RESUMO
Polyhydroxyalkanoate (PHA) is one of the biocompatible and biodegradable plastics that can be produced and accumulated as granules inside microorganisms. In this study, a new approach to rapidly quantify a short-chain-length PHA, polyhydroxybutyrate (PHB), produced from genetically engineered Escherichia coli containing phaCAB is presented. The mass of each bacterial cell was measured using a laser-induced radio frequency (rf) plasma charge detection quadrupole ion trap mass spectrometer (LIRFP CD QIT-MS), and then, the PHB contents were determined by calculating the change in cellular mass. The quantitative results showed that the PHB contents measured by LIRFP CD QIT-MS were consistent with those by reference analysis, gas chromatography (GC). The PHB content of each bacterial sample can be obtained within 20 min from sampling using LIRFP CD QIT-MS while GC analysis takes 2 days. In addition, LIRFP CD QIT-MS does not use any hazardous chemicals in cellular mass quantification as compared to GC. This indicates that LIRFP CD QIT-MS has potential in routine monitoring of PHB production.
Assuntos
Escherichia coli , Polímeros , Cromatografia Gasosa-Espectrometria de Massas , Hidroxibutiratos , Espectrometria de Massas/métodos , PoliésteresRESUMO
A laser-induced rf plasma (LIRFP) ion source was developed to ionize submicrometer-sized particles for the first time. The LIRFP ion source can increase the charge of those particles to several thousand charges via charge exchange reactions so that those particles can be trapped and analyzed with a charge detection quadrupole ion trap-mass spectrometer (CD QIT-MS). Different reagent gases for charge exchange reaction were investigated, viz. argon, nitrogen, oxygen, methane, helium, krypton, xenon, argon/methane (with ratios of 10:1 and 2:1), argon/nitrogen (with a ratio of 1:1), nitrogen/oxygen (10:1), krypton/methane (10:1), and air. The average charge of 0.75 µm polystyrene particles could reach 1631 using an argon/methane mixture with a ratio of â¼10:1. The average charges for freeze-dried Escherichia coli EC11303, Escherichia coli strain W, and Staphylococcus aureus were 842, 1112, and 971, respectively, with a mass-to-charge ratio ( m/ z) range from 107 to 108; and the average masses were 3.5 × 1010 Da, 6.0 × 1010 Da, and 5.6 × 1010 Da, respectively. The average mass and charge of the vaccinia virus were â¼9.1 × 109 Da and â¼708 with a m/ z of â¼107. This LIRFP CD QIT-MS method was rapid with only 20 min for each sample measurement.
Assuntos
Gases/química , Íons/química , Escherichia coli/química , Lasers , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Tamanho da Partícula , Poliestirenos/química , Ondas de Rádio , Staphylococcus aureus/química , Eletricidade Estática , Vaccinia virus/químicaRESUMO
Cupriavidus necator is well known for its ability to accumulate polyhydroxybutyrate (PHB). When supplemented with propionic acid (or sodium propionate) in the growth medium, the bacterium is also able to synthesize polyhydroxybutyrate-co-hydroxyvalerate (PHBV). In order to increase the fraction of 3-hydroxyvalerate (3HV) in PHBV, we cloned the propionate permease gene prpP from C. necator and the propionyl-CoA synthase gene prpE from Cupriavidus taiwanensis and transformed into an Escherichia coli containing phaCAB operon of C. necator. The effects on PHBV accumulation in cells co-expressed with phaCAB and prpE or prpP in the media contained mixed carbon sources (glucose and sodium propionate) were evaluated. The HV fraction in PHBV increased when prpE or prpP was overexpressed in the cells. Concentrations of yeast extracts could also affect the fraction of HV. In addition, when glucose was replaced by sodium pyruvate, sodium succinate, or sodium gluconate, only PHB were detected in the recombinant strains.
Assuntos
Proteínas de Bactérias/genética , Coenzima A Ligases/genética , Cupriavidus necator/genética , Proteínas de Membrana Transportadoras/genética , Ácidos Pentanoicos/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Coenzima A Ligases/metabolismo , Cupriavidus/genética , Cupriavidus/metabolismo , Cupriavidus necator/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glucose/metabolismo , Hidroxibutiratos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Óperon , Poliésteres/metabolismo , Propionatos/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Polyhydroxyalkanoates are polyesters synthesized by numerous microorganisms. These polyesters are biodegradable and have similar properties to those of conventional plastics. Cupriavidus taiwanensis strain 184 is phylogenetically related to the well-known polyhydroxybutyrate (PHB) producer Ralstonia eutropha (Cupriavidus necator) and is also shown to be able to accumulate significant amounts of PHB. In this study, we cloned the PHB synthesis genes (phaCAB) from C. taiwanensis 184 into Escherichia coli for biosynthesis of PHB. The recombinant E. coli strains were able to synthesize significant amounts of PHB. The PHB amounted to about 66~70% of total cell material of these recombinant strains.