Plasmid size can affect the ability of Escherichia coli to produce high-quality plasmids

Large molecular weight plasmids are often used in gene therapy and DNA vaccines. To investigate the effect of plasmid size on the performance of Escherichia coli host strains during plasmid preparation, we employed E. coli JM109 and TOP10 cells to prepare four plasmids ranging from 4.7 to 16.8?kb in size. Each plasmid was extracted from JM109 and TOP10 cells using an alkaline lysis mini-preparation method. However, when commercial kits were used to extract the same plasmids from JM109 cells, the large molecular weight plasmids substantially degraded, compared with their smaller counterparts. No degradation was observed when the four plasmids were extracted from E. coli TOP10 cells using the same commercial kit. We conclude, therefore, that the performance of E. coli in high quality plasmid preparations can be affected by plasmid size.     

Vesicle-Mediated Transfer of Virulence Genes from Escherichia coli O157:H7 to Other Enteric Bacteria

Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109. Electron micrographs of purified DNA from E. coli O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (hly, L7095, mobA, and gfp), chromosomal DNA (uidA and eaeA), and phage DNA (stx1 and stx2). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (E. coli JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 10³ transformants μg of DNA⁻¹) and demonstrated that vesicles can deliver antibiotic resistance. Transformed E. coli JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.     

Efficiency of the pTF-FC2 pas Poison-Antidote Stability System in Escherichia coli Is Affected by the Host Strain, and Antidote Degradation Requires the Lon Protease

The stabilization of a test plasmid by the proteic, poison-antidote plasmid addiction system (pas) of plasmid pTF-FC2 was host strain dependent, with a 100-fold increase in stability in Escherichia coli CSH50, a 2.5-fold increase in E. coli JM105, and no detectable stabilization in E. coli strains JM107 and JM109. The lethality of the PasB toxin was far higher in the E. coli strains in which the pas was most effective. Models for the way in which poison-antidote systems stabilize plasmids require that the antidote have a much higher rate of turnover than that of the toxin. A decrease in host cell death following plasmid loss from an E. coli lon mutant and a decrease in plasmid stability suggested that the Lon protease plays a role in the rate of turnover of PasA antidote.     

Production of l-phenylalanine from trans-cinnamic acids by high-level expression of phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli

A combined promoter expression vector pBV–PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed. Pal gene was cloned and inserted into the region between SalI and PstI restriction sites of expression vector pBV220 (containing P_LP_R promoter) to obtain recombinant expression vector pBV220–PAL. The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220–PAL to construct expression vector pBV–PAL. The recombinant plasmid pBV220–PAL and pBV–PAL were introduced into Escherichia coli JM109 by transformation. The result showed that the transformant E. coli JM109 (pBV–PAL) gave a much higher PAL activity than that transformant E. coli JM109 (pBV220–PAL). Recombinant PAL expression level of the transformant JM109 (pBV–PAL) was about 9.6% of total cellular protein, specific enzyme activity was 2.3-fold higher than that of the transformant JM109 (pBV220–PAL), reached 35 U/g (dry cells weight, DCW). PAL specific activity of 123 U/g (DCW) could be achieved in a 5-l fermentor. 80.5% conversion rate of trans-cinnamic acid to l-phenylalanine and 5.12 g/l l-phenylalanine were obtained after 3 h bioconversion using the transformant JM109 (pBV–PAL). The recombinant strain JM109 containing the combined promoter expression vector pBV–PAL was shown to be effective and practical to product l-phenylalanine.     

Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran

A gene encoding the carboxymethylcellulase (CMCase) of a marine bacterium, Bacillus subtilis subsp. subtilis A-53, was cloned in Escherichia coli JMB109 and the recombinant strain was named as E. coli JMB109/A-53. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth, extracted by Design Expert Software based on response surface methodology, were 100.0 g/l, 7.5 g/l, and 7.0, respectively, whereas those for production of CMCase were 100.0 g/l, 7.5 g/l, and 8.0. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/A-53 were found to be and 40 and 35 °C, respectively. The optimal agitation speed and aeration rate of a 7 l bioreactor for cell growth were 400 rpm and 1.5 vvm, whereas those for production of CMCase were 400 rpm and 0.5 vvm. The optimal inner pressure for cell growth was 0.06 MPa, which was the same as that for production of CMCase. The production of CMCase by E. coli JM109/A-53 under optimized conditions was 880.2 U/ml, which was 2.9 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen source for production of CMCase by a recombinant E. coli JM109/A-53 and the productivity of E. coli JM109/A-53 was 5.9 times higher than that of B. subtilis subp. subtilis A-53.     

Comparison of optimal conditions for mass production of carboxymethylcellulase by <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 with other recombinants in pilot-scale bioreactor

The optimal conditions for mass production of carboxymethylcellulase (CMCase) by E. coli JM109/A-68 were investigated and compared with other E. coli JM109 recombinants producing CMCase. The optimal agitation speed and aeration rate for cell growth of E. coli JM109/A- 68 were 500 rpm and 0.50 vvm in a 7 L bioreactor, whereas those for production of CMCase were 416 rpm and 0.95 vvm. The optimal vessel pressures for cell growth as well as production of CMCase in a 100 L bioreactor were 0.04 MPa. The maximal production of CMCase by E. coli JM109/A-68 under the optimized conditions in a 100 L bioreactor was 11.0 times higher than its wild type, B. velezensis A-68. Optimal conditions for mass production of CMCase by recombinants were different from those for wild strains. The higher production of CMCase by E. coli JM109/A-68 and other recombinant of E. coli seemed to result from its higher cell growth under the optimal conditions for dissolved oxygen and its mixed-growth associated production pattern compared to the growthassociated production of B. velezensis A-68.     

Extracellular recombinant protein production under continuous culture conditions with Escherichia coli using an alternative plasmid selection mechanism

The secretion of recombinant proteins into the extracellular space by Escherichia coli presents advantages like easier purification and protection from proteolytic degradation. The controlled co-expression of a bacteriocin release protein aids in moving periplasmic proteins through the outer membrane. Since such systems have rarely been applied in continuous culture it seemed to be attractive to study the interplay between growth-phase regulated promoters controlling release protein genes and the productivity of a chemostat process. To avoid the use of antibiotics and render this process more sustainable, alternative plasmid selection mechanisms were required. In the current study, the strain E. coli JM109 harboring plasmid p582 was shown to stably express and secrete recombinant β-glucanase in continuous culture using a minimal medium. The segregational instability of the plasmid in the absence of antibiotic selection pressure was demonstrated. The leuB gene, crucial in the leucine biosynthetic pathway, was cloned onto plasmid p582 and the new construct transformed into an E. coli Keio (ΔleuB) knockout strain. The ability of the construct to complement the leucine auxotrophy was initially tested in shake-flasks and batch cultivation. Later, this strain was successfully grown for more than 200 h in a chemostat and was found to be able to express the recombinant protein. Significantly, it showed a stable maintenance of the recombinant plasmid in the absence of any antibiotics. The plasmid stability in a continuously cultivated E. coli fermentation, in the absence of antibiotics, with extracellular secretion of recombinant protein provides an interesting model for further improvements.     

用于质粒DNA规模化生产的大肠杆菌发酵培养基的筛选

为降低质粒DNA的生产成本,在标准LB培养基的基础上,利用国产试剂配制成十种大肠杆菌液体培养基,以pEGFPC3、pcDNAlacZ和pcDNKLYZ质粒转化的JM109和DH5α大肠杆菌为指示菌进行小规模发酵培养,定时采样测量OD600值及质粒产量,获得一种高性价比培养基。用该培养基培养重组大肠肝菌,绘制生长曲线,并于其对数生长中期进行42℃诱导。结果表明经42℃诱导后,重组大肠肝菌JM109和DH5α的质粒产量均有提高,重组JM109的产量比重组DH5α约提高20％,为低成本、大规模生产重组质粒提供了良好的技术保障。     

Degradation of trichloroethylene by recombinant E. coli in continuous culture

Trichloroethylene (TCE) degradation by the recombinant E. coli JM109 harboring a TCE-degradative plasmid (pIO720 or pIO72K) in continuous culture was studied. The ampicillin-resistant plasmid, pIO720, contained the cumene dioxygenase genes and the dimethyl sulfide monooxygenase genes. pIO72K was constructed according to replacement of an ampicillin resistance gene on pIO720 by a kanamycin resistance gene. In the case of E. coli JM109 (pIO720) in continuous culture, TCE degradation activity decreased rapidly after continuous culture started, and the remaining number of host cells harboring pIO720 also decreased rapidly. In the case of E. coli JM109 (pIO72K) in continuous culture, TCE degradation activity was stable during continuous culture for at least 300 h and the number of the host cells harboring pIO72K did not decrease. TCE degradation activity of E. coli JM109 (pIO72K) was the highest at a dilution rate of 0.2 h^–1.     

Engineering the growth pattern and cell morphology for enhanced PHB production by <Emphasis Type="Italic">Escherichia coli</Emphasis>

E. coli JM109?envC?nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109?minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109?envC?nlpD and E. coli JM109?minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109?minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109?envC?nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.     

Construction of a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 for production of carboxymethylcellulase and comparison of its production with its wild type, <Emphasis Type="Italic">Bacillus velezensis</Emphasis> A-68 in a pilot-scale bioreactor

A gene encoding carboxymethylcellulase (CMCase) of Bacillus velezensis A-68 had been cloned in Escherichia coli JM109. Based on productivity and economic aspect, rice bran and ammonium chloride were chosen to be optimal carbon and nitrogen sources for production of CMCase by E. coli JM109/A-68. The optimal conditions for rice bran, ammonium chloride, and initial pH of medium for production of CMCase were established by the response surface methodology (RSM). The concentrations of four salts in the medium, K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄, for production of CMCase also were optimized. The optimal temperatures for cell growth and production of CMCase were 37°C. The maximal production of CMCase by E. coli JM109/A-68 was 880.2 U/mL, which was 10.5 time higher than its wild type, B. velezensis A-68. The production of CMCase by E. coli JM109/A-68 was compared with that by B. velezensis A-68 in a 100 L pilot-scale bioreactor under the optimized conditions. The production of CMCase by E. coli JM109/A-68 was found to be the mixed-growth associated unlike the growthassociated production of CMCase by B. velezensis A-68.     

Expression of a deleted variant of human plasminogen in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A recombinant plasmid carrying a modified gene of human plasminogen (mini-plasminogen), lacking four kringle domains and an amino terminal fragment, and containing an additional oligopeptide of six N-terminal histidine residues has been constructed. The plasmid was used for transformation of E. coli JM 109 cells to obtain a strain producing a recombinant modified human plasminogen. The target protein is superexpressed in a form of inclusion bodies and is composed of more than 50% insoluble protein. The renaturated and chromatographically purified protein exhibits amidolytic activity specific for plasminogen proenzyme in a fibrinolytic system.     

Cloning and expression in Escherichia coli of the BanI and BanIII restriction-modification systems from Bacillus aneurinolyticus

The genes coding for the GGPyPuCC-specific (BanI) and ATCGAT-specific (BanIII) restriction-modification systems of Bacillus aneurinolyticus IAM1077 were cloned and expressed in Escherichia coli using pBR322 as a vector. The plasmids carrying the BanI and BanIII restriction-modification genes were designated pBanIRM8 and pBanIIIRM12, respectively. The restriction maps of these recombinant plasmids were constructed. These two plasmids were stably maintained in E. coli HB101. However, when E. coli JM109 was used as a host, pBanIIIRM12 was efficiently propagated but pBanIRM8 was not. The HB101 cells carrying only the restriction gene of BanIII were viable, but the BanI restriction gene carrier could not form colonies on agar plates. The growth of bacteriophage λ was strongly restricted only in the F. coli HB101 cells harboring pBanIRM8. These facts indicate that the BanI restriction enzyme is expressed and functions more efficiently than BanI modification enzyme in E. coli.     

Identification of Pseudomonas bacteriophage PaP3 lysozyme

The complete genome of bacteriophage PaP3 was sequenced in a previous study by our laboratory; however, the PaP3 lysozyme gene could not be identified by homology search. In this study, based on bioinformatic analysis of its secondary structure, we have determined that the protein encoded by the p02 gene of PaP3 is likely to be a lysin. To confirm the function of the p02 gene, a recombinant expression plasmid was constructed by inserting the p02 gene into a pQE-31 plasmid; the recombinant construct was cloned and expressed in Escherichia coli JM109. The lytic activity of the expressed, purified product was observed by gel diffusion assay. The result showed that the recombinant plasmid successfully expressed 6 × his-tagged p02 protein. The expressed product had a growth inhibitory effect on Staphylococcus aureus but not on Pseudomonas aeruginosa or E. coli. However, it retained lytic activity against peptidoglycan from cell walls of P. aeruginosa and E. coli. Therefore, it is supposed that this lysozyme requires the help of holin or other punching proteins to exert lytic effects on live gram-negative bacteria. The results suggest that the p02 protein of PaP3 is a new member of the lysozyme family, which is not completely host-specific and might serve as an anti-staphylococcal agent.     

Extension of Chronological Lifespan by ScEcl1 Depends on Mitochondria in Saccharomyces cerevisiae

We constructed two plasmids that have a strong tac promoter and a structural gene for tryptophanase of Enterohacter aerogenes SM-18 (pKT901EA) or Escherichia coli K-12 (pKT951EC). The tryptophanase activity of E. coli JM109 transformed with pKT90lEA (JM109/pKT901EA) was inducible with isopropyl-β-D-thiogalactopyranoside, and 3.6 times higher than that of E. aerogenes SM-18. Cells of JM109/pKT901EA induced for tryptophanase synthesized L-tryptophan from indole, ammonia, and pyruvate more efficiently than E. aerogenes SM-18. Although JM109/pKT951EC expressed a similar level of tryptophanase activity to that of JM109/pKT901EA, the synthesis of L-tryptophan by the cells of JM109/pKT951EC did not proceed well compared with JM109/pKT901EA. Tryptophanases from E. aerogenes and E. coli K-12 were purified, and their properties were investigated. The purified E. aerogenes tryptophanase showed higher stability against heat inactivation than E. coli tryptophanase.     

Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli

Glutathione (GSH) is one of the most ubiquitous non-protein thiols that is involved in numerous cellular activities. The gene coding for a novel bifunctional enzyme catalyzing the reaction for glutathione synthesis, gshF, was cloned from Streptococcus thermophilus SIIM B218 and expressed in Escherichia coli JM109. In the presence of the precursor amino acids and ATP, the induced cells of E. coli JM109 (pTrc99A-gshF) could accumulate 10.3 mM GSH in 5 h. The S. thermophilus GshF was insensitive to feedback inhibition caused by GSH even at 20 mM. At elevated concentrations of the precursor amino acids and ATP, E. coli JM109 (pTrc99A-gshF) produced 36 mM GSH with a molar yield of 0.9 mol/mol based on added cysteine and of 0.45 mol/mol based on added ATP. When ATP was replaced with glucose, E. coli JM109 (pTrc99A-gshF) produced 7 mM in 3 h. Saccharomyces cerevisiae was used to generate ATP for GSH production. In the presence of glucose and the pmr1 mutant of S. cerevisiae BY4742, JM109 (pTrc99A-gshF) produced 33.9 mM GSH in 12 h with a yield of 0.85 mol/mol based on added l-cysteine. It is shown that the S. thermophilus GshF can be successfully used for GSH production.     

Metabolic enhancement due to plasmid maintenance

Summary Plasmid maintenance allows the strain JM109 of Escherichia coli to grow in a minimal defined medium (M9). JM109 carrying no plasmid can hardly grow in M9 whereas JM109 carrying one, two and three plasmids have a clear metabolic advantage over the untransformed strain. In a complex medium like LB (Luria-Bertani Broth) all strains grow well and despite the number of plasmids carried by the host maximum specific growth rates are not severely affected. Our results suggest that the glucose metabolism is an essential factor contributing to this behavior.