FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3127002952> ?p ?o ?g. }

• W3127002952 abstract "Abstract The nicotinamide cofactor specificity of enzymes plays a key role in regulating metabolic processes and attaining cellular homeostasis. Multiple studies have used enzyme engineering tools or a directed evolution approach to switch the cofactor preference of specific oxidoreductases. However, whole-cell adaptation towards the emergence of novel cofactor regeneration routes was not previously explored. To address this challenge, we used an Escherichia coli NADPH-auxotroph strain. We continuously cultivated this strain under selective conditions. After 500-1100 generations of adaptive evolution using different carbon sources, we isolated several strains capable of growing without an external NADPH source. Most isolated strains were found to harbor a mutated NAD-dependent malic enzyme (MaeA). A single mutation in MaeA was found to switch cofactor specificity while lowering enzyme activity. Most mutated MaeA variants also harbored a second mutation that restored the catalytic efficiency of the enzyme. Remarkably, the best MaeA variants identified this way displayed overall superior kinetics relative to the wildtype variant with NAD + . In other evolved strains, the dihydrolipoamide dehydrogenase (Lpd) was mutated to accept NADP + thus enabling the pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase complexes to regenerate NADPH. Interestingly, no other central metabolism oxidoreductase seems to evolve towards reducing NADP + , which we attribute to several biochemical constraints such as unfavorable thermodynamics. This study demonstrates the potential and biochemical limits of evolving oxidoreductases within the cellular context towards changing cofactor specificity, further showing that long-term adaptive evolution can optimize enzyme activity beyond what is achievable via rational design or directed evolution using small libraries. Importance In the cell, NAD(H) and NADP(H) cofactors have different functions. The former mainly accepts electrons from catabolic reactions and carries them to respiration, while the latter provides reducing power for anabolism. Correspondingly, the ratio of the reduced to the oxidized form differs for NAD (low) and NADP (high), reflecting their distinct roles. We challenged the flexibility of E. coli’s central metabolism in multiple adaptive evolution experiments using an NADPH-auxotroph strain. We found several mutations in two enzymes, changing the cofactor preference of malic enzyme and dihydrolipoamide dehydrogenase. Upon deletion of their corresponding genes we performed additional evolution experiments which did not lead to the emergence of any additional mutants. We attribute this restricted number of mutational targets to intrinsic thermodynamic barriers: The high ratio of NADPH to NADP + limits metabolic redox reactions which can regenerate NADPH, mainly by mass action constraints." @default.
• W3127002952 created "2021-02-15" @default.
• W3127002952 creator A5014885322 @default.
• W3127002952 creator A5016671567 @default.
• W3127002952 creator A5018879367 @default.
• W3127002952 creator A5022872476 @default.
• W3127002952 creator A5022971719 @default.
• W3127002952 creator A5026953755 @default.
• W3127002952 creator A5037021002 @default.
• W3127002952 creator A5038401866 @default.
• W3127002952 creator A5040274654 @default.
• W3127002952 creator A5044028530 @default.
• W3127002952 creator A5073847402 @default.
• W3127002952 creator A5080526636 @default.
• W3127002952 creator A5081179224 @default.
• W3127002952 date "2021-02-10" @default.
• W3127002952 modified "2023-09-27" @default.
• W3127002952 title "Emergence of NADP+-reducing enzymes in Escherichia coli central metabolism via adaptive evolution" @default.
• W3127002952 cites W1945922467 @default.
• W3127002952 cites W1983784785 @default.
• W3127002952 cites W2004235182 @default.
• W3127002952 cites W2011861065 @default.
• W3127002952 cites W2022374283 @default.
• W3127002952 cites W2024244537 @default.
• W3127002952 cites W2093057648 @default.
• W3127002952 cites W2094476648 @default.
• W3127002952 cites W2104222134 @default.
• W3127002952 cites W2116976883 @default.
• W3127002952 cites W2119964499 @default.
• W3127002952 cites W2131708560 @default.
• W3127002952 cites W2144779133 @default.
• W3127002952 cites W2288141281 @default.
• W3127002952 cites W2395503246 @default.
• W3127002952 cites W2523360402 @default.
• W3127002952 cites W2610567633 @default.
• W3127002952 cites W2754615371 @default.
• W3127002952 cites W2767900770 @default.
• W3127002952 cites W2792274852 @default.
• W3127002952 cites W2811140905 @default.
• W3127002952 cites W2885800232 @default.
• W3127002952 cites W2902826360 @default.
• W3127002952 cites W3035144923 @default.
• W3127002952 cites W3037033469 @default.
• W3127002952 doi "https://doi.org/10.1101/2021.02.08.430375" @default.
• W3127002952 hasPublicationYear "2021" @default.
• W3127002952 type Work @default.
• W3127002952 sameAs 3127002952 @default.
• W3127002952 citedByCount "2" @default.
• W3127002952 countsByYear W31270029522021 @default.
• W3127002952 crossrefType "posted-content" @default.
• W3127002952 hasAuthorship W3127002952A5014885322 @default.
• W3127002952 hasAuthorship W3127002952A5016671567 @default.
• W3127002952 hasAuthorship W3127002952A5018879367 @default.
• W3127002952 hasAuthorship W3127002952A5022872476 @default.
• W3127002952 hasAuthorship W3127002952A5022971719 @default.
• W3127002952 hasAuthorship W3127002952A5026953755 @default.
• W3127002952 hasAuthorship W3127002952A5037021002 @default.
• W3127002952 hasAuthorship W3127002952A5038401866 @default.
• W3127002952 hasAuthorship W3127002952A5040274654 @default.
• W3127002952 hasAuthorship W3127002952A5044028530 @default.
• W3127002952 hasAuthorship W3127002952A5073847402 @default.
• W3127002952 hasAuthorship W3127002952A5080526636 @default.
• W3127002952 hasAuthorship W3127002952A5081179224 @default.
• W3127002952 hasBestOaLocation W31270029521 @default.
• W3127002952 hasConcept C104317684 @default.
• W3127002952 hasConcept C143065580 @default.
• W3127002952 hasConcept C181199279 @default.
• W3127002952 hasConcept C197957613 @default.
• W3127002952 hasConcept C20950039 @default.
• W3127002952 hasConcept C2776317432 @default.
• W3127002952 hasConcept C2777344606 @default.
• W3127002952 hasConcept C2779268744 @default.
• W3127002952 hasConcept C547475151 @default.
• W3127002952 hasConcept C55493867 @default.
• W3127002952 hasConcept C75520062 @default.
• W3127002952 hasConcept C86803240 @default.
• W3127002952 hasConcept C9418097 @default.
• W3127002952 hasConceptScore W3127002952C104317684 @default.
• W3127002952 hasConceptScore W3127002952C143065580 @default.
• W3127002952 hasConceptScore W3127002952C181199279 @default.
• W3127002952 hasConceptScore W3127002952C197957613 @default.
• W3127002952 hasConceptScore W3127002952C20950039 @default.
• W3127002952 hasConceptScore W3127002952C2776317432 @default.
• W3127002952 hasConceptScore W3127002952C2777344606 @default.
• W3127002952 hasConceptScore W3127002952C2779268744 @default.
• W3127002952 hasConceptScore W3127002952C547475151 @default.
• W3127002952 hasConceptScore W3127002952C55493867 @default.
• W3127002952 hasConceptScore W3127002952C75520062 @default.
• W3127002952 hasConceptScore W3127002952C86803240 @default.
• W3127002952 hasConceptScore W3127002952C9418097 @default.
• W3127002952 hasLocation W31270029521 @default.
• W3127002952 hasOpenAccess W3127002952 @default.
• W3127002952 hasPrimaryLocation W31270029521 @default.
• W3127002952 hasRelatedWork W1653909930 @default.
• W3127002952 hasRelatedWork W1656364975 @default.
• W3127002952 hasRelatedWork W1797418561 @default.
• W3127002952 hasRelatedWork W1953994264 @default.
• W3127002952 hasRelatedWork W1963967386 @default.
• W3127002952 hasRelatedWork W2071151402 @default.
• W3127002952 hasRelatedWork W2089731752 @default.

• next