scholarly journals Toward Engineering the Substrate Specificity of a PHA Synthase (PhaC)

2021 ◽  
Author(s):  
◽  
Alex Kane
Keyword(s):  
Screening Method ◽  
Nile Red ◽  
Pha Synthase ◽  
High Grade ◽  
Wild Type ◽  
Aromatic Substrates ◽  
Type Activity ◽  
Coa Ligase ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

<p>Manufacturing of high-grade plastics from petroleum-based feedstocks is a high-cost, unsustainable process resulting in expensive products. My overall goal was to engineer the pathway of bacterial bio-polyester formation, in order to produce high-grade bioplastics. More specifically, the aim was to introduce aromatic rings into the main-chain of the polyhydroxyalkanoate (PHA) polymer currently produced by specialist bacteria. This research aimed to create these bio-plastics from renewable resources, rather than relying on petroleum-based sources.  A key enzyme for this process is the polyhydroxyalkanoate synthase, PhaC. This enzyme is capable of polymerizing activated hydroxybutyrate-CoA monomers. I began with the establishment of a system that allowed the use of directed evolution. I constructed a minimal plasmid for the expression of PhaC and a second plasmid with the CoA ligase genes required for substrate activation. I generated error-prone PCR libraries of the Cupriavidus necator phaCa, Chromobacterium sp. USM2 phaCb and an ancestrally reconstructed phaCb-LCA that contained differing spectra of mutations. A life-or-death selection was employed to select for PhaC variants able to polymerise aromatic substrates based upon the toxicity of the un-polymerized aromatic hydroxyacid monomers. I determined the minimum inhibitory concentrations (MICs) for six of these monomers in Escherichia coli for downstream selection. Lastly, I adapted a Nile red screening method to test wild-type PHA accumulation of PhaC enzymes.  Selections for mutants capable of polymerizing aromatic monomers were implemented on the libraries generated from phaCa and phaCb. Whereas, the library generated from phaCb-LCA was screened for variants with increased wild-type activity. Selections yielded no candidates for further testing. However, the screen isolated several variants with increased wild-type activity. These variants may serve as a new scaffold for further mutagenesis experiments to achieve the overall goal; to produce a high-grade bioplastic.</p>

scholarly journals Toward Engineering the Substrate Specificity of a PHA Synthase (PhaC)

2021 ◽  
Author(s):  
◽  
Alex Kane
Keyword(s):  
Screening Method ◽  
Nile Red ◽  
Pha Synthase ◽  
High Grade ◽  
Wild Type ◽  
Aromatic Substrates ◽  
Type Activity ◽  
Coa Ligase ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

<p>Manufacturing of high-grade plastics from petroleum-based feedstocks is a high-cost, unsustainable process resulting in expensive products. My overall goal was to engineer the pathway of bacterial bio-polyester formation, in order to produce high-grade bioplastics. More specifically, the aim was to introduce aromatic rings into the main-chain of the polyhydroxyalkanoate (PHA) polymer currently produced by specialist bacteria. This research aimed to create these bio-plastics from renewable resources, rather than relying on petroleum-based sources.  A key enzyme for this process is the polyhydroxyalkanoate synthase, PhaC. This enzyme is capable of polymerizing activated hydroxybutyrate-CoA monomers. I began with the establishment of a system that allowed the use of directed evolution. I constructed a minimal plasmid for the expression of PhaC and a second plasmid with the CoA ligase genes required for substrate activation. I generated error-prone PCR libraries of the Cupriavidus necator phaCa, Chromobacterium sp. USM2 phaCb and an ancestrally reconstructed phaCb-LCA that contained differing spectra of mutations. A life-or-death selection was employed to select for PhaC variants able to polymerise aromatic substrates based upon the toxicity of the un-polymerized aromatic hydroxyacid monomers. I determined the minimum inhibitory concentrations (MICs) for six of these monomers in Escherichia coli for downstream selection. Lastly, I adapted a Nile red screening method to test wild-type PHA accumulation of PhaC enzymes.  Selections for mutants capable of polymerizing aromatic monomers were implemented on the libraries generated from phaCa and phaCb. Whereas, the library generated from phaCb-LCA was screened for variants with increased wild-type activity. Selections yielded no candidates for further testing. However, the screen isolated several variants with increased wild-type activity. These variants may serve as a new scaffold for further mutagenesis experiments to achieve the overall goal; to produce a high-grade bioplastic.</p>

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

scholarly journals Screening Method for Polyhydroxyalkanoate Synthase Mutants Based on Polyester Degree of Polymerization Using High-Performance Liquid Chromatography

2021 ◽  
Vol 9 (9) ◽  
pp. 1949
Author(s):  
Manami Ishii-Hyakutake ◽  
Tetsuo Sakurai ◽  
Takeharu Tsuge
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Benzyl Alcohol ◽  
High Performance ◽  
Cultured Cells ◽  
Screening Method ◽  
Degree Of Polymerization ◽  
Pha Synthase ◽  
Wild Type ◽  
Wild Type Enzyme

A high-throughput screening method based on the degree of polymerization (DP) of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC). In this method, PHA production was achieved using recombinant Escherichia coli supplemented with benzyl alcohol as a chain terminal compound. The cultured cells containing benzyl alcohol-capped PHA were decomposed by alkaline treatment, and the peaks of the decomposed monomer and benzyl alcohol were detected using HPLC. The DP of PHA could be determined from the peak ratio of the decomposed monomer to terminal benzyl alcohol. The measured DP was validated by other instrumental analyses using purified PHA samples. Using this system, mutants of PHA synthase from Bacillus cereus YB-4 (PhaRCYB4) were screened, and some enzymes capable of producing PHA with higher DP than the wild-type enzyme were obtained. The PHA yields of two of these enzymes were equivalent to the yield of the wild-type enzyme. Therefore, this screening method is suitable for the selection of beneficial mutants that can produce high molecular weight PHAs.

Application of an optimized electroporation procedure for replacement of the polyhydroxyalkanoate synthase I gene inNocardia corallina

1996 ◽  
Vol 42 (7) ◽  
pp. 715-719 ◽  
Author(s):  
Henry E. Valentin ◽  
Douglas Dennis
Keyword(s):  
Growth Conditions ◽  
Gene Replacement ◽  
Kanamycin Resistance ◽  
Pha Synthase ◽  
Wild Type ◽  
Kanamycin Resistance Gene ◽  
Gene Encoding ◽  
Nocardia Corallina ◽  
Polyhydroxyalkanoate Synthase ◽  
Type Gene

To develop a system for gene replacement in Nocardia corallina, a protocol for electroporation was optimized by systematic alterations of growth conditions, field strength, time constant and the electroporation buffer. Transformation efficiencies of 0.5 × 106–3 × 106transformants/μg plasmid DNA were obtained routinely. The gene encoding the polyhydroxyalkanoate (PHA) synthase I of N. corallina was cloned and interrupted by insertion of a kanamycin-resistance gene. The resulting plasmid was introduced into N. corallina by electroporation to inactivate the wild-type gene by homologous recombination. Kanamycin-resistant clones were screened by Southern hybridization for the absence of the wild-type gene and analyzed for PHA accumulation.Key words: gene replacement, Nocardia, polyhydroxyalkanoates (PHAs).

Arabidopsis 4-COUMAROYL-COA LIGASE 8 contributes to the biosynthesis of the benzenoid ring of coenzyme Q in peroxisomes

2019 ◽  
Vol 476 (22) ◽  
pp. 3521-3532
Author(s):  
Eric Soubeyrand ◽  
Megan Kelly ◽  
Shea A. Keene ◽  
Ann C. Bernert ◽  
Scott Latimer ◽  
...  
Keyword(s):  
Oxidative Metabolism ◽  
Time Course ◽  
Reverse Genetics ◽  
De Novo ◽  
Coenzyme Q ◽  
Control Level ◽  
Wild Type ◽  
Fluorescent Reporters ◽  
Coa Ligase ◽  
Peroxisomal Targeting

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the β-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the β-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate β-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the β-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m−2 s−1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.

scholarly journals Modified Monosaccharides Content of Xanthan Gum Impairs Citrus Canker Disease by Affecting the Epiphytic Lifestyle of Xanthomonas citri subsp. citri

2021 ◽  
Vol 9 (6) ◽  
pp. 1176
Author(s):  
Simone Cristina Picchi ◽  
Laís Moreira Granato ◽  
Maria Júlia Festa Franzini ◽  
Maxuel Oliveira Andrade ◽  
Marco Aurélio Takita ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Xanthan Gum ◽  
Wild Type Strain ◽  
Natural Infection ◽  
Citrus Canker ◽  
Wild Type ◽  
Xanthomonas Citri ◽  
Canker Disease ◽  
Key Enzyme ◽  
Citrus Canker Disease

Xanthomonas citri subsp. citri (X. citri) is a plant pathogenic bacterium causing citrus canker disease. The xanA gene encodes a phosphoglucomutase/phosphomannomutase protein that is a key enzyme required for the synthesis of lipopolysaccharides and exopolysaccharides in Xanthomonads. In this work, firstly we isolated a xanA transposon mutant (xanA::Tn5) and analyzed its phenotypes as biofilm formation, xanthan gum production, and pathogenesis on the sweet orange host. Moreover, to confirm the xanA role in the impaired phenotypes we further produced a non-polar deletion mutant (ΔxanA) and performed the complementation of both xanA mutants. In addition, we analyzed the percentages of the xanthan gum monosaccharides produced by X. citri wild-type and xanA mutant. The mutant strain had higher ratios of mannose, galactose, and xylose and lower ratios of rhamnose, glucuronic acid, and glucose than the wild-type strain. Such changes in the saccharide composition led to the reduction of xanthan yield in the xanA deficient strain, affecting also other important features in X. citri, such as biofilm formation and sliding motility. Moreover, we showed that xanA::Tn5 caused no symptoms on host leaves after spraying, a method that mimetics the natural infection condition. These results suggest that xanA plays an important role in the epiphytical stage on the leaves that is essential for the successful interaction with the host, including adaptive advantage for bacterial X. citri survival and host invasion, which culminates in pathogenicity.

scholarly journals Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

2010 ◽  
Vol 76 (9) ◽  
pp. 2884-2894 ◽  
Author(s):  
Efraín Manilla-Pérez ◽  
Alvin Brian Lange ◽  
Stephan Hetzler ◽  
Marc Wältermann ◽  
Rainer Kalscheuer ◽  
...  
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Marine Bacterium ◽  
Neutral Lipids ◽  
Nile Red ◽  
Dry Weight ◽  
Wax Ester ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Key Enzyme

ABSTRACT In many microorganisms, the key enzyme responsible for catalyzing the last step in triacylglycerol (TAG) and wax ester (WE) biosynthesis is an unspecific acyltransferase which is also referred to as wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT; AtfA). The importance and function of two AtfA homologues (AtfA1 and AtfA2) in the biosynthesis of TAGs and WEs in the hydrocarbon-degrading marine bacterium Alcanivorax borkumensis SK2 have been described recently. However, after the disruption of both the AtfA1 and AtfA2 genes, reduced but substantial accumulation of TAGs was still observed, indicating the existence of an alternative TAG biosynthesis pathway. In this study, transposon-induced mutagenesis was applied to an atfA1 atfA2 double mutant to screen for A. borkumensis mutants totally defective in biosynthesis of neutral lipids in order to identify additional enzymes involved in the biosynthesis of these lipids. At the same time, we have searched for a totally TAG-negative mutant in order to study the function of TAGs in A. borkumensis. Thirteen fluorescence-negative mutants were identified on Nile red ONR7a agar plates and analyzed for their abilities to synthesize lipids. Among these, mutant 2 M131 was no longer able to synthesize and accumulate TAGs if pyruvate was used as the sole carbon source. The transposon insertion was localized in a gene encoding a putative cytochrome c family protein (ABO_1185). Growth and TAG accumulation experiments showed that the disruption of this gene resulted in the absence of TAGs in 2 M131 but that growth was not affected. In cells of A. borkumensis SK2 grown on pyruvate as the sole carbon source, TAGs represented about 11% of the dry weight of the cells, while in the mutant 2 M131, TAGs were not detected by thin-layer and gas chromatography analyses. Starvation and lipid mobilization experiments revealed that the lipids play an important role in the survival of the cells. The function of neutral lipids in A. borkumensis SK2 is discussed.

Sign in / Sign up

Export Citation Format

Share Document