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1.
Nat Chem Biol ; 19(2): 168-175, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36470994

RESUMO

Anaplerosis is an essential feature of metabolism that allows the continuous operation of natural metabolic networks, such as the citric acid cycle, by constantly replenishing drained intermediates. However, this concept has not been applied to synthetic in vitro metabolic networks, thus far. Here we used anaplerotic strategies to directly access the core sequence of the CETCH cycle, a new-to-nature in vitro CO2-fixation pathway that features several C3-C5 biosynthetic precursors. We drafted four different anaplerotic modules that use CO2 to replenish the CETCH cycle's intermediates and validated our designs by producing 6-deoxyerythronolide B (6-DEB), the C21-macrolide backbone of erythromycin. Our best design allowed the carbon-positive synthesis of 6-DEB via 54 enzymatic reactions in vitro at yields comparable to those with isolated 6-DEB polyketide synthase (DEBS). Our work showcases how new-to-nature anaplerotic modules can be designed and tailored to enhance and expand the synthetic capabilities of complex catalytic in vitro reaction networks.


Assuntos
Dióxido de Carbono , Policetídeo Sintases , Policetídeo Sintases/metabolismo , Macrolídeos , Eritromicina , Antibacterianos
2.
Cell Chem Biol ; 25(7): 833-839.e4, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-29731424

RESUMO

The incorporation of different extender units generates structural diversity in polyketides. There is significant interest in engineering substrate specificity of polyketide synthases (PKSs) to change their chemical structure. Efforts to change extender unit selectivity are hindered by the lack of simple screening methods and easily available atypical extender units. Here, we present a chemo-biosynthetic strategy that employs biocatalytic proofreading and allows access to a large variety of extender units. First, saturated acids are chemically coupled to free coenzyme A (CoA). The corresponding acyl-CoAs are then converted to alkylmalonyl-CoAs in a "one-pot" reaction through the combined action of an acyl-CoA oxidase and enoyl-CoA carboxylase/reductase. We synthesized six different extender units and used them in in vitro competition screens to investigate active site residues conferring extender unit selectivity. Our results show the importance of an uncharacterized glutamine in extender unit selectivity and open the possibility for comprehensive studies on extender incorporation in PKSs.


Assuntos
Acil-CoA Desidrogenases/metabolismo , Acil-CoA Oxidase/metabolismo , Policetídeos/metabolismo , Estrutura Molecular , Policetídeos/química
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