Chemically Intractable No More: In Vivo Incorporation of “Click”-Ready Fatty Acids into Poly-[(<i>R</i>)-3-hydroxyalkanoates] in <i>Escherichia coli</i>

Pinto, Atahualpa; Ciesla, Jessica H.; Palucci, Adriana; Sutliff, Bradley P.; Nomura, Christopher T.

doi:10.1021/acsmacrolett.5b00823

Cited by 19 publications

(17 citation statements)

References 51 publications

(69 reference statements)

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“…Additionally, during the review process of this paper, Nomura et al reported an in vivo synthesis of clickable PHA polymers utilizing ω-azido fatty acids as the feedstocks. 62 Since HHxCoA could be metabolically generated from 3-( R )-hydroxy-5-hexynoic acid 63 and exhibits high reactivity toward PhaC Cs (22% activity of HBCoA), it is currently used as a probe to investigate the formation of PHA granules.…”

Section: Resultsmentioning

confidence: 99%

Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain

et al. 2016

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Polyhydroxyalkanoates (PHAs) are carbon and energy storage polymers produced by a variety of microbial organisms under nutrient-limited conditions. They have been considered as an environmentally friendly alternative to oil-based plastics due to their renewability, versatility and biodegradability. PHA synthase (PhaC) plays a central role in PHA biosynthesis, in which its activity and substrate specificity are major factors in determining the productivity and properties of the produced polymers. However, the effects of modifying the substrate side chain are not well understood because of the difficulty to accessing the desired analogs. In this report, a series of 3-(R)-hydroxyacyl coenzyme A (HACoA) analogs were synthesized and tested with class I synthases from Chromobacterium sp. USM2 (PhaCCs and A479S-PhaCCs) and Caulobacter crescentus (PhaCCc) as well as class III synthase from Allochromatium vinosum (PhaECAv). It was found that, while different PHA synthases displayed distinct preference with regard to the length of the alkyl side chains, they could withstand moderate side chain modifications such as terminal unsaturated bonds and the azide group. Specifically, the specific activity of PhaCCs toward propynyl analog (HHxyCoA) was only 5-fold less than that toward the classical substrate HBCoA. The catalytic efficiency (kcat/Km) of PhaECAv toward azide analog (HABCoA) was determined to be 2.86 × 105 M−1s−1, which was 6.2% of the value of HBCoA (4.62 × 106 M−1s−1) measured in the presence of bovine serum albumin (BSA). These side chain modifications may be employed to introduce new material functions to PHAs as well as to study PHA biogenesis via click-chemistry, in which the latter remains unknown and is important for metabolic engineering to produce PHAs economically.

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Section: Resultsmentioning

confidence: 99%

Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain

et al. 2016

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“…Samples were concentrated to relative dryness using a rotary evaporator, and redissolved in 1 mL chloroform. Crude polymers were purified by non-solvent precipitation in cold methanol as described previously (Pinto et al 2016), with several modifications. Briefly, dissolved samples were added dropwise to 10 mL of ice-cold methanol (4 °C) with rapid stirring.…”

Section: Methodsmentioning

confidence: 99%

“…The weight average (M w ) and number average (M n ) molecular weights for each sample were determined by gel permeation chromatography (GPC) as described previously (Pinto et al 2016). Briefly, PHA solutions of approximately 1.0 g L −1 were prepared by dissolution in chloroform and passed through a syringe filter (0.45 μm PTFE).…”

Section: Methodsmentioning

confidence: 99%

“…1). This system allowed for strict control of repeating unit composition which enables great control over the physical properties of PHA polymers produced using this system, unlocking the potential for tailoring PHA materials for click-chemistry modifications (Levine et al 2015, 2016; Pinto et al 2016). Although these previous studies addressed control of the monomer composition and thus physical and chemical properties of PHA polymers, overall polymer yields were still relatively low and some fatty acid substrates had poor incorporation into either PHA homo- or copolymers.…”

Section: Introductionmentioning

confidence: 99%

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

Scheel

Lundgren

et al. 2016

AMB Expr

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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.

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“…Particularly, recent protein engineering of the synthase or structural proteins demonstrated that various functional proteins can be attached to the PHA granule surface and stably maintained outside the cell as bio‐beads. Therefore, using genetically engineered PHA synthase could aid in the introduction of specific functionalities into PHA granules, which will further broaden its applications in advanced delivery systems …”

Section: Pha‐based Nanovehicles As Therapeutic Delivery Carriermentioning

confidence: 99%

Recent advances of using polyhydroxyalkanoate‐based nanovehicles as therapeutic delivery carriers

Liu

Loh

2016

WIREs Nanomed Nanobiotechnol

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Polyhydroxyalkanoates (PHAs) are a class of diverse biodegradable polyesters that can be produced either by natural bioconversion process or by chemical synthesis via the ring-opening polymerization of β-lactones. Because of the excellent biodegradability and biocompatibility, the development of PHA-based nanovehicles as therapeutic delivery carriers, including nanoparticles, micelles, liposomes, and vesicles, has received considerable attention in recent years, and these sophisticated materials have demonstrated significant impact on the drug bioavailability, better encapsulation, and less toxic properties of biodegradable polymers. In this review, the most recent advances of using PHA-based nanovehicles as therapeutic delivery carriers are summarized with respect to different material types including intrinsic bulk PHA and functionalized PHA through biological and chemical approaches. The bio-significance of using different carriers in the controlled and targeted delivery of hydrophobic drugs, proteins, vaccines, nucleic acids (DNA and siRNA), and biological macromolecules in cancer therapy is also discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1429. doi: 10.1002/wnan.1429 For further resources related to this article, please visit the WIREs website.

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Chemically Intractable No More: In Vivo Incorporation of “Click”-Ready Fatty Acids into Poly-[(R)-3-hydroxyalkanoates] in Escherichia coli

Cited by 19 publications

References 51 publications

Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain

Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain

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

Recent advances of using polyhydroxyalkanoate‐based nanovehicles as therapeutic delivery carriers

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