TY - JOUR
T1 - An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichia coli
AU - Olavarria, Karel
AU - Carnet, Alexandre
AU - van Renselaar, Joachim
AU - Quakkelaar, Caspar
AU - Cabrera, Ricardo
AU - Guedes da Silva, Leonor
AU - Smids, Aron L.
AU - Villalobos, Pablo Andres
AU - van Loosdrecht, Mark C.M.
AU - Wahl, S. Aljoscha
PY - 2020
Y1 - 2020
N2 - Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (kcatNADH/KMNADH)/(kcatNADPH/KMNADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli, opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB.
AB - Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (kcatNADH/KMNADH)/(kcatNADPH/KMNADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli, opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB.
KW - acetoacetyl-CoA reductase
KW - cofactor specificity
KW - NADH
KW - NADPH
KW - oxygen limitation
KW - Polyhydroxybutyrate
UR - http://www.scopus.com/inward/record.url?scp=85097059196&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2020.10.022
DO - 10.1016/j.jbiotec.2020.10.022
M3 - Article
C2 - 33122026
AN - SCOPUS:85097059196
SN - 0168-1656
VL - 325
SP - 207
EP - 216
JO - Journal of Biotechnology
JF - Journal of Biotechnology
ER -