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2.
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. 相似文献
3.
Pseudomonas putida KT2442 commonly produces medium-chain-length polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (C6) to 3-hydroxydodecanoate (C12) when grown in glucose or even number fatty acid. When two of the beta-oxidation genes fadBA were deleted, the P. pudida KT2442 mutant named KTOY06 accumulated a homopolymer of poly-3-hydroxyheptanoate (P3HHp) up to 71 wt% of its cell dry weight in the presence of heptanoate as a single carbon source. P3HHp contents in the cell dry weight were in direct proportional to Na-heptanoate concentration up to 10 g/L. In contrast, under the same cultivation conditions, the wild type P. putida KT2442 produced a copolymer consisting of 3-hydroxyheptanoate (3HHp) and 5.3–8.4 mol% 3-hydroxynonanoate (3HN). Gas chromatography (GC), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and gel permeation chromatography (GPC) were used to characterize the homopolymer P3HHp, respectively. It was found that the P3HHp with an average molecular weight of 455 kDa was a completely amorphous homopolymer without crystallinity. P3HHp is thermo-degradable at around 250 °C. 相似文献
4.
Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two- to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant. 相似文献
5.
The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated. When olive oil, corn oil, or palm oil was fed
as a sole carbon source, the wild-type strain of A. eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during
its stationary growth phase. In addition, a recombinant strain of A. eutrophus PHB −4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high
cellular content (approximately 80% w/w). The mole fraction of 3-hydroxyhexanoate units was 4–5 mol% whatever the structure
of the triglycerides fed. The polyesters produced by the A. eutrophus strains from olive oil were 200–400 kDa (the number-average molecular mass). The results demonstrate that renewable and inexpensive
plant oils are excellent carbon sources for efficient production of PHA using A. eutrophus strains.
Received: 3 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997 相似文献
6.
Bdellovibrio bacteriovorus HD100 is an obligate predator that invades and grows within the periplasm of Gram‐negative bacteria, including mcl‐polyhydroxyalkanoate (PHA) producers such as Pseudomonas putida. We investigated the impact of prey PHA content on the predator fitness and the potential advantages for preying on a PHA producer. Using a new procedure to control P. putida KT2442 cell size we demonstrated that the number of Bdellovibrio progeny depends on the prey biomass and not on the viable prey cell number or PHA content. The presence of mcl‐PHA hydrolysed products in the culture supernatant after predation on P. putida KT42Z, a PHA producing strain lacking PhaZ depolymerase, confirmed the ability of Bdellovibrio to degrade the prey's PHA. Predator motility was higher when growing on PHA accumulating prey. External addition of PHA polymer (latex suspension) to Bdellovibrio preying on the PHA minus mutant P. putida KT42C1 restored predator movement, suggesting that PHA is a key prey component to sustain predator swimming speed. High velocities observed in Bdellovibrio preying on the PHA producing strain were correlated to high intracellular ATP levels of the predator. These effects brought Bdellovibrio fitness benefits as predation on PHA producers was more efficient than predation on non‐producing bacteria. 相似文献
7.
Conversion of 3-hydroxypropionate (3HP) from 1,3-propanediol (PDO) was improved by expressing dehydratase gene ( dhaT) and aldehyde dehydrogenase gene ( aldD) of Pseudomonas putida KT2442 under the promoter of phaCAB operon from Ralstonia eutropha H16. Expression of these genes in Aeromonas hydrophila 4AK4 produced up to 21 g/L 3HP in a fermentation process. To synthesize homopolymer poly(3-hydroxypropionate) (P3HP), and copolymer poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P3HP4HB), dhaT and aldD were expressed in E. coli together with the phaC1 gene encoding polyhydroxyalkanoate (PHA) synthase gene of Ralstonia eutropha, and pcs' gene encoding the ACS domain of the tri-functional propionyl-CoA ligase (PCS) of Chloroflexus aurantiacus. Up to 92 wt% P3HP and 42 wt% P3HP4HB were produced by the recombinant Escherichia coli grown on PDO and a mixture of PDO+1,4-butanediol (BD), respectively. 相似文献
8.
We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain ( P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation ( P. oleovorans GPo500) revealed that the molecular mass (M w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA – mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt 相似文献
9.
The synthesis of poly(3-hydroxyalkanoates) (PHA) by Pseudomonas putida KT2442 growing on long-chain fatty acids was studied in continuous cultures. The effects of the growth rate on the biomass
and polymer concentration were determined and it was found that the PHA concentrations decreased with increasing growth rates.
The highest volumetric productivity was 0.13 g PHA l -1 h -1 at a specific growth rate (μ) of 0.1 h -1. The molecular mass of the polymer remained constant at all growth rates but changes in the monomeric composition of the
PHA synthesized were observed. Variation of the carbon to nitrogen (C/N) ratio of the substrate feed at μ=0.1 h -1 revealed optimal PHA formation at C/N=20 mol/mol. In order to optimize PHA production P. putida KT2442 was cultivated to high cell densities in oxygen-limited continuous cultures. In this way a maximum biomass concentration
of 30 g/l containing approximately 23% PHA was achieved. This corresponds to a volumetric productivity of 0.69 g l -1 h -1.
Received: 14 December 1995 / Received revision: 18 April 1996 / Accepted: 22 April 1996 相似文献
10.
Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ
Ac
cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC ( phaPCJ
A.c
) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as
one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block
polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC
studies showed the polymer to possess two glass transition temperatures ( T
g), one melting temperature ( T
m) and one cool crystallization temperature ( T
c). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer
to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random
copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young’s
modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio.
Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length
block copolymer PHB- b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community. 相似文献
11.
Medium-chain-length polyhydroxyalkanoates (mcl-PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate,
3-hydroxydodecanoate, and high-content 3-hydroxytetradecanoate (HTD) was produced by knockout mutant Pseudomonas putida KT2442 termed P. putida KTOY06. When grown on 6 to14 g/L single-carbon-source tetradecanoic acid, P. putida KTOY06, which β-oxidation pathway was weakened by deleting genes of 3-ketoacyl-coenzyme A (CoA) thiolase ( fadA) and 3-hydroxyacyl-CoA dehydrogenase ( fadB), for the first time, produced several mcl-PHA including 31 to 49 mol% HTD as a major monomer. HHx contents in these mcl-PHAs
remained approximately constant at less than 3 mol%. In addition, large amounts of oligo-HTD were detected in cells, indicating
the limited ability of P. putida KTOY06 in polymerizing long-chain-length 3-hydroxyalkanoates. The mcl-PHA containing high HTD monomer contents was found
to have both higher crystallinity and improved tensile strength compared with that of typical mcl-PHA. 相似文献
12.
拥有Ⅰ型聚羟基脂肪酸酯(PHA)舍酶基因的嗜水气单胞菌CGMCC 0911株可利用月桂酸而不能利用葡萄糖作为碳源积累PHBHHx。将氯霉素抗性基因(cm)插入到该基因中,获得带有Ⅰ型PHA合酶断裂基因(phaC::Cm)的自杀质粒pFH10。自杀质粒DFH10通过接合作用转入嗜水气单胞菌CGMCC 0911株中并发生体内同源重组,Cm被整合到基因组上,获得Ⅰ型PHA合酶缺失突变株。DNA序列测定证明了这一结果。GC分析表明,突变株不再产生PHBHHx,但却可利用月桂酸或葡萄糖积累中长链PHA,明显表明野生型嗜水气单胞菌基因组中存在另一个编码Ⅱ型PHA合酶的基因,且只有Ⅰ型PHA合酶被钝化后,这个功能被隐藏的Ⅱ型PHA合酶才可在细胞中发挥作用。 相似文献
13.
4-Hydroxybutyrate (4HB) was produced by Aeromonas hydrophila 4AK4, Escherichia coli S17-1, or Pseudomonas putida KT2442 harboring 1,3-propanediol dehydrogenase gene dhaT and aldehyde dehydrogenase gene aldD from P. putida KT2442 which are capable of transforming 1,4-butanediol (1,4-BD) to 4HB. 4HB containing fermentation broth was used for production
of homopolymer poly-4-hydroxybutyrate [P(4HB)] and copolymers poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)]. Recombinant
A. hydrophila 4AK4 harboring plasmid pZL-dhaT-aldD containing dhaT and aldD was the most effective 4HB producer, achieving approximately 4 g/l 4HB from 10 g/l 1,4-BD after 48 h of incubation. The strain
produced over 10 g/l 4HB from 20 g/l 1,4-BD after 52 h of cultivation in a 6-L fermenter. Recombinant E. coli S17-1 grown on 4HB containing fermentation broth was found to accumulate 83 wt.% of intracellular P(4HB) in shake flask study.
Recombinant Ralstonia eutropha H16 grew to over 6 g/l cell dry weight containing 49 wt.% P(3HB-13%4HB) after 72 h. 相似文献
14.
Pseudomonas putida KT2442, a natural producer of polyhydroxyalkanoate, spends a lot of energy and carbon sources to form flagella and pili; therefore, deleting the genes involved in the biosynthesis and assembly of flagella and pili might improve PHA productivity. In this study, two novel deletion systems were constructed in order to efficiently remove the 76 genes involved in the biosynthesis and assembly of flagella and pili in P. putida KT2442. Both systems combine suicide-plasmid-based homologous recombination and mutant lox site-specific recombination and involve three plasmids. The first includes pK18mobsacB, pWJW101, and pWJW102; and the second includes pZJD29c, pDTW202, and pWJW103. These newly constructed systems were successfully used to remove different gene clusters in P. putida KT2442 and showed a high deletion efficiency (above 90%) whether for the second-round or the third-round recombination. Both systems could efficiently delete the gene PP4378 encoding flagellin in putida KT2442, resulting in the mutant strain WJPP01. The second system was used to remove the pili-forming gene cluster PP2357-PP2363 in putida KT2442, resulting in the mutant strain WJPP02, and also used to remove the flagella-forming gene cluster PP4329-PP4397 in WJPP02, resulting in the mutant strain WJPP03. Compared with the wild-type KT2442, the 1.2% genome reduction mutant WJPP03 grew faster, lacked flagella and motility, showed sharply decreased biofilm and 3′,5′-cyclic diguanylic acid (c-di-GMP), but accumulated more polyhydroxyalkanoate. The biomass, polyhydroxyalkanoate yield, and content of WJPP03 increased 19.1, 73.4, and 45.6%, respectively, with sodium hexanoate supplementation, and also increased 11.4, 53.6, and 37.9%, respectively, with lauric acid supplementation. 相似文献
15.
Medium-chain-length (mcl)-polyhydroxyalkanoates (PHAs), elastomeric polyesters synthesized by Genus Pseudomonas bacteria, generally have many different monomer components. In this study, PHAs biosynthesized by four type strains of Pseudomonas ( P. putida, P. citronellolis, P. oleovorans, and P. pseudoalcaligenes) and a typical PHA producer ( P. putida KT2440) were characterized in terms of the monomer structure and composition by gas chromatography-mass spectrometry (GC-MS) analysis. With a thiomethyl pretreatment of PHA methanolysis derivatives, two unsaturated monomers, 3-hydroxy-5-dodecenoate (3H5DD) and 3-hydroxy-5-tetradecenoate (3H5TD), were identified in mcl-PHAs produced by P. putida and P. citronellolis. The quantitative analysis of PHA monomers was performed by employing GC-MS with methanolysis derivatives, and the results coincided with those obtained by performing nuclear magnetic resonance spectroscopy. Only poly(3-hydroxybutyrate) was detected from the P. oleovorans and P. pseudoalcaligenes type strains. These analytical results would be useful as a reference standard for phenotyping of new PHA-producing bacteria. 相似文献
16.
The generation and characterization of Pseudomonas putida KT2442 mutants affected in poly-3-hydroxyalkanoate (PHA) synthesis are reported. The mutants from P. putida KT2442 carrying several copies of the PHA-polymerase-encoding gene ( phaC) were isolated via N-methyl- N′-nitro- N-nitrosoguanidine chemical mutagenesis and contained mutation(s) on genes that are involved in PHA accumulation other than
the phaC genes. No PHA-free mutants were obtained, suggesting that there must be various routes for the synthesis of PHA polymerase
precursors. One of the isolated mutants (GPp120) accumulated more PHA than the parental strain, and there was virtually no
down-regulation of PHA formation by growth in non-limiting amounts of nitrogen, which normally block or reduce formation of
PHA. Compared to the parental strain, GPp120 exhibited significant changes in physiology and morphology when grown in minimal
medium: the growth rate was reduced more than twofold and cells formed filaments. The other four groups of isolated mutants,
with P. putida strains GPp121 to GPp124 as characteristic type strains, exhibited morphological characteristics similar to those of the
parental strain. However, they showed reduced PHA production compared to the parental PHA + strain, and especially GPp121 and GPp122 showed PHA formation tightly controlled by nutrient conditions. All of these mutants
provide starting points for genetically dissecting the biosynthesis and regulation of PHA precursors.
Received: 10 November 1997 / Received revision: 6 February 1998 / Accepted: 6 February 1998 相似文献
17.
Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h −1 but accumulated almost no poly-3-hydroxyalkanoates (PHA). Subsequent nitrogen limitation on nonanoic acid resulted in the
accumulation of only 27% medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited growth ( μ = 0.15 h −1) produced 70 g l −1 biomass containing 75% PHA. At a higher exponential feed rate ( μ = 0.25 h −1), the overall productivity was increased but less biomass (56 g l −1) was produced due to higher oxygen demand, and the biomass contained less PHA (67%). It was concluded that carbon-limited
exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA. Nitrogen limitation is unnecessary. 相似文献
18.
A polyhydroxyalkanoate (PHA) synthase gene phaC2
Ps from Pseudomonas stutzeri strain 1317 was introduced into a PHA synthase gene phbC
Re negative mutant, Ralstonia eutropha PHB −4. It conferred on the host strain the ability to synthesize PHA, the monomer compositions of which varied widely when grown
on different carbon sources. During cultivation on gluconate, the presence of phaC2
Ps in R. eutropha PHB −4 led to the accumulation of polyhydroxybutyrate (PHB) homopolymer in an amount of 40.9 wt% in dry cells. With fatty acids,
the recombinant successfully produced PHA copolyesters containing both short-chain-length and medium-chain-length 3-hydroxyalkanoate
(3HA) of 4–12 carbon atoms in length. When cultivated on a mixture of gluconate and fatty acid, the monomer composition of
accumulated PHA was greatly affected and the monomer content was easily regulated by the addition of fatty acids in the cultivation
medium. After the ( R)-3-hydroxydecanol-ACP:CoA transacylase gene phaG
Pp from Pseudomonas putida was introduced into phaC2
Ps-containing R. eutropha PHB −4, poly(3HB- co-3HA) copolyester with a very high 3-hydroxybutyrate (3HB) fraction (97.3 mol%) was produced from gluconate and the monomer
compositions of PHA synthesized from fatty acids were also altered. This study clearly demonstrated that PhaC2 Ps cloned from P. stutzeri 1317 has extraordinarily low substrate specificity in vivo, though it has only 54% identity in comparison to a previously described low-substrate-specificity PHA synthase PhaC1 Ps from Pseudomonas sp. 61–3. This study also indicated that the monomer composition and content of the synthesized PHA can be effectively modulated
by controlling the addition of carbon sources or by modifying metabolic pathways in the hosts. 相似文献
19.
Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate
(3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbA Re) and NADPH-dependent acetoacetyl-CoA reductase (PhbB Re) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1
Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB −4 to produce copolyesters [P(3HB- co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into
GPp104 strain conferred the ability to synthesize P(3HB- co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated
at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1
Ps gene only. In addition, recombinant strains of R. eutropha PHB −4 produced P(3HB- co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant
strains, R. eutropha PHB −4/pJKSc46- pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB- co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that
the copolyesters obtained here were random copolymers of 3HB and 3HA units.
Received: 12 July 1999 / Received revision: 1 October 1999 / Accepted: 2 October 1999 相似文献
20.
Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two- to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant. 相似文献
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