Inactivation of type I polyhydroxyalkanoate synthase in Aeromonas hydrophila resulted in discovery of another potential PHA synthase

Hu, Fengqing; You, Song

doi:10.1007/s10295-006-0180-6

Cited by 4 publications

(2 citation statements)

References 26 publications

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“…has a complex regulation, which is controlled at the enzymatic level by cofactor inhibition and metabolite availability; at transcriptional level by specific and global transcriptional regulatory factors; and at translational level by global post-transcriptional regulators [47]. Up to now, only few reports have shown the presence of genes associated with the synthesis of scl and mclPHAs in the same organism [48], [49]. For this reason, the physiological and regulatory mechanisms that govern the production of both types of polymers in the same bacterium are still poorly understood.…”

Section: Discussionmentioning

confidence: 99%

High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

et al. 2014

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Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases.

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Section: Discussionmentioning

confidence: 99%

High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

et al. 2014

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“…Cells were harvested by centrifugation after 72 h and lyophilized. Gas chromatographic (GC) analysis of intracellular PHA content and PHA composition was performed as described previously [12] .…”

Section: Production Of Phbhhxmentioning

confidence: 99%

Characterization of Anti-Cancer Drug Materials Loaded Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Microspheres for Drug Delivery System in Biochemical Material System

Jing

Xiao

Zhang

et al. 2012

AMR

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Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is one of the components of polyhydroxyalkanoates (PHAs) and some of its mechanical properties have been shown to improve over poly (3-hydroxybutyrate) (PHB) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The investigation of PHBHHx microspheres as a drug delivery system was prepared by emulsion-solvent evaporation method for the sustained release of anti-cancer drug 5-fluorouracil (5-FU) and cyclosporin A (CsA). The mean diameter of the PHBHHx microspheres ranged from 5.24 to 22.10 μm dependent on the different processing parameters. The PHBHHx concentration, emulsifier concentration, anti-cancer drug dosage, and agitation speed, were optimized according to the encapsulation efficiency of 4% PHBHHx, 0.5% SDS, 10 mg anti-cancer drug, and 500 rpm. Under optimized conditions, the encapsulation efficiency of 5-FU and CsA microspheres were 7.19% and 96.44%, respectively. The morphologies of scanning electron microscope (SEM) suggested that PHBHHx microspheres were relatively smooth that provided better dispersion compared to PHB microspheres. The in vitro release profiles indicated 32.42% of 5-FU and 30.61% of CsA were released from PHBHHx microspheres during the initial burst phase, and the drug release from PHBHHx microsphere could be detected even after one month. The characteristics of PHBHHx microspheres demonstrated the feasibility of PHBHHx microsphere as a novel matrix for drug release system. With positive maintenance of the therapeutic concentrations of the drug, side effects can be reduced and patient compliance can be improved.

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Site-Directed Mutagenesis of Aeromonas hydrophila Enoyl Coenzyme A Hydratase Enhancing 3-Hydroxyhexanoate Fractions of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Cao

Fang

et al. 2007

Curr Microbiol

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The aim of this study is to enhance 3-hydroxyhexanoate (3HHx) fractions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), abbreviated as PHBHHx, through site-directed mutagenesis of Aeromonas hydrophila enoyl Coenzyme A hydratase (PhaJ(Ah)). Two amino acids (Leu-65 and Val-130) were selected as a substitutional site based on the structural information of PhaJ(Ah). The purified proteins from the wild-type enzyme and mutants were used to determine hydratase activities. Hydratase activities of four single-mutation enzymes were similar to those of the wild type PhaJ(Ah), while hydratase activities of two double-mutation enzymes were much lower. In addition, the mutated phaJ (Ah) was individually co-transformed into E. coli BL21 (DE3) together with pFH21, which carried the PHA synthase (PhaC(Ah)) gene from A. hydrophila. The recombinant E. coli harboring plasmid pETJ1 (L65A), pETJ2 (L65V) or plasmid pETJ3 (V130A) synthesized the enhanced 3HHx fractions of PHBHHx from dodecanoate, indicating that Leu-65 and Val-130 of PhaJ(Ah) play an important role in determining the acyl chain length substrate specificity. The mutated PhaJ(Ah) (L65A, L65V, or V130A) provided higher 3HHx precursors for PHA synthase, resulting in the enhanced 3HHx fractions of PHBHHx. It is possible to change the acyl chain length substrate specificity of PhaJ through site-directed mutagenesis and produce PHBHHx with a wider range of alterable monomer composition.

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Inactivation of type I polyhydroxyalkanoate synthase in Aeromonas hydrophila resulted in discovery of another potential PHA synthase

Cited by 4 publications

References 26 publications

High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

Characterization of Anti-Cancer Drug Materials Loaded Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Microspheres for Drug Delivery System in Biochemical Material System

Site-Directed Mutagenesis of Aeromonas hydrophila Enoyl Coenzyme A Hydratase Enhancing 3-Hydroxyhexanoate Fractions of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

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