In Vitro Reconstitution of the Entire Enterocin Biosynthetic Pathway: New Insights into Type II PKS Enzymology

Persistent Link:
http://hdl.handle.net/10150/195469
Title:
In Vitro Reconstitution of the Entire Enterocin Biosynthetic Pathway: New Insights into Type II PKS Enzymology
Author:
Cheng, Qian
Issue Date:
2007
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Type II polyketide synthases (PKSs) are responsible for the generation of structurally diverse and clinically important aromatic polyketides. The bacteriostatic agent enterocin (enc) isolated from the marine microbe "Streptomyces maritimus" is derived from a rare benzoate primer unit and contains a unique nonaromatic caged core structure resulting from a Favorskii-like carbon skeleton rearrangement. The apparent diversion between enterocin biosynthesis and all other type II PKS pathways offered the opportunity to discover novel enzymatic strategies that may be exploited to diversify the chemical structures of polyketides. A comprehensive biochemical analysis was performed in order to characterize the key steps in enterocin biosynthesis and finally to reconstitute the whole pathway in vitro using purified recombinant enzymes.A nonribosomal peptide synthetase (NRPS)-like priming mechanism was discovered for the selective activation of a benzoic acid starter unit and its subsequent attachment to the enc PKS to initiate polyketide biosynthesis. This is the first example of a type II PKS that employs an NPRS-like priming mechanism to utilize alternative non- acetate starter units. Secondly, the minimal enc PKS was reconstituted in vitro to give three novel acetate-primed metabolites that had never been identified by heterologous in vivo expression of recombinant enc PKS gene sets. The minimal enc PKS was then merged with the NRPS-like chain initiation module and the resulting multienzyme complex catalyzed the formation of benzoate-primed natural products wailupemycin F and wailupemycin G. Favorskii-like rearrangement of the nascent polyketide chain was replicated in vitro and the flavin-dependent oxygenase EncM was confirmed to be solely responsible for catalyzing this unprecedented rearrangement. Other biosynthetic steps in the late stage of the enc pathway were also replicated in vitro, including the methylation of desmethyl-5-deoxyenterocin to 5-deoxyenterocin and the hydroxylation of 5-deoxyenterocin to enterocin.Finally, the entire enc type II PKS pathway was successfully assembled in vitro using ten recombinant proteins and three commercial enzymes. Five enc-based natural products were generated from benzoic acid and malonyl-coenzyme A. This biochemical investigation on enterocin biosynthesis represents the first complete in vitro reconstitution of a type II PKS system and also provides an alternative strategy to create complex natural products by multienzyme synthesis.
Type:
text; Electronic Dissertation
Keywords:
Biosynthesis; Type II Polyketide Synthase; Enzymology; In Vitro Reconstitution; Mutasynthesis; Favorskii Rearrangement
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Pharmaceutical Sciences; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Moore, Bradley S.
Committee Chair:
Moore, Bradley S.

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleIn Vitro Reconstitution of the Entire Enterocin Biosynthetic Pathway: New Insights into Type II PKS Enzymologyen_US
dc.creatorCheng, Qianen_US
dc.contributor.authorCheng, Qianen_US
dc.date.issued2007en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractType II polyketide synthases (PKSs) are responsible for the generation of structurally diverse and clinically important aromatic polyketides. The bacteriostatic agent enterocin (enc) isolated from the marine microbe "Streptomyces maritimus" is derived from a rare benzoate primer unit and contains a unique nonaromatic caged core structure resulting from a Favorskii-like carbon skeleton rearrangement. The apparent diversion between enterocin biosynthesis and all other type II PKS pathways offered the opportunity to discover novel enzymatic strategies that may be exploited to diversify the chemical structures of polyketides. A comprehensive biochemical analysis was performed in order to characterize the key steps in enterocin biosynthesis and finally to reconstitute the whole pathway in vitro using purified recombinant enzymes.A nonribosomal peptide synthetase (NRPS)-like priming mechanism was discovered for the selective activation of a benzoic acid starter unit and its subsequent attachment to the enc PKS to initiate polyketide biosynthesis. This is the first example of a type II PKS that employs an NPRS-like priming mechanism to utilize alternative non- acetate starter units. Secondly, the minimal enc PKS was reconstituted in vitro to give three novel acetate-primed metabolites that had never been identified by heterologous in vivo expression of recombinant enc PKS gene sets. The minimal enc PKS was then merged with the NRPS-like chain initiation module and the resulting multienzyme complex catalyzed the formation of benzoate-primed natural products wailupemycin F and wailupemycin G. Favorskii-like rearrangement of the nascent polyketide chain was replicated in vitro and the flavin-dependent oxygenase EncM was confirmed to be solely responsible for catalyzing this unprecedented rearrangement. Other biosynthetic steps in the late stage of the enc pathway were also replicated in vitro, including the methylation of desmethyl-5-deoxyenterocin to 5-deoxyenterocin and the hydroxylation of 5-deoxyenterocin to enterocin.Finally, the entire enc type II PKS pathway was successfully assembled in vitro using ten recombinant proteins and three commercial enzymes. Five enc-based natural products were generated from benzoic acid and malonyl-coenzyme A. This biochemical investigation on enterocin biosynthesis represents the first complete in vitro reconstitution of a type II PKS system and also provides an alternative strategy to create complex natural products by multienzyme synthesis.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectBiosynthesisen_US
dc.subjectType II Polyketide Synthaseen_US
dc.subjectEnzymologyen_US
dc.subjectIn Vitro Reconstitutionen_US
dc.subjectMutasynthesisen_US
dc.subjectFavorskii Rearrangementen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmaceutical Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMoore, Bradley S.en_US
dc.contributor.chairMoore, Bradley S.en_US
dc.contributor.committeememberMontfort, William R.en_US
dc.contributor.committeememberJacobson, Myron K.en_US
dc.contributor.committeememberYang, Danzhouen_US
dc.contributor.committeememberCordes, Matthew H. J.en_US
dc.identifier.proquest2416en_US
dc.identifier.oclc659748318en_US
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