Escherichia coli transformation

Fraser, P.D. and Sandmann, G., In vitro assays of three carotenogenic membrane-bound enzymes from Escherichia coli transformed with different crt genes, Biochem. Biophys. Res. Commun. 185, 9, 1992. 

Sandmann, G., Carotenoid analysis in mutants from Escherichia coli transformed with carotenogenic gene cluster and Scenedesmus obliquus mutant C-6D, Meth. Enzymol. 214, 341, 1993. 

Azospmlhtm strains were grown on LB medium containing 1% (w/v) pectin at 30°C for seven days, and Escherichia coli transformants for two days. Culture media were centrifuged at 10.000 g for 10 minutes. The supernatant constituted the enzyme preparation. 

The regioselectivity of a Rhodococcus rhodochrous nitrilase has been demonstrated for the conversion of 5-fluoro-l,3-dicyanobenzene to 5-fluoro-3-cyano-benzoic acid [62]. The nitrilase was expressed in an Escherichia coli transformant, and a cell-free extract was employed as catalyst (0.14wt% cell-free extract) in 0.1m sodium phosphate buffer (pH 7.2) at 25 °C containing 0.18 m 5-fluoro-l,3-dicyanobenzene. After 72 h, the conversion was >98% and the reaction was stopped by addition of phosphoric acid (pH 2.4) to yield 5-fluoro-3-cyano-benzoic acid as a crystalline product (97% isolated yield). 

Hirayae, K. Hirata, A. Akutsu, K. Hara, S. Havukkala, I. Nishizawa, Y. Hibi, T. (1996)/n vitro growth inhibition of plant pathog ic fungi, Botrytis spp., by Escherichia coli transformed with a chitinolytic enzyme gene from a marine bactmum, Alteromonas sp. Strain 79401. Ann. Phytopathol. Soc. Jpn., 62, 30-6. 

Yokoyama, A. Shizuri, Y. Misawa, N. (1998) Production of new carotenoids, astaxanthin glucosides, by Escherichia coli transformants carrying carotenoid biosynthaic genes. Tetrahedron Lett., 39,3709-12. 

D192G <1> (<1> the mutation causes an enhanced feedback-resistant y-glutamyl kinase activity and conferrs an analogue-resistant phenotype to an Escherichia coli transformant containing the mutated gene [1]) [1]... 

Massarelli, L Forlani, G. Ricca, E. De Felice, M. Enhanced and feedback-resistant y-glutamyl kinase activity of an Escherichia coli transformant carrying a mutated proB gene of Streptococcus thermophilus. FEMS Microbiol. Lett., 182, 143-147 (2000)... 

M. Wada, K. Kita, H. Yanase, and S. Shimizu, Stereoselective reduction of of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes, Appl. Microbiol. Biotechnol. 1999, 53, 486-490. 

Escherichia coli transformed by such expression vectors... 

Chung, C. T., Niemela, S. L and Miller, R. H. (1989) One-step preparation of competent Escherichia coli transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86, 2172-2175. 

Comparison of the molecular size of native enzymes estimated by gel filtration and that of purified enzymes on SDS-PAGE indicates that native enzymes of winter squash [59] and zucchini [67] are present in a dimeric form, whereas those of tomato [51] and apple fruits [61] are in a monomers. However, Satoh et al. [68] reported that ACC synthases expressed in Escherichia coli transformed with cDNAs for tomato enzymes (LE-ACS2 and LE-ACS4) were present as dimers as were the winter squash enzymes similarly expressed in E. coli from two different cDNAs [CMW33 (CM-ACSl) and CMAlOl (CM-ACS2)]. It is possible that although, in its primary structure ACC synthase tends to dimerize, tomato ACC synthase is so modified after translation in vivo that dimerization is prevented. The exact nature of the modification is not known. Li and Mattoo [69] reported that dimerization of tomato enzyme expressed in E. coli was prevented when the 52 amino acid residues at the carboxyl terminal were deleted. 

ACC synthase produced in Escherichia coli transformed with a cDNA which was cloned from apple fruits was purified and crystallised [69a]. The intact protein contained subunits with molecular masses of 52 kDa, but during storage of the purified enzyme, the size of the subunit was reduced to 47 kDa although enzyme activity was retained. The crystallised enzyme contained the smaller subunit of 47 kDa exclusively. X-ray analysis of this crystalline enzyme showed that the enzyme was a homodimer [69a]. Since the truncated enzyme retained activity probably the C-terminal 5 kDa portion had been deleted as had been observed in ACC synthase from other sources. This indicates that the C-terminal portion does not contribute to dimerization of the subunits. Because Satoh et al. [68] observ ed that the apple enzyme produced by transformed E. coli was present as a dimer whereas the enzyme extracted from apple tissues appears to be a monomer, it is still not clear whether the native enzyme of apple is present in a dimeric or monomeric form. A possible modification of the enzyme that might prevent dimerization in apple tissue, but not in E. coli is worth examining. 

Manufacture of high-density protein arrays presents a greater challenge due to the inherent heterogeneity in the physico-chemical properties and stability of proteins. However, arrays of recombinant proteins representing all yeast open reading frames (ORFs) have been manufactured and used for identification of novel binding activities [6], Escherichia coli transformed with cDNA libraries to express mammalian proteins have been arrayed to identify autoantibodies in serum from the mouse model of systemic lupus erythematosus [7]. 

Fusarium solani [114], Arthrobacter sp. [115], Escherichia coli, transformed with a KLehsiella ozaenae plasmid DNA [116], Rhodococcus rhodochrous J1 [117,118], and Alcaligenes faecalis JM3 [119, 120]. The enzyme of Rhodococcus rhodochrous J1 was employed for the production of p-aminobenzoic acid from p-aminobenzonitrile [121], and nicotinic acid from 3-cyanopyridine [122]. The conversion of 3 anopyridine to nicotinic add, by a nitrilase of Nocardia rhodochrous IjL100-2, was also reported by Vaughan et al. [123]. These nitrilases were usually inactive on aliphatic nitriles. More recently, a new nitrilase, that acts preferentially on aliphatic nitriles, was purified and characterized in Rhodococcus rhodochrous K22 [124]. 

Ruther, A., Misawa, N., Boger, R, and Sandmann, G. (1997) Production of zeaxanthin in Escherichia coli transformed with different carotenogenic plasmids. Appl. Microbiol Biotechnol, 48, 162-167. 

Kizaki, N Yasohara, Y, Hasegawa, J Wada, M Kataoka, M and Shimizu, S. (2001) Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxyhutanoate by Escherichia coli transformant cells coexpressing the carbonyl reductase and glucose dehydrogenase genes. Appl. Mkorbiol. Biotechnol., 55, 590-595. 

In the kinetic resolution of amino acid amides with the use of amidases, such as DAP and DaaA, it is possible to synthesize D-amino acids by kinetic resolution, selectively from racemic acid amides [16]. An Escherichia coli transformant highly expressing DAP catalyzed the synthesis of 2.5M (about 220g/l) D-alanine from 5M racemic alanine amide in a 4.5-h reaction, d-2-Amino butyric acid, D-methionine, D-norvaline, and D-norleucine were S5mthesized in a similar manner. We have been successful in the evolution of DAP [17] and DaaA by mutations [18]. 

Shinoda, S., Matsuoka, H., Tsuchie, T., Miyoshi, S. I., Yamamoto, S., Taniguchi, H. and Mizuguchi, Y, 1991, Purification and characterization of a lecithin-dependent haemolysin from Escherichia coli transformed by a Vibrio parahemolyticus gene. J. Gen. Microbiol. 137 2705-2711. 

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