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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 PHA yield as a percentage of dry weight between LSBJ and RSC02 at 24 h and 48 h, utilizing either sodium butyrate (C4) or decanoic acid (C10). LSBJ saw insignificant changes in PHB production between 24 and 48 h, while RSC02 increased slightly from 8.2 to 12.3% of cell dry weight. PHD yield from LSBJ increased from 22.6 to 31.7% between 24 and 48 h, an increase of ~40%, while PHD yield from RSC02 increased from 37.2 to 45.0%, an increase of ~20%. Regardless of the time, RSC02 produced more polymer than LSBJ in all cases. All values are the averages of triplicate experiments plus or minus the standard deviation about those averages
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Fig4: Comparison of PHA yield as a percentage of dry weight between LSBJ and RSC02 at 24 h and 48 h, utilizing either sodium butyrate (C4) or decanoic acid (C10). LSBJ saw insignificant changes in PHB production between 24 and 48 h, while RSC02 increased slightly from 8.2 to 12.3% of cell dry weight. PHD yield from LSBJ increased from 22.6 to 31.7% between 24 and 48 h, an increase of ~40%, while PHD yield from RSC02 increased from 37.2 to 45.0%, an increase of ~20%. Regardless of the time, RSC02 produced more polymer than LSBJ in all cases. All values are the averages of triplicate experiments plus or minus the standard deviation about those averages

Mentions: To determine whether the increased lag phase in RSC02 had a negative effect on PHA yield earlier in the production cycle, small subsamples were removed from both LSBJ and RSC02 shake flasks after 24 h and 48 h during the growth profile experiments. Similar to the data shown in Table 3, LSBJ produced very little PHB at either 24 or 48 h, and there was very little change between the two time points (Fig. 4). However, LSBJ yields of PHD increased from 22.6 to 31.7% of cell dry weight, an increase of approximately 40% (Fig. 4). RSC02 produced significantly more polymer than LSBJ regardless of the time or fatty acid substrate; PHB yield increased from 8.2 to 12.3% from 24 to 48 h, while PHD yield increased by ~20% between time points from 37.2 to 45.0% (Fig. 4).Fig. 4


Enhancing poly(3-hydroxyalkanoate) production in Escherichia coli by the removal of the regulatory gene arcA
Comparison of PHA yield as a percentage of dry weight between LSBJ and RSC02 at 24 h and 48 h, utilizing either sodium butyrate (C4) or decanoic acid (C10). LSBJ saw insignificant changes in PHB production between 24 and 48 h, while RSC02 increased slightly from 8.2 to 12.3% of cell dry weight. PHD yield from LSBJ increased from 22.6 to 31.7% between 24 and 48 h, an increase of ~40%, while PHD yield from RSC02 increased from 37.2 to 45.0%, an increase of ~20%. Regardless of the time, RSC02 produced more polymer than LSBJ in all cases. All values are the averages of triplicate experiments plus or minus the standard deviation about those averages
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120623&req=5

Fig4: Comparison of PHA yield as a percentage of dry weight between LSBJ and RSC02 at 24 h and 48 h, utilizing either sodium butyrate (C4) or decanoic acid (C10). LSBJ saw insignificant changes in PHB production between 24 and 48 h, while RSC02 increased slightly from 8.2 to 12.3% of cell dry weight. PHD yield from LSBJ increased from 22.6 to 31.7% between 24 and 48 h, an increase of ~40%, while PHD yield from RSC02 increased from 37.2 to 45.0%, an increase of ~20%. Regardless of the time, RSC02 produced more polymer than LSBJ in all cases. All values are the averages of triplicate experiments plus or minus the standard deviation about those averages
Mentions: To determine whether the increased lag phase in RSC02 had a negative effect on PHA yield earlier in the production cycle, small subsamples were removed from both LSBJ and RSC02 shake flasks after 24 h and 48 h during the growth profile experiments. Similar to the data shown in Table 3, LSBJ produced very little PHB at either 24 or 48 h, and there was very little change between the two time points (Fig. 4). However, LSBJ yields of PHD increased from 22.6 to 31.7% of cell dry weight, an increase of approximately 40% (Fig. 4). RSC02 produced significantly more polymer than LSBJ regardless of the time or fatty acid substrate; PHB yield increased from 8.2 to 12.3% from 24 to 48 h, while PHD yield increased by ~20% between time points from 37.2 to 45.0% (Fig. 4).Fig. 4

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.