Enhancing poly(3-hydroxyalkanoate) production in Escherichia coli by the removal of the regulatory gene arcA

Abstract

Recombinant Escherichia coli is a desirable platform for the production of many biological compounds including poly(3-hydroxyalkanoates), a class of naturally occurring biodegradable polyesters with promising biomedical and material applications. Although the controlled production of desirable polymers is possible with the utilization of fatty acid feedstocks, a central challenge to this biosynthetic route is the improvement of the relatively low polymer yield, a necessary factor of decreasing the production costs. In this study we sought to address this challenge by deleting arcA and ompR, two global regulators with the capacity to inhibit the uptake and activation of exogenous fatty acids. We found that polymer yields in a ΔarcA mutant increased significantly with respect to the parental strain. In the parental strain, PHV yields were very low but improved 64-fold in the ΔarcA mutant (1.92–124 mg L−1) The ΔarcA mutant also allowed for modest increases in some medium chain length polymer yields, while weight average molecular weights improved by approximately 1.5-fold to 12-fold depending on the fatty acid substrate utilized. These results were supported by an analysis of differential gene expression, which showed that the key genes (fadD, fadL, and fadE) encoding fatty acid degradation enzymes were all upregulated by 2-, 10-, and 31-fold in an ΔarcA mutant, respectively. Additionally, the short chain length fatty acid uptake genes atoA, atoE and atoD were upregulated by 103-, 119-, and 303-fold respectively, though these values are somewhat inflated due to low expression in the parental strain. Overall, this study demonstrates that arcA is an important target to improve PHA production from fatty acids.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0291-z) contains supplementary material, which is available to authorized users.

Low Carbon Concentration Feeding Improves Medium-Chain-Length Polyhydroxyalkanoate Production in Escherichia coli Strains With Defective β-Oxidation

“…Low cell mass concentration was obtained in this work and was thought to be the reason for low polymer production. Similarly, low P(3HD) production was acquired when cultures were grown in a medium without a co-carbon source (Scheel et al, 2016). When glycerol was supplemented as a co-carbon source at 10 g/L, a high mass concentration of 6.04 g/L was reached in accordance with an improvement in the polymer yield (Hiroe et al, 2016).…”

“…The production system designed with direct conversion of MCL-PHAs from related fatty acids accumulated 0.26 g/L of P(3HD) (Tappel et al, 2012). Thereafter, P(3HD) production was improved up to 0.60 g/L by removing the regulatory gene arcA from the LSBJ strain (Scheel et al, 2016). Following a recent study, we further enhanced the production of the near P(3HD) homopolymer to 1.47 g/L after growing cells in a mineral-based medium supplemented with 5 g/L yeast extract (Hiroe et al, 2016).…”

Increased Production of the Value-Added Biopolymers Poly(R-3-Hydroxyalkanoate) and Poly(γ-Glutamic Acid) From Hydrolyzed Paper Recycling Waste Fines

“…The yields and repeating unit compositions of PHA polymers were determined using GC, as previously described with slight modification (Braunegg et al, 1978;Scheel et al, 2016). ACS HPLC-grade chloroform and methanol were used for gas chromatography (GC) sample preparation.…”

Deletion of arcA, iclR, and tdcC in Escherichia coli to improve l‐threonine production

“…For example, arcA deletion has been used in E. coli to activate the TCA cycle and aerobic respiration [36]; a higher flux at the entrance of the TCA was observed due to the arcA deletion, resulting in a reduced production of acetate and less carbon loss [6]. Enhancing the flux of TCA by the arcA deletion in E. coli has been used to produce l-serine [13], fumaric acid [14], poly(3hydroxyalkanoate) [16,37], acetyl-CoA-derived chemicals [17], and 1,3-propanediol [18]. The arcA deletion could not further increase the production of succinate in anaerobic fermentation [38], possibly because the major effect of arcA is on the enzymes that control the aerobic respiration [39].…”

“…Overexpression of arcA and deletion of ptsG can significantly decrease glucose consumption and hence decrease the acetate accumulation [11]; however, deletion of arcA gene can also decrease the acetate accumulation because of the high TCA cycle flux [12]. Therefore, deletion of arcA to enhance the flux of TCA cycle in E. coli has been used to improve the production of l-serine [13], fumaric acid [14], poly(3-hydroxyalkanoate) [15,16], and acetyl-CoA-derived chemicals [17]. Furthermore, disruption of arcA can increase NADH/NAD + ratio, leading to the improved production of 1,3-propanediol [18].…”