Biosynthesis of Rhamnosylated Anthraquinones in Escherichia coli.

Rhamnose is a naturally occurring deoxysugar present as a glycogenic component of plant and microbial natural products. A recombinant mutant Escherichia coli strain was developed by overexpressing genes involved in the TDP-L-rhamnose biosynthesis pathway of different bacterial strains and Saccharothrix espanaensis rhamnosyl transferase to conjugate intrinsic cytosolic TDP-L-rhamnose with anthraquinones supplemented exogenously. Among the five anthraquinones (alizarin, emodin, chrysazin, anthrarufin, and quinizarin) tested, quinizarin was biotransformed into a rhamoside derivative with the highest conversion ratio by whole cells of engineered E. coli. The quinizarin glycoside was identified by various chromatographic and spectroscopic analyses. The anti-proliferative property of the newly synthesized rhamnoside, quinizarin-4-O-α-L-rhamnoside, was assayed in various cancer cells.

One-Pot Multienzyme Cofactors Recycling (OPME-CR) System for Lactose and Non-natural Saccharide Conjugated Polyphenol Production.

A one-pot multienzyme cofactors recycling (OPME-CR) system was designed for the synthesis of UDP-α-d-galactose, which was combined with LgtB, a ß-(1,4) galactosyltransferase from Neisseria meningitidis, to modify various polyphenol glycosides. This system recycles one mole of ADP and one mole of UDP to regenerate one mole of UDP-α-d-galactose by consuming two moles of acetylphosphate and one mole of d-galactose in each cycle. The ATP additionally used to generate UDP from UMP was also recycled at the beginning of the reaction. The engineered cofactors recycling system with LgtB efficiently added a d-galactose unit to a variety of sugar units such as d-glucose, rutinose, and 2-deoxy-d-glucose. The temperature, pH, incubation time, and divalent metal ions for the OPME-CR system were optimized. The maximum number of UDP-α-d-galactose regeneration cycles (RCmax) was 18.24 by fed batch reaction. The engineered system generated natural and non-natural polyphenol saccharides efficiently and cost-effectively.

Overexpression of a pathway specific negative regulator enhances production of daunorubicin in bldA deficient Streptomyces peucetius ATCC 27952.

The dnrO gene is the first regulator to be activated in the daunorubicin (DNR) biosynthesis pathway of Streptomyces peucetius ATCC 27952. DnrO is known for its self-repression capability while it activates rest of the DNR biosynthesis pathway through cascades of regulatory events. S. peucetius was found to contain no functional copy of bldA-tRNA while a detailed examination of dnrO codons reveals the presence of TTA codon, which is rarely encoded by bldA-tRNA. Therefore, for evaluating the role of dnrO in DNR production, multiple engineered strains of S. peucetius were generated by heterologously expressing bldA, dnrO and combination of bldA and dnrO. Using these strains, the effects of heterologously expressed bldA and overexpressed dnrO were evaluated on pathway specific regulators, mycelial densities and production of DNR. The results showed that the transcription level of dnrO and master regulator dnrI, was found to be elevated in bldA containing strain in comparison to dnrO overexpressed strain. The bldA containing strain produces 45.7% higher DNR than bldA deficient wild type strain from culture broth with OD600 of 1.45 at 72h. Heterologous expression of bldA-tRNA is accounted for increased transcription levels of the DNR pathway specific regulators and enhanced DNR production.

Hydroxylation of long chain fatty acids by CYP147F1, a new cytochrome P450 subfamily protein from Streptomyces peucetius.

Cytochrome P450 (CYP) 147F1 from Streptomyces peucetius is a new CYP subfamily of that has been identified as ω-fatty acid hydroxylase. We describe the identification of CYP147F1 as a fatty acid hydroxylase by screening for the substrate using a substrate binding assay. Screening of substrates resulted in the identification of fatty acid groups of compounds as potential hits for CYP147F1 substrates. Fatty acids from C10:0 to C18:0 all showed type I shift spectra indicating their potential as substrates. Among several fatty acids tested, lauric acid, myrsitic acid, and palmitic acid were used to characterize CYP147F1. CYP147F1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida as surrogate electron transfer partners. Kinetic parameters, including the dissociation constant, Km, NADH consumption assay, production formation rate, and coupling efficiency for CYP147F1 were also determined.

Homology modeling and docking studies of Streptomyces peucetius CYP147F1 as limonene hydroxylase.

Homology modeling of Streptomyces peucetius CYP147F1 was constructed using three cytochrome P450 structures, CYP107L1, CYPVdh, and CYPeryF, as templates. The lowest energy SPCYP147F1 model was then assessed for stereochemical quality and side-chain environment by Accelrys Discovery Studio 3.1 software. Further activesite optimization of the SPCYP147F1 was performed by molecular dynamics to generate the final SPCYP147F1 model. The substrate limonene was then docked into the model. The model-limonene complex was used to validate the active-site architecture, and functionally important residues within the substrate recognition site were identified by subsequent characterization of the secondary structure. The docking of limonene suggested that SPCYP147F1 would have broad specificity with the ligand based on the two different orientations of limonene within the active site facing to the heme. Limonene with C7 facing the heme with distance of 3.4 Angstrom from the Fe was predominant.

Role of σ-factor (orf21) in clavulanic acid production in Streptomyces clavuligerus NRRL3585.

A putative sigma factor gene, orf21, was disrupted or overexpressed in the wild-type clavulanic acid (CA) producer Streptomyces clavuligerus NRRL3585 and characterized. An orf21 mutant (Streptomyces clavuligerus HN14) of S. clavuligerus was obtained by insertional inactivation via double-crossover. Although there was little reduction of sporulation in the mutant, the growth pattern was similar between mutant and wild-type. The production was reduced by 10-15% in S. clavuligerus HN14 compared to that in wild-type. Overexpression of orf21 in wild-type cells caused hyperproduction of spores on solid medium and increased clavulanic acid production by 1.43-fold. The overexpression of orf21 in wild-type S. clavuligerus stimulated the expression of the early clavulanic acid genes, ceas2 and cas2, and the regulatory gene, ccaR, as demonstrated by RT-PCR. The elevation of the ceas2, cas2 and ccaR transcripts was consistent with the enhanced production of clavulanic acid.

Biosynthesis of ribostamycin derivatives by reconstitution and heterologous expression of required gene sets.

Ribostamycin is a 4,5-disubstituted 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics and naturally produced by Streptomyces ribosidificus ATCC 21294. It is also an intermediate in the biosynthesis of butirosin and neomycin. In the biosynthesis of ribostamycin, DOS is glycosylated to generate paromamine which is converted to neamine by successive dehydrogenation followed by amination, and finally ribosylation of neamine gives ribostamycin. Here, we report the biosynthesis of 6'-deamino-6'-hydroxyribostamycin (a ribostamycin derivative or pseudoribostamycin) in Streptomyces venezuelae YJ003 by reconstructing gene cassettes for direct ribosylation of paromamine. A trace amount of pseudoribostamycin was detected with ribostamycin in the isolates of ribostamycin cosmid heterologously expressed in Streptomyces lividans TK24. It has also indicated that the ribosyltransferase can accept both neamine and paromamine. Thus, the present in vivo modification of ribostamycin could be useful for the production of hybrid compounds to defend against bacterial resistance to aminoglycosides.

Homology modeling, binding site identification and docking in flavone hydroxylase CYP105P2 in Streptomyces peucetius ATCC 27952.

Homology models of cytochrome P450 105P2 (CYP105P2) were constructed using four P450 structures, CYP105A1, CYP105, CYP165B3 and CYP107L1, as templates for the model building. Using Accelrys Discovery Studio 2.1 software, the lowest energy CYP105P2 model was then assessed for stereochemical quality and side-chain environment. Further active site optimization of the CYP105P2 model built using these templates was performed by molecular dynamics to generate the final CYP105P2 model. The substrates, flavone, flavanone, quercetin and naringenin, were docked into the model. The model-flavone complex was used to validate the active site architecture, and structurally and functionally important residues were identified by subsequent characterization of the secondary structure.

Heterologous production of ribostamycin derivatives in engineered Escherichia coli.

Aminoglycosides are a class of important antibiotic compounds used for various therapeutic indications. In recent times, their efficacy has been curtailed due to the rapid development of bacterial resistance. There is a need to develop novel derivatives with an improved spectrum of activity and higher sensitivity against pathogenic bacteria. Although efforts have been focused on the development of newer therapeutic agents by chemical synthesis, to our knowledge, there has been no attempt to harness the potential of microorganisms for this purpose. Escherichia coli affords a widely studied cellular system that could be utilized not only for understanding but also for attempting to engineer the biosynthetic pathway of secondary metabolites. The primary metabolic pathway of E. coli can be engineered to divert the precursor pool required for the biosynthesis of secondary metabolites. Utilizing this approach previously, we engineered E. coli host and generated E. coli M1. Here, we produced a ribostamycin derivative in the engineered host by heterologous expression of the recombinants constructed from the genes encoding the biosynthetic pathway in aminoglycoside-producing strains. The products obtained from the transformants were isolated, analyzed and verified to be ribostamycin derivatives. The study further demonstrated the importance of E. coli as surrogate antibiotic producer and also offered future possibility for the production of other aminoglycoside derivatives through genetic engineering and expression in a heterologous background.

Limitations in doxorubicin production from Streptomyces peucetius.

Doxorubicin (DXR), produced by Streptomyces peucetius ATCC 27952, exhibits potent antitumor activity against various cancer cell lines. Considerable time has lapsed since the biosynthesis of DXR and its overproduction was first summarized. Based on biosynthetic studies and product analysis, various factors affecting its production by the parental strain, S. peucetius ATCC 27952, are reviewed to better circumvent any bottlenecks in DXR production, thereby providing ideas to genetically engineered industrial strains of S. peucetius.

Improvement in doxorubicin productivity by overexpression of regulatory genes in Streptomyces peucetius.

Biosynthesis of doxorubicin (DXR) is tightly regulated, limiting its production in Streptomyces peucetius cultures. The regulatory genes dnrN, dnrI, afsR, and metK1-sp from S. peucetius ATCC 27952 were cloned into the pIBR25 expression vector under the control of the strong ermE* promoter to enhance DXR production. The constructed expression plasmids, pNI25 (with dnrN-dnrI), pNIS25 (with dnrN-dnrI-metK1-sp), pNIR25 (with dnrN-dnrI-afsR), pRS25 (with afsR-metK1-sp) and pNIRS25 (with dnrN-dnrI-afsR-metK1-sp), were transformed into S. peucetius. The recombinant strains NI, NIS and NIR produced greater amounts of DXR than the parental strain with an increment of 1.2-fold by pNI25, 1.4-fold by pNIS25 and 4.3-fold by pNIR25, whereas pRS25 and pNIRS25 had no significant effect on DXR production. We also studied the transcriptional level of overexpressed regulatory genes and relative production of DXR, daunorubicin (DNR) and epsilon-rhodomycinone (RHO) in each recombinant strain.

Self-resistance mechanism in Streptomyces peucetius: overexpression of drrA, drrB and drrC for doxorubicin enhancement.

The resistance genes drrABC from Streptomyces peucetius ATCC 27952 were cloned into the pIBR25 expression vector under a strong ermE* promoter to enhance doxorubicin (DXR) production. The recombinant expression plasmids, pDrrAB25, pDrrC25 and pDrrABC25, were constructed to overexpress drrAB, drrC and drrABC, respectively, in S. peucetius ATCC 27952. The recombinant strains produced more DXR than the parental strain: a 2.2-fold increase with pDrrAB25, a 5.1-fold increase with pDrrC25, and a 2.4-fold increase with pDrrABC25. We also studied the relative ratios of doxorubicin, daunorubicin and epsilon-rhodomycinone produced in these recombinant strains.

Enhancement of doxorubicin production by expression of structural sugar biosynthesis and glycosyltransferase genes in Streptomyces peucetius.

To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes desIII and desIV from Streptomyces venezuelae ATCC 15439 and the glycosyltransferase pair dnrS/dnrQ from Streptomyces peucetius ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong ermE promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of dnrS and the dnrS/dnrQ pair, whereas pDesSD25 and pDesQS25 were constructed to express desIII/desIV and dnrS/dnrQ-desIII/desIV, respectively. All of these recombinant plasmids were introduced into S. peucetius ATCC 27952. The recombinant strains produced more DXR than the S. peucetius parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.

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.

Functional characterization of kanB by complementing in engineered Streptomyces fradiae Deltaneo6::tsr.

A putative aminotransferase gene, kanB, lies in the biosynthetic gene cluster of Streptomyces kanamyceticus ATCC 12853 and has 66% identity with neo6 in neomycin biosynthesis. Streptomyces fradiae Deltaneo6::tsr was generated by disrupting neo6 in the neomycin producer Streptomyces fradiae. Neomycin production was completely abolished in the disruptant mutant but was restored through self-complementation of neo6. S. fradiae HN4 was generated through complementation with kanB in Streptomyces fradiae neo6::tsr. Based on metabolite analysis by ESI/MS and LC/MS, neomycin production was restored in Streptomyces fradiae HN4. Thus, like neo6, kanB also functions as a 2-deoxy-scyllo-inosose aminotransferase that has dual functions in the formation of 2-deoxy-scyllo-inosose (DOS).

Expanding substrate specificity of GT-B fold glycosyltransferase via domain swapping and high-throughput screening.

Glycosyltransferases (GTs) are crucial enzymes in the biosynthesis and diversification of therapeutically important natural products, and the majority of them belong to the GT-B superfamily, which is composed of separate N- and C-domains that are responsible for the recognition of the sugar acceptor and donor, respectively. In an effort to expand the substrate specificity of GT, a chimeric library with different crossover points was constructed between the N-terminal fragments of kanamycin GT (kanF) and the C-terminal fragments of vancomycin GT (gtfE) genes by incremental truncation method. A plate-based pH color assay was newly developed for the selection of functional domain-swapped GTs, and a mutant (HMT31) with a crossover point (N-kanF-669 bp and 753 bp-gtfE-C) for domain swapping was screened. The most active mutant HMT31 (50 kDa) efficiently catalyzed 2-DOS (aglycone substrate for KanF) glucosylation using dTDP-glucose (glycone substrate for GtfE) with k(cat)/K(m) of 162.8 +/- 0.1 mM(-1) min(-1). Moreover, HMT31 showed improved substrate specificity toward seven more NDP-sugars. This study presents a domain swapping method as a potential means to glycorandomization toward various syntheses of 2-DOS-based aminoglycoside derivatives.

Identification of a cryptic type III polyketide synthase (1,3,6,8-tetrahydroxynaphthalene synthase) from Streptomyces peucetius ATCC 27952.

We identified a 1,134-bp putative type III polyketide synthase from the sequence analysis of Streptomyces peucetius ATCC 27952, named Sp-RppA, which is characterized as 1,3,6,8-tetrahydroxynaphthalene synthase and shares 33% identity with SCO1206 from S. coelicolor A3(2) and 32% identity with RppA from S. griseus. The 1,3,6,8-tetrahydroxynaphthalene synthase is known to catalyze the sequential decarboxylative condensation, intramolecular cyclization, and aromatization of an oligoketide derived from five units of malonyl-CoA to give 1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidizes to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). In this study, we report the in vivo expression and in vitro synthesis of flaviolin from purified gene product (Sp-RppA).

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.

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.

A two-component regulatory system involved in clavulanic acid production.

A pair of genes encoding the bacterial two-component regulatory system, orf22 and orf23, was found next to the clavulanic acid gene cluster of Streptomyces clavuligerus NRRL3585. Orf23 was deleted for the construction of S. clavuligerus/Deltaorf23. Although growth and morphological analyses showed no differences between S. clavuligerus/Deltaorf23 and wild-type, the production of clavulanic acid in S. clavuligerus/Deltaorf23 was found to be decreased. In addition, the co-overexpression of orf22/orf23 in wild-type resulted in an enhanced 1.49-fold production of clavulanic acid, and the complementation of orf22/orf23 in S. clavuligerus/Deltaorf23 restored clavulanic acid production about 80% as normal levels. These results demonstrate that the orf22/orf23 two-component regulatory system participates as a positive regulator of the biosynthesis of clavulanic acid and increased levels of orf22/orf23 can contribute to enhanced production of clavulanic acid in S. clavuligerus.