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

Author(s)

Olavarria, Karel ^[1] ; Carnet, Alexandre ^[1] ; van Renselaar, Joachim ^[1] ; Quakkelaar, Caspar ^[1] ; Cabrera, Ricardo ^[2] ; da Silva, Leonor Guedes ^[1] ; Smids, Aron L. ^[1] ; Andres Villalobos, Pablo ^[2] ; van Loosdrecht, Mark C. M. ^[1] ; Wahl, S. Aljoscha ^[1]

Affiliation

^[1] Delft Univ Technol, Fac Tech Nat Wetenschappen, Dept Biotechnol, Van der Maasweg 9, NL-2629 HZ Delft - Netherlands

^[2] Univ Chile, Fac Ciencias, Dept Biol, Las Palmeras 3425, Nunoa, Region Metropol - Chile

• Document type: Journal article
• Source: Journal of Biotechnology; v. 325, p. 215-224, JAN 10 2021.
• Web of Science Citations: 1

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 (k(cat)(NADH)/K-M(NADH))/(k(cat)(NADPH)/K-M(NADPH))>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. (AU)

• FAPESP's process: 13/50357-2 - Programmable balancing of growth and formation of poyhydroxyalkanoates in Escherichia coli
• Grantee: José Gregório Cabrera Gomez
• Support Opportunities: Regular Research Grants