Language：English   Publishing type：Research paper (scientific journal)  

4-Hydroxybutyrate (4HB) and the related compounds are important in the chemical industry, and microbial metabolic engineering for 4HB production has been pioneered via the TCA cycle route employing clostridial enzymes. This study focused on an alternative, energy-efficient route for 4HB-CoA production using a unique enzyme from a different domain of life. The [4Fe-4S] cluster-containing 4HB-CoA dehydratase (4HcD), catalyzing radical-based atypical dehydration/hydration, is one of the enzymes difficult to apply in biotechnological applications due to its high sensitivity to reactive oxygen species. The present results demonstrated that an oxygen-tolerant 4HcD derived from an aerobic archaeon enabled conversion of crotonyl-CoA to 4HB-CoA in polyhydroxyalkanoate (PHA)-producing Cupriavidus necator. This archaeal enzyme contributed to establishing an energetically favorable ATP-generating biosynthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) under low-aeration conditions. By rewiring C4-acyl-CoA metabolism in the host and modifying the N-terminus of 4HcD to potentially enhance its translation efficiency, the 4HB molar fraction of up to 12.6 mol % was obtained while inherent 3-hydroxyhexanoate incorporation was suppressed. This pathway was further extended to chemolithoautotrophic metabolism via gas fermentation, which enabled the direct production of the copolyester from CO2 and H2. Together, these findings highlight the potential of integrating archaeal catalytic functions within hydrogen-oxidizing bacteria as a promising strategy for advancing carbon capture and the sustainable biosynthesis of functional biopolymers.

DOI： 10.1021/acssynbio.6c00154

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of (R)-3-hydroxybutyrate (3HB) and (R)-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaCAR, a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaCAR was applied in the natural PHA-producing bacterium, R. eutropha. During the investigation, (R/S)-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha. This was probably due to formation of excess 2-ketobutyrate (2KB) from (R/S)-2HB and the consequent L-valine depletion caused by dominant L-isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent D-lactate dehydrogenase (Dld) and [Fe-S] protein-dependent L-lactate dehydrogenase as the enzymes responsible for sensitivity to (R)-2HB and (S)-2HB, respectively. The engineered R. eutropha strain (phaCAR+, ldhACd-hadACd+ encoding clostridial (R)-2-hydroxyisocaproate dehydrogenase and (R)-2-hydoroxyisocaproate CoA transferase, ∆dld) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium (RS)-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaCAR. The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB-co-3HV) and P(2HB) segments, suggesting that PhaCAR functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell. KEY POINTS: (R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha (R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identified The engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB.

DOI： 10.1007/s00253-023-12797-6

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