ArticleApplied and environmental microbiology2013
Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.
Article in Applied and environmental microbiology, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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14 citing papers in PubMed, 54 citations in OpenAlex.
- Formate-strengthened NADH supply in Salinivibrio sp. TGB4 improves polyhydroxyalkanoates production.World journal of microbiology & biotechnology · 2026Article
- Phages-bacteria interactions underlying the dynamics of polyhydroxyalkanoate-producing mixed microbial cultures via meta-omics study.mSystems · 2025Article
- Assay development and inhibition of theMicrobiology (Reading, England) · 2023Article
- Adaptive Laboratory Evolution ofMolecules (Basel, Switzerland) · 2022Article
- Engineering an acetoacetyl-CoA reductase from Cupriavidus necator toward NADH preference under physiological conditions.Scientific reports · 2022Article
- Development ofFrontiers in bioengineering and biotechnology · 2021Article
- With Chitosan and PLGA as the Delivery Vehicle,Frontiers in immunology · 2021Article
- Modification of acetoacetyl-CoA reduction step in Ralstonia eutropha for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from structurally unrelated compounds.Microbial cell factories · 2019Article
- Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation.Applied microbiology and biotechnology · 2019Article
- Crystal structure and kinetic analyses of a hexameric form of (S)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium acetobutylicum.Acta crystallographica. Section F, Structural biology communications · 2018Article
- Applying asymptotic methods to synthetic biology: Modelling the reaction kinetics of the mevalonate pathway.Journal of theoretical biology · 2018Article
- Anaerobic poly-3-D-hydroxybutyrate production from xylose in recombinant Saccharomyces cerevisiae using a NADH-dependent acetoacetyl-CoA reductase.Microbial cell factories · 2016Article
- MtgA Deletion-Triggered Cell Enlargement of Escherichia coli for Enhanced Intracellular Polyester Accumulation.PloS one · 2015Article
- Improved production of poly(lactic acid)-like polyester based on metabolite analysis to address the rate-limiting step.AMB Express · 2014Article
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9 authors at 2 institutions in 1 country.
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Abstract
NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.
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