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1.
Strains of the fission yeast Schizosaccharomyces pombe have been constructed containing single or multiple chromosomally integrated copies of an expression cassette for production
of human gastric lipase. Integrant strains of S. pombe secrete active lipase and are stable for lipase production over a minimum of 50 generations in non-selective media. Lipase
activity levels for integrant strains containing up to three tandem copies of the expression cassette are strongly correlated
with copy number of the cassette in both complete and minimal media. Lipase activity is higher in complete medium than in
minimal medium. Strains carrying three chromosomally integrated expression cassette copies can be grown without selection
in complete medium and are capable of significantly higher lipase activities than strains containing the expression cassette
on a multicopy plasmid.
Received: 27 March 1997 / Received revision: 13 August 1997 / Accepted: 25 August 1997 相似文献
2.
Abstract
The survival of a plasmid-containing Bacillus subtilis released into mushroom compost was investigated. The indigenous Bacillus population of mushroom compost exhibited an antibiotic-resistance profile that was distinguished by almost complete absence
of chloramphenicol resistance. Bacillus subtilis containing the chloramphenicol-resistance plasmid pC194 was released into mushroom compost microcosms and populations were
monitored at different incubation temperatures. The organism colonized both sterile and untreated compost at 37°C, and to
a lesser extent at 50°C, but was eliminated after 30 d at 65°C. Although sporulation of the B. subtilis population occurred within compost, the population was maintained for up to 13 weeks at 50°C, largely as vegetative cells.
Experiments in which the B. subtilis host strain, without plasmid, was released demonstrated that plasmid carriage had no effect on the ability of the bacterium
to colonize and survive in compost. Furthermore, the size and composition of the indigenous bacterial population was unaffected
by the presence of the introduced B. subtilis strain. Virtually no loss of plasmid pC194 from the B. subtilis population in compost was observed, and experiments at low growth rates in chemostats confirmed the stability of this host/vector
system in the absence of positive selection pressure.
Received: 9 July 1997; Accepted: 20 October 1997 相似文献
3.
P.R. Patnaik 《Biotechnology letters》2000,22(21):1719-1725
The rates of growth of recombinant bacteria depend on their plasmid content. This is modelled by expressing the specific growth rate in terms of the number of copies of the plasmid per cell. Three models in common use have been tested with different Escherichia coli strains and one strain of Bacillus stearothermophilus containing different plasmids. While no particular model was decisively better than others for all data, that of Bentley & Quiroga (Biotechnol. Bioeng. 1993, 42: 222–234) was the best for specific growth rates which vary inversely with the plasmid copy number, and a modified form of the model of Satyagal & Agarwal (Biotechnol. Bioeng. 1989, 33: 1135–1144) was the best for growth rates which increase with the copy number. The differences appear to be linked to the plasmid replication mechanisms. Contrary to some claims, no model portrayed the experimentally observed inflection points. 相似文献
4.
Plasmid R1 drd-19 and two of its copy mutants (pKN102 and pKN103) were transferred from Escherichia coli to Salmonella typhimurium, where the expression of the copy mutations was studied further. The copy number (ratio of plasmid DNA to chromosomal DNA) was the same in S. typhimurium and in E. coli. The activities of the plasmid-coded antibiotic-metabolizing enzymes β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylyltransferase as well as the resistances to ampicillin and streptomycin were proportional to the gene dosage up to at least a threefold increase in the steady state plasmid copy number, whereas resistance to chloramphenicol showed no increase with increased number of plasmid copies per chromosome equivalent. Also the resistance to rifampicin was affected since S. typhimurium cells became more sensitive the higher the copy number of the resident plasmid. Furthermore, plasmid R1 showed molecular instability in S. typhimurium cells since there was a tendency to dissociate into resistance transfer factors and resistance determinants and also to form miniplasmids. This tendency to instability was more pronounced the higher the plasmid copy number. 相似文献
5.
G. Gellissen M. Piontek U. Dahlems V. Jenzelewski J. E. Gavagan R. DiCosimo D. L. Anton Z. A. Janowicz 《Applied microbiology and biotechnology》1996,46(1):46-54
The methylotrophic yeast Hansenula polymorpha has been developed as an efficient production system for heterologous proteins. The system offers the possibility to cointegrate
heterologous genes in anticipated fixed copy numbers into the chromosome. As a consequence coproduction of different proteins
in stoichiometric ratios can be envisaged. This provides options to design this yeast as an industrial biocatalyst in procedures
where several enzymes are required for the efficient conversion of a given inexpensive compound into a valuable product. To
this end recombinant strains have been engineered with multiple copies of expression cassettes containing the glycolate oxidase
(GO) gene from spinach and the catalase T (CTT1) gene from S. cerevisiae. The newly created strains produce high levels of the peroxisomal glycolate oxidase and the cytosolic catalase T. The strains
efficiently convert glycolate into glyoxylic acid, oxidizing the added substrate and decomposing the peroxide formed during
this reaction into water and oxygen.
Received: 31 October 1995/Received last revision: 23 February 1996/Accepted: 4 March 1996 相似文献
6.
Four isogenic strains (himAhimDdouble mutant,himAandhimDsingle mutants, and their wild type counterpart) harboringorip15A plasmid (pACYC184 or pACYC184Amp or pACYC177) show different copy numbers of that plasmid in the early stationary phase of cultivation. The copy number oforip15A plasmid increases about four times in thehimAhimDdouble (65–70 copies per cell) andhimDsingle mutant cells (50–56 copies per cell) and was almost the same inhimAmutant (17–18 copies per cell) and wild type cells (14–16 copies per cell). The results suggest that HimD can form homodimers, which are functionally competent for the regulation oforip15A plasmid copy number. Complementation experiments ofhimAhimDdouble mutant cells using plasmid carryinghimAandhimDgenes (pPLhiphimA-5) confirm the effect of integration host factor (IHF) absence on increasing the copy number oforip15A plasmid (plasmid producing IHF complemented the defect of IHF mutant). The absence of IHF (usinghimAhimDdouble mutant as host) had no effect on the copy number of the pBR322 (oripMB1) plasmid. 相似文献
7.
R W Newbert B Barton P Greaves J Harper G Turner 《Journal of industrial microbiology & biotechnology》1997,19(1):18-27
Several commercially improved strains of Penicillium chrysogenum have been shown to carry amplifications of the entire penicillin biosynthesis gene cluster. Analysis previously carried
out using the strain BW 1890 has here been extended to the characterisation of other members of the SmithKline Beecham strain
improvement series. We have determined the length of the amplicon to be 57.4 kb and shown a general increase in copy number
and penicillin titre through the series. Sequence analyses of the promoter regions of the acvA, ipnA and aat genes in the high titre strain BW 1901, and comparisons with wild-type sequences have not identified any potentially titre-enhancing
mutations. In addition, cDNA screening has failed to identify any further transcribed elements within the co-amplified region.
The homogeneity of hybridisation patterns and the identification and analysis of a single copy revertant has shown that the
amplification is of a direct tandem nature and we propose a model of chromatid misalignment and recombination as its mode
of generation. Hybridisation analysis of penicillin non-producing mutants has indicated the loss, in all those investigated,
of the entire penicillin biosynthesis gene cluster, similarities between the deletion junctions in these strains and comparison
with previously published data indicating the presence of recombinogenic regions flanking the penicillin biosynthesis gene
cluster.
Received 05 November 1996/ Accepted in revised form 25 April 1997 相似文献
8.
We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid
pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded
(ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid.
Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid
DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our
results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in
the yeast genome.
Received: 9 February 1996/Accepted: 7 July 1996 相似文献
9.
Vivien Jessica Klein Luciana Fernandes Brito Fernando Perez-Garcia Trygve Brautaset Marta Irla 《Microbial biotechnology》2023,16(5):1011-1026
The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L−1 in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains. 相似文献
10.
Cserjan-Puschmann M Kramer W Duerrschmid E Striedner G Bayer K 《Applied microbiology and biotechnology》1999,53(1):43-50
The aim of this work was the establishment of a novel method to determine the metabolic load on host-cell metabolism resulting
from recombinant protein production in Escherichia coli. This tool can be used to develop strategies to optimise recombinant fermentation processes through adjustment of recombinant-protein
expression to the biosynthetic capacity of the host-cell. The signal molecule of the stringent-response network, guanosine
tetraphosphate (ppGpp), and its precursor nucleotides were selected for the estimation of the metabolic load relating to recombinant-protein
production. An improved analytical method for the quantification of nucleotides by ion-pair, high-performance liquid chromatography
was established. The host-cell response upon overexpression of recombinant protein in fed-batch fermentations was investigated
using the production of human superoxide dismutase (rhSOD) as a model system. E. coli strains with different recombinant systems (the T7 and pKK promoter system) exerting different loads on host-cell metabolism
were analysed with regard to intracellular nucleotide concentration, rate of product formation and plasmid copy number.
Received: 30 April 1999 / Received revision: 26 July 1999 / Accepted: 1 August 1999 相似文献
11.
I. A. van Gemeren A. Beijersbergen W. Musters R. J. Gouka C. A. M. J. J. van den Hondel C. T. Verrips I. A. van Gemeren 《Applied microbiology and biotechnology》1996,45(6):755-763
A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency
of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated
at the pyrG locus. Transformants containing a construct encoding a direct, in-frame fusion of the xylanase pre-peptide to the mature
cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase
pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains
with different numbers of a cutinase construct containing its own pre-prosequence. The multicopy strains showed a 6- to 12-fold
increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the
number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated
with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation
or secretion.
Received: 3 August 1995/Received revision: 20 December 1995/Accepted: 8 January 1996 相似文献
12.
R. W. Herzog N. K. Singh D. W. Urry H. Daniell 《Applied microbiology and biotechnology》1997,47(4):368-372
A gene for a synthetic protein-based polymer, G-(VPGVG)119-VPGV, coding for the EG-120mer (elastomer), was cloned into a fungal expression vector to allow constitutive expression of
the polymer controlled by the gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter sequence of Aspergillus nidulans. Stable transformants of A. nidulans showed plasmid integration with varying copy number when analyzed by Southern-blot hybridization. Expression of the synthetic
gene was demonstrated by Northern-blot hybridization. However, the translational efficiency for production of the polymer
polypeptide was low, presumably because of certain codons in the polymer gene (CCG and GUA) that are rarely used by A. nidulans. Partial purification by reversible phase transition followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis
revealed the presence of polymer protein in a transformant that contained multiple copies of the polymer gene. This study
represents the first attempt to express a synthetic gene (with no natural analog) in a fungus.
Received: 23 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996 相似文献
13.
Yoshiki Tsuchida Sakurako Kimura Nobuaki Suzuki Masayuki Inui Hideaki Yukawa 《Applied microbiology and biotechnology》2010,87(5):1855-1866
A 24-kb plasmid with 21 open reading frames (ORFs) was newly isolated from Corynebacterium glutamicum ATCC 14997 and named pCGR2. Three of its ORFs were indispensable for stable autonomous replication of pCGR2 in C. glutamicum: in the absence of selective pressure, deletion derivatives of pCGR2 containing the three ORFs showed stability in C. glutamicum for over 50 generations. The first of these ORFs encoded replicase repA whose gene product revealed high amino acid sequence similarity to corresponding gene products of C. glutamicum pCG1-family plasmids in general, and to that of pTET3 plasmid repA in particular. The other two ORFs were located upstream of repA and exhibited high sequence similarity to pTET3 parA and parB, respectively. Interestingly, plasmids based on the pCGR2 were compatible not only with those based on different family plasmids
(pBL1, pCASE1) but also with those based on pCG1-family plasmid. Plasmids comprising pCGR2 repA showed a copy number of four in C. glutamicum. The number increased to 240 upon introduction of a mutation within the repA origin of the putative promoter for counter-transcribed RNA. This 60-fold increase in copy number should immensely contribute
towards enhanced expression of desired genes in C. glutamicum. 相似文献
14.
Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and
21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II
fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative
strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level
was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent
areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from
the bioluminescent jellyfish Aequorea victoria.
Received: 18 November 1996/ Revision received: 19 February 1997/ Accepted: 20 November 1997 相似文献
15.
Two δ-integration vectors were evaluated for the insertion of an inducible expression cassette (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomycin-resistance gene (neo) into the genome of Saccharomyces cerevisiae via homologous recombination. Cells containing integrations were selected by resistance to the aminoglycoside G418. The first
vector was a traditional construct containing only one δ sequence; with this vector, the transformation efficiency and the
number of integrations per cell were quite low. The second carried two δ sequences flanking the desired insert, and the unneeded
bacterial sequences were removed by restriction-enzyme digestion immediately before transformation. When this double δ vector
was employed, the integrated copy number was more than doubled relative to the single δ system and final β-galactosidase levels
exceeded those obtained with the 2μ-based plasmid. Furthermore, the integrations appeared more stable in long-term sequential
culture (both with and without induction of the lacZ gene) than those obtained via the single δ vector.
Received: 2 December 1996 / Received revision: 21 March 1997 / Accepted: 13 April 1997 相似文献
16.
K. Leenhouts G. Buist A. Bolhuis A. ten Berge J. Kiel I. Mierau M. Dabrowska G. Venema J. Kok 《Molecular & general genetics : MGG》1996,253(1-2):217-224
A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with
antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid
pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive
and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance.
Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration
of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can
be used to introduce multiple mutations in one strain. A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial
species.
Received: 2 April 1996 / Accepted: 15 July 1996 相似文献
17.
D. Borthakur J. A. Downie A. W. B. Johnston J. W. Lamb 《Molecular & general genetics : MGG》1985,200(2):278-282
Summary A strain of R. phaseoli cured of its symbiotic plasmid, pRP2JI, retained the ability to make exopolysaccharide (EPS). However, a region of pRP2JI, when cloned at an increased copy number in wide host-range vectors and transferred to this and other strains of Rhizobium, inhibited EPS synthesis. The gene responsible was termed psi (polysaccharide inhibition) and was located in a region of the symbiotic plasmid close to nodulation and nitrogen fixation genes. psi is important in the symbiosis since a wild-type strain containing psi cloned on a multicopy plasmid failed to form Phaseolus nodules, and mutant strains containing psi::Tn5 mutations failed to fix nitrogen in Phaseolus nodules. It is proposed that the function of psi may be to repress in the bacteriod the expression of genes such as those for EPS synthesis which are normally expressed in free-living culture. 相似文献
18.
The effect of plasmid content on growth of Lactococcus lactis ssp. diacetylactis harboring different plasmids and on plasmid stability was studied. Strain DRC-2C is a plasmid Lac+- and Prt+-free strain. Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity. The plasmid-free strain DRC-2C exhibited
none of these features. Plasmid-encoded properties were clearly identified. Results showed that plasmid content decreased
bacterial growth in terms of the specific growth rate determined. Slightly lower specific growth rate and lactic acid production
were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host
cell. The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the
same. This indicated that the plasmids were stably maintained and unchanged during the fermentation.
Received: 27 July 2002 / Accepted: 27 August 2002 相似文献
19.
The stability of pKD1-based vectors in the yeast Kluyveromyces lactis was investigated during short- and long-term culture. The vectors carried an expression/secretion cassette consisting of
the Saccharomyces cerevisiaeSUC2 gene under the control of the S. cerevisiaeα-factor promoter and leader. The first set of vectors contained the entire pKD1 sequence linearized at either the unique
EcoRI or the unique SphI site of the pKD1 plasmid. During long-term sequential batch culture in selective medium with either vector, invertase activity
rapidly dropped while the plasmid-bearing population increased from 60% to 100%. This apparently contradictory behavior was due to structural instability. The
enzyme restriction patterns of recovered plasmid DNA retained the pKD1 band while the band containing the SUC2 cassette had decreased substantially in size. To overcome this structural instability, a vector carrying the pKD1 replication
origin and the cis-acting stability locus (lacking the inverted repeats) was employed in a pKD1+ (but otherwise isogenic) strain. With this plasmid, invertase activity remained constant (for at least 70 generations). While
the new vector was significantly more stable, initial invertase activity was substantially lower than that for the vectors
containing the full pKD1 sequence. Southern hybridization confirmed that this decrease was primarily due to reduced copy number.
The results indicate that full-pKD1 vectors may be preferred for batch culture, while partial-pKD1 vectors are more suitable
for long-term (e.g. fed-batch or continuous) culture.
Received: 24 June 1997 / Received revision: 14 November 1997 / Accepted: 29 November 1997 相似文献
20.
The isolation of strains of Bacillus subtilis showing improved plasmid stability characteristics by means of selective chemostat culture 总被引:1,自引:0,他引:1
A pUB110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature alpha-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chlorampheniol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain. 相似文献