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.

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

“…In a DarcA background, central metabolic pathways are unregulated, and TCA cycle reactions are still operative under conditions of restricted oxygen supply (Pettinari et al 2008). These mutants are unable to grow in minimal medium if it is not supplemented with high levels of complex nutrients, such as yeast extract (Nikel et al 2006(Nikel et al , 2008b, probably reflecting a regulatory deficiency in the partitioning of pyruvate-derived carbon skeletons between competing requirements for NAD(P)H oxidation and biosynthesis. However, we reported that a creC(Con) mutation allowed growth of arcA mutants even in semi-synthetic media (Nikel et al 2008b).…”

“…Biomass (expressed as CDW) was determined gravimetrically from the pellet fraction of appropriate culture aliquots, after two rounds of centrifugation (8000 g, 10 min) and washing of the cells with 150 mmol l )1 NaCl (Nikel et al 2006). Ethanol concentration was measured by using an enzymatic kit based on alcohol dehydrogenase (Randox Laboratories Ltd., Oceanside, CA, USA).…”

“…MYA medium contained (in g l )1 ): Na 2 HPO 4 , 6AE0; KH 2 PO 4 , 3AE0; (NH 4 ) 2 SO 4 , 1AE4; NaCl, 0AE5; yeast extract, 10AE0; casein amino acids, 5AE0; and MgSO 4 AE7H 2 O, 0AE2; initial pH = 7AE2 (Nikel et al 2006). SGA medium contained the same salts as MYA, but yeast extract was suppressed and casein amino acids concentration was lowered to 0AE3 g l )1 .…”

“…Two-stage bioreactor cultivations were carried out in a 5AE6-l stirred tank reactor equipped with six flat-bladed disk turbines (BioFlo 110; New Brunswick Scientific Co., Edison, NJ, USA) essentially as described by Nikel et al (2006), with a 4-l working volume and pH controlled at 7AE20 ± 0AE05 by automatic addition of 3 mol l )1 KOH or 1AE5 mol l )1 H 2 SO 4 . To prevent foam formation, 30 ll l )1 Antifoam 289 (Sigma-Aldrich, St Louis, MO, USA) was manually added at the onset of the run.…”

GacS-Dependent Regulation of Polyhydroxyalkanoate Synthesis in Pseudomonas putida CA-3

“…Signal transduction systems associated with the detection of extracellular conditions and the coordination of central metabolic pathways involved have also been implicated in PHA production control. The arc system in Escherichia coli, which regulates the expression of several operons relative to the redox state of the environment, has previously been manipulated to improve E. coli as a host strain for poly-␤-hydroxybutyrate (PHB) biosynthesis (16). In addition, arc gene mutants enable the expression of TCA cycle components under microaerobic conditions, elevating levels of reducing equivalents, which can be channeled into PHB production.…”