RESUMEN
Medium-chain-length polyhydroxyalkanoates (mcl-PHAs), widely used in medical area, are commonly synthesized by Pseudomonas spp. This study tries to use ß-oxidation pathways engineered P. entomophila to achieve single source of a series of mcl-monomers for microbial production of PHA homopolymers. The effort is proven successful for the first time to obtain a wide range of mcl-PHA homopolymers from engineered P. entomophila LAC23 grown on various fatty acids, respectively, ranging from poly(3-hydroxyheptanoate) to poly(3-hydroxytetradecanoate). Effects of a PHA monomer chain length on thermal and crystallization properties including the changes of Tm , Tg , and Td5% are investigated. Additionally, strain LAC23 is used to synthesize random copolymers of 3-hydroxyoctanoate (3HO) and 3-hydroxydodecanoate (3HDD) or 3-hydroxytetradecanoates, their compositions could be controlled by adjusting the ratios of two related fatty acids. Meanwhile, block copolymer P(3HO)-b-P(3HDD) is synthesized by the same strain. It is found for the first time that even- and odd number mcl-PHA homopolymers have different physical properties. When the gene of the PHA synthase in the engineered P. entomophila is replaced by phaC from Aeromonas hydrophila 4AK4, poly(3-hydroxybutyrate-co-30 mol%-3-hydroxyhexanoate) is synthesized. Therefore, P. entomophila can be used to synthesize the whole range of PHA (C7-C14) homopolymers, random- and block copolymers.
Asunto(s)
Aciltransferasas , Proteínas Bacterianas , Polihidroxialcanoatos , Pseudomonas , Aciltransferasas/biosíntesis , Aciltransferasas/genética , Aeromonas hydrophila/enzimología , Aeromonas hydrophila/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Ingeniería Genética , Polihidroxialcanoatos/biosíntesis , Polihidroxialcanoatos/genética , Pseudomonas/genética , Pseudomonas/metabolismoRESUMEN
3-Hydroxyalkanoic acids (3HA) are precious precursors for synthesis of value added chemicals. According to their carbon chain lengths, 3HA can be divided into two groups: short-chain-length (SCL) 3HA consisting of 3-5 carbon atoms and medium-chain-length (MCL) 3HA containing 6-14 carbon atoms. To produce MCL 3HA, a metabolic engineered pathway expressing tesB gene, a thioesterase encoding gene that has been reported to catalyze acyl-CoA to free fatty acids, was constructed in Pseudomonas entomophila L48. When tesB of Escherichia coli encoding thioesterase II was introduced into polyhydroxyalkanoate (PHA) synthase and ß-oxidation pathway deleted mutant of P. entomophila LAC31 derived from wild type P. entomophila L48, 6.65g/l 3-hydroxytetradecanoic acid (3HTD) and 4.6g/l 3-hydroxydodecanoic acid (3HDD) were obtained, respectively, when tetradecanoic acid or dodecanoic acid as related carbon sources was added in shake flask cultures. Moreover, 1.8g/l of 3-hydroxydecanoic (3HD) acid was also produced by P. entomophila LAC31 harboring PTE1 gene cloned from Saccharomyces cerevisiae using corresponding fatty acid decanoic acid. Interestingly, shake flask studies indicated that PTE1 harboring strain showed advantages over tesB expressing one for 3HDD and 3HD production, while tesB favored 3HTD production by P. entomophila LAC31. For the first time our study revealed that fine chemicals 3HTD, 3HDD or 3HD could be efficiently produced by metabolic engineered ß-oxidation in Pseudomonas spp grown on related fatty acids.
Asunto(s)
Eliminación de Gen , Mejoramiento Genético/métodos , Hidroxiácidos/metabolismo , Operón/genética , Oxidorreductasas/metabolismo , Pseudomonas/enzimología , Pseudomonas/genética , Hidroxiácidos/química , Hidroxiácidos/aislamiento & purificación , Ingeniería Metabólica/métodos , Peso Molecular , Oxidación-Reducción , Oxidorreductasas/genética , Pseudomonas/clasificación , Especificidad de la EspecieRESUMEN
A medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) producer Pseudomonas entomophila L48 was investigated for microbial production of 3-hydroxydodecanote homopolymer. Pseudomonas entomophila L48 was found to produce MCL PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), and 3-hydroxydodecanoate (3HDD) from related carbon sources fatty acids. In this study, some of the genes encoding key enzymes in ß-oxidation cycle of P. entomophila such as 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and acetyl-CoA acetyltransferase were deleted to study the relationship between ß-oxidation and PHA synthesis in P. entomophila. Among the mutants constructed, P. entomophila LAC26 accumulated over 90 wt % PHA consisting of 99 mol % 3HDD. A fed-batch fermentation process carried out in a 6 L automatic fermentor produced 7.3 g L(-1) PHA consisting of over 97 mol % 3HDD fraction. Properties of MCL PHA were significantly improved along with increasing 3HDD contents. P(2.1 mol % 3HD-co-97.9 mol % 3HDD) produced by P. entomophila LAC25 had the widest temperature range between T(g) and T(m), which were -49.3 and 82.4 °C, respectively, in all MCL PHA reported so far. The new type of PHA also represented high crystallinity caused by side-chain crystallization compared with short side chain PHA. For the first time, P(3HDD) homopolymers were obtained.
Asunto(s)
Proteínas Bacterianas/metabolismo , Microbiología Industrial/métodos , Organismos Modificados Genéticamente/genética , Polihidroxialcanoatos , Pseudomonas/enzimología , 3-Hidroxiacil-CoA Deshidrogenasas/deficiencia , 3-Hidroxiacil-CoA Deshidrogenasas/genética , Acetil-CoA C-Acetiltransferasa/deficiencia , Acetil-CoA C-Acetiltransferasa/genética , Acetil-CoA C-Aciltransferasa/deficiencia , Acetil-CoA C-Aciltransferasa/genética , Proteínas Bacterianas/genética , Reactores Biológicos , Cromatografía de Gases , Cristalización , Ácidos Grasos/metabolismo , Fermentación , Eliminación de Gen , Espectroscopía de Resonancia Magnética , Organismos Modificados Genéticamente/crecimiento & desarrollo , Oxidación-Reducción , Polihidroxialcanoatos/biosíntesis , Polihidroxialcanoatos/genética , Polihidroxialcanoatos/aislamiento & purificación , Pseudomonas/genéticaRESUMEN
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.
Asunto(s)
Aciltransferasas/metabolismo , Materiales Biocompatibles/química , Cupriavidus necator/genética , Genética Microbiana/métodos , Polihidroxialcanoatos/biosíntesis , Pseudomonas putida/genética , Ingeniería de Tejidos/métodos , 4-Butirolactona/metabolismo , Aciltransferasas/química , Aciltransferasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Materiales Biocompatibles/farmacología , Biodegradación Ambiental , Rastreo Diferencial de Calorimetría , Clonación Molecular , Cupriavidus necator/enzimología , Hidroxibutiratos/metabolismo , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Plásmidos , Poliésteres/metabolismo , Polihidroxialcanoatos/análisis , Pseudomonas putida/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Resistencia a la Tracción , Transformación BacterianaRESUMEN
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.
Asunto(s)
Ácido 3-Hidroxibutírico/metabolismo , Aeromonas hydrophila/metabolismo , Polímeros/metabolismo , Ácido 3-Hidroxibutírico/química , Aeromonas hydrophila/genética , PlásmidosRESUMEN
To produce extracellular chiral 3-hydroxyacyl acids (3HA) by fermentation, a novel pathway was constructed by expressing tesB gene encoding thioesterase II into Pseudomonas putida KTOY01, which was a polyhydroxyalkanoate (PHA) synthesis operon knockout mutant. 3HA mixtures of 0.35 g/l consisting of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate (3HDD) were produced in shake-flask study using dodecanoate as a sole carbon source. Additional knockout of fadB and fadA genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KTOY01 led to the weakening of the beta-oxidation pathway. The fadBA and PHA synthesis operon knockout mutant P. putida KTOY07 expressing tesB gene produced 2.44 g/l 3HA, significantly more than that of the beta-oxidation intact mutant. The 3HA mixture contained 90 mol% 3HDD as a dominant component. A fed-batch fermentation process carried out in a 6-l automatic fermentor produced 7.27 g/l extracellular 3HA containing 96 mol% fraction of 3HDD after 28 h of growth. For the first time, it became possible to produce 3HDD-dominant 3HA monomers.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Láuricos/metabolismo , Mutación , Operón , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/genética , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Acetil-CoA C-Aciltransferasa/genética , Acetil-CoA C-Aciltransferasa/metabolismo , Proteínas Bacterianas/genética , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Pseudomonas putida/enzimología , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismoRESUMEN
Production of R-3-hydroxybutyric acid (3HB) was observed when genes of beta-ketothiolase (PhbA), acetoacetyl CoA reductase (PhbB), and thioesterase II (TesB) were jointly expressed in Escherichia coli. TesB, generally regarded as a medium chain length acyl CoA thioesterase, was found, for the first time, to play an important role for transforming short chain length 3-hydroxybutyrate-CoA to its free fatty acid, namely, 3HB. E. coli BW25113 (pSPB01) harboring phbA, phbB, and tesB genes produced approximately 4 g/l 3HB in shake flask culture within 24 h with glucose used as a carbon source. Under anaerobic growth conditions, 3HB production was found to be more effective, achieving 0.47 g 3HB/g glucose compared with only 0.32 g 3HB/g glucose obtained from aerobic process. When growth was conducted on sodium gluconate, 6 g/l 3HB was obtained. In a 24-h fed-batch growth process conducted in a 6-l fermentor containing 3 l glucose mineral medium, 12 g/l 3HB was produced from 17 g/l cell dry weight (CDW). This was the highest 3HB productivity achieved by a one-stage fermentation process for 3HB production.
Asunto(s)
Ácido 3-Hidroxibutírico/metabolismo , Acetil-CoA C-Aciltransferasa/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Biotecnología/métodos , Escherichia coli/metabolismo , Ácido Graso Sintasas/metabolismo , Tioléster Hidrolasas/metabolismo , Acetil-CoA C-Aciltransferasa/genética , Oxidorreductasas de Alcohol/genética , Escherichia coli/genética , Ácido Graso Sintasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometría Ultravioleta , Tioléster Hidrolasas/genéticaRESUMEN
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.
