scholarly journals Polyhydroxyalkanoate (PHA) Production in Pseudomonas sp. phDV1 Strain Grown on Phenol as Carbon Sources

2021 ◽  
Vol 9 (8) ◽  
pp. 1636
Author(s):  
Iliana Kanavaki ◽  
Athina Drakonaki ◽  
Ermis Dionisios Geladas ◽  
Apostolos Spyros ◽  
Hao Xie ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Carbon Sources ◽  
Biodegradable Plastics ◽  
Pha Synthase ◽  
Pseudomonas Sp ◽  
Degrading Bacterium ◽  
Cell Extracts ◽  
Transmission Electron ◽  
Key Enzyme ◽  
Pha Production

Pseudomonas strains have a variety of potential uses in bioremediation and biosynthesis of biodegradable plastics. Pseudomonas sp. strain phDV1, a Gram-negative phenol degrading bacterium, has been found to utilize monocyclic aromatic compounds as sole carbon source via the meta-cleavage pathway. The degradation of aromatic compounds comprises an important step in the removal of pollutants. The present study aimed to investigate the ability of the Pseudomonas sp. strain phDV1 to produce polyhydroxyalkanoates (PHAs) and examining the effect of phenol concentration on PHA production. The bacterium was cultivated in minimal medium supplemented with different concentrations of phenol ranging from 200–600 mg/L. The activity of the PHA synthase, the key enzyme which produces PHA, was monitored spectroscopically in cells extracts. Furthermore, the PHA synthase was identified by mass spectrometry in cell extracts analyzed by SDS-PAGE. Transmission electron micrographs revealed abundant electron-transparent intracellular granules. The isolated biopolymer was confirmed to be polyhydroxybutyrate (PHB) by FTIR, NMR and MALDI-TOF/TOF analyses. The ability of strain Pseudomonas sp. phDV1 to remove phenol and to produce PHB makes the strain a promising biocatalyst in bioremediation and biosynthesis of biodegradable plastics.

scholarly journals Key Aromatic-Ring-Cleaving Enzyme, Protocatechuate 3,4-Dioxygenase, in the Ecologically Important MarineRoseobacter Lineage

2000 ◽  
Vol 66 (11) ◽  
pp. 4662-4672 ◽  
Author(s):  
Alison Buchan ◽  
Lauren S. Collier ◽  
Ellen L. Neidle ◽  
Mary Ann Moran
Keyword(s):  
Aromatic Compounds ◽  
Southern Hybridization ◽  
Ring Cleavage ◽  
Degenerate Pcr ◽  
Cell Extracts ◽  
Aromatic Compound Degradation ◽  
Genes Encoding ◽  
Cleavage Enzyme ◽  
Key Enzyme ◽  
Dioxygenase Activity

ABSTRACT Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the α-proteobacteria was investigated. Initial screens suggested that isolates in theRoseobacter lineage can degrade aromatic compounds via the β-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the β-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3,4-dioxygenase activity in cell extracts when grown onp-hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates,Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the β-ketoadipate pathway (pcaC,pcaQ, and pobA) were found to cluster withpcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the twoRoseobacter group isolates than between genes from eitherRoseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additionalRoseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3,4-dioxygenase and the β-ketoadipate pathway was found in all six Roseobacterisolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.

scholarly journals Developing Bioprospecting Strategies for Bioplastics Through the Large-Scale Mining of Microbial Genomes

2021 ◽  
Vol 12 ◽  
Author(s):  
Paton Vuong ◽  
Daniel J. Lim ◽  
Daniel V. Murphy ◽  
Michael J. Wise ◽  
Andrew S. Whiteley ◽  
...  
Keyword(s):  
Large Scale ◽  
High Salinity ◽  
Production Systems ◽  
Genome Mining ◽  
Pha Synthase ◽  
Ecological Group ◽  
Microbial Genomes ◽  
Medium Chain Length ◽  
Key Enzyme ◽  
Pha Production

The accumulation of petroleum-based plastic waste has become a major issue for the environment. A sustainable and biodegradable solution can be found in Polyhydroxyalkanoates (PHAs), a microbially produced biopolymer. An analysis of the global phylogenetic and ecological distribution of potential PHA producing bacteria and archaea was carried out by mining a global genome repository for PHA synthase (PhaC), a key enzyme involved in PHA biosynthesis. Bacteria from the phylum Actinobacteria were found to contain the PhaC Class II genotype which produces medium-chain length PHAs, a physiology until now only found within a few Pseudomonas species. Further, several PhaC genotypes were discovered within Thaumarchaeota, an archaeal phylum with poly-extremophiles and the ability to efficiently use CO2 as a carbon source, a significant ecological group which have thus far been little studied for PHA production. Bacterial and archaeal PhaC genotypes were also observed in high salinity and alkalinity conditions, as well as high-temperature geothermal ecosystems. These genome mining efforts uncovered previously unknown candidate taxa for biopolymer production, as well as microbes from environmental niches with properties that could potentially improve PHA production. This in silico study provides valuable insights into unique PHA producing candidates, supporting future bioprospecting efforts toward better targeted and relevant taxa to further enhance the diversity of exploitable PHA production systems.

scholarly journals Fructose-Based Production of Short-Chain-Length and Medium-Chain-Length Polyhydroxyalkanoate Copolymer by Arctic Pseudomonas sp. B14-6

Polymers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1398
Author(s):  
Tae-Rim Choi ◽  
Ye-Lim Park ◽  
Hun-Suk Song ◽  
Sun Mi Lee ◽  
Sol Lee Park ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Chain Length ◽  
Average Molecular Weight ◽  
Short Chain ◽  
Pha Synthase ◽  
Pseudomonas Sp ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Pha Production

Arctic bacteria employ various mechanisms to survive harsh conditions, one of which is to accumulate carbon and energy inside the cell in the form of polyhydroxyalkanoate (PHA). Whole-genome sequencing of a new Arctic soil bacterium Pseudomonas sp. B14-6 revealed two PHA-production-related gene clusters containing four PHA synthase genes (phaC). Pseudomonas sp. B14-6 produced poly(6% 3-hydroxybutyrate-co-94% 3-hydroxyalkanoate) from various carbon sources, containing short-chain-length PHA (scl-PHA) and medium-chain-length PHA (mcl-PHA) composed of various monomers analyzed by GC-MS, such as 3-hydroxybutyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecenoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid. By optimizing the PHA production media, we achieved 34.6% PHA content using 5% fructose, and 23.7% PHA content using 5% fructose syrup. Differential scanning calorimetry of the scl-co-mcl PHA determined a glass transition temperature (Tg) of 15.3 °C, melting temperature of 112.8 °C, crystallization temperature of 86.8 °C, and 3.82% crystallinity. In addition, gel permeation chromatography revealed a number average molecular weight of 3.6 × 104, weight average molecular weight of 9.1 × 104, and polydispersity index value of 2.5. Overall, the novel Pseudomonas sp. B14-6 produced a polymer with high medium-chain-length content, low Tg, and low crystallinity, indicating its potential use in medical applications.

scholarly journals Expression of 3-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Genes Enhances Production of Polyhydroxyalkanoate Copolymer from Glucose in Recombinant Escherichia coli JM109

2005 ◽  
Vol 71 (8) ◽  
pp. 4297-4306 ◽  
Author(s):  
Christopher T. Nomura ◽  
Kazunori Taguchi ◽  
Zhihua Gan ◽  
Kazuhiro Kuwabara ◽  
Tomoyo Tanaka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chain Length ◽  
Acyl Carrier Protein ◽  
Carbon Sources ◽  
Pha Synthase ◽  
Carrier Protein ◽  
Substrate Specificities ◽  
E Coli ◽  
Pha Production ◽  
Pha Copolymers

ABSTRACT Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.

scholarly journals In Vivo Characterization and Application of the PHA Synthase from Azotobacter vinelandii for the Biosynthesis of Polyhydroxyalkanoate Containing 4-Hydroxybutyrate

Polymers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1576
Author(s):  
Pei-Shze Mok ◽  
Jo-Ann Chuah ◽  
Nazalan Najimudin ◽  
Pauline-Woan-Ying Liew ◽  
Bor-Chyan Jong ◽  
...  
Keyword(s):  
Azotobacter Vinelandii ◽  
Carbon Sources ◽  
Amino Acid Sequences ◽  
Pha Synthase ◽  
Short Chain Length ◽  
Monomer Composition ◽  
Key Enzyme ◽  
Granule Morphology ◽  
In Vivo Characterization

Polyhydroxyalkanoate (PHA) is a biodegradable thermoplastic naturally synthesized by many microorganisms, and the PHA synthase (PhaC) is known to be the key enzyme involved in determining the material properties and monomer composition of the produced PHA. The ability to exploit widely distributed, commonly found soil microorganisms such as Azotobacter vinelandii to synthesize PHA containing the lipase-degradable 4-hydroxybutyrate (4HB) monomer will allow for convenient production of biocompatible and flexible PHA. Comparisons between the A. vinelandii wild type and mutant strains, with and without a surface layer (S-layer), respectively, in terms of gene or amino acid sequences, synthase activity, granule morphology, and PHA productivity, revealed that the S-layer is the sole factor affecting PHA biosynthesis by A. vinelandii. Based on PHA biosynthesis using different carbon sources, the PhaC of A. vinelandii showed specificity for short-chain-length PHA monomers, making it a member of the Class I PHA synthases. In addition, it was proven that the PhaC of A. vinelandii has the inherent ability to polymerize 4-hydroxybutyrate (4HB) and the mediated accumulation of PHA with 4HB fractions ranging from 10 mol% to as high as 22 mol%. The synthesis of biocompatible PHA containing tailorable amounts of 4HB with an expanded range of elasticity and lipase-degradability will enable a wider range of applications in the biomedical field.

Liver Toxicity in Rats Fed A Mixture of Pcb Congeners and 2,3,7,8-Tcdd

1997 ◽  
Vol 3 (S2) ◽  
pp. 51-52
Author(s):  
B.J. Cornell ◽  
A. Singh ◽  
I. Chu
Keyword(s):  
Aromatic Compounds ◽  
Liver Toxicity ◽  
Corn Oil ◽  
Morphological Changes ◽  
Test Substance ◽  
Sprague Dawley ◽  
Morphological Alterations ◽  
Pcb Congeners ◽  
Transmission Electron ◽  
Sprague Dawley Rats

Polyhalogenated aromatic compounds such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (PCDDs) continue to be environmental contaminants because of their bioaccumulation in the food chain and high resistance to biodegradation. The current study was undertaken to determine if a mixture of PCB congeners (WHO-IPCS) were interactive with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in producing morphological changes in the rat liver. Both compounds are known to produce a broad range of biochemical and morphological alterations including enzyme induction.Groups (N=5) of female Sprague-Dawley rats were administered TCDD (0, 2.5, 25, 250, 1000 ng/kg bw/day) or PCB (0, 2, 20 μg/kg bw/day) alone, or in combination with each concentration of both compounds. Incorrect concentrations were published in a previous abstract. The test substance was mixed with corn oil and given by gavage at 2 ml/kg daily for 28 days. At the end of the experiment, the rats were killed and liver samples were prepared for transmission electron microscopy.

scholarly journals V2O3/C composite fabricated by carboxylic acid-assisted sol–gel synthesis as anode material for lithium-ion batteries

2021 ◽  
Author(s):  
G. S. Zakharova ◽  
E. Thauer ◽  
A. N. Enyashin ◽  
L. F. Deeg ◽  
Q. Zhu ◽  
...  
Keyword(s):  
Electrochemical Performance ◽  
Lithium Ion Batteries ◽  
Sol Gel ◽  
Carbon Sources ◽  
Lithium Ion ◽  
X Ray Diffraction ◽  
Electron Microscopies ◽  
Transmission Electron ◽  
Battery Electrode ◽  
Sol Gel Synthesis

AbstractThe potential battery electrode material V2O3/C has been prepared using a sol–gel thermolysis technique, employing vanadyl hydroxide as precursor and different organic acids as both chelating agents and carbon sources. Composition and morphology of resultant materials were characterized by X-ray diffraction, Raman spectroscopy, scanning and transmission electron microscopies, physical sorption, and elemental analysis. Stability and electronic properties of model composites with chemically and physically integrated carbon were studied by means of quantum-chemical calculations. All fabricated composites are hierarchically structured and consist of carbon-covered microparticles assembled of polyhedral V2O3 nanograins with intrusions of amorphous carbon at the grain boundaries. Such V2O3/C phase separation is thermodynamically favored while formation of vanadium (oxy)carbides or heavily doped V2O3 is highly unlikely. When used as anode for lithium-ion batteries, the nanocomposite V2O3/C fabricated with citric acid exhibits superior electrochemical performance with an excellent cycle stability and a specific charge capacity of 335 mAh g−1 in cycle 95 at 100 mA g−1. We also find that the used carbon source has only minor effects on the materials’ electrochemical performance.

Cloning of the Nocardia corallina polyhydroxyalkanoate synthase gene and production of poly-(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly-(3-hydroxyvalerate-co-3-hydroxyheptanoate)

1998 ◽  
Vol 44 (7) ◽  
pp. 687-691 ◽  
Author(s):  
Brian Hall ◽  
Jennifer Baldwin ◽  
Ho Gun Rhie ◽  
Douglas Dennis
Keyword(s):  
Fatty Acids ◽  
Ralstonia Eutropha ◽  
Carbon Sources ◽  
Pha Synthase ◽  
Broad Host Range ◽  
Coding Region ◽  
Reading Frame ◽  
Nocardia Corallina ◽  
Odd Chain Fatty Acids ◽  
Chain Fatty Acids

The polyhydroxyalkanoate (PHA) synthase gene (phaCNc) from Nocardia corallina was identified in a lambda library on a 6-kb BamHI fragment. A 2.8-kb XhoII subfragment was found to contain the ntact PHA synthase. This 2.8-kb fragment was subjected to DNA sequencing and was found to contain the coding region for the PHA synthase and a small downstream open reading frame of unknown function. On the basis of DNA sequence, phaCNc is closest in homology to the PHA synthases (phaCPaI and phaCPaII) of Pseudomonas aeruginosa (approximately 41% identity and 55% similarity). The 2.8-kb XhoII fragment containing phaCNc was subcloned into broad host range mobilizable plasmids and transferred into Escherichia coli, Klebsiella aerogenes (both containing a plasmid bearing phaA and phaB from Ralstonia eutropha), and PHA-negative strains of R. eutropha and Pseudomonas putida. The recombinant strains were grown on various carbon sources and the resulting polymers were analyzed. In these strains, the PHA synthase from N. corallina was able to mediate the production of poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) containing high levels of 3-hydroxyhexanoate when grown on hexanoate and larger even-chain fatty acids and poly(3-hydroxyvalerate-co-3-hydroxyheptanoate) containing high levels of 3-hydroxyheptanoate when grown on heptanoate or larger odd-chain fatty acids. Key words: polyhydroxyalkanoates (PHAs), Nocardia corallina, biodegradable, polyester.

scholarly journals Polyhydroxyalkanoates production by a bacterium isolated from mangrove soil samples collected from Quang Ninh province

2012 ◽  
Vol 3 (2) ◽  
pp. 76-79 ◽  
Author(s):  
Thuoc Van Doan ◽  
Binh Thi Nguyen
Keyword(s):  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
Culture Medium ◽  
Carbon Sources ◽  
Nile Red ◽  
Soil Samples ◽  
Mangrove Soil ◽  
Transmission Electron ◽  
Bacterium Strain ◽  
Quang Ninh

A PHA producing bacterium (strain QN271) was selected from mangrove soil samples collected from Quang Ninh province by using the Nile red dying technique. PHA accumulation in the selected bacterium strain was confirmed by transmission electron microscope. With the exception of maltose or sucrose, the bacterium strain was found to be able to synthesize PHA from various carbon sources (glucose, xylose, fructose, glycerol, and glucose plus propionate). The strain accumulated poly(3-hydroxybutyrate) from glucose, fructose, xylose, and glycerol whereas poly(3-hydroxybutyrate-co-3-hydroxyvalarate) was produced when a combination of glucose and propionate was included in the culture medium. Fructose was found to be most suitable substrate for PHA synthesis by strain QN271. PHA content of 63.3% and CDW of 6 g/L were obtained after 32 hrs of cultivation in fructose medium. Chủng vi khuẩn có khả năng sinh tổng hợp PHA đã được phân lập từ đất rừng ngập mặn tỉnh Quảng Ninh nhờ kỹ thuật nhuộm với Nile red. Ảnh quan sát dưới kính hiển vi điện tử dẫn truyền chứng tỏ rằng chủng vi khuẩn này có khả năng tích lũy lượng lớn PHA trong tế bào. Chủng vi khuẩn tuyển chọn có khả năng sinh tổng hợp PHA từ nhiều nguồn các bon khác nhau như glucose, xylose, fructose, glucerol, glucose và propionate nhưng không có khả năng tổng hợp PHA từ maltose hoặc saccharose. Chủng vi khuẩn tuyển chọn tổng hợp poly (3-hydroxybutyrate) từ các nguồn các-bon như glucose, xylose, fructose, hay glycerol, trong khi đó poly (3-hydroxybutyrate-co-3-hydroxyvalarate) sẽ được tổng hợp khi phối hợp sử dụng hai nguồn các-bon (glucose và propionate). Fructose là nguồn các-bon tốt nhất cho chủng QN271 sinh tổng hợp PHA, khi nuôi cấy trong môi trường có fructose chủng vi khuẩn này có thể tạo ra lượng sinh khối là 6 g/L trong đó có chứa 63.3% PHA sau 32 giờ.

scholarly journals Linking the Salmonella enterica 1,2-propanediol utilization bacterial microcompartment shell to the enzymatic core via the shell protein PduB

2021 ◽  
Author(s):  
Nolan W Kennedy ◽  
Carolyn E Mills ◽  
Charlotte H Abrahamson ◽  
Andre Archer ◽  
Michael C Jewett ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Carbon Sources ◽  
Enzyme Concentration ◽  
Fluorescent Reporters ◽  
Shell Protein ◽  
Bacterial Microcompartment ◽  
Enzyme Encapsulation ◽  
Transmission Electron ◽  
Serovar Typhimurium ◽  
Enzymatic Machinery

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is the sole protein responsible for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies also reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and systems-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis.

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