The heterologous expression of Vitreoscilla hemoglobin (VHb) is known to enhance short-chain-length (scl) and short-chain-length-medium-chain-length (scl-mcl) polyhydroxyalkanoate (PHA) production in Escherichia coli, Aeromonas hydrophila and Pseudomonas putida, but has not been evaluated for the production of medium-chain-length (mcl) PHA in pseudomonads. Three recombinant strains of P. putida KT2440 were constructed, harboring pBBR1MCS-2 expression plasmids containing 0, 1 and 2 copies of the vgb gene. The vgb gene was placed under the control of the lac promoter, which conferred constitutive expression of vgb, since no lac repressor was produced. Mcl-PHA production was assessed in nonanoic acid-limited, fed-batch fermentations with exponential feeding. Although the VHb levels in the engineered strains were comparable to those observed in vgb-bearing pseudomonads, the recombinant strains had lower biomass production, PHA accumulation, volumetric productivity and product yield when compared to the wild-type strain. The performance of the plasmid-harboring strains decreased with increasing vgb copy numbers, presumably due to an increased metabolic burden arising from plasmid expression. All the fermentations ended at similar times due to insufficient oxygen supply and subsequent uncontrollable foaming, demonstrating that VHb production did not alleviate oxygen limitation. These results indicate that the effectiveness of VHb engineering must be assessed on a case-by-case basis.
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