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Current manufacturing of most bulk chemicals through petrochemical routes considerably contributes to common concerns over the depletion of fossil carbon sources and greenhouse gas emissions. Sustainable future production of commodities thus requires the shift to renewable feedstocks in combination with established or newly developed synthesis routes. In this study, the potential of Cupriavidus necator H16 for autotrophic synthesis of the building block chemical 2-hydroxyisobutyric acid (2-HIBA) is evaluated. A novel biosynthetic pathway was implemented by heterologous expression of the 2-hydroxyisobutyryl-coenzyme A (2-HIB-CoA) mutase from Aquincola tertiaricarbonis L108, relying on a main intermediate of strain H16's C4 overflow metabolism, 3-hydroxybutyryl-CoA. The intention was to direct the latter to 2-HIBA instead or in addition to poly-3-hydroxybutyrate (PHB). Autotrophic growth and 2-HIBA (respectively, PHB) synthesis of wild-type and PHB-negative mutant strains were investigated producing maximum 2-HIBA titers of 3.2 g L(-1) and maximum specific 2-HIBA synthesis rates (q 2-HIBA) of about 16 and 175 μmol g(-1) h(-1), respectively. The obtained specific productivity was the highest reported to date for mutase-dependent 2-HIBA synthesis from heterotrophic and autotrophic substrates. Furthermore, expression of a G protein chaperone (MeaH) in addition to the 2-HIB-CoA mutase subunits yielded improved productivity. Analyzing the inhibition of growth and product synthesis due to substrate availability and product accumulation revealed a strong influence of 2-HIBA, when cells were cultivated at high titers. Nevertheless, the presented results imply that at the time the autotrophic synthesis route is superior to thus far established heterotrophic routes for production of 2-HIBA with C. necator.

) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of thioesterase II, are discussed. Carbon skeleton rearrangement of carboxylic acids via a chemically challenging radical mechanism is catalyzed by coenzyme B 12 -dependent acyl-coenzyme A (acyl-CoA) mutases (1). During catalysis, both acyl-CoA and B 12 molecules are completely buried within the enzyme. This extensive interaction is mediated by highly conserved amino acid residues, forming a characteristic triose phosphate isomerase (TIM) barrel and a Rossman fold. The best-studied member of this enzyme family is methylmalonylCoA mutase (MCM), specifically catalyzing the isomerization of succinyl-and (R)-methylmalonyl-CoA (2). Several genetic defects impairing mitochondrial MCM activity are associated with methylmalonic aciduria, an inborn error of branched-chain amino acid metabolism (3, 4). Another mutase playing a role in central carbon metabolism is ethylmalonyl-CoA mutase (ECM), involved in acetic acid assimilation in bacteria lacking the glyoxylate cycle (5). In addition, isobutyryl-CoA mutase (ICM) appears to function mainly in secondary metabolism, e.g., the bacterial synthesis of polyketide antibiotics (6). Recently, a fourth subfamily of coenzyme B 12 -dependent acyl-CoA mutases has been characterized, specifically catalyzing the interconversion of 3-hydroxybutyrylCoA enantiomers and 2-hydroxyisobutyryl-CoA (7) (Fig. 1). Initially, the 2-hydroxyisobutyryl-CoA mutase (HCM) has been discovered in the bacterial strains Aquincola tertiaricarbon...

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.

Background The synthesis of 2-hydroxyisobutyric acid (2-HIB), a promising building block for, e.g., Plexiglas® production, is described as an example for a clean and sustainable bioproduction. Methods A derivative strain of Cupriavidus necator H16, impaired in the poly-ß-hydroxybutyrate synthesis pathway and equipped with xenogenic 2-hydroxyisobutyryl-coenzyme A mutase from Aquincola tertiaricarbonis L108, was applied. Batch cultivation was performed in the presence of vitamin B12 by supplying a gas mixture comprising hydrogen, oxygen, and carbon dioxide. Results Exploiting the chemo-litho-autotrophic potential of this so-called knallgas bacterium, 2-HIB was synthesized and excreted into the cultivation broth under aerobic conditions when inorganic nitrogen-limited conditions allowed an overflow metabolism of carbon metabolites. 2-HIB synthesis proceeded at a rate of 8.58 mg/[(g bacterial dry mass)·h]. Approximately 400 mg/L in total was obtained. The results were subsequently compared to calculated model data to evaluate the efficiency of the conversion of the substrates into the product. To achieve overall yield data regarding the substrate conversion, the model describes an integral process which includes both 2-HIB synthesis and biomass formation. Conclusions This study has confirmed the feasibility of the microbial synthesis of the bulk chemical 2-HIB from hydrogen and carbon dioxide by exploiting the chemo-litho-autotrophic metabolism of C. necator H16 PHB−4, additionally expressing the foreign 2-HIB-coenzyme A mutase. The product synthesis was satisfying as a proof of principle but does not yet approach the maximum value as derived from the model data. Furthermore, the biosynthesis potential of an optimized process is discussed in view of its technical application.