Abstract
The gene from Bacillus licheniformis coding for a thermostable α-amylase was subcloned into the broad-host-range plasmid pKT210 in Escherichia coli. The recombinant plasmid pGNB6 was transferred into Zymomonas mobilis ATCC 31821 by conjugation. Plasmid pGNB6 was stably maintained in E. coli and unstable in Z. mobilis. The amylase gene was expressed in Z. mobilis at a lower level (25%) than in E. coli and regulation of enzyme biosynthesis was different in the host cells. Almost all the α-amylase activity was recovered in the culture medium of Z. mobilis. This enzyme localization seemed to be the result of protein secretion rather than cell lysis. Integration of the amylase gene into a cryptic plasmid of Z. mobilis was observed. The amylase gene was still expressed, although at a lower level, and the α-amylase activity, associated with a protein of molecular mass 62,000 daltons, was immunologically identical in Z. mobilis, E. coli and B. licheniformis.
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Bagdasarian M, Lurz R, Ruckert B, Franklin FCH, Bagdasarian MM, Frey J, Timmis KN (1981) Specific-purpose plasmid cloning vectors II. Broad host range, high copy number RSF 1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16: 237–247
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7: 1513–1523
Brestic-Goachet N, Gunasekaran P, Cami B, Baratti J (1987) Transfer and expression of broad-host-range plasmids in Zymomonas mobilis. Biotechnol Lett 9: 13–18
Brestic-Goachet N, Gunasekaran P, Cami B, Baratti J (1989) Transfer and expression of an Erwinia chrysanthemi cellulase gene in Zymomonas mobilis. J Gen Microbiol 135: 893–902
Bringer S, Sahm H, Swyzen W (1984) Ethanol production by Zymomonas mobilis and its application on an industrial scale. Biotechnol Bioeng Symp 14: 311–319
Byun MOK, Kaper JB, Ingram LO (1986) Construction of a new vector for the expression of foreign genes in Zymomonas mobilis. J Ind Microbiol 1: 9–15
Cami B, Bregerere F, Abastado JP, Kourilsky P (1981) Multiple sequences related to classical histocompatibility antigenes in the mouse genomes. Nature 291: 763–775
Datta N, Hedges RW, Shaw EJ, Sykes RB, Richmond MH (1971) Properties of an R. factor from Pseudomonas aeruginosa. J Bacteriol 108: 1244–1249
Dostalek M, Haeggstroem MH (1983) Mixed culture of Saccharomycopsis fibuligera and Zymomonas mobilis on starch. Use of oxygen as a regulator. Eur J Appl Microbiol Biotechnol 17: 269–274
Favela-Torres E, Baratti J (1988) Ethanol production from wheat flour by Zymomonas mobilis. J Ferm Technol 66: 167–172
Filloux A, Joyet P, Murgier M, Lazdunski A (1985) Cloning and expression of a Bacillus licheniformis α-amylase gene in Pseudomonas aeruginosa. FEMS Microbiol Lett 30: 203–207
Harboe N, Ingild A (1973) Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol 2: 161–164
Joyet P, Guerineau M, Heslot H (1984) Cloning of a thermostable α-amylase gene from Bacillus licheniformis and its expression in Escherichia coli and Bacillus subtilis. FEMS Microbiol Lett 21: 353–358
Kushner SR, Bayer HW, Nicosia S (1978) In: Genetic engineering. Elsevier North-Holland, Amsterdam, pp 17–23
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Lee JK, Pagan RJ, Rogers PL (1983) Continuous simultaneous saccharification and fermentation of starch using Zymomonas mobilis. Biotechnol Bioeng 25: 659–669
Lee GM, Kim CH, Lee KJ, Yusof ZAM, Ham MH, Rhee SK (1986) Simultaneous saccharification and ethanol fermentation of sago starch using immobilized Zymomonas mobilis. J Ferm Technol 64: 293–297
Maniatis T, Fritsh EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Preziosi L, Michel GPF, Baratti J (1990) Characterization of sucrose-hydrolyzing enzymes of Zymomonas mobilis. Arch Microbiol 153: 181–186
Saito N, Yamamoto K (1975) Regulatory factors affecting α-amylase production in Bacillus licheniformis. J Bacteriol 121: 848–856
Skotnicki ML, Tribe DE, Rogers PL (1980) R-plasmid transfer in Zymomonas mobilis. Appl Environ Microbiol 40: 7–12
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517
Willemot K, Cornelis P (1983) Growth defects of Escherichia coli cells which contain the gene of an α-amylase from Bacillus coagulans on a multicopy plasmid. J Gen Microbiol 129: 311–319
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Brestic-Goachet, N., Gunasekaran, P., Cami, B. et al. Transfer and expression of a Bacillus licheniformis α-amylase gene in Zymomonas mobilis . Arch. Microbiol. 153, 219–225 (1990). https://doi.org/10.1007/BF00249071
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DOI: https://doi.org/10.1007/BF00249071