RESUMO
Polyhydroxyalkanoates (PHA) synthesis genes phbC and orfZ cloned from Ralstonia eutropha H16 were transformed into beta-oxidation weakened Pseudomonas putida KTOY08ΔGC, a mutant of P. putida KT2442. The recombinant P. putida strain termed KTHH06 was able to produce a short-chain-length PHA block copolymer consisting of poly(3-hydroxybutyrate) (P3HB) as one block and poly(4-hydroxybutyrate) (P4HB) as another block. One-dimensional and two-dimensional nuclear magnetic resonance (NMR) clearly indicated the polymer was a diblock copolymer consisting of 20 mol % P3HB as one block and 80 mol % P4HB as another one. Differential scanning calorimetric (DSC) showed that P3HB block melting temperatures (T(m)) in the block copolymer P3HB-b-P4HB was shift to low temperature compared with homopolymer P3HB and a blend of P3HB and P4HB. The block copolymer with a number average molecular weight of 50000 Da and a polydispersity of 3.1 demonstrated a better yield and tensile strength compared with that of its related random copolymer and blend of homopolymers of P3HB and P4HB.
Assuntos
Aciltransferases/metabolismo , Materiais Biocompatíveis/química , Cupriavidus necator/genética , Genética Microbiana/métodos , Poli-Hidroxialcanoatos/biossíntese , Pseudomonas putida/genética , Engenharia Tecidual/métodos , 4-Butirolactona/metabolismo , Aciltransferases/química , Aciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Materiais Biocompatíveis/farmacologia , Biodegradação Ambiental , Varredura Diferencial de Calorimetria , Clonagem Molecular , Cupriavidus necator/enzimologia , Hidroxibutiratos/metabolismo , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Plasmídeos , Poliésteres/metabolismo , Poli-Hidroxialcanoatos/análise , Pseudomonas putida/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Resistência à Tração , Transformação BacterianaRESUMO
A mutant termed Aeromonas hydrophila AKLF was constructed by deleting acetic acid pathway related genes pta and ackA in A. hydrophila 4AK4. Accumulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) in A. hydrophila AKLF was increased by 47% from 2.11 to 3.10g/L associated with a reduction on acetic acid formation compared with A. hydrophila 4AK4 when lauric acid was used as carbon resource. A. hydrophila AKLF harboring pVGAB encoding Vitreoscilla hemoglobin, ß-ketothiolase and acetoacetyl-CoA reductase was found to produce 85% more PHBHHx compared to its wild type. Expression of plasmid pD(Ec)L(Pp) harboring genes related to fatty acid metabolism in A. hydrophila AKLF led to 63% more PHBHHx production than A. hydrophila 4AK4. Replacing phaC in A. hydrophila AKLF with a mutant phaC2 from Pseudomonas stutzeri 1317 resulted in enhanced production of copolymers of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates compared to A. hydrophila 4AK4 harboring the mutant phaC2 in the chromosome as control.
Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Aeromonas hydrophila/metabolismo , Polímeros/metabolismo , Ácido 3-Hidroxibutírico/química , Aeromonas hydrophila/genética , PlasmídeosRESUMO
Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate (HD), and 3-hydroxydodecanoate (HDD) from a wide-range of carbon sources. In this study, fadA and fadB genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KT2442 were knocked out to weaken the beta-oxidation pathway. Two-step culture was proven as the optimal method for PHA production in the mutant termed P. putida KTOY06. In a shake-flask culture, when dodecanoate was used as a carbon source, P. putida KTOY06 accumulated 84 wt % PHA, much higher than 50 wt % PHA in its wild type KT2442. The PHA monomer composition was completely different: the HDD fraction in PHA produced by KTOY06 was 41 mol %, much higher compared with 7.5 mol % only in KT2442. The fermentor-scale culture indicated the HDD fraction in PHA decreased during the culture time from 35 to 25 mol % in a one-step fermentation process or from 75 to 49 mol % in a two-step fermentation process. It is for the first time that PHA with a dominant HDD fraction was produced. Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than PHA with a low HDD fraction did, demonstrating an improved application property.