Poly(3-hydroxybutyrate) synthesis by recombinant Escherichia coli arcA mutants in microaerobiosis

Abstract

We assessed the effects of different arcA mutations on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli strains carrying the pha synthesis genes from Azotobacter sp. strain FA8. The arcA mutations used were an internal deletion and the arcA2 allele, a leaky mutation for some of the characteristics of the Arc phenotype which confers high respiratory capacity. PHB synthesis was not detected in the wild-type strain in shaken flask cultures under low-oxygen conditions, while ArcA mutants gave rise to polymer accumulation of up to 24% of their cell dry weight. When grown under microaerobic conditions in a bioreactor, the arcA deletion mutant reached a PHB content of 27% +/- 2%. Under the same conditions, higher biomass and PHB concentrations were observed for the strain bearing the arcA2 allele, resulting in a PHB content of 35% +/- 3%. This strain grew in a simple medium at a specific growth rate of 0.69 +/- 0.07 h(-1), whereas the deletion mutant needed several nutritional additives and showed a specific growth rate of 0.56 +/- 0.06 h(-1). The results presented here suggest that arcA mutations could play a role in heterologous PHB synthesis in microaerobiosis.

FIG. 1.

Diamide sensitivity test for E. coli SP314 carrying either pQE32 (A) or pJP24 (B) grown on medium B-xylose plates. Filter paper disks containing 0.1 M (left) and 0.5 M (right) diamide were applied over a bacterial lawn, and the sensitivity was scored as the diameter of the growth inhibition zone.

FIG. 2.

Bioreactor cultures of K1060/pJP24 (ArcA⁺) (A) and CT1062/pJP24 (ΔarcA) (B) were carried out in MYAG in microaerobiosis. No air was supplied during the cultivation, and a constant agitation speed of 75 rpm was applied to maintain homogeneous conditions.

FIG. 3.

Bioreactor cultures of CT1061/pJP24 (arcA2) were carried out in SMAG in microaerobiosis. No air was supplied during cultivation, and a constant agitation speed of 75 rpm was applied to maintain homogeneous conditions.