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Title (Primary) Synthesis of the building block 2-hydroxyisobutyrate from fructose and butyrate by Cupriavidus necator H16
Author Przybylski, D.; Rohwerder, T.; Harms, H.; Yaneva, N.; Müller, R.H.;
Journal Applied Microbiology and Biotechnology
Year 2013
Department UMB;
Volume 97
Issue 20
Language englisch;
POF III (all) T15;
Keywords 2-Hydroxyisobutyryl-CoA mutase; 2-Hydroxyisobutyric acid; Cupriavidus necator; H16 PHB−4; Fructose; Butyrate; Overflow metabolism
UFZ wide themes RU4;

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 PHB4 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 PHB4 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.

ID 14137
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Przybylski, D., Rohwerder, T., Harms, H., Yaneva, N., Müller, R.H. (2013):
Synthesis of the building block 2-hydroxyisobutyrate from fructose and butyrate by Cupriavidus necator H16
Appl. Microbiol. Biotechnol. 97 (20), 8875 - 8885