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

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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 material: The online version of this article (doi:10.1186/s13568-016-0291-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Comparison of poly(3-hydroxydecanoate) (PHD) yield between LSBJ and mutant strains RSC02, RSC04, and RSC06. The average yield achieved by RSC02 is significantly greater than that of LSBJ, while RSC04 and RSC06 are not significantly different from LSBJ. All values are averages of triplicate experiments plus or minus the standard deviation around those averages. An asterisk denotes a statistically significant difference compared to LSBJ (Student’s t-test, two-tailed, α = 0.05)
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Fig2: Comparison of poly(3-hydroxydecanoate) (PHD) yield between LSBJ and mutant strains RSC02, RSC04, and RSC06. The average yield achieved by RSC02 is significantly greater than that of LSBJ, while RSC04 and RSC06 are not significantly different from LSBJ. All values are averages of triplicate experiments plus or minus the standard deviation around those averages. An asterisk denotes a statistically significant difference compared to LSBJ (Student’s t-test, two-tailed, α = 0.05)

Mentions: Our goal was to develop a strain capable of producing PHA polymers with controlled repeating unit compositions and increased yields. To achieve this, the mutant strains RSC02, RSC04, and RSC06 were derived from E. coli LSBJ by the deletion of arcA, ompR, and a tandem arcA/ompR deletion, respectively. The amount of poly(3-hydroxydecanoate) (PHD) produced by these strains was then compared to E. coli LSBJ while expressing PhaJ4 and PhaC1(STQK) in a set of preliminary experiments. RSC02 produced significantly more PHD than other strains, with a yield of 0.353 g L−1 (Fig. 2). Strains RSC04 and RSC06 were not found to be significantly different from LSBJ (Fig. 2; Additional file 1: Table S1).Fig. 2


Enhancing poly(3-hydroxyalkanoate) production in Escherichia coli by the removal of the regulatory gene arcA
Comparison of poly(3-hydroxydecanoate) (PHD) yield between LSBJ and mutant strains RSC02, RSC04, and RSC06. The average yield achieved by RSC02 is significantly greater than that of LSBJ, while RSC04 and RSC06 are not significantly different from LSBJ. All values are averages of triplicate experiments plus or minus the standard deviation around those averages. An asterisk denotes a statistically significant difference compared to LSBJ (Student’s t-test, two-tailed, α = 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120623&req=5

Fig2: Comparison of poly(3-hydroxydecanoate) (PHD) yield between LSBJ and mutant strains RSC02, RSC04, and RSC06. The average yield achieved by RSC02 is significantly greater than that of LSBJ, while RSC04 and RSC06 are not significantly different from LSBJ. All values are averages of triplicate experiments plus or minus the standard deviation around those averages. An asterisk denotes a statistically significant difference compared to LSBJ (Student’s t-test, two-tailed, α = 0.05)
Mentions: Our goal was to develop a strain capable of producing PHA polymers with controlled repeating unit compositions and increased yields. To achieve this, the mutant strains RSC02, RSC04, and RSC06 were derived from E. coli LSBJ by the deletion of arcA, ompR, and a tandem arcA/ompR deletion, respectively. The amount of poly(3-hydroxydecanoate) (PHD) produced by these strains was then compared to E. coli LSBJ while expressing PhaJ4 and PhaC1(STQK) in a set of preliminary experiments. RSC02 produced significantly more PHD than other strains, with a yield of 0.353 g L−1 (Fig. 2). Strains RSC04 and RSC06 were not found to be significantly different from LSBJ (Fig. 2; Additional file 1: Table S1).Fig. 2

View Article: PubMed Central - PubMed

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 material: The online version of this article (doi:10.1186/s13568-016-0291-z) contains supplementary material, which is available to authorized users.

No MeSH data available.