Synthesis of the building block 2-hydroxyisobutyrate from fructose and butyrate by Cupriavidus necator H16

Abstract

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.

Structural Basis of the Stereospecificity of Bacterial B12-dependent 2-Hydroxyisobutyryl-CoA Mutase

“…In the future, tailor-made mutases might be developed for the rearrangement of carboxylic acids relevant for industrial and pharmaceutical production, in particular for the generation or identification of enzymes with efficient HCM, PCM, and ICM activity (7,(33)(34)(35). Detailed structural information of the substrate interaction helps in the rational enzyme design of these industrially relevant biocatalysts.…”

Thermophilic Coenzyme B 12 -Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of ( R )-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA

“…2), such as the oxidation of butyric acid, which has already been tested for the HCMdependent building block production in C. necator DSM 428 (13). Considering the temperature optimum of RCM, however, expression in a thermophilic production strain should be favored in the future to tap the full potential of the enzyme.…”

“…Carboxylic acids, including 2-hydroxyisobutyric and 3-hydroxybutyric acid, were routinely quantified using high-performance liquid chromatography with refractive index (RI) detection (13) applying an eluent of 0.01 N sulfuric acid at 0.5 ml per min and a Nucleogel ION 300 OA column (300 by 7.7 mm; Macherey-Nagel). In addition, 2-hydroxyisobutyric acid was identified as the corresponding methyl ester by gas chromatography (GC) as described before (8) by applying mass detection (5973 mass selective detector [MSD]; Agilent).…”

“…Currently most interesting, however, is a biotechnological synthesis route employing HCM for the industrial production of the poly(methyl methacrylate) precursor 2-hydroxyisobutyric acid from renewable carbon (11), having great potential to completely replace the established petrochemical processes. Accordingly, mutase-dependent synthesis of this important building block from simple sugars and carboxylic acids as well as from carbon dioxide has already been demonstrated at the lab scale (12)(13)(14)(15). Implementation of an industrial HCM process seems to be particularly feasible, as the metabolic route delivering the mutase substrate 3-hydroxybutyryl-CoA is part of a well-studied overflow metabolism in bacteria.…”

Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

“…As a proof of principle, production of 2-HIBA by Cupriavidus necator H16 strains expressing HCM genes has already been demonstrated with fructose, butyric acid, or carbon dioxide plus knallgas as the substrates (8)(9)(10)(11). However, product yields were very low in these early studies, an observation that was subsequently ascribed to the fact that HCM from Aquincola tertiaricarbonis L108 is highly specific for (S)-3-hydroxybutyryl-CoA and thus poorly compatible with the PHB cycle (3).…”