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Cornelissen S Julsing MK Schmid A Bühler B 《Journal of industrial microbiology & biotechnology》2012,39(2):275-287
Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis.
In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression
levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U gCDW−1, respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol gCDW−1). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in
order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical
production, but are sufficient for preparative-scale drug metabolite synthesis. 相似文献
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Hemolysis is a major symptom of diseased eels infected by Edwardsiella tarda. The hemolysin gene of E. tarda strain ET16 was cloned into plasmid pSK and expressed in Escherichia coli. The mol. mass of the functional β-hemolysin was estimated to be approximately 34 kDa by gel filtration and by SDS-PAGE followed
by in situ hemolysin activity analysis. The cloned fragment containing the β-hemolysin locus from E. tarda strain ET16 expressed in E. coli was estimated to be 5.3 kb in length; the deduced gene product was identical in mol. mass and properties to the extracellular
products of E. tarda strain ET16. The presence of EcoRI and XbaI sites within the β-hemolysin gene of E. tarda was determined from the loss of hemolytic activity in subclones. Analysis of the DNA sequence of a 2,436-bp HaeIII-HindIII fragment that included EcoRI and XbaI sites revealed three ORFs organized as an operon that encoded three predicted polypeptides of 15,874, 7,055, and 34,804
Da. A 34-kDa polypeptide expressing hemolytic activity in cell lysates of the clone DH5α(pETH3E) is very likely the β-hemolysin
encoded by the third ORF. The observation that hemolytic activity appeared in the culture medium of E. tarda, but not in that of E. coli strain DH5α(pETH3E) indicates the existence of a mechanism for transporting the hemolysin across the cell envelope in E. tarda that is different from that of E. coli.
Received: 7 July 1995 / Accepted: 24 October 1995 相似文献
5.
The recognition of the potential efficacy of plasmid DNA (pDNA) molecules as vectors in the treatment and prevention of emerging
diseases has birthed the confidence to combat global pandemics. This is due to the close-to-zero safety concern associated
with pDNA vectors compared to viral vectors in cell transfection and targeting. Considerable attention has been paid to the
potential of pDNA vectors but comparatively less thought has been given to the practical challenges in producing large quantities
to meet current rising demands. A pilot-scale fermentation scheme was developed by employing a stoichiometrically-designed
growth medium whose exceptional plasmid yield performance was attested in a shake flask environment for pUC19 and pEGFP-N1
transformed into E. coliDH5α and E. ColiJM109, respectively. Batch fermentation of E. coliDH5α-PUC19 employing the stoichiometric medium displayed a maximum plasmid volumetric and specific yield of 62.6 mg/L and
17.1 mg/g (mg plasmid/g dry cell weight), respectively. Fed-batch fermentation of E. coliDH5α-pUC19 on a glycerol substrate demonstrated one of the highest ever reported pilot-scale plasmid specific yield of 48.98
mg/g and a volumetric yield of 0.53 g/L. The attainment of high plasmid specific yields constitutes a decrease in plasmid
manufacturing cost and enhances the effectiveness of downstream processes by reducing the proportion of intracellular impurities.
The effect of step-rise temperature induction was also considered to maximize CoIE1-origin plasmid replication. 相似文献
6.
Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain. 相似文献
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P. L. Schuerman J. S. Liu H. Mou A. M. Dandekar 《Applied microbiology and biotechnology》1997,47(5):560-565
Escherichia coli strains that did not have the ability to use sucrose as a sole carbon source gained this ability after receiving a cloned
fragment of DNA from Agrobacterium tumefaciens. No invertase was detected in the sucrose-metabolizing E. coli, but evidence for the activity of certain enzymes, known to be produced by biotype 1 strains of Agrobacterium, were found. Evidence was found for the presence of d-glucoside 3-dehydrogenase (G3DH) and α-3-ketoglucosidase. The activity of enzyme extracts on 3-ketosucrose also indicated
that 3-ketoglucose reductase, or some enzyme that acts on 3-ketoglucose, was present in the Suc+
E. coli as well. The fragment was found to complement a G3DH mutant of A. tumefaciens and was also found to confer chemotaxis towards sucrose in E. coli.
Received: 13 September 1996 / Received revision: 15 January 1997 / Accepted: 24 January 1997 相似文献
8.
Reinhard Breitling Dieter Gerlach Manfred Hartmann Detlev Behnke 《Molecular & general genetics : MGG》1989,217(2-3):384-391
Summary A DNA segment covering the signal sequence coding region, the ribosome binding site, and the promoter of the staphylokinase
(sak) 42D gene (Behnke and Gerlach 1987) was cloned into pUC19 to form a portable expression-secretion unit (ESU). Fusion of human
interferon α1 (hIFNα1) and hybrid hIFNα1/2 genes to thissak ESU resulted in secretory expression of the two gene products in bothEscherichia coli andBacillus subtilis. While most of the IFNα was exported to the periplasmic space ofE. coli, about 99% was secreted to the culture medium by recombinantB. subtilis strains. The total yield inE. coli was 1.2×105 IU/ml. This level of expression and export led to instability of the recombinant strains that was spontaneously relieved
in vivo by inactivation of thesak ESU through insertion of an IS1 element. No such instability was observed withB. subtilis although expression and secretion levels reached even 3×106 IU/ml. Proteolytic degradation of IFNα by extracellular proteases was avoided by a combination of constitutive expression
and secretion during the logarithmic growth phase and the use of exoprotease-reduced host strains. The IFNα1 protein purified fromB. subtilis culture supernatant was correctly processed, carried the expected 11 amino acid N-terminal elongation that resulted from
DNA manipulations and proved to be homogenous in Western blotting experiments. The same recombinant plasmid that directed
efficient secretion of hIFNα1 inB. subtilis gave poor yields when introduced intoStreptococcus sanguis. 相似文献
9.
The sweet protein monellin gene was expressed in Saccharomyces cerevisiae under the control of the GAL1 promoter and α-factor signal peptide sequence of S. cerevisiae. The gene, which was obtained through mutation of the synthesized single-chain monellin gene, was cloned into an E. coli-yeast shuttle vector pYES2.0 which carries the galactose-inducible promoter GAL1. Then the α-factor signal peptide of S. cerevisiae was linked also, resulting in the secreting expression vector pYESMTA. The recombinant plasmid was subsequently transformed
into strain S. cerevisiae INVsc1. The peptide efficiently directed the secretion of monellin from the recombinant yeast cell. A maximum yield of active
monellin was 0.41 g l−1 of the supernatant from INVsc1 harboring pYESMTA. 相似文献
10.
M. L. Rúa H. Atomi C. Schmidt-Dannert R. D. Schmid 《Applied microbiology and biotechnology》1998,49(4):405-410
An efficient expression system for the previously only weakly expressed thermophilic lipase BTL2 (Bacillus thermocatenulatus lipase 2) was developed for the production of large amounts of lipase in Escherichia coli. Therefore, the gene was subcloned in the pCYTEXP1 (pT1) expression vector downstream of the temperature-inducible λ promoter
PL. Three different expression vectors were constructed: (i) pT1-BTL2 containing the mature lipase gene, (ii) pT1-preBTL2 containing the prelipase gene and (iii) pT1-OmpABTL2 containing the mature lipase gene fused to the signal peptide of the OmpA protein, the major outer membrane
protein of E. coli. With pT1-BTL2 and pT1-preBTL2, comparable expression levels of 7000–9000 U/g cells were obtained independently of the E. coli host. In contrast, with E. coli JM105 harbouring pT1-OmpABTL2, 660 000 soluble lipase U/g cells was produced, whereas, with E. coli DH5α and BL321, production levels of 30 000 U/g cells were achieved. However, most of the lipase remained insoluble but active
after cell breakage because of the unprocessed OmpA signal peptide. A simple cholate extraction followed by proteinase K cleavage
and ultrafiltration allowed the isolation of 1.15 × 106 units of 90% pure mature lipase/wet cells.
Received: 29 August 1997 / Received revision: 17 November 1997 / Accepted: 18 November 1997 相似文献
11.
The phoA expression system is an efficient one and is successfully used in foreign gene expression. In a previous study, it was found
that pH during the expression phase had a significant effect on extracellular hEGF production under control of the phoA promoter by Escherichia coli DA19, an acetate-tolerant strain of E. coli DH5α, in a chemically defined medium, but the level of hEGF production was only 75.5 mg/L. E. coli DB15 is another acetate tolerant mutant of DH5α. In the present study, production of hEGF under control of the phoA promoter by DB15 was further investigated. When transition from the growth phase, where phosphate was abundant, to the expression
phase where phosphate was limited, was performed based on cell density, the extracellular hEGF reached 165 mg/L, twice that
when transition was based on dissolved oxygen. Furthermore, adding 0.22 g/L of CaCl2 during the growth phase, further increased hEGF production to 228 mg/L, which is 3-fold the level produced by DA19 (pAET-8)
cultured in the same medium. 相似文献
12.
Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 × 10–6 recepient–1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5 cells at a frequency of 1 × 10–8 recepient–1. The growth rate of E. coli DH5; (pUPI199) was slower as compared with E. coli DH5. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5 (pUPI199) effluxed 63% of the accumulated silver ions. 相似文献
13.
Axenic aerobic biofilms inhibit corrosion of SAE 1018 steel through oxygen depletion 总被引:1,自引:0,他引:1
A. Jayaraman E. T. Cheng J. C. Earthman T. K. Wood 《Applied microbiology and biotechnology》1997,48(1):11-17
Corrosion inhibition of SAE 1018 steel by pure-culture biofilms of Pseudomonas fragi and Escheri-chia coli DH5α has been evaluated in complex Luria-Bertani medium, seawater-mimicking medium, and modified Baar's medium at 30 °C.
In batch cultures, both bacteria inhibited corrosion three to six fold compared to sterile controls, and the corrosion was
comparable to that observed in anaerobic sterile media. To corroborate this result, a continuous reactor and electrochemical
impedance spectroscopy were used to show that both P. fragi K and E. coli DH5α decreased the corrosion rate by 4- to 40-fold as compared to sterile controls; this matched the decrease in corrosion
found with sterile medium in the absence of oxygen and with E. coli DH5α grown anaerobically. In addition, the requirement for live respiring cells was demonstrated by the increase in the corrosion
rate that was observed upon killing the P. fragi K biofilm in continuous cultures, and it was shown that fermentation products do not cause an increase in corrosion. Hence,
pure-culture biofilms inhibit corrosion of SAE 1018 steel by depleting oxygen at the metal surface.
Received: 16 December 1996 / Received revision: 18 March 1997 / Accepted: 27 March 1997 相似文献
14.
Elicitation can lead to overproduction of secondary metabolites in plants and microbes. Potential changes in cytosolic Ca2+ levels in bacteria were studied in response to elicitation. We report, for the first time, the effect of oligosaccharide
elicitors on intracellular Ca2+ levels. The apoaequorin gene was cloned into Escherichia coli DH5α and Bacillus subtilis 1604 cultures. Addition of elicitors, oligoguluronate and mannan oligosaccharides, to the cultures caused up to 11-fold increase
in cytosolic Ca2+ in E. coli and tenfold increase in B. subtilis. These increases in Ca2+ levels could therefore contribute to the enhancement of secondary metabolite levels. 相似文献
15.
To clone bifunctional vectors in streptomycetes, it was necessary to define the restriction-modification system ofStreptomyces flavopersicus. Plasmid DNA from bifunctional vectors pIJ699 and pXED3-13, isolated fromE. coli strains with different methylation systems:E. coli DH5α (dam
+
dcm
+),E. coli MB5386(dam dcm), E. coli CB51 (dam dcm
+),E. coli NM544 (dam
+
dcm), was used for transformation of protoplasts from strainS. flavopersicus. Restriction ofdcm-methylated DNA fromS. flavopersicus was established. As a host in the intermediate cloning strainE. coli NM544 (dam
+
dcm) should be used, as thedcm-transmethylase-dependent strainS. flavopersicus does not process DNA from this strain. 相似文献
16.
Sukhija K Pyne M Ali S Orr V Abedi D Moo-Young M Chou CP 《Molecular biotechnology》2012,51(2):109-118
Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method
was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets
(FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P
trc
and P
araB
), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol)
under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation
using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive
genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic
engineering. 相似文献
17.
The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows 总被引:1,自引:0,他引:1
G. Basnakova E. R. Stephens M. C. Thaller G. M. Rossolini L. E. Macaskie 《Applied microbiology and biotechnology》1998,50(2):266-272
A Citrobacter sp. originally isolated from metal-polluted soil accumulates heavy metals via metal-phosphate deposition utilizing inorganic
phosphate liberated via PhoN phosphatase activity. Further strain development was limited by the non-transformability of this
environmental isolate. Recombinant Escherichia coli DH5α bearing cloned phoN or the related phoC acquired metal-accumulating ability, which was compared with that of the Citrobacter sp. with respect to removal of uranyl ion (UO2
2+) from dilute aqueous flows and its deposition in the form of polycrystalline hydrogen uranyl phosphate (HUO2PO4). Subsequently, HUO2PO4-laden cells removed Ni2+ from dilute aqueous flows via intercalation of Ni2+ into the HUO2PO4 lattice. Despite comparable acid phosphatase activity in all three strains, the E. coli DH5α (phoN) construct was superior to Citrobacter N14 in both uranyl and nickel accumulation, while the E. coli DH5α (phoC) construct was greatly inferior in both respects. Expression of phosphatase activity alone is not the only factor that permits
efficient and prolonged metal phosphate accumulation, and the data highlight possible differences in the PhoN and PhoC phosphatases,
which are otherwise considered to be related in many respects.
Received: 30 December 1997 / Received revision: 25 March 1998 / Accepted: 26 March 1998 相似文献
18.
Jong Hwa Yum Eun Young Lee Sung-Ho Hur Seok Hoon Jeong Hyukmin Lee Dongeun Yong Yunsop Chong Eun-Woo Lee Patrice Nordmann Kyungwon Lee 《Journal of microbiology (Seoul, Korea)》2010,48(3):358-364
This study was performed to characterize the chromosomal metallo-β-lactamases (MBLs) of Elizabethkingia meningoseptica isolated from Korea and to propose a clustering method of BlaB and GOB MBLs based on their amino acid similarities. Chromosomal
MBL genes were amplified by PCR from 31 clinical isolates of E. meningoseptica. These PCR products were then cloned into a vector and electrotransformed into E. coli DH5α. Nucleotide sequencing was performed by the dideoxy chain termination method using PCR products or cloned DNA fragments.
Antimicrobial susceptibilities were determined by the agar dilution method. PCR experiments showed that all 31 E. meningoseptica isolates contained both the blaB and the bla
GOB genes. DNA sequence analysis revealed that E. meningoseptica isolates possessed seven types of blaB gene, including five novel variants (blaB-9 to blaB-13) and 11 types of bla
GOB gene, including 10 novel variants (bla
GOB-8 to bla
GOB-17). The most common combination of MBL was BlaB-12 plus GOB-17 (n=19). Minimum inhibitory concentrations of imipenem and meropenem
for the electrotransformants harboring novel BlaB and GOB MBLs were two- or four-fold higher than those for the recipient
E. coli DH5α. BlaB and GOB MBLs were grouped in three and six clusters including fifteen novel variants, respectively, based on amino
acid similarities. 相似文献
19.
Prostaglandins (PGs) are important local messenger molecules in many tissues and organs of animals including human. For applications
in medicine and animal care, PGs are mostly purified from animal tissues or chemically synthesized. To generate a clean, reliable,
and inexpensive source for PGs, we have now engineered expression of a suitable cyclooxygenase gene in Escherichia coli and achieved production levels of up to 2.7 mg l−1 PGF2α. The cyclooxygenase gene cloned from the red alga Gracilaria vermiculophylla appears to be fully functional without any eukaryotic modifications in E. coli. A crude extract of the recombinant E. coli cells is able to convert in vitro the substrate arachidonic acid (AA) to PGF2α. Furthermore, these E. coli cells produced PGF2α in a medium supplemented with AA and secreted the PGF2α product. To our knowledge, this is the first report of the functional expression of a cyclooxygenase gene and concomitant
production of PGF2α in E. coli. The successful microbial synthesis of PGs with reliable yields promises a novel pharmaceutical tool to produce PGF2α at significantly reduced prices and greater purity. 相似文献
20.
The gene encoding creatininase from Pseudomonas putida RS65 was cloned, sequenced and expressed in Escherichia coli. One plasmid containing a 7.0-kb HindIII insert was selected by its ability to express creatininase activity. After deletion of the adjacent restriction fragments,
a 1.1-kb SphI fragment, which contained the full length of the creatininase gene, was subcloned into a pUC18 vector and the nucleotide
sequence of the creatininase gene was determined. The gene consists of 771 base pairs and encodes a protein of 257 amino acids.
The constitutive creatininase productivity of E. coli DH5α (pCRN741) cultured in broth was about 8.5-fold higher than that of P. putida RS65 cultured in a creatinine-containing medium. The creatininase gene was expressed efficiently in E. coli from its own promoter. Journal of Industrial Microbiology & Biotechnology (2000) 24, 2–6.
Received 02 April 1999/ Accepted in revised form 31 July 1999 相似文献