Overexpression of a heterologous protein, haloalkane dehalogenase, in a poly-beta-hydroxybutyrate-deficient strain of the facultative methylotroph Methylobacterium extorquens AM1.
Using an expression vector containing p(mxaF'), a strong native promoter, expression of a model heterologous protein, haloalkane dehalogenase, from Xanthobacter autotrophicus GJ10 was achieved in the methylotrophic bacterium, Methylobacterium extorquens AM1. Although expression using the wild-type strain was <5% of total cell protein, expression at a level of 10% of the total cell protein was achieved in a mutant unable to synthesize poly-beta-hydroxybutyrate granules. Two other tested heterologous proteins, catechol dioxygenase and green fluorescent protein, were expressed at moderate levels in both wild-type and the PHB-negative strain. These results suggest that the M. extorquens PHB-negative strain is a possible platform for overexpression of heterologous proteins with labeled or unlabeled methanol as a starting material.
FitzGerald
Kelly A
KA
Departments of Chemical Engineering, University of Washington, Box 351750, Seattle, Washington 98195-1750, USA.
Lidstrom
Mary E
ME
D050548:Catechol 1,2-Dioxygenase; D002478:Cells, Cultured; D003001:Cloning, Molecular; D049308:Dioxygenases; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins; D006867:Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006885:Hydroxybutyrates / Q000378:metabolism*; D008164:Luminescent Proteins / Q000096:biosynthesis; D000432:Methanol / Q000378:metabolism; D020580:Methylobacterium / Q000235:genetics* / Q000378:metabolism*; D010105:Oxygenases / Q000096:biosynthesis; D011091:Polyesters / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012016:Reference Values; D013045:Species Specificity; D000094122:Polyhydroxybutyrates
D050548:Catechol 1,2-Dioxygenase; D049308:Dioxygenases; D049452:Green Fluorescent Proteins; D006867:Hydrolases; D006885:Hydroxybutyrates; D008164:Luminescent Proteins; D000432:Methanol; D010105:Oxygenases; D011091:Polyesters; D011994:Recombinant Proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C046137:haloalkane dehalogenase