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1.
Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied. 相似文献
2.
Ingo Schmidt 《Current microbiology》2009,59(2):130-138
The ammonia oxidizers Nitrosomonas europaea and Nitrosomonas eutropha are able to grow chemoorganotrophically under anoxic conditions with pyruvate, lactate, acetate, serine, succinate, α-ketoglutarate,
or fructose as substrate and nitrite as terminal electron acceptor. The growth yield of both bacteria is about 3.5 mg protein
(mmol pyruvate)−1 and the maximum growth rates of N. europaea and N. eutropha are 0.094 d−1 and 0.175 d−1, respectively. In the presence of pyruvate and CO2 about 80% of the incorporated carbon derives from pyruvate and about 20% from CO2. Pyruvate is used as energy and only carbon source in the absence of CO2 (chemoorganoheterotrophic growth). CO2 stimulates the chemoorganotrophic growth of both ammonia oxidizers and the expression of ribulose bisphosphate carboxylase/oxygenase
is down-regulated at increasing CO2 concentration. Ammonium, although required as nitrogen source, is inhibitory for the chemoorganotrophic metabolism of N. europaea and N. eutropha. In the presence of ammonium pyruvate consumption and the expression of the genes aceE, ppc, gltA, odhA, and ppsA (energy conservation) as well as nirK, norB, and nsc (denitrification) are reduced. 相似文献
3.
Diane E. Darlington Chiu-Yueh Hung Jiahua Xie 《Plant Cell, Tissue and Organ Culture》2009,99(2):157-165
Agrobacterium
tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The
optimal concentration of kanamycin that could effectively inhibit cell growth and division in non-transgenic tissues was 50 mg l−1 and thus all putative transgenic plants were obtained on induction medium containing 50 mg l−1 kanamycin. The verification of transformants was achieved by both histochemical GUS assay and PCR amplification of nptII gene. Southern blot analysis was performed to further confirm that transgene nptII was stably integrated into the A. racemosus genome. A transformation frequency of approximately 10% was achieved using this protocol, but no beneficial effect from the
addition of acetosyringone (50 μM) was observed. This transformation system will be a useful tool for future studies of genes
responsible for Se-accumulation in A. racemosus. 相似文献
4.
PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA Cma , 1179 bp), acetoacetyl-CoA reductase (phaB Cma , 738 bp), and PHA synthase (phaC Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD600, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa. 相似文献
5.
A genetic transformation system has been developed for callus cells of Crataegus
aronia using Agrobacterium
tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with
5 mg l−1 Indole-3-butyric acid (IBA) and 0.5 mg l−1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different
types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red
colored callus was obtained on MS medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli
were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l−1 hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this
is the first time to report an Agrobacterium-mediated transformation system in Crataegus
aronia. 相似文献
6.
Alejandro López-Cortés Oliverio Rodríguez-Fernández Hever Latisnere-Barragán Humberto C. Mejía-Ruíz Getzabeth González-Gutiérrez Carlos Lomelí-Ortega 《World journal of microbiology & biotechnology》2010,26(1):109-118
Polyhydroxyalakanote (PHA) was produced by the marine bacteria Paracoccus seriniphilus Strain E71. Three methods were used for screening PHA in this strain: (1) microscopic analysis, (2) specifically designed
primers for amplify fragments of phaC gene from Gram negative bacteria, and (3) measurements using spectroscopy, calorimetry, thermogravimetry, and rheology. The
polyhydroxyalkanoic acid synthase gene (phaC) sequence had 77% identity with the phaC gene of P. denitrificans PD1222 strain. Additionally, the translated sequence showed an 86% similarity with the amino acid sequence of the phaC gene N-terminal portion of the P. denitrificans PD1222 strain. Our phaC sequence was closely related to two phaC sequences that correspond to P. denitrificans; therefore, this is the first report of a sequence of phaC that codifies a poly-(3-hydroxyalkanoate) synthase class I, specifically a poly-beta-hydroxybutyrate polymerase from the
marine bacteria Paracoccus seriniphilus. The polymer PHA of E71 melts at 167.86°C (T
m), which corresponded to the fusion of the crystalline polymer and thermally degrades at 296.52°C, indicating that the biopolymer
has good thermal stability. Rheology showed that this polymer behaves as a nonNewtonian fluid. All these characteristics suggest
that the E71 strain produces a PHA that corresponds to the crystalline thermoplastic polymer PHB type. 相似文献
7.
Xiao-Xing Wei Zhen-Yu Shi Mei-Qing Yuan Guo-Qiang Chen 《Applied microbiology and biotechnology》2009,82(4):703-712
Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (P
adhE
) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P
adhE
and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production
under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium,
E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight
of PHB was observed to be much higher under microaerobic culture conditions. The high activity of P
adhE
appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed
to the static culture condition. 相似文献
8.
9.
10.
Fernanda Laroza Paganelli Eliana Gertrudes de Macedo Lemos Lúcia Maria Carareto Alves 《World journal of microbiology & biotechnology》2011,27(4):773-778
Polyhydroxyalkanoates (PHAs) are hydroxyalkanoate polymers that are produced and accumulate by many kinds of bacteria. These
polymers act as an energy store for bacteria. Polyhydroxybutyrate (PHB) is the most studied polymer in the PHA family. These
polymers have awakened interest in the environmental and industrial research areas because they are biodegradable and have
thermoplastic qualities, like polypropylene. In this work, we analyzed the PHB production in Bradyrhizobium sp., Rhizobium leguminosarum bv. phaseoli, and Rhizobium huautlense cultured with two different carbon sources. We did biochemical quantification of PHB production during the three phases of
growth. Moreover, these samples were used for RNA extraction and phbC gene expression analysis via real-time PCR. The bacteria showed different manner of growth, PHB accumulation and phbC gene expression when different quantity and quality of carbon sources were used. These results showed that under different
growth media conditions, the growth and metabolism of different species of bacteria were influenced. These differences reflect
the increase or decrease in PHB accumulation. 相似文献
11.
Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying
enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates
HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and
GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes
in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa
formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates. 相似文献
12.
A novel lipase gene, lipJ08, was cloned from Candida rugosa ATCC14830, along with the already reported five lipase genes (lip1–lip5). Nucleotide sequencing indicated that the lipJ08 gene contains a 1650 bp open reading frame (ORF) without introns. The deduced amino acid sequence corresponds to 534 amino
acid residues, including a putative signal sequence of 15 amino acid residues. Seventeen of the non-universal serine codons
(CTG) of lipJ08 were converted into universal serine codons (TCT) by PCR-based mutagenesis. The native and codon-optimized lipJ08 genes were expressed in Pichia
pastoris. The hydrolytic activity of the recombinant LIPJ08 was 4.7 U/ml, whereas the activity of the recombinant wild-type lipase
could not be detected. 相似文献
13.
Pan W Perrotta JA Stipanovic AJ Nomura CT Nakas JP 《Journal of industrial microbiology & biotechnology》2012,39(3):459-469
Sugar maple hemicellulosic hydrolysate containing 71.9 g/l of xylose was used as an inexpensive feedstock to produce polyhydroxyalkanoates
(PHAs) by Burkholderia cepacia ATCC 17759. Several inhibitory compounds present in wood hydrolysate were analyzed for effects on cell growth and PHA production
with strong inhibition observed at concentrations of 1 g/l furfural, 2 g/l vanillin, 7 g/l levulinic acid, and 1 M acetic
acid. Gradual catabolism of lower concentrations of these inhibitors was observed in this study. To increase the fermentability
of wood hydrolysate, several detoxification methods were tested. Overliming combined with low-temperature sterilization resulted
in the highest removal of total inhibitory phenolics (65%). A fed-batch fermentation exhibited maximum PHA production after
96 h (8.72 g PHA/L broth and 51.4% of dry cell weight). Compositional analysis by NMR and physical–chemical characterization
showed that PHA produced from wood hydrolysate was composed of polyhydroxybutyrate (PHB) with a molecular mass (M
N) of 450.8 kDa, a melting temperature (T
m) of 174.4°C, a glass transition temperature (T
g) of 7.31°C, and a decomposition temperature (T
decomp) of 268.6°C. 相似文献
14.
Deutch CE 《Antonie van Leeuwenhoek》2011,99(4):781-793
Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ1-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ1-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this
strain had l-proline dehydrogenase activity as detected both by reaction of Δ1-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min−1 mg−1) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min−1 mg−1). A soluble fraction from this strain had Δ1-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min−1 mg−1) as determined by the NAD+-dependent oxidation of dl-Δ1-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during
osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial
therapy for this organism. 相似文献
15.
Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were
obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l−1 phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT
(2–6 mg l−1). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l−1) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern
analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants
followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency
as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew
on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization. 相似文献
16.
Atlić A Koller M Scherzer D Kutschera C Grillo-Fernandes E Horvat P Chiellini E Braunegg G 《Applied microbiology and biotechnology》2011,91(2):295-304
Poly(hydroxyalkanoates) (PHAs) constitute biodegradable polyesters and are considered among the most promising candidates
to replace common petrochemical plastics in various applications. To date, all commercial processes for PHA production employ
microbial discontinuous fed-batch fermentations. These processes feature drawbacks such as varying product quality and the
inevitable periods of downtime for preparation and post-treatment of the bioreactor equipment. An unprecedented approach to
PHA production was chosen in the presented work using a multistage system consisting of five continuous stirred tank reactors
in series (5-SCR), which can be considered as a process engineering substitute of a continuous tubular plug flow reactor.
The first stage of the reactor cascade is the site of balanced bacterial growth; thereafter, the fermentation broth is continuously
fed from the first into the subsequent reactors, where PHA accumulation takes place under nitrogen-limiting conditions. Cupriavidus necator was used as production strain. The focus of the experimental work was devoted to the development of a PHA production process
characterized by high productivity and high intracellular polymer content. The results of the experimental work with the reactor
cascade demonstrated its potential in terms of volumetric and specific productivity (1.85 g L−1 h−1 and 0.100 g g−1 h−1, respectively), polymer content (77%, w/w) and polymer properties (M
w = 665 kg/mol, PDI = 2.6). Thus, implementing the technology for 5-SCR production of PHB results in an economically viable
process. The study compares the outcome of the work with literature data from continuous two-stage PHA production and industrial
PHA production in fed-batch mode. 相似文献
17.
Qi Zhu Fengtao Wu Feng Ding Dong Ye Yongqin Chen Yi Li Yang Zhifan 《Plant Cell, Tissue and Organ Culture》2009,96(3):317-324
Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important
steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and
female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive
to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation
medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three
days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for
formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh
selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of
the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to
produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated
from initial infected callus explants. 相似文献
18.
Pramila Shah N. K. Singh Neeraj Khare Meenal Rathore S. Anandhan M. Arif Rupesh Kumar Singh S. C. Das Z. Ahmed Narendra Kumar 《Plant Cell, Tissue and Organ Culture》2008,95(3):363-371
Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore,
experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic co-transformation.
6-benzylaminopurine at 0.05 mg l−l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets
in indole-3-aceticacid 0.5 mg l−l. Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after
co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by
polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent. 相似文献
19.
20.
The F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaCRe). We have now carried out site-directed saturation mutagenesis of F420 of PhaCRe and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaCRe enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaCRe enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant. 相似文献