Polyketide synthesis in vitro on a modular polyketide synthase

Wiesmann, Kirsten E.H.; Cortés, Jesús M.; Brown, Murray J. B.; Cutter, Annabel L.; Staunton, James; Leadlay, Peter F.

doi:10.1016/1074-5521(95)90122-1

Cited by 94 publications

(88 citation statements)

References 18 publications

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“…[13][14][15][16][17][18][19][20][21] In these studies, proof of structure was often achieved by molecular weight determination using GC/MS or ESI. It was of interest to see if mass spectrometry could also be used to further characterise the structure and stereochemistry of these highly functionalised molecules.…”

Section: Discussionmentioning

confidence: 99%

“…[13][14][15][16][17][18][19][20][21] In order to facilitate studies into erythromycin biosynthesis, the erythromycin PKS (MW $1 MDa) has been truncated to yield significantly smaller model systems (MW $400 kDa) called DEBS 1-TE 22 ( Fig. 1(b)) and DEBS 1 TE.…”

Section: -12mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: -12mentioning

confidence: 99%

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confidence: 99%

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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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“…Extender AT domains have a high degree of substrate specificity, whereas the AT L domain in DEBS1 exhibits considerable flexibility. Acetyl-CoA, butyryl-CoA and isobutyryl-CoA can serve as starter units in addition to propionyl-CoA in a heterologous host or in vitro (Kao et al, 1994;Lau et al, 2000;Wiesmann et al, 1995). When the DEBS genes are heterologously expressed in Streptomyces coelicolor CH999, 6dEB and 15-nor-6dEB (Fig.…”

Section: Introductionmentioning

confidence: 99%

A specific role of the Saccharopolyspora erythraea thioesterase II gene in the function of modular polyketide synthases

Hu¹,

Pfeifer

Chao

et al. 2003

Microbiology

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Bacterial modular polyketide synthase (PKS) genes are commonly associated with another gene that encodes a thioesterase II (TEII) believed to remove aberrantly loaded substrates from the PKS. Co-expression of the Saccharopolyspora erythraea ery-ORF5 TEII and eryA genes encoding 6-deoxyerythronolide B synthase (DEBS) in Streptomyces hosts eliminated or significantly lowered production of 8,89-deoxyoleandolide [15-nor-6-deoxyerythronolide B (15-nor-6dEB)], which arises from an acetate instead of a propionate starter unit. Disruption of the TEII gene in an industrial Sac. erythraea strain caused a notable amount of 15-norerythromycins to be produced by utilization of an acetate instead of a propionate starter unit and also resulted in moderately lowered production of erythromycin compared with the amount produced by the parental strain. A similar behaviour of the TEII gene was observed in Escherichia coli strains that produce 6dEB and 15-methyl-6dEB. Direct biochemical analysis showed that the ery-ORF5 TEII enzyme favours hydrolysis of acetyl groups bound to the loading acyl carrier protein domain (ACP L ) of DEBS. These results point to a clear role of the TEII enzyme, i.e. removal of a specific type of acyl group from the ACP L domain of the DEBS1 loading module.

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“…The information, which the DEBS PKS has revealed in regards to functional domains and module organization, has laid the foundation for virtually all analyses of modular PKSs (79). One of the key factors which has made the DEBS PKS such a well studied system is the ability for either all or part of the DEBS biosynthetic gene cluster to be successfully expressed in several heterologous hosts other than Saccharopolyspora erythraea, including S. venezuelae (75,(80)(81)(82)(83) with much lower production levels (55,56,80,84,85,87). The observation of novel erythromycin analogs not only means that the AT L has relaxed substrate specificity but also that the catalytic domains in downstream modules to some extent do as well.…”

Section: Introductionmentioning

confidence: 99%

Functional Separation of Multimodular Type I PKS Polypeptides by Utilizing Matched Docking Domains From a Heterologous PKS System

Yan¹

2000

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Bacterial type I modular polyketide synthases (PKS) are large multifunctional enzyme systems responsible for the biosynthesis of complex polyketide natural products such as erythromycin, pikromycin, and borrelidin. Type I systems are comprised of a loading module which generally selects an appropriate acyl group starter unit, and multiple discrete extension modules, responsible for each single round of acyl group incorporation into the final polyketide core structure. These modules can exist naturally as either single discrete polypeptides, such as modules 5 and 6 from the pikromycin PKS (PikA3 and PikA4 respectively), or as multimodular polypeptides fused together by short intrapolypeptide linkers such as the loading module and the first and second extension modules of the erythromycin and pikromycin PKSs (DEBS1 and PikAI respectively). While short peptide linkers between modules on the same polypeptide facilitate the transfer of polyketide intermediates from one module to the next via their close proximity to one another, docking domains found at the Cterminus of one module and the N-terminus of the next subsequent module facilitate the needed protein-protein interactions for the passage of biosynthetic intermediates between modules on separate polypeptides.The ability to utilize docking domains in place of intrapolypeptide linkers was explored in the pikromycin and erythromycin PKSs by dissecting the tri-modular PikAI and DEBS1 polypeptides with matched docking domains. It has been shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains from a heterologous modular phoslactomycin PKS are used in place of the intrapolypeptide linker. In both cases the yields of pikromycin ii produced by the S. venezuelae host mutant, which is a PikAI deletion strain were 50% of that of an S. venezuelae strain expressing the native trimodular PikAI.Additionally, expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a 10-fold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate.The utilization of docking domains to separate linked modules was also demonstrated in the erythromycin PKS. Expression of the first protein involved in erythromycin biosynthesis (DEBS1) with the DEBS thioesterase fused to the Cterminal (DEBS1-TE) in S. venezuelae results in the production of triketide lactone products. Separation of DEBS1-TE resulted in 50% triketide lactone production, consistent with the observations in the pikromycin system. Published work has shown that the DEBS loading module has relaxed substrate specificity, and is capable of incorporating acetate, butyrate and isobutyrate in addition to the normally observed propionate starter unit, which typically predominates. However, in the current study when the DEBS loading module is separated from module 1 with matched docking domains, a dramatic shift in the starter unit, favoring the isobutyrate derive...

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Polyketide synthesis in vitro on a modular polyketide synthase

Abstract: We have obtained a purified polyketide synthase system, derived from DEBS, which retains catalytic activity. This approach opens the way for mechanistic and structural analyses of active multienzymes derived from any modular polyketide synthase.

Cited by 94 publications

References 18 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

A specific role of the Saccharopolyspora erythraea thioesterase II gene in the function of modular polyketide synthases

Functional Separation of Multimodular Type I PKS Polypeptides by Utilizing Matched Docking Domains From a Heterologous PKS System

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