scholarly journals Role of PhaC Type I and Type II Enzymes during PHA Biosynthesis

Polymers ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 910 ◽  
Author(s):  
Valeria Mezzolla ◽  
Oscar D’Urso ◽  
Palmiro Poltronieri
Keyword(s):  
Chain Length ◽  
Ralstonia Eutropha ◽  
Chemical Properties ◽  
Class I ◽  
Type I ◽  
Class Iii ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length

PHA synthases (PhaC) are grouped into four classes based on the kinetics and mechanisms of reaction. The grouping of PhaC enzymes into four classes is dependent on substrate specificity, according to the preference in forming short-chain-length (scl) or medium-chain-length (mcl) polymers: Class I, Class III and Class IV produce scl-PHAs depending on propionate, butyrate, valerate and hexanoate precursors, while Class II PhaC synthesize mcl-PHAs based on the alkane (C6 to C14) precursors. PHA synthases of Class I, in particular PhaCCs from Chromobacterium USM2 and PhaCCn/RePhaC1 from Cupriavidus necator/Ralstonia eutropha, have been analysed and the crystal structures of the C-domains have been determined. PhaCCn/RePhaC1 was also studied by X-ray absorption fine-structure (XAFS) analysis. Models have been proposed for dimerization, catalysis mechanism, substrate recognition and affinity, product formation, and product egress route. The assays based on amino acid substitution by mutagenesis have been useful to validate the hypothesis on the role of amino acids in catalysis and in accommodation of bulky substrates, and for the synthesis of PHB copolymers and medium-chain-length PHA polymers with optimized chemical properties.

scholarly journals Role of PhaC Type I and Type II Enzymes during PHA Biosynthesis

2018 ◽  
Author(s):  
Valeria Mezzolla ◽  
Oscar Fernando D'Urso ◽  
Palmiro Poltronieri
Keyword(s):  
Chain Length ◽  
Chemical Properties ◽  
Product Formation ◽  
Class I ◽  
Type I ◽  
Class Iii ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length

PHA synthases (PhaC) are grouped into four classes based on the kinetics and mechanisms of reaction. The grouping of PhaC enzymes into four classes is dependent on substrate specificity, according to the preference in forming short chain length (scl) or medium chain length (mcl) polymers: class I, class III, and class IV produce scl-PHAs depending on propionate, butyrate, valerate and hexanoate precursors, while class II phaC synthesize mcl-PHAs based on the alkane (C6 to C14) precursors. PHA synthases of class I, in particular PhaCCs from Chromobacterium USM2 and PhaCCn/RePhaC1 from Cupriavidus necator/R. eutropha, have been analysed and the crystal structures of the C-domains have been determined. PhaCCn/RePhaC1 was also studied by small angle X-ray scattering (SAXS) analysis. Models have been proposed for dimerization, catalysis mechanism, substrate recognition and affinity, product formation and product egress route. The assays based on amino acid substitution by mutagenesis have been useful to validate the hypothesis on the role of amino acids in catalysis and in accommodation of bulky substrates, for the synthesis of PHB co-polymers and medium chain length-PHA polymers with optimized chemical properties.

scholarly journals Increasing the Carbon Flux toward Synthesis of Short-Chain-Length—Medium-Chain-Length Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae through Modification of the β-Oxidation Cycle

2004 ◽  
Vol 70 (9) ◽  
pp. 5685-5687 ◽  
Author(s):  
Valeria Cora de Oliveira ◽  
Isamu Maeda ◽  
Syndie Delessert ◽  
Yves Poirier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dehydrogenase Activity ◽  
Chain Length ◽  
Carbon Flux ◽  
Ralstonia Eutropha ◽  
Short Chain ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Aeromonas Caviae

ABSTRACT Short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in Saccharomyces cerevisiae from intermediates of the β-oxidation cycle by expressing the polyhydroxyalkanoate synthases from Aeromonas caviae and Ralstonia eutropha in the peroxisomes. The quantity of polymer produced was increased by using a mutant of the β-oxidation-associated multifunctional enzyme with low dehydrogenase activity toward R-3-hydroxybutyryl coenzyme A.

scholarly journals Transcriptome Changes in Pseudomonas putida KT2440 during Medium-Chain-Length Polyhydroxyalkanoate Synthesis Induced by Nitrogen Limitation

2020 ◽  
Vol 22 (1) ◽  
pp. 152
Author(s):  
Dorota Dabrowska ◽  
Justyna Mozejko-Ciesielska ◽  
Tomasz Pokój ◽  
Slawomir Ciesielski
Keyword(s):  
Chain Length ◽  
Nitrogen Limitation ◽  
Stringent Response ◽  
Wild Type ◽  
Pseudomonas Putida Kt2440 ◽  
Medium Chain ◽  
Environmental Stimuli ◽  
Medium Chain Length ◽  
In The Wild

Pseudomonas putida’s versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs’ metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

Role of carboxyl pendant groups of medium chain length poly(3-hydroxyalkanoate)s in biomedical temporary applications

2010 ◽  
Vol 117 (4) ◽  
pp. 1888-1896 ◽  
Author(s):  
E. Renard ◽  
L. Timbart ◽  
G. Vergnol ◽  
V. Langlois
Keyword(s):  
Chain Length ◽  
Medium Chain ◽  
Medium Chain Length

scholarly journals Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase:In VivoandIn VitroStudies of Enzymatic Activity and Substrate Specificity

2013 ◽  
Vol 79 (12) ◽  
pp. 3813-3821 ◽  
Author(s):  
Jo-Ann Chuah ◽  
Satoshi Tomizawa ◽  
Miwa Yamada ◽  
Takeharu Tsuge ◽  
Yoshiharu Doi ◽  
...  
Keyword(s):  
Chain Length ◽  
Fold Increase ◽  
Short Chain ◽  
Pha Synthase ◽  
Wild Type ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length

ABSTRACTSaturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase fromChromobacteriumsp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCsfor 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity.In vitroactivities for polymerization of 3HV and 3HHx monomers were consistent within vivosubstrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

scholarly journals Characterization of a Novel Subgroup of Extracellular Medium-Chain-Length Polyhydroxyalkanoate Depolymerases from Actinobacteria

2012 ◽  
Vol 78 (20) ◽  
pp. 7229-7237 ◽  
Author(s):  
Joana Gangoiti ◽  
Marta Santos ◽  
María Auxiliadora Prieto ◽  
Isabel de la Mata ◽  
Juan L. Serra ◽  
...  
Keyword(s):  
Chain Length ◽  
De Novo ◽  
Hydrolysis Product ◽  
Maximum Activity ◽  
Short Chain Length ◽  
Great Ability ◽  
Content Type ◽  
Pha Depolymerase ◽  
Medium Chain ◽  
Medium Chain Length

ABSTRACTNineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters,Streptomyces roseolusSL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin andp-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured withpNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of severalActinobacteriastrains, includingS. roseolusSL3, were identified on the basis of the peptidede novosequencing of theStreptomyces venezuelaeSO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases.

scholarly journals Medium Chain and Long Chain Alkanes Hydroxylase Producing Whole Cell Biocatalyst From Marine Bacteria

2018 ◽  
Vol 22 (1) ◽  
pp. 12
Author(s):  
Ahmad Thontowi ◽  
Elvi Yetti ◽  
Yopi Yopi
Keyword(s):  
Chain Length ◽  
Alkane Hydroxylase ◽  
16S Rdna Gene ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Degrading Bacteria ◽  
Whole Cell Biocatalyst ◽  
Test Result ◽  
Long Chain

Alkanes are  major component of crude oil that could be hydrolyzed by the enzyme of alkane hydroxylase. The are three types of alkane hydroxylase based on the chain length of alkane such as short-chain length/SCL (C2-C4), medium-chain length/MCL (C5-C17), and long-chain length/LCL (C>18). The aims of this study were to characterize and identify alkanes-degrading bacteria from these bacteria. The 30 strains from marine were grown on MCL (Pentane-C5H12, Decane-C10H22, and Pentadecane-C15H32) and LCL (n-Paraffin-C12H19C17 and branch of Pristane-C19H40). The study showed twenty-nine isolates have the ability to degrade alkanes compounds, whereas 14 isolates have grown ability on MCL and LCL medium, 11 isolates have the ability to grow on MCL and n-LCL, 3 isolates have the ability only to grow on MCL medium and 1 isolate has the ability only grow on n-LCL medium. The growth test result indicated that 29 isolates have medium-chain alkane monooxygenase and long-chain alkane hydroxylase. Based on 16S rDNA gene analysis, we obtained twenty nine of oil- degrading bacteria, namely a-proteobacteria (57 %), g-proteobacteria (30 %), Flavobacteria (7 %), Bacilli (3%) and Propionibacteriales (3 %). g-Proteobacteria and a-proteobacteria which seems to play an important role in the alkane biodegradation.

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