A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha.
Ralstonia eutropha (formerly Alcaligenes eutrophus) is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and molecular engineering of this organism. We report here a versatile gene expression method for the genetic engineering of R. eutropha. This method, based on a simplified electroporation protocol, uses a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). Using this method, we successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB(-)4, with various pBS29-P2-based recombinants. A transformation frequency as high as 4x10(3) Km-resistance colonies/mug DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor alpha-galactosidase in R. eutropha.
Solaiman
Daniel K Y
DK
Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, PA 19038, USA. dan.solaiman@ars.usda.gov
Swingle
Bryan M
BM
Ashby
Richard D
RD
D001704:Biopolymers / Q000096:biosynthesis; D001709:Biotechnology / Q000379:methods*; D020118:Cupriavidus necator / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D018274:Electroporation; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D044224:Pseudomonas syringae / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000235:genetics; D014169:Transformation, Bacterial; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism
D001704:Biopolymers; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000520:alpha-Glucosidases