An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichia coli

Karel Olavarria, Alexandre Carnet, Joachim van Renselaar, Caspar Quakkelaar, Ricardo Cabrera, Leonor Guedes da Silva, Aron L. Smids, Pablo Andres Villalobos, Mark C.M. van Loosdrecht, S. Aljoscha Wahl

Research output: Contribution to journalArticleScientificpeer-review

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)207-216
Number of pages10
JournalJournal of Biotechnology
Volume325
DOIs
Publication statusPublished - 2020

Keywords

  • acetoacetyl-CoA reductase
  • cofactor specificity
  • NADH
  • NADPH
  • oxygen limitation
  • Polyhydroxybutyrate

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