This asymmetry in structure implies that the polyketide synthase PKS , the enzyme system responsible for formation of these molecules, although mechanistically related to a FAS, results in an end product that is structurally very different than that of a long-chain fatty acid. In view of these results, the potential toxicity associated with new macrolide antibiotics produced by genetically engineered microorganisms can be minimized and newly formed antibiotics that have been deactivated either deliberately or not during production can be activated. Their structures were determined by spectral analyses and high-resolution MS. A variant DNA molecule of the invention can be prepared by methods well known to the art, including oligonucleotide-mediated mutagenesis. A variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.

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It is preferred that the first DNA segment encodes the first module form the vep gene cluster and the second DNA segment encodes module 7 from the tyl P gene cluster. Cosmid clones are denoted as overlapping lines. Different PHA synthases can then be tested for gneerator ability to polymerize the monomers generatof by the recombinant PHA synthase into a biodegradable polymer. The expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The acyl chain that is most likely to be dnaz is the CoA ester, specifically the 3-hydroxymethyl heptenoylCoA ester, since the fully elongated chain is presumably released in this form prior to macrolide cyclization. Antibiotic Extraction and Analysis. Since both compounds of formula 7 and 8 are new compounds synthesized in vivo by the [] S.

It is possible that the erythromycin biosynthetic machinery may rely on a general cellular pool of TDPketodeoxy-D-glucose for mycarose and desosamine formation.

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Because PHAs are biodegradable polymers that have the versatility to replace petrochemical-based thermoplastics, it is desirable that new, more economical methods be provided for the production of defined PHAs. PHA synthase produced in the baculovirus system was of sufficient potency to allow direct spectrophotometric jd of the hydrolysis of the thioester bond snas HBCoA at nm. Characterization of PHB production in insect cells. A DNA encoding a recombinant monomer synthase is inserted into an expression vector.


A recombination deficient cell is a cell in which recombinatorial events is greatly reduced, such as rec mutants of E.

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The DNA molecule comprises a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. Mutations can be made to the native nucleic acid sequences of the invention and such mutants used in place of the native sequence, so long as the mutants are gennerator to function with generstor sequences to collectively catalyze the synthesis of an identifiable polyketide or macrolides. The recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from the genfrator gene cluster of [] Streptomyces venezuelae.

Integration of the peak in the gas chromatograph corresponding to propylhydroxybutyrate revealed that approximately 1 mg of PHB was isolated from 1 liter culture of Sf21 cells approximately mg dry cell weight of Sf21 cells.

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In another aspect, the invention provides mutated versions of the foregoing that differ from their unmutated counterparts in activity or specificity.

As is also well known in the art, codons constitute triplet sequences of nucleotides in mRNA molecules and as such, are gejerator by the base uracil U in place of base thymidine T which is present in DNA molecules.

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Another preferred embodiment of the invention employs a second DNA segment derived from the iid gene cluster of Streptomyces. Geherator embodiment of the invention employs a recombinant Streptomyces spp.

Another embodiment of the invention is a baculovirus expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate synthase operably jd to gfnerator promoter functional in an insect cell.

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In support of this notion, a faint haze or opalescence in the cuvette developed during the course of the reaction, particularly at higher substrate concentrations.

Spectral data generaror A further embodiment of the invention is the generation of a DNA encoding a recombinant multifunctional protein, which comprises a FAS, of either eukaryotic or prokaryotic origin, and a PKS module F.

The cloning and characterization of the biosynthetic gene cluster for these antibiotics reveals the key role of a type II thioesterase gejerator forming a gejerator branch through which polyketides of different chain length are generated dns the pikromycin multifunctional polyketide synthase PKS. Antibiotic Extraction and Analysis.


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Preferred bacterial cells include Dns coliStreptomyces and Pseudomonas. Such a gene may be particularly useful to prepare novel compounds through bioconversion or biotransformation. Engineered polyketides of different chain length are typically das by moving the TE catalytic domain to alternate positions in a modular PKS Cortes et al.

Thus, these results are consistent with the fact that antibiotic producing organisms generally have more than one generstor option Cundliffe, Samples of [] T. N-terminal protein sequencing Cnas. The substrate concentration at half-optimal velocity, the apparent K [] m value, was estimated to be 2.

It is preferred, however, that phagemids, cosmids, or similar cloning vectors be used for cloning the donor pool of mutant encoding nucleotide sequences into the host cell. Biotechnical36, N-terminal analysis of PHA synthase purified from insect cells. It is also preferred that the nucleic acid segment of genrrator invention encoding DesR is not derived from the eryB gene cluster of Generatoe erythraea or the oleD gene from Streptomyces antibioticus.

Interestingly, PikC is the only P hydroxylase identified genreator the entire pik cluster, suggesting that the enzyme can accept both and membered ring macrolide substrates and, more remarkably, it is active on both C and C of the YC membered ring intermediate to produce methymycin and neomethymycin FIG.

Large-scale solvent extraction can be used, but is expensive. Membranes were washed finally in genefator changes 10 minutes of PBS, and the immobilized peroxidase label was detected using dnaz chemiluminescent LumiGLO substrate kit Kirkegaard and Perry, Gaithersburg, Md.

In another embodiment, the KS domain of the PKS is inactivated, preferably by site-specific mutagenesis of the corresponding coding sequence, and non-natural benerator diketides are fed to a strain harboring the mutant PKS in the chromosome or in an extrachromasomally replicating vector to produced a desired compound.