The Experts below are selected from a list of 3204 Experts worldwide ranked by ideXlab platform
Anthony J. Sinskey - One of the best experts on this subject based on the ideXlab platform.
-
Applications of Polyhydroxyalkanoates in the Medical Industry
International Journal of Biotechnology for Wellness Industries, 2012Co-Authors: Christopher J. Brigham, Anthony J. SinskeyAbstract:The bio-based, biodegradable family of polymers, Polyhydroxyalkanoates (PHA), is an attractive candidate for an environmentally friendly replacement of petroleum-based plastics in many applications. In the past decade, many groups have examined the biodegradability and biocompatibility of PHA in cell culture systems or in an animal host. Findings suggest that PHA is a suitable material for fabrication of resorbable medical devices, such as sutures, meshes, implants, and tissue engineering scaffolds. The degradation kinetics of some PHA polymers is also suggestive of drug release applications. In this review, we examine the progress, potential applications, challenges and outlook in the medical Polyhydroxyalkanoate field.
-
manipulation of ralstonia eutropha carbon storage pathways to produce useful bio based products
Sub-cellular biochemistry, 2012Co-Authors: Christopher J. Brigham, T. G. Volova, N. O. Zhila, Ekaterina I Shishatskaya, Anthony J. SinskeyAbstract:Ralstonia eutrophais a Gram-negative betaproteobacterium found natively in soils that can utilize a wide array of carbon sources for growth, and can store carbon intracellularly in the form of Polyhydroxyalkanoate. Many aspects of R. eutrophamake it a good candidate for use in biotechnological production of Polyhydroxyalkanoate and other bio-based, value added compounds. Manipulation of the organism’s carbon flux is a cornerstone to success in developing it as a biotechnologically relevant organism. Here, we examine the methods of controlling and adapting the flow of carbon in R. eutrophametabolism and the wide range of compounds that can be synthesized as a result. The presence of many different carbon utilization pathways and the custom genetic toolkit for manipulation of those pathways gives R. eutrophaa versatility that allows it to be a biotechnologically important organism.
-
elucidation of β oxidation pathways in ralstonia eutropha h16 by examination of global gene expression
Journal of Bacteriology, 2010Co-Authors: Christopher J. Brigham, Chokyun Rha, Charles F Budde, Jason Wyatt Holder, Qiandong Zeng, Alison E Mahan, Anthony J. SinskeyAbstract:Ralstonia eutropha H16 is capable of growth and Polyhydroxyalkanoate production on plant oils and fatty acids. However, little is known about the triacylglycerol and fatty acid degradation pathways of this bacterium. We compare whole-cell gene expression levels of R. eutropha H16 during growth and Polyhydroxyalkanoate production on trioleate and fructose. Trioleate is a triacylglycerol that serves as a model for plant oils. Among the genes of note, two potential fatty acid β-oxidation operons and two putative lipase genes were shown to be upregulated in trioleate cultures. The genes of the glyoxylate bypass also exhibit increased expression during growth on trioleate. We observed that single β-oxidation operon deletion mutants of R. eutropha could grow using palm oil or crude palm kernel oil as the sole carbon source, regardless of which operon was present in the genome, but a double mutant was unable to grow under these conditions. A lipase deletion mutant did not exhibit a growth defect in emulsified oil cultures but did exhibit a phenotype in cultures containing nonemulsified oil. Mutants of the glyoxylate shunt gene for isocitrate lyase were able to grow in the presence of oils, while a malate synthase (aceB) deletion mutant grew more slowly than wild type. Gene expression under Polyhydroxyalkanoate storage conditions was also examined. Many findings of this analysis confirm results from previous studies by our group and others. This work represents the first examination of global gene expression involving triacylglycerol and fatty acid catabolism genes in R. eutropha.
-
new insight into the role of the phap phasin of ralstonia eutropha in promoting synthesis of polyhydroxybutyrate
Journal of Bacteriology, 2001Co-Authors: Gregory M York, Joanne Stubbe, Anthony J. SinskeyAbstract:Phasins are proteins that are proposed to play important roles in Polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with Polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.
-
pha synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo
Nature Biotechnology, 1997Co-Authors: Sang Jun Sim, Kristi D Snell, Scott A Hogan, Joanne Stubbe, Chokyun Rha, Anthony J. SinskeyAbstract:A synthetic operon for Polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding beta-ketothiolase and acetoacetyl-CoA reductase behind a modified synthase gene containing an Escherichia coli promoter and ribosome binding site. Plasmids containing the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E. coli DH5 alpha and analyzed for polyhydroxybutyrate production. The molecular weight of polymer isolated from recombinant E. coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E. coli containing the native A. eutrophus operon. A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of Polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.
Paul S Freemont - One of the best experts on this subject based on the ideXlab platform.
-
al pha beads bioplastic based protease biosensors for global health applications
Materials Today, 2021Co-Authors: Richard Kelwick, Alexander J Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M Emery, Michael R Templeton, Paul S FreemontAbstract:Abstract Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector Polyhydroxyalkanoates (AL-PHA) beads – a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured Polyhydroxyalkanoate beads. In the presence of a specific protease, superfolder green fluorescent reporter proteins are cleaved from the AL-PHA beads – resulting in a loss of bead fluorescence. The Tobacco Etch Virus (TEV) AL-PHA biosensor detected the proteolytic activity of at least 1.85 pM of AcTEV. AL-PHA beads were also engineered to detect cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be further developed for use in resource-limited settings.
-
al pha beads bioplastic based protease biosensors for global health applications
bioRxiv, 2020Co-Authors: Richard Kelwick, Alexander J Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M Emery, Michael R Templeton, Paul S FreemontAbstract:ABSTRACT Proteases are multi-functional, proteolytic enzymes that have complex roles in human health and disease. Detecting the activities of proteases can lead to important insights into communicable and non-communicable diseases. Therefore, the development of protease detection strategies can be beneficial to an array of global health applications. To this end, we developed Advanced proteoLytic detector Polyhydroxyalkanoates (AL-PHA) beads – a library of low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured Polyhydroxyalkanoate (PHA) bioplastic beads. These PhaC-fusions also incorporate modular specific protease cleavage sites. In the presence of a specific protease, superfolder green fluorescent (sfGFP) reporter proteins are cleaved off of the AL-PHA beads - resulting in a loss of bead fluorescence. These AL-PHA biosensors were initially optimised using a commercially available Tobacco Etch Virus (TEV) protease. Our third generation TEV biosensor (PhaC-112L-T-G) detected 0.5 U (1.85 pM) of AcTEV activity and 10 units of AcTEV protease activity resulted in a visually noticeable loss in AL-PHA bead fluorescence. AL-PHA beads also detected cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be adapted towards a low-cost and high-throughput protease detection assay for global health applications.
-
Cell-free prototyping strategies for enhancing the sustainable production of Polyhydroxyalkanoates bioplastics
2017Co-Authors: Richard Kelwick, Alexander J Webb, Luca Ricci, Soo Mei Chee, Derek Bell, Paul S FreemontAbstract:The Polyhydroxyalkanoates are a group of microbially-produced biopolymers that have been proposed as sustainable alternatives to several oil-derived plastics. However, Polyhydroxyalkanoates are currently more expensive to produce than oil-derived plastics and therefore, more efficient production processes would be desirable. Cell-free transcription-translation-based metabolic engineering strategies have been previously used to optimise several different biosynthetic pathways but not the Polyhydroxyalkanoates biosynthetic pathways. Here we have developed several Escherichia coli cell-free transcription-translation-based systems for in vitro prototyping of Polyhydroxyalkanoates biosynthetic operons, and also for screening relevant metabolite recycling enzymes. These cell-free transcription-translation reactions were customised through the addition of whey permeate, an industrial waste that has been previously used as a low-cost feedstock for optimising in vivo Polyhydroxyalkanoates production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ~20% compared to control reactions that did not include whey permeate. An analysis of pH in our cell-free reactions suggests that the observed increase in GFPmut3b production was likely through enhanced ATP generation, as a consequence of the glycolytic processing of lactose present in whey permeate. We also found that whey permeate enhanced cell-free reactions produced ~3μM (R)-3HB-CoA, whilst, coupled cell-free biotransformation/transcription-translation reactions produced a ten-fold greater yield of (R)-3HB-CoA. These reactions were also used to characterise a Clostridium propionicum propionyl CoA transferase enzyme that can recycle Acetyl-CoA. Together our data demonstrate that cell-free approaches can be used to complement in vivo workflows for identifying additional strategies for optimising Polyhydroxyalkanoates production.
Richard Kelwick - One of the best experts on this subject based on the ideXlab platform.
-
al pha beads bioplastic based protease biosensors for global health applications
Materials Today, 2021Co-Authors: Richard Kelwick, Alexander J Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M Emery, Michael R Templeton, Paul S FreemontAbstract:Abstract Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector Polyhydroxyalkanoates (AL-PHA) beads – a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured Polyhydroxyalkanoate beads. In the presence of a specific protease, superfolder green fluorescent reporter proteins are cleaved from the AL-PHA beads – resulting in a loss of bead fluorescence. The Tobacco Etch Virus (TEV) AL-PHA biosensor detected the proteolytic activity of at least 1.85 pM of AcTEV. AL-PHA beads were also engineered to detect cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be further developed for use in resource-limited settings.
-
al pha beads bioplastic based protease biosensors for global health applications
bioRxiv, 2020Co-Authors: Richard Kelwick, Alexander J Webb, Yizhou Wang, Amelie Heliot, Fiona Allan, Aidan M Emery, Michael R Templeton, Paul S FreemontAbstract:ABSTRACT Proteases are multi-functional, proteolytic enzymes that have complex roles in human health and disease. Detecting the activities of proteases can lead to important insights into communicable and non-communicable diseases. Therefore, the development of protease detection strategies can be beneficial to an array of global health applications. To this end, we developed Advanced proteoLytic detector Polyhydroxyalkanoates (AL-PHA) beads – a library of low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured Polyhydroxyalkanoate (PHA) bioplastic beads. These PhaC-fusions also incorporate modular specific protease cleavage sites. In the presence of a specific protease, superfolder green fluorescent (sfGFP) reporter proteins are cleaved off of the AL-PHA beads - resulting in a loss of bead fluorescence. These AL-PHA biosensors were initially optimised using a commercially available Tobacco Etch Virus (TEV) protease. Our third generation TEV biosensor (PhaC-112L-T-G) detected 0.5 U (1.85 pM) of AcTEV activity and 10 units of AcTEV protease activity resulted in a visually noticeable loss in AL-PHA bead fluorescence. AL-PHA beads also detected cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be adapted towards a low-cost and high-throughput protease detection assay for global health applications.
-
Cell-free prototyping strategies for enhancing the sustainable production of Polyhydroxyalkanoates bioplastics
2017Co-Authors: Richard Kelwick, Alexander J Webb, Luca Ricci, Soo Mei Chee, Derek Bell, Paul S FreemontAbstract:The Polyhydroxyalkanoates are a group of microbially-produced biopolymers that have been proposed as sustainable alternatives to several oil-derived plastics. However, Polyhydroxyalkanoates are currently more expensive to produce than oil-derived plastics and therefore, more efficient production processes would be desirable. Cell-free transcription-translation-based metabolic engineering strategies have been previously used to optimise several different biosynthetic pathways but not the Polyhydroxyalkanoates biosynthetic pathways. Here we have developed several Escherichia coli cell-free transcription-translation-based systems for in vitro prototyping of Polyhydroxyalkanoates biosynthetic operons, and also for screening relevant metabolite recycling enzymes. These cell-free transcription-translation reactions were customised through the addition of whey permeate, an industrial waste that has been previously used as a low-cost feedstock for optimising in vivo Polyhydroxyalkanoates production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ~20% compared to control reactions that did not include whey permeate. An analysis of pH in our cell-free reactions suggests that the observed increase in GFPmut3b production was likely through enhanced ATP generation, as a consequence of the glycolytic processing of lactose present in whey permeate. We also found that whey permeate enhanced cell-free reactions produced ~3μM (R)-3HB-CoA, whilst, coupled cell-free biotransformation/transcription-translation reactions produced a ten-fold greater yield of (R)-3HB-CoA. These reactions were also used to characterise a Clostridium propionicum propionyl CoA transferase enzyme that can recycle Acetyl-CoA. Together our data demonstrate that cell-free approaches can be used to complement in vivo workflows for identifying additional strategies for optimising Polyhydroxyalkanoates production.
Marek Kowalczuk - One of the best experts on this subject based on the ideXlab platform.
-
3d printed polyester based prototypes for cosmetic applications future directions at the forensic engineering of advanced polymeric materials
Materials, 2019Co-Authors: Joanna Rydz, Wanda Sikorska, Marta Musiol, Henryk Janeczek, Jakub Wlodarczyk, Marlena Misiurskamarczak, Justyna łeczycka, Marek KowalczukAbstract:Knowledge of degradation and impairment phenomena of (bio)degradable polymeric materials under operating conditions, and thus the selection of test procedures and prediction of their behavior designates the scope and capabilities as well as possible limitations of both: the preparation of the final product and its durability. The main novelty and objective of this research was to determine the degradation pathways during testing of polylactide and polylactide/Polyhydroxyalkanoate materials made with three-dimensional printing and the development of a new strategy for the comprehensive characterization of such complex systems including behavior during waste disposal. Prototype objects were subjected to tests for damage evolution performed under simulating operating conditions. The reference samples and the tested items were characterized by gel permeation chromatography and differential scanning calorimetry to determine changes in material properties. The studies showed that: Polyhydroxyalkanoate component during accelerated aging and degradation in environments rich in microorganisms accelerated the degradation of the material; paraffin accelerates polylactide degradation and slows degradation of Polyhydroxyalkanoate-based material; under the influence of an environment rich in enzymes, paraffin contamination accelerates biodegradation; under the influence of natural conditions, paraffin contamination slowed degradation; the processing conditions, in particular the printing orientation of individual parts of the container, influenced the material properties in its various regions, affecting the rate of degradation of individual parts.
-
3D-Printed Polyester-Based Prototypes for Cosmetic Applications—Future Directions at the Forensic Engineering of Advanced Polymeric Materials
MDPI AG, 2019Co-Authors: Joanna Rydz, Wanda Sikorska, Henryk Janeczek, Marta Musioł, Jakub Włodarczyk, Marlena Misiurska-marczak, Justyna Łęczycka, Marek KowalczukAbstract:Knowledge of degradation and impairment phenomena of (bio)degradable polymeric materials under operating conditions, and thus the selection of test procedures and prediction of their behavior designates the scope and capabilities as well as possible limitations of both: the preparation of the final product and its durability. The main novelty and objective of this research was to determine the degradation pathways during testing of polylactide and polylactide/Polyhydroxyalkanoate materials made with three-dimensional printing and the development of a new strategy for the comprehensive characterization of such complex systems including behavior during waste disposal. Prototype objects were subjected to tests for damage evolution performed under simulating operating conditions. The reference samples and the tested items were characterized by gel permeation chromatography and differential scanning calorimetry to determine changes in material properties. The studies showed that: Polyhydroxyalkanoate component during accelerated aging and degradation in environments rich in microorganisms accelerated the degradation of the material; paraffin accelerates polylactide degradation and slows degradation of Polyhydroxyalkanoate-based material; under the influence of an environment rich in enzymes, paraffin contamination accelerates biodegradation; under the influence of natural conditions, paraffin contamination slowed degradation; the processing conditions, in particular the printing orientation of individual parts of the container, influenced the material properties in its various regions, affecting the rate of degradation of individual parts
Dieter Jendrossek - One of the best experts on this subject based on the ideXlab platform.
-
Polyhydroxyalkanoate pha granules have no phospholipids
Scientific Reports, 2016Co-Authors: Stephanie Bresan, Anna Sznajder, Waldemar Hauf, Karl Forchhammer, Daniel Pfeiffer, Dieter JendrossekAbstract:Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of Polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only.
-
Polyhydroxyalkanoate granules are complex subcellular organelles (carbonosomes).
Journal of bacteriology, 2009Co-Authors: Dieter JendrossekAbstract:Polyhydroxyalkanoates (PHAs) such as poly(3-hydroxybutyrate) (PHB) or poly(3-hydroxyoctanoate), are universal prokaryotic storage compounds of carbon and energy. PHAs are accumulated intracellularly in form of inclusion bodies (PHA granules) during times of oversupply with carbon sources (for
-
Microbial degradation of Polyhydroxyalkanoates.
Annual review of microbiology, 2002Co-Authors: Dieter Jendrossek, René HandrickAbstract:Polyesters such as poly(3-hydroxybutyrate) (PHB) or other Polyhydroxyalkanoates (PHA) have attracted commercial and academic interest as new biodegradable materials. The ability to degrade PHA is widely distributed among bacteria and fungi and depends on the secretion of specific extracellular PHA depolymerases (e-PHA depolymerases), which are carboxyesterases (EC 3.1.1.75 and EC 3.1.1.76), and on the physical state of the polymer (amorphous or crystalline). This contribution provides a summary of the biochemical and molecular biological characteristics of e-PHA depolymerases and focuses on the intracellular mobilization of storage PHA by intracellular PHA depolymerases (i-PHA depolymerases) of PHA-accumulating bacteria. The importance of different assay systems for PHA depolymerase activity is also discussed.
-
biosynthesis of copolyesters consisting of 3 hydroxybutyric acid and medium chain length 3 hydroxyalkanoic acids from 1 3 butanediol or from 3 hydroxybutyrate by pseudomonas sp a33
Applied Microbiology and Biotechnology, 1995Co-Authors: Eun Yeol Lee, Dieter Jendrossek, A Schirmer, Chunho Choi, Alexander SteinbüchelAbstract:Pseudomonas sp. A33 and other isolates of aerobic bacteria accumulated a complex copolyester containing 3-hydroxybutyric acid (3HB) and various medium-chain-length 3-hydroxyalkanoic acids (3HAMCL) from 3-hydroxybutyric acid or from 1,3-butanediol under nitrogen-limitated culture conditions. 3HB contributed to 15.1 mol/100 mol of the constituents of the polyester depending on the strain and on the cultivation conditions. The accumulated polymer was a copolyester of 3HB and 3HAMCL rather than a blend of poly(3HB) and poly(3HAMCL) on the basis of multiple evidence. 3-Hydroxyhexadecenoic acid and 3-hydroxyhexadecanoic acid were detected as constituents of Polyhydroxyalkanoates, which have hitherto not been described, by13C nuclear magnetic resonance or by gas chromatography/mass spectrometric analysis. In total, ten different constituents were detected in the polymer synthesized from 1,3-butanediol by Pseudomonas sp. A33:besides seven saturated (3HB, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydrohexadecanoate) three unsaturated (3-hydroxydodecenoate, 3-hydroxytetradecenoate and 3-hydrohexadecanoate) hydroxyalkanoic acid constituents occured. The Polyhydroxyalkanoate synthase of Pseudomonas sp. A33 was cloned, and its substrate specificity was evaluated by heterologous expression in various strains of P. putida, P. oleovorans and Alcaligenes eutrophus.