genetic engineering approach for the production of rhamnosyl and allosyl flavonoids from Escherichia coli.

The main functions of glycosylation are stabilization, detoxification and solubilization of substrates and products. To produce glycosylated products, Escherichia coli was engineered by overexpression of TDP-L-rhamnose and TDP-6-deoxy-D-allose biosynthetic gene clusters, and flavonoids were glycosylated by the overexpression of the glycosyltransferase gene from Arabidopsis thaliana. For the glycosylation, these flavonoids (quercetin and kaempferol) were exogenously fed to the host in a biotransformation system. The products were isolated, analyzed and confirmed by HPLC, LC/MS, and ESI-MS/MS analyses. Several conditions (arabinose, IPTG concentration, OD(600), substrate concentration, incubation time) were optimized to increase the production level. We successfully isolated approximately 24 mg/L 3-O-rhamnosyl quercetin and 12.9 mg/L 3-O-rhamnosyl kaempferol upon feeding of 0.2 mM of the respective flavonoids and were also able to isolate 3-O-allosyl quercetin. Thus, this study reveals a method that might be useful for the biosynthesis of rhamnosyl and allosyl flavonoids and for the glycosylation of related compounds.

Enhancement of clavulanic acid production by expressing regulatory genes in gap gene deletion mutant of Streptomyces clavuligerus NRRL3585.

Streptomyces clavuligerus NRRL3585 produces a clinically important ss-lactamase inhibitor, clavulanic acid (CA). In order to increase the production of CA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was deleted in S. clavuligerus NRRL3585 to overcome the limited glyceraldehyde-3-phosphate pool; the replicative and integrative expressions of ccaR (specific regulator of the CA biosynthetic operon) and claR (Lys-type transcriptional activator) genes were transformed together into deleted mutant to improve clavulanic acid production. We constructed two recombinant plasmids to enhance the production of CA in the gap1 deletion mutant of S. clavuligerus NRRL3585: pHN11 was constructed for overexpression of ccaR-claR, whereas pHN12 was constructed for their chromosomal integration. Both pHN11 and pHN12 transformants enhanced the production of CA by 2.59-fold and 5.85-fold, respectively, compared to the gap1 deletion mutant. For further enhancement of CA, we fed the pHN11 and pHN12 transformants ornithine and glycerol. Compared to the gap1 deletion mutant, ornithine increased CA production by 3.24- and 6.51-fold in the pHN11 and pHN12 transformants, respectively, glycerol increased CA by 2.96- and 6.21-fold, respectively, and ornithine and glycerol together increased CA by 3.72- and 7.02-fold, respectively.

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.

A novel Ca2+-dependent phospholipase D from Streptomyces tendae, possessing only hydrolytic activity.

An extracellular phospholipase D (PLD(St)) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLD(St) was estimated to be approximately 43 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60 degrees C, and the enzyme was stable at or below 60 degrees C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca(2+), and the maximum activity was at 2 mM CaCl(2). The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 micromol min(-1) mg(-1), respectively. More importantly, PLD(St) could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol and ethanolamine, which have been extensively used to evaluate the activity. The result strongly suggests that PLD( St ) does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLD(St) may therefore be a novel type of PLD enzyme.

Metabolic engineering of Escherichia coli for the biological synthesis of 7-O-xylosyl naringenin.

Flavonoids are a group of polyphenolic compounds that have been recognized as important due to their physiological and pharmacological roles and their health benefits. Glycosylation of flavonoids has a wide range of effects on flavonoid solubility, stability, and bioavailability. We previously generated the E. coli BL21 (DE3) Deltapgi host by deleting the glucose-phosphate isomerase (Pgi) gene in E. coli BL21 (DE3). This host was further engineered for whole-cell biotransformation by integration of galU from E. coli K12, and expression of calS8 (UDP-glucose dehydrogenase) and calS9 (UDP-glucuronic acid decarboxylase) from Micromonospora echinospora spp. calichensis and arGt-4 (7-O-glycosyltransferase) from Arabidopsis thaliana to form E. coli (US89Gt-4), which is expected to produce glycosylated flavonoids. To test the designed system, the engineered host was fed with naringenin as a substrate, and naringenin 7-O-xyloside, a glycosylated naringenin product, was detected. Product was verified by HPLCLC/MS and ESI-MS/MS analyses. The reconstructed host can be applied for the production of various classes of glycosylated flavonoids.

Improved squalene production via modulation of the methylerythritol 4-phosphate pathway and heterologous expression of genes from Streptomyces peucetius ATCC 27952 in Escherichia coli.

Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.

Mass spectrometric screening of transcriptional regulators involved in antibiotic biosynthesis in Streptomyces coelicolor A3(2).

DNA-affinity capture assay (DACA) coupled with liquid chromatography-tandem mass spectrometry analysis was applied to identify the transcriptional regulators involved in the biosynthesis of actinorhodin (Act) and undecylprodigiosin (Red) in Streptomyces coelicolor. The aim of this analysis was to determine the specific transcriptional regulators binding to the promoter region of actII-ORF4 or redD. The results of the DACA, as the first screening tool, identified eight proteins, including AdpA, as candidate regulators binding to those promoter regions. To show the direct physical relationship between the regulators and promoters, we purified four regulators over-expressed in soluble form in Escherichia coli and subjected these to an electrophoretic mobility shift assay (EMSA). The results of the EMSA appeared to be compatible with the DACA results for those regulators. A null mutant was also constructed for one of these regulators, SCO6008, which showed early Red production and quite delayed Act production in R5(-) medium. These observations suggest that DACA can be widely used to find new regulators and that the regulator SCO6008 may be involved in antibiotic production through its binding to the redD promoter.

Characterization of CalS9 in the biosynthesis of UDP-xylose and the production of xylosyl-attached hybrid compound.

The gene cluster of calicheamicin contains calS9, which encodes UDP-GlcA decarboxylase that converts UDP-GlcA to UDP-xylose. calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21 (DE3) to characterize its putative function. The reaction product was analyzed by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by gene replacement to generate Streptomyces sp. GerSM2 mutant, which was unable to produce dihydrochalcomycin. calS7, calS8, and calS9 UDP-xylose biosynthetic genes were cloned in an integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in Streptomyces sp. GerSM2. Finally, novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was isolated and analyzed by HPLC and liquid chromatography-mass spectrometry.

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.

Squalene-hopene cyclase (Spterp25) from Streptomyces peucetius: sequence analysis, expression and functional characterization.

Squalene-hopene cyclase, which catalyzes the complex cyclization of squalene to the pentacyclic triterpene, hopene, is a key enzyme in the biosynthesis of hopanoids. The deduced amino acid sequence of the Streptomyces peucetius gene (spterp25) had significant similarity to other prokaryotic squalene-hopene cyclases. Like other triterpene cyclases, the S. peucetius squalene-hopene cyclase contains eight so-called QW-motifs with an aspartate-rich domain. The 2,025-bp squalene-hopene cyclase-encoding gene was expressed in Escherichia coli BL21(DE3)pLySs, and the in vitro activity of the recombinant cyclase was demonstrated using purified membrane protein. The cyclization product hopene was identified by gas chromatography/mass spectrometry (GC/MS).

A novel low molecular weight phospholipase D from Streptomyces sp. CS684.

With the aim of isolating economically viable enzymes from a microbial source, a novel phospholipase D (PLD) was purified from Streptomyces sp. CS684 (PLD(684)). PLD(684) had molecular weight of 29 kDa, which makes it the second smallest PLD reported so far. The enzyme activity was optimum at pH 6 and 45 degrees C, and enhanced by various detergents. It was stable from pH 7 to 9 and at or below 45 degrees C when assayed after 40 h and 2h, respectively. The K(m) and V(max) values for phosphatidylcholine were 1.16 mM and 1453.74 micromol min(-1)mg(-1), respectively. It catalyzed the transphosphatidylation of glycerol, but not that of l-serine, myo-inositol or ethanolamine. Low molecular weight PLD(684) with transphosphatidylation activity may be utilized in the industrial production of rare and commercially important phospholipids.

Cloning and functional characterization of the germacradienol synthase (spterp13) from Streptomyces peucetius ATCC 27952.

Sequence analysis of the metabolically rich genome of Streptomyces peucetius ATCC 27952 revealed a 2,199 bp sesquiterpene alcohol (germacradienol) synthase-encoding gene from the germacradienol synthase/terpene cyclase gene cluster. The gene was named spterp13, and its putative function is as a germacradienol synthase/terpene cyclase. The amino acid sequence of Spterp13 shows 66% identity with SAV2163 (GeoA) from S. avermitilis MA- 4680 and 65% identity with SCO6073 from S. coelicolor A3(2), which produces germacradienol/geosmin. The fulllength recombinant protein was heterologously expressed as a his-tagged fusion protein in Escherichia coli, purified, and shown to catalyze the Mg2+-dependent conversion of farnesyl diphosphate to the germacradienol, which was verified by gas chromatography/mass spectrometry.

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.

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.

Enhancement of clavulanic acid by replicative and integrative expression of ccaR and cas2 in Streptomyces clavuligerus NRRL3585.

Clavulanic acid (CA) is an inhibitor of beta-lactamase that is produced from Streptomyces clavuligerus NRRL3585 and is used in combination with other antibiotics in clinical treatments. In order to increase the production of CA, the replicative and integrative expressions of ccaR (encoding for a specific regulator of the CA biosynthetic operon) and cas2 (encoding for the rate-limiting enzyme in the CA biosynthetic pathway) were applied. Six recombinant plasmids were designed for this study. The pIBRHL1, pIBRHL3, and pIBRHL13 were constructed for overexpression, whereas pNQ3, pNQ2, and pNQ1 were constructed for chromosomal integration with ccaR, cas2, and ccaR-cas2, respectively. All of these plasmids were transformed into S. clavuligerus NRRL3585. CA production in transformants resulted in a significantly enhanced amount greater than that of the wild type, a 2.25-fold increase with pIBRHL1, a 9.28-fold increase with pNQ3, a 5.06-fold increase with pIBRHL3, a 2.93-fold increase with pNQ2 integration, a 5.79-fold increase with pIBRHL13, and a 23.8-fold increase with pNQ1. The integrative pNQ1 strain has been successfully applied to enhance production.

Enhanced proteomic analysis of Streptomyces peucetius cytosolic protein using optimized protein solubilization protocol.

Improvements in the dissolution of proteins in two-dimensional gel electrophoresis have greatly advanced the ability to analyze the proteomes of microorganisms under a wide variety of physiological conditions. This study examined the effect of various combinations of chaotropic agents, a reducing agent, and a detergent on the dissolution of the Streptomyces peucetius cytosolic proteins. The use of urea alone in a rehydration buffer as a chaotropic agent gave the proteome a higher solubility than any of the urea and thiourea combinations, and produced the highest resolution and clearest background in two-dimensional gel electrophoresis. Two % CHAPS, as a detergent in a rehydration buffer, improved the protein solubility. After examining the effect of several concentrations of reducing agent, 50 mM DTT in a rehydration buffer was found to be an optimal condition for the proteome analysis of Streptomyces. Using this optimized buffer condition, more than 2,000 distinct and differentially expressed soluble proteins could be resolved using two-dimensional gel electrophoresis with a pI ranging from 4-7. Under this optimized condition, 15 novel small proteins with low-level expression, which could not be analyzed under the non-optimized conditions, were identified. Overall, the optimized condition helped produce a better reference gel for Streptomyces peucetius.

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.