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The culture fluorescence of two recombinant Escherichia coli strains with high plasmid copy number were studied and compared to both the host and low copy number varieties of the corresponding strains. Culture fluorescence data are related to the concentration of reduced intracellular nicotinamide adenine dinucleotide within a cell, and can therefore be used as a means for detecting changes in metabolic states. Correlation curves relating culture fluorescence to biomass show that the recombinant system maintains a larger pool of intracellular NADH at high plasmid copy numbers than either the host or the recombinant system at low copy numbers. These results demonstrate the ability of a fluorescence probe to detect differences in the metabolic demands made on an over-producing recombinant organism. 相似文献
3.
Relative levels of many individual proteins in Escherichia coli HB101 strains with 0, 37, 56, and 240 plasmids per chromosome were determined by computer image analysis of two-dimensional gel electrophoresis patterns. The plasmids investigated had very similar sequences except for small domains encoding the represser of plasmid replication. At the intermediate plasmid copy number of 56, levels of several of the TCA cycle enzymes (oxoglutarate dehydrogenase complex, succinate thiokinase, and succinate dehydrogenase) as well as in aspartate transcarbamoylase increased. At a plasmid copy number of 240, higher amounts of PEP carboxylase as well as several of the heat shock proteins were observed. Furthermore, at high plasmid levels, significant decreases occurred in growth rate, pyruvate kinase I, pyruvate dehydrogenase complex, unadenylated glutamine synthetase, aspartate transcarbamoylase as well as in several of the proteins involved in translation. Decreases in ribosome content as well as in the free 30S and 50S ribosomal subunit pool fractions were also observed in separate analyses. These results indicate that recombinant DNA manipulations can cause major alterations in numerous host cell properties which could significantly influence cloned protein production or metabolic engineering endeavors. 相似文献
4.
Chromosomal mutants were isolated in Escherichia coli that altered carotenoid production from transformed carotenoid biosynthesis genes on a pACYC-derived plasmid (pPCB15). The mutations were mapped by sequencing. One group of mutations appeared to affect the cell metabolism without changing the copy number of the carotenoid synthesis plasmid. The other group of mutations either increased or decreased the copy number of the pPCB15 plasmid as determined by real-time PCR. The copy number change in most mutants was likely specific for ColE1-type plasmids for which copy number is controlled by a small antisense RNA. This collection of host strains would be useful for fine tuning expression of proteins and adjusting production of desired molecules without recloning to different vectors. 相似文献
5.
The instantaneous specific growth rate of a recombinant bacterial culture is directly calculated using a simple structured kinetic modeling approach. Foreign plasmid replication and foreign protein expression represent metabolic burdens to the host cell. The individual effects of these plasmid-mediated activities on the growth rate of plasmid-bearing cells are estimated separately. The dynamic and steady state simulations of the model equations show remarkable agreement with widely observed experimental trends in plasmid copy number and foreign protein content. The model provides an important tool for understanding and controlling plasmid instability in recombinant bacterial fermentations. The modeling framework employed here is suitable for studying the metabolism and growth of a variety of microbial cultures. 相似文献
6.
E. Bahreini R. Abbasalipourkabir M. T. Goodarzi M. Saidijam S. S. Safavieh 《Preparative biochemistry & biotechnology》2013,43(5):510-528
The gram-negative bacterium Escherichia coli (E. coli) offers a means for rapid, high-yield, and economical production of recombinant proteins. Here, a protocol for optimization of parameters involved in bacterial expression conditions is described. L-Asparaginase (ASNase II) was chosen as a model protein for our experiments. ASNase II gene (ansB) was cloned into the pAED4 plasmid and transformed into E. coli BL21pLysS (DE3)-competent cells. It was assumed that high cell density and high copy number of recombinant plasmid in the bacteria host could result in very high production of the recombinant protein. Circumstances for the overproduction of recombinant ASNase II including cell growth conditions, isopropyl β-D-1-thiogalactopyranoside (IPTG) level, ampicillin (Amp) concentration before and during IPTG induction, and cell density were optimized. Regarding the final optimization, overexpression of ASNase II was assessed on a large scale in LB medium. Periplasmic ASNase II was extracted using an alkaline lysis method. The extracted protein was purified by one-step DEAE-Sepharose fast-flow chromatography. ASNase II activity was considered an index for the protein expression. Applying the optimized practical protocol, protein production was significantly enhanced in comparison to the traditional IPTG induction method in the absence of a fermentor and can be applied for overexpression of other recombinant proteins. 相似文献
7.
A set of eight closely related plasmid constructs carrying CI857-controlled recombinant genes has been used as a model to study plasmid stability in Escherichia coli, in the absence of antibiotic selection. Plasmid loss rates and relative interdivision times of plasmid-bearing cells and plasmid-free cells have been analyzed throughout prolonged cultures. Whereas the calculated plasmid loss rates are not consistent for a given plasmid and set of conditions, the relative growth fitness of plasmid-bearing cells is highly reproducible. In the absence of gene expression, plasmid maintenance is influenced by the length of the cloned segment, the growth temperature, and the plasmid copy number, but not by the plasmid size. At high, inducing temperatures, the effects of the metabolic burden are eclipsed by the toxicity exhibited by the different proteins produced, which is determined by structural features. Despite the multifactorial nature of the negative pressures acting independently on plasmid-bearing cells, the relative cell fitness in a mixed cell population is very reproducible for a given vector, resulting in a monotonous spread of the plasmid-free cells in recombinant cultures. 相似文献
8.
Random mutagenesis and directed evolution has been successfully used to improve desired properties of enzymes for biocatalysis and metabolic engineering. Here we employ the method to increase copy number of a pBBR-based broad host range plasmid, which can be used to express desired enzymes in a variety of microbial hosts. Localized random mutagenesis was performed in the replication control region of a pBBR-derived plasmid containing a beta-carotene reporter. Mutant plasmids were isolated that showed increased beta-carotene production. Real-time PCR analysis confirmed that the copy number of the mutant plasmids increased 3-7 fold. Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions. Single amino acid change of serine to leucine at codon 100 of the replication protein and single nucleotide change of C to T at 46 bp upstream of the rep gene caused the increase of plasmid copy number. The utility of the mutant plasmids for metabolic engineering were further demonstrated by increased beta-carotene production, when an isoprenoid pathway gene (dxs) was co-expressed on a compatible plasmid. The mutant plasmids were tested in Agrobacterium tumefaciens. Increase of plasmid copy number and beta-carotene production was also observed in the non-Escherichia coli host. 相似文献
9.
Cranenburgh RM Lewis KS Hanak JA 《Journal of molecular microbiology and biotechnology》2004,7(4):197-203
The Escherichia coli strain DH1lacdapD enables plasmid selection and maintenance that is free from antibiotics and selectable marker genes. This is achieved by using only the lac operator sequence as a selectable element. This strain is currently used to generate high copy number plasmids with no antibiotic resistance genes for use as DNA vaccines and for expression of recombinant proteins. Until now these have been limited to pUC-based plasmids containing a high copy number pMB1-derived origin of replication, and the principle lacO(1) and auxiliary lacO(3) operators. In this study we have shown that this system can also be used to select and maintain pBR322-based plasmids with the lower copy number pMB1 origin of replication, and that lacO(1) alone or a palindromic version of lacO(1) can provide a sufficient level of repressor titration for plasmid selection. This is advantageous for recombinant protein production, where low copy number plasmids are often used and plasmid maintenance is important. The degree of repressor titration due to these plasmids was measured using the natural lactose operon in E. coli DH1 as a model. 相似文献
10.
Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli 总被引:1,自引:0,他引:1
Plasmid-host cell interactions have been investigated experimentally using Escherichia coli HB101, plasmid RSF1050 which contains the origin of replication of pMB1, and four other closely related copy number mutant plasmids. Growth characteristics of these recombinant strains and beta-lactamase activity expressed from a plasmid gene were investigated in Luria broth (LB) and in minimal medium (M9) containing in some cases casamino acids or different concentrations of alpha-methylglucoside, a competitive inhibitor of glucose transport. Maximum specific growth rates in LB and minimal media were reduced for increasing plasmid content per cell. Plasmid copy number increased when specific growth rate was reduced by changing medium composition. Growth rates of high copy number strains were less sensitive to alpha-methylglucoside than lower copy number strains and the plasmidfree host. The overall efficiency of plasmid gene expression, measured as the ratio of beta-lactamase specific activity to plasmid content, decreased significantly with increasing plasmid content in LB medium. 相似文献
11.
Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli 总被引:1,自引:0,他引:1
Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene. 相似文献
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13.
《Enzyme and microbial technology》2009,44(7):507-512
The influence of proteolysis over recombinant protein quality has been studied using rhamnulose 1-phosphate aldolase (RhuA) production as case example. Progressive induction by means of continuous isopropyl-β-d-thiogalactopyranoside (IPTG) dosage in Escherichia coli fed-batch cultures led to high specific levels of recombinant protein. However, the specific activity profile did not correlate to the specific protein content when the process was run at 37 °C and there was a decrease of the enzyme activity along the induction phase. Specific activity loss depending on the presence of an energy source was observed at short term, but protein degradation due to the action of energy-independent metalloproteases occurred after a longer time period. The effects of lowering the temperature were analysed on both mechanisms, and a reduction of the specific activity loss was observed when the process temperature was decreased to 28 °C. Lower plasmid copy number and specific production rates probably alleviated the metabolic load on host cell during recombinant protein overexpression, and a high increase of the enzyme activity was achieved in high cell density fed-batch cultures under these conditions. 相似文献
14.
The potential for production of penicillin G-acylase (PGA), encoded by the chromosomal genepga
i, of four strains belonging to a genealogical line derived from the strainEscherichia coli W, was evaluated in a medium with and without the inducer phenylacetic acid (PA). These strains were used as hosts of the
recombinant plasmid pKA18, in which the structural genepga
c isolated from the strain RE3, the best host strain of a line giving the highest production, was cloned. The presence of the
inducer reduced the copy number of the plasmid in all recombinant strains. Only in recombinant strain RE3 (pKA18) the reduction
of the gene dosage ofpga
c resulted also in the reduction of the amount of PGA synthesized by the cells. The reduced activity of the cells did not result
from a segregation of plasmid-free clones. Also the growth rate was decreased by 20 and 40% in the host and recombinant strains,
respectively. The host strain RE3 showing the highest production of PGA was also the best host of the recombinant plasmid
in terms of the segregational stability and copy number (198 copies per chromosome). The recombinant strain RE3 (pKA18) also
provided the highest production of PGA. 相似文献
15.
Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems. 相似文献
16.
Efficient expression of a foreign protein product by the yeastSaccharomyces cerevisiaerequires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromereplasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, evenin a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that amaximum of 4-6 centromere plasmids per cell can be stably partitionedat cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation. 相似文献
17.
The effect of plasmid multimerization on segregational instability was investigated using a structured, segregated model of genetically modified Escherichia coli cells. By including the multimerization of plasmids, the model can predict the proportion of each multimer in the total plasmid population. Simulation results suggest that the plasmid copy number is controlled by the total plasmid content (i.e., total number of plasmid origins) in the host cell and that multimerization reduces the total number of independent, monomeric segregation units. However, multimerization is found to have a minor effect on decreasing plasmid segregational stability for multicopy plasmids with average copy number per cell greater than about 25. Also model predictions were used to test whether or not a nonrandom plasmid distribution at cell fission could cause segregational instability. Even in the case of severely biased partitioning, plasmids whose copy number is above 45 per cell do not show significant segregational instability. The results suggest that when the ColE1-type plasmid does not encode and express any large or disruptive foreign proteins, the copy number of 45 per cell may be the threshold at which only growth rate-dependent instability is responsible for overall plasmid instability. 相似文献
18.
Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria 总被引:10,自引:0,他引:10
Multicopy plasmids are often chosen for the expression of recombinant genes in Escherichia coli. The high copy number is generally desired for maximum gene expression; however, the metabolic burden effects that usually result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications. In this study, low-copy mini-F plasmids were compared to high-copy pMB1-based plasmids for production of two metabolites in E. coli: polyphosphate (polyP) and lycopene derived from isopentenyl diphosphate (IPP). The stationary-phase accumulation of polyP on a per cell basis was enhanced approximately 80% when either high- or low-copy plasmids were used, from 120 micromol/g DCW without augmented polyP kinase (PPK) activity to approximately 220 micromol/g DCW. The cell density of the high-copy plasmid-containing culture at stationary phase was approximately 24% lower than the low-copy culture and 30% lower than the control culture. This difference in cell density is likely a metabolic burden effect and resulted in a lower overall product concentration for the high-copy culture (approximately 130 micromol/L culture) relative to the low-copy culture (approximately 160 micromol/L culture). When the gene for DXP (1-deoxy-D-xylulose 5-phosphate) synthase, the first enzyme in the IPP mevalonate-independent biosynthetic pathway, was expressed from the tac promoter on multicopy and low-copy plasmids, lycopene production was enhanced two- to threefold over that found in cells expressing the chromosomal copy only. Cell growth and lycopene production decreased substantially when isopropyl beta-D-thiogalactosidase (IPTG) was added to the high-copy plasmid-containing culture, suggesting that overexpression of DXP synthase was a significant metabolic burden. In the low-copy plasmid-containing culture, no differences in cell growth or lycopene production were observed with any IPTG concentrations. When dxs was placed under the control of the arabinose-inducible promoter (P(BAD)) on the low-copy plasmid, the amount of lycopene produced was proportional to the arabinose concentration and no significant changes in cell growth resulted. These results suggest that low-copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism. 相似文献
19.
Jaume Pinsach Carles de Mas Josep Lpez-Santín Gerald Striedner Karl Bayer 《Enzyme and microbial technology》2008,43(7):507-512
The influence of proteolysis over recombinant protein quality has been studied using rhamnulose 1-phosphate aldolase (RhuA) production as case example. Progressive induction by means of continuous isopropyl-β-d-thiogalactopyranoside (IPTG) dosage in Escherichia coli fed-batch cultures led to high specific levels of recombinant protein. However, the specific activity profile did not correlate to the specific protein content when the process was run at 37 °C and there was a decrease of the enzyme activity along the induction phase. Specific activity loss depending on the presence of an energy source was observed at short term, but protein degradation due to the action of energy-independent metalloproteases occurred after a longer time period. The effects of lowering the temperature were analysed on both mechanisms, and a reduction of the specific activity loss was observed when the process temperature was decreased to 28 °C. Lower plasmid copy number and specific production rates probably alleviated the metabolic load on host cell during recombinant protein overexpression, and a high increase of the enzyme activity was achieved in high cell density fed-batch cultures under these conditions. 相似文献
20.
Although plasmid copy number varies widely among different plasmid species, normally copy number is maintained within a narrow range for any given plasmid. Such copy number control has been shown to occur by regulation of the rate of plasmid DNA replication. Here we report a novel mechanism by which the pSC101 plasmid also can detect an imbalance between the cellular level of its replication protein, RepA, and plasmid-borne RepA binding sites to inhibit bacterial DNA replication and delay host cell division when RepA is in relative excess. We show that delayed cell division occurs by RepA-mediated induction of the SOS response and can be reversed by over-expression of the host DNA primase, DnaG. The effects of RepA excess are prevented by introducing a surfeit of RepA binding sites. The mechanism reported here may help to limit variation in plasmid copy number and allow repopulation of cells with plasmids when copy number falls--potentially pre-empting plasmid loss in cultures of dividing cells. 相似文献