Synthesis of the building block 2-hydroxyisobutyrate from fructose and butyrate by Cupriavidus necator H16.
2-Hydroxyisobutyryl-coenzyme A mutase, originally discovered in the context of methyl tert-butyl ether degradation in Aquincola tertiaricarbonis L108, catalyzes the isomerization of 3-hydroxybutyryl-coenzyme A (3-HB-CoA) to 2-hydroxyisobutyryl-CoA. It thus constitutes the basis for a biotechnological route from practically any renewable carbon to 2-hydroxyisobutyrate (2-HIB) via the common metabolite 3-hydroxybutyrate. At first sight, recombinant Cupriavidus necator H16 expressing the mutase seems to be well suited for such a synthesis process, as a strong overflow metabolism via (R)-3-HB-CoA is easily induced in this bacterium possessing the poly-3-hydroxybutyrate metabolism. However, the recently established stereospecificity of the mutase, dominantly preferring the (S)-enantiomer of 3-HB-CoA, calls for a closer investigation of C. necator as potential 2-HIB production strain and raised the question about the strain's potential to yield 2-HIB from substrates directly providing (S)-3-HB-CoA. We compared two mutase-expressing C. necator H16 strains for their capability to synthesize 2-HIB from fructose and butyrate, delivering either (R)- or (S)-3-HB-CoA. Our results indicate that due to the enantiospecificity of the mutase, fructose is a weaker substrate for 2-HIB synthesis than butyrate. Production rates achieved with the PHB-negative strain H16 PHB(-)4 on butyrate were higher than on fructose. Using the wild-type did not significantly improve the production rates as the latter showed a 34-fold and a 5-fold lower 2-HIB synthesis rate compared to H16 PHB(-)4 on fructose and butyrate, respectively. Moreover, both strains showed concomitant excretion of undesired side products, such as pyruvate and 3-hydroxybutyrate, significantly decreasing the 2-HIB yield.
Department of Environmental Microbiology, Helmholtz Centre for Environmental Research (UFZ), Permoserstrasse 15, 04318, Leipzig, Germany.
Denise
D
Przybylski
Thore
T
Rohwerder
Hauke
H
Harms
Nadya
N
Yaneva
Roland H
RH
Müller
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020562:Betaproteobacteria / Q000201:enzymology; D002087:Butyrates / Q000378:metabolism*; D020118:Cupriavidus necator / Q000235:genetics / Q000378:metabolism*; D005632:Fructose / Q000378:metabolism*; D005818:Genetic Engineering; D006885:Hydroxybutyrates / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002087:Butyrates; D006885:Hydroxybutyrates; D005632:Fructose; C008039:2-hydroxyisobutyric acid; D019751:Intramolecular Transferases