Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase

Weissman, Kira J.; Bycroft, Matthew; Cutter, Annabel L.; Hanefeld, Ulf; Frost, Elizabeth J.; Timoney, Máire C.; Harris, Robert G.; Handa, Sandeep; Roddis, Marc; Staunton, James; Leadlay, Peter F.

doi:10.1016/s1074-5521(98)90666-4

Cited by 48 publications

(40 citation statements)

References 48 publications

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“…Biosynthetic studies with mini-PKSs such as DEBS 1-TE and DEBS 1 TE both in vivo and in vitro have yielded various structural and stereochemical analogues of lactones 2 and 3. 13,15,16,21,24,25 Analysis of these compounds by gas chromatography/mass spectrometry (GC/MS) has provided a powerful means of structural proof without the requirement for large quantities of analyte. With one exception, 16 however, these studies have not explored whether mass spectrometry can be used to probe the functionality or stereochemistry of these complex molecules.…”

mentioning

confidence: 99%

Structural elucidation studies of polyketide tetrasubstituted δ-lactones by gas chromatography/tandem mass spectrometry and electrospray mass spectrometry

Weissman

Kearney

Leadlay

et al. 1999

Rapid Commun. Mass Spectrom.

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A series of tetrasubstituted polyketide delta-lactones were used to evaluate whether gas chromatography/tandem mass spectrometry (GC/MS/MS) and electrospray mass spectrometry (ESI-MS) are useful techniques for probing the structure and stereochemistry of such highly functionalised molecules. Analyses were performed with two commercially available mass spectrometers: a Finnigan/MAT GCQ instrument (CI source) and a Q-TOF Hybrid quadrupole time-of-flight instrument (ESI source). The analyses revealed that a range of variation in the structure and stereochemistry of the lactones did not affect the fragmentation pathway common to these molecules. By accurate mass determination (ESI-MS), the first two fragmentations were assigned to losses of water. Although it was anticipated that the initial dehydration would include the hydroxyl group at the 3-position of the lactones, evidence from deuterium- and (18)O-labelling studies suggests that the losses of water instead involve the oxygen atoms in the ester bond. Attempts to identify further the structures of daughter ions by GC/MS/MS were complicated by extensive rearrangements and non-specific hydrogen/deuterium migrations within the lactones. Together, these results illustrate the limitations of mass spectrometry in the structural elucidation of complex molecules. Copyright 1999 John Wiley& Sons, Ltd.

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confidence: 99%

Structural elucidation studies of polyketide tetrasubstituted δ-lactones by gas chromatography/tandem mass spectrometry and electrospray mass spectrometry

Weissman

Kearney

Leadlay

et al. 1999

Rapid Commun. Mass Spectrom.

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“…In vitro experiments comparing "natural diketide" and "propyl diketide" thioesters as substrates for purified DEBS have shown that the enzyme has a modest preference for the "propyl diketide" thioester over the "natural diketide" thioester (3,27). The "natural diketide"-thioglycolate and the "propyl diketide"-SNPC were first compared in the E. coli strain expressing DEBS1-mod2 and DEBS2 and -3 ( Table 4).…”

Section: Diketide Feeding With Alternatives To Snac Methyl S-[(2r‫3mentioning

confidence: 99%

Chemobiosynthesis of Novel 6-Deoxyerythronolide B Analogues by Mutation of the Loading Module of 6-Deoxyerythronolide B Synthase 1

Murli¹,

MacMillan²,

Hu³

et al. 2005

Appl Environ Microbiol

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Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler "monoketide" acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1 o for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono-and diketide feeding in both E. coli and S. coelicolor.The polyketide class of natural products comprises numerous compounds with great diversity in both structure and biological activity. Many of these compounds have found uses in medicine and agriculture (20,26). The increasing prevalence of antibiotic-resistant pathogens has made the search for new and better antimicrobial compounds more urgent. One traditional avenue of research is the chemical modification of existing antibiotics to generate more-potent molecules. For example, the macrolide antibiotic erythromycin can be chemically converted to semisynthetic derivatives called ketolides (e.g., ABT773, telithromycin) that are active against macrolide-resistant bacteria.The erythromycin precursor polyketide 6-deoxyerythronolide B (6-dEB) is produced from one propionyl-coenzyme A (CoA) starter unit and six (2S)-methylmalonyl-CoA extender units by a series of condensation and reduction steps catalyzed by 6-dEB synthase (DEBS) (Fig. 1) (5). The loading module of DEBS consists of an acyltransferase (AT L ) and an acyl carrier protein (ACP L ). Starter unit selection is determined by the specificity of the AT L , and, with DEBS, propionate is the preferred starter unit. Attempts to change the specificity of the loading module by genetic engineering have produced strains that are able to produce specific 6-dEB and erythromycin analogues such as 14-nor erythromycin (16,17,28), although typically at much reduced titers. Chemobiosynthesis, the feeding of a diketide-S-N-acetyl cysteamine (SNAC) or diketide-S-N-propionyl cysteamine (SNPC) to a version of DEBS deficient in the first ketosynthase (KS) step, is an important and established route for the production of 6-dEB analogues (6, 9). This requires the expression of a modified DEBS1 which either carries an inactivating mutation in KS1 (DEBS1 KS1 O ) (9) or completely lacks both the loading module and module 1 of DEBS1 (DEBS1-mod2) (8,23,25). Subsequent bioconversion and ch...

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“…However, production can be restored by addition of a synthetic thioester diketide that mimics the structure of the intermediate generated from the Wrst condensation cycle. The diketide enters the cell and can form the required thioester linkage with the second module of the PKS [3,11,27]. By varying the structure of the diketide employed, this process has opened up an avenue for production of a variety of novel analogues.…”

Section: Introductionmentioning

confidence: 99%

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues is improved by removal of the initial catalytic sites of the polyketide synthase

Ward¹,

Desai²,

Hu³

et al. 2006

J Ind Microbiol Biotechnol

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Precursor-directed biosynthesis has been shown to be a powerful tool for the production of polyketide analogues that would be difficult or cost prohibitive to produce from medicinal chemistry efforts alone. It has been most extensively demonstrated using a KS1 null mutation (KS1(0)) to block the first round of condensation in the biosynthesis of the erythromycin polyketide synthase (DEBS) for the production of analogues of its aglycone, 6-deoxyerythronolide B (6-dEB). Here we show that removing the DEBS loading domain and first module (mod1Delta), rather than using the KS1(0) system, can lead to an increase in the utilization of some chemical precursors and production of 6-dEB analogues (R-6dEB) in both Streptomyces coelicolor and Saccharopolyspora erythraea. While the difference in utilization of the precursor was diketide specific, in strains fed (2R*, 3S*)-5-fluoro-3-hydroxy-2-methylpentanoate N-propionylcysteamine thioester, twofold increases in both utilization of the diketide and 15-fluoro-6dEB (15F-6dEB) production were observed in S. coelicolor, and S. erythraea exhibited a tenfold increase in production of 15-fluoro-erythromycin when utilizing the mod1Delta rather than the KS1(0) system.

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Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase

Cited by 48 publications

References 48 publications

Structural elucidation studies of polyketide tetrasubstituted δ-lactones by gas chromatography/tandem mass spectrometry and electrospray mass spectrometry

Structural elucidation studies of polyketide tetrasubstituted δ-lactones by gas chromatography/tandem mass spectrometry and electrospray mass spectrometry

Chemobiosynthesis of Novel 6-Deoxyerythronolide B Analogues by Mutation of the Loading Module of 6-Deoxyerythronolide B Synthase 1

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues is improved by removal of the initial catalytic sites of the polyketide synthase

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