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

doi:10.1016/j.jbiotec.2020.10.022

Karel Olavarria, Alexandre Carnet + Show 8 more

Open Access

https://doi.org/10.1016/j.jbiotec.2020.10.022

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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.

Highlights

(poly)-3-hydroxybutyrate (PHB) was discovered almost 100 years ago (Lemoigne, 1926), studies focused on this polymer have recently burgeoned in response to increasing interest in environment-friendly materials to replace non-biodegradable plastics
Cell-free extracts obtained from E. coli K-12 MG1655 cultures, with or without the plasmid pBBRMCS-2-phaCABCnecator, were employedascontrols.GiventhekineticparametersoftheAARCn(KMA cAcCoA(NADPH) = 9 μM, KMNADPH = 19 μM (28)), the conditions of the assay should be saturating for this enzyme
A. phosphatis-enriched mixed culture, activity using NADH was 60 times higher than activity with NADPH. This NADH-depending activity was four times higher than the NADPH-dependent activity observed in the cell-free extract from E. coli cells carrying several episomal copies of the phaCAB genes from C. necator per cell. These results suggested that (i) acetoacetyl-CoA reductase is expressed at high levels in Ca

Summary

Introduction

(poly)-3-hydroxybutyrate (PHB) was discovered almost 100 years ago (Lemoigne, 1926), studies focused on this polymer have recently burgeoned in response to increasing interest in environment-friendly materials to replace non-biodegradable plastics. PHB production cost is still high if compared with fossil-fuel based plastics. One of the factors affecting PHB production cost is the required oxygen supply (Fig. 1). Carlson and co-workers studied the maximum theoretical PHB yield under anaerobic conditions, given the glycolytic pathways available in Escherichia coli (Carlson et al, 2005). They concluded that co-feeding glucose and acetate with a 2:1 ratio, it is possible to co-generate two

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