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1.
We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from
fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4
Pa from Pseudomonas aeruginosa, termed phaJ4
Pp, was identified. The gene products of phaJ1
Pp, which was identified previously, and phaJ4
Pp were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase
activity included PhaJ4Pp, as revealed by western blot analysis. These results strongly suggest that PhaJ4Pp forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1Pp was suggested to be different from that of PhaJ1Pa from P. aeruginosa although these two proteins share 67% amino acid sequence identity. 相似文献
2.
3.
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
4.
A novel nicotine-degrading Pseudomonas sp. strain, HZN6, was isolated from a pesticide-wastewater treatment facility in Hangzhou. The strain could grow on nicotine
as its sole source of carbon, nitrogen, and energy. The strain’s main intermediate metabolites were determined to be pseudooxynicotine,
3-succinoyl-pyridine (SP), and 6-hydroxy-3-succinoyl-pyridine (HSP). A Tn5 transposon mutant was generated in which the degradation
pathway was blocked at the SP. A 4,583-bp DNA fragment flanking the transposon insertion site was obtained through self-formed
adaptor PCR and analyzed. The mutant gene orfC displays 89% deduced amino acid sequence identity with the sirA-like gene (sirA2, a sulfurtransferase homologue gene) of Pseudomonas stutzeri A1501. The orfC-disrupted strain lost the ability to degrade SP, and the complementation strains with the orfC from the Pseudomonas sp. HZN6 and the sirA2 (PP_1233) from Pseudomonas putida KT2440 recovered the degradation ability. Though the orfC-disrupted strain also lost the xanthine dehydrogenase activity, the effects of tungsten on the degradation of SP and hypoxanthine
revealed that the hydroxylation of SP to HSP was not a xanthine dehydrogenase type. These results demonstrated that the orfC gene was essential for the SP metabolism involved in the nicotine metabolic pathway in the Pseudomonas sp. HZN6 strain. This study might advance the understanding of the nicotine metabolic mechanism in Pseudomonas. 相似文献
5.
The previously unknown sequences of several pyoverdines (PVD) produced by a biotechnologically-relevant bacterium, namely, Pseudomonas taiwanensis VLB120, were characterized by high performance liquid chromatography (HPLC)–high resolution mass spectrometry (HRMS). The same structural characterization scheme was checked before by analysis of Pseudomonas sp. putida KT2440 samples with known PVDs. A new sample preparation strategy based on solid-phase extraction was developed, requiring significantly reduced sample material as compared to existing methods. Chromatographic separation was performed using hydrophilic interaction liquid chromatography with gradient elution. Interestingly, no signals for apoPVDs were detected in these analyses, only the corresponding aluminum(III) and iron(III) complexes were seen. The chromatographic separation readily enabled separation of PVD complexes according to their individual structures. HPLC-HRMS and complementary fragmentation data from collision-induced dissociation and electron capture dissociation enabled the structural characterization of the investigated pyoverdines. In Pseudomonas sp. putida KT2240 samples, the known pyoverdines G4R and G4R A were readily confirmed. No PVDs have been previously described for Pseudomonas sp. taiwanensis VLB120. In our study, we identified three new PVDs, which only differed in their acyl side chains (succinic acid, succinic amide and malic acid). Peptide sequencing by MS/MS provided the sequence Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. Of particular interest is the presence of OHAsn, which has not been reported as PVD constituent before. 相似文献
6.
Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful
chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid
(DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after
24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in
a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC
production rate was 0.356 g L−1 h−1. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by
nuclear magnetic resonance. 相似文献
7.
A multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) gene was cloned from Pseudomonas sp. Lm10. Sequence analysis showed that the rAHAS regulatory subunit was identical to that of Pseudomonas
putida KT2440 (sensitive AHAS, sAHAS), whereas six different sites [H134→N (rAHAS→sAHAS), A135→P, S136→T, I210→V, F264→Y, and S486→W]
were found in the catalytic subunit. The rAHAS and sAHAS were over expressed, purified and characterized. rAHAS showed higher
resistance to four kinds of AHAS-inhibitor herbicides than sAHAS. The resistance factor of rAHAS was 56.0-fold, 12.6-fold,
6.5-fold, and 9.2-fold as compared with sAHAS when metsulfuron-methyl, imazethapyr, flumetsulam, and pyriminobac-methyl used
as inhibitor, respectively. The specific activity of rAHAS was lower than that of sAHAS and the K
m value of rAHAS for pyruvate was approximately onefold higher than the corresponding value for sAHAS. Data from site-directed
mutagenesis demonstrated that alteration at A135, F264, and S486 resulted in resistance reduction, while the mutation at H134,
S136, and I210 has little effect on the resistance. A135 was mainly responsible for resistance to imidazolinone; F264 conferred
resistance to sulfonylurea and triazolopyrimidine sulfonamide; and S486 showed multiple herbicides resistance to the four
herbicides. 相似文献
8.
Jia Mi Hendrik Schewe Markus Buchhaupt Dirk Holtmann Jens Schrader 《World journal of microbiology & biotechnology》2016,32(7):112
In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield YP/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids. 相似文献
9.
Yao Yang Ting Chen Pengjuan Ma Guangdong Shang Yijun Dai Sheng Yuan 《Biodegradation》2010,21(4):593-602
A nicotinate dehydrogenase (NaDH) gene cluster was cloned from Comamonas testosteroni JA1. The enzyme, termed NaDHJA1, is composed of 21, 82, and 46 kDa subunits, respectivley containing [2Fe2S], Mo(V) and cytochrome c domains. The recombinant NaDHJA1 can catalyze the hydroxylation of nicotinate and 3-cyanopyridine. NaDHJA1 protein exhibits 52.8% identity to the amino acid sequence of NaDHKT2440 from P. putida KT2440. Sequence alignment analysis showed that the [2Fe2S] domain in NaDHJA1 had a type II [2Fe-2S] motif and a type I [2Fe-2S] motif, while the same domain in NaDHKT2440 had only a type II [2Fe-2S] motif. NaDHKT2440 had an additional hypoxanthine dehydrogenase motif that NaDHJA1 does not have. When the small unit of NaDHJA1 was replaced by the small subunit from NaDHKT2440, the hybrid protein was able to catalyze the hydroxylation of nicotinate, but lost the ability to catalyze hydroxylation
of 3-cyanopyridine. In contrast, after replacement of the small subunit of NaDHKT2440 with the small subunit from NaDHJA1, the resulting hybrid protein NaDHJAS+KTL acquired the ability to hydroxylate 3-cyanopyridine. The subunits swap results indicate the [2Fe2S] motif determines the
3-cyanopyridine hydroxylation ability, which is evidently different from the previous belief that the Mo motif determines
substrate specificity. 相似文献
10.
Yang TH Jung YK Kang HO Kim TW Park SJ Lee SY 《Applied microbiology and biotechnology》2011,90(2):603-614
Previously, we have developed metabolically engineered Escherichia coli strains capable of producing polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] by employing evolved Clostridium propionicum propionate CoA transferase (Pct
Cp
) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1
Ps6-19). Introduction of mutations four sites (E130, S325, S477, and Q481) of PhaC1
Ps6-19 have been found to affect the polymer content, lactate mole fraction, and molecular weight of P(3HB-co-LA). In this study, we have further engineered type II Pseudomonas PHA synthases 1 (PhaC1s) from Pseudomonas chlororaphis, Pseudomonas sp. 61-3, Pseudomonas putida KT2440, Pseudomonas resinovorans, and Pseudomonas aeruginosa PAO1 to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates by site-directed
mutagenesis of four sites (E130, S325, S477, and Q481). All PhaC1s having mutations in these four sites were able to accept
lactyl-CoA as a substrate and supported the synthesis of P(3HB-co-LA) in recombinant E. coli, whereas the wild-type PhaC1s could not accumulate polymers in detectable levels. The contents, lactate mole fractions, and
the molecular weights of P(3HB-co-LA) synthesized by recombinant E. coli varied depending upon the source of the PHA synthase and the mutants used. PLA homopolymer could also be produced at ca.
7 wt.% by employing the several PhaC1 variants containing E130D/S325T/S477G/Q481K quadruple mutations in wild-type E. coli XL1-Blue. 相似文献
11.
Zahir Ahmad Zahir Usman Ghani Muhammad Naveed Sajid Mahmood Nadeem Hafiz Naeem Asghar 《Archives of microbiology》2009,191(5):415-424
Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains
isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under
axenic conditions at 1, 5, 10 and 15 dS m−1. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m−1. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain
yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m−1. Similarly, chlorophyll content and K+/Na+ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of
these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene.
The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction. 相似文献
12.
Pratiksha Bhadauriya Radha Gupta Surendra Singh Prakash Singh Bisen 《World journal of microbiology & biotechnology》2008,24(1):139-141
n-Alkanes pattern in response to NaCl stress has been studied in the cyanobacterium Anabaena cylindrica. Saturated hydrocarbons were separated and identified by gas chromatography-mass spectrometry (GC-MS) using serially coupled
capillary column. Light chain n-alkanes in the range of C9–C17 (43%) and heavy chain n-alkanes in range of C17–C23 (34%) and C23–C31 (23%) were identified as the major components of total hydrocarbons in the NaCl adapted cells of A. cylindrica. In contrast, NaCl-untreated cells of A. cylindrica had dominance of only long chain n-alkanes in the range of C23–C31 comprising about 94% of its total n-alkanes. The persistence of high level (43%) of short chain n-alkanes (C9–C17) in NaCl adapted cells of A. cylindrica as compared to its negligible level (0.2%) in NaCl untreated counterpart clearly indicates that NaCl stress causes the A. cylindrica to shift towards the synthesis of short chain n-alkanes. 相似文献
13.
The fadBA operon in the fatty acid β-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA).
Succinate at 150 mg l−1 stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation
of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l−1 with a cell dry weight of 10.3 g l−1. 相似文献
14.
15.
Taylor B. Cook Jacqueline M. Rand Wasti Nurani Dylan K. Courtney Sophia A. Liu Brian F. Pfleger 《Journal of industrial microbiology & biotechnology》2018,45(7):517-527
Pseudomonas putida is a promising bacterial host for producing natural products, such as polyketides and nonribosomal peptides. In these types of projects, researchers need a genetic toolbox consisting of plasmids, characterized promoters, and techniques for rapidly editing the genome. Past reports described constitutive promoter libraries, a suite of broad host range plasmids that replicate in P. putida, and genome-editing methods. To augment those tools, we have characterized a set of inducible promoters and discovered that IPTG-inducible promoter systems have poor dynamic range due to overexpression of the LacI repressor. By replacing the promoter driving lacI expression with weaker promoters, we increased the fold induction of an IPTG-inducible promoter in P. putida KT2440 to 80-fold. Upon discovering that gene expression from a plasmid was unpredictable when using a high-copy mutant of the BBR1 origin, we determined the copy numbers of several broad host range origins and found that plasmid copy numbers are significantly higher in P. putida KT2440 than in the synthetic biology workhorse, Escherichia coli. Lastly, we developed a λRed/Cas9 recombineering method in P. putida KT2440 using the genetic tools that we characterized. This method enabled the creation of scarless mutations without the need for performing classic two-step integration and marker removal protocols that depend on selection and counterselection genes. With the method, we generated four scarless deletions, three of which we were unable to create using a previously established genome-editing technique. 相似文献
16.
The macrolide antibiotics are biosynthesized by initial assembly of a macrolactone ring, followed by a series of post-polyketide
(PKS) modifications. In general, the additional hydroxyl or epoxy groups are installed by cytochrome P450 enzymes, improving
the bioactivity profile through structural diversification of natural products. The biosynthetic gene cluster for the 16-membered
macrolide antibiotic dihydrochalcomycin (DHC) has been cloned from Streptomyces sp. KCTC 0041BP. Three cytochrome P450 genes are found in the DHC biosynthetic gene (ger) cluster. Two P450 enzymes were characterized from this cluster. Disruption of gerPI accumulated predominantly 12,13-de-epoxydihydrochalcomycin while disruption of gerPII accumulated 8-dehydroxy-12,13-de-epoxydihydrochalcomycin; DHC production was abolished in both cases. The results suggest
that GerPII P450 catalyzes hydroxylation at the C8 position followed by an epoxidation reaction catalyzed by GerPI P450 at the C12–C13 position. 相似文献
17.
Srivastava S Yadav A Seem K Mishra S Chaudhary V Nautiyal CS 《Current microbiology》2008,56(5):453-457
Pseudomonas is an efficient plant growth–promoting rhizobacteria; however, among the limiting factors for its commercialization, tolerance
for high temperature is the most critical one. After screening 2,500 Pseudomnas sp. strains, a high temperature tolerant–strain Pseudomonas putida NBRI0987 was isolated from the drought-exposed rhizosphere of chickpea (Cicer arietinum L. cv. Radhey), which was grown under rain-fed conditions. P. putida NBRI0987 tolerated a temperature of 40°C for ≤ 5 days. To the best of our knowledge, this is the first report of a Pseudomnas sp. demonstrating survival estimated by counting viable cells under such a high temperature. P. putida NBRI0987 colony-forming unit (CFU)/ml on day 10 in both the absence and presence of MgSO4.7H2O (MgSO4) in combination with glycerol at 40°C were 0.0 and 1.7 × 1011, respectively. MgSO4 plus glycerol also enhanced the ability of P. putida NBRI0987 to tolerate high temperatures by inducing its ability to form biofilm. However, production of alginate was not critical
for biofilm formation. The present study demonstrates overexpression of stress sigma factor σ
S
(RpoS) when P. putida NBRI0987 is grown under high-temperature stress at 40°C compared with 30°C. We present evidence, albeit indirect, that the
adaptation of P. putida NBRI0987 to high temperatures is a complex multilevel regulatory process in which many different genes can be involved. 相似文献
18.
Pseudomonas putida E41 was isolated from oil-contaminated soil and showed its ability to grow on ethyl-benzene as the sole carbon and energy
source. Moreover, P. putida E41 show the activity of biodegradation of ethylbenzene in the batch culture. E41 showed high efficiency of biodegradation
of ethylbenzene with the optimum conditions (a cell concentration of 0.1 g wet cell weight/L, pH 7.0, 25°C, and ethylbenzene
concentration of 50 mg/L) from the results of the batch culture. The maximum degradation rate and specific growth rate (μmax) under the optimum conditions were 0.19+0.03 mg/mg-DCW (Dry Cell Weight)/h and 0.87+0.13 h−1, respectively. Benzene, toluene and ethylbenzene were degraded when these compounds were provided together; however, xylene
isomers persisted during degradation by P. putida E41. When using a bioreactor batch system with a binary culture with P. putida BJ10, which was isolated previously in our lab, the degradation rate for benzene and toluene was improved in BTE mixed medium
(each initial concentration: 50 mg/L). Almost all of the BTE was degraded within 4 h and 70–80% of m-, p-, and o-xylenes within 11 h in a BTEX mixture (initial concentration: 50 mg/L each). In summary, we found a valuable new strain of
P. putida, determined the optimal degradation conditions for this isolate and tested a mixed culture of E41 and BJ10 for its ability
to degrade a common sample of mixed contaminants containing benzene, toluene, and xylene. 相似文献
19.
Valsartan orodispersible tablets have been developed at 40-mg dose, with the intention of facilitating administration to patients
experiencing problems with swallowing and hopefully, improving its poor oral bioavailability. Work started with selecting
drug compatible excipients depending on differential scanning calorimetric analysis. A 33 full factorial design was adopted for the optimization of the tablets prepared by freeze-drying technique. The effects of
the filler type, the binder type, and the binder concentration were studied. The different tablet formulas were characterized
for their physical properties, weight variation, disintegration time, surface properties, wetting properties, and in vitro dissolution. Amongst the prepared 27 tablet formulas, formula number 6 (consisting of 4:6 valsartan:mannitol and 2% pectin)
was selected to be tested in vivo. Oral bioavailability of two 40 mg valsartan orodispersible tablets was compared to the conventional commercial tablets after
administration of a single dose to four healthy volunteers. Valsartan was monitored in plasma by high-performance liquid chromatography.
The apparent rate of absorption of valsartan from the prepared tablets (C
max = 2.879 μg/ml, t
max = 1.08 h) was significantly higher than that of the conventional tablets (C
max = 1.471 μg/ml, t
max = 2.17 h), P ≤ 0.05. The relative bioavailability calculated as the ratio of mean total area under the plasma concentration–time curve
for the orodispersible tablets relative to the conventional ones was 135%. The results of the in vivo study revealed that valsartan orodispersible tablets would be advantageous with regards to improved patient compliance, rapid
onset of action, and increase in bioavailability. 相似文献
20.
Pyrimidine salvage pathways are vital for all bacteria in that they share in the synthesis of RNA with the biosynthetic pathway
in pyrimidine prototrophs, while supplying all pyrimidine requirements in pyrimidine auxotrophs. Salvage enzymes that constitute
the pyrimidine salvage pathways were studied in 13 members of Pseudomonas and former pseudomonads. Because it has been established that all Pseudomonas lack the enzyme uridine/cytidine kinase (Udk) and all contain uracil phosphoribosyl transferase (Upp), these two enzymes
were not included in this experimental work. The enzymes assayed were: cytosine deaminase [Cod: cytosine + H2O → uracil + NH3], cytidine deaminase [Cdd: cytidine + H2O → uridine + NH3], uridine phosphorylase [Udp: uridine + Pi ↔ uracil + ribose – 1 - P], nucleoside hydrolase [Nuh: purine/pyrimidine nucleoside + H2O → purine/pyrimidine base + ribose], uridine hydrolase [Udh: uridine/cytidine + H2O → uracil/cytosine + ribose]. The assay work generated five different Pyrimidine Salvage Groups (PSG) designated PSG1 – PSG5
based on the presence or absence of the five enzymes. These enzymes were assayed using reverse phase high-performance liquid
chromatography techniques routinely carried out in our laboratory. Escherichia coli was included as a standard, which contains all seven of the above enzymes. 相似文献