Engineering an acetoacetyl‑CoA reductase from Cupriavidus necator toward NADH preference under physiological conditions
Author
dc.contributor.author
Olavarría Gamez, Karel
Author
dc.contributor.author
Pijman, Yared O.
Author
dc.contributor.author
Cabrera Paucar, Ricardo Mauricio
Author
dc.contributor.author
Van Loosdrecht, Mark
Author
dc.contributor.author
Wahl, S. Aljoscha
Admission date
dc.date.accessioned
2023-03-08T14:49:30Z
Available date
dc.date.available
2023-03-08T14:49:30Z
Publication date
dc.date.issued
2022
Cita de ítem
dc.identifier.citation
Scientific Reports (2022) 12:3757
es_ES
Identifier
dc.identifier.other
10.1038/s41598-022-07663-w
Identifier
dc.identifier.uri
https://repositorio.uchile.cl/handle/2250/191967
Abstract
dc.description.abstract
The coupling of PHB generation with NADH reoxidation is required to generate PHB as a fermentation product. A fundamental trait to accomplish this feature is to express a functional NADH-preferring acetoacetyl-CoA reductase, engaged in PHB accumulation. One way to obtain such a reductase is by engineering the cofactor preference of the acetoacetyl-CoA reductase encoded by the phaB1 gene from Cupriavidus necator (AAR(Cn1)). Aiming to have a deeper understanding of the structural determinants of the cofactor preference in AAR(Cn1), and to obtain an NADH-preferring acetoacetyl-CoA reductase derived from this protein, some engineered enzymes were expressed, purified and kinetically characterized, together with the parental AAR(Cn1). One of these engineered enzymes, Chimera 5, experimentally showed a selectivity ratio ((k(cat)/K-M)(NADH)/(k(cat)/K-M)(NADPH)) approximate to 18, which is 160 times higher than the selectivity ratio experimentally observed in the parental AAR(Cn1). A thermodynamic-kinetic approach was employed to estimate the cofactor preference and flux capacity of Chimera 5 under physiological conditions. According to this approach, Chimera 5 could prefer NADH over NADPH between 25 and 150 times. Being a derivative of AAR(Cn1), Chimera 5 should be readily functional in Escherichia coli and C. necator. Moreover, with the expected expression level, its activity should be enough to sustain PHB accumulation fluxes similar to the fluxes previously observed in these biotechnologically relevant cell factories.
es_ES
Patrocinador
dc.description.sponsorship
joint research program NWO-FAPESP of The Netherlands Organization for Scientific Research (NWO)
Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) NWO: BBE.2017.013-FAPESP: 2017/50249-6
Netherlands Ministry of Education, Culture and Science (OCW) 024.002.002
Netherlands Organization for Scientific Research (NWO)
es_ES
Lenguage
dc.language.iso
en
es_ES
Publisher
dc.publisher
Scientific Reports
es_ES
Type of license
dc.rights
Attribution-NonCommercial-NoDerivs 3.0 United States