Production of heterologous protein by Methylobacterium extorquens in high cell density fermentation.
The green fluorescent protein (GFP) was used as a model protein to study the recombinant protein production by the strain Methylobacterium extorquens ATCC 55366. Scale-up from shake flasks to 20 l fed-batch fermentation was achieved using methanol as a sole carbon and energy source and a completely minimal culture medium. Two different expression vectors were used to express GFP. Clone PCM-GFP containing the vector pCM110 with native promoter of the methanol dehydrogenase PmxaF produced approximately 100-fold more GFP than the clone PRK-GFP containing the vector pRK310 with the heterogeneous promoter Plac. Several fed-batch fermentations with and without selective pressure (tetracycline) were run in a 20 l stirred tank fermenter using the two different clones of M. extorquens. The methanol concentration was monitored with an on-line semiconductor gas sensor in the culture broth. It was maintained at a non-toxic level of 1.4 g l(-1) with an adaptative control which regulates the methanol feed rate. The same growth profile was achieved in all fermentations. The maximum growth rate (micro(max)) was 0.18 h(-1) with an overall yield (Y(X/S)) of 0.3 g g(-1) methanol. With this high cell density fermentation process, we obtained high levels (up to 4 g l(-1)) of GFP with the clone PCM-GFP. The maximum specific GFP production (Y(GFP/X)) with this clone was 80 mg g(-1) representing approximately 16% of the total cell protein. Additional feeding of pure oxygen to the fermenter permitted a longer phase of exponential growth but had no effect on the total yields of biomass and GFP. The specific GFP production of clone PCM-GFP remained unaffected in the presence or absence of selective pressure (tetracycline), within the initial 50 h of the fermentation culture. These results suggest that M. extorquens ATCC 55366 could be an interesting candidate for overexpression of recombinant proteins.
Bélanger
L
L
Department of Natural Resource Sciences, Microbiology Unit, McGill University, 21111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9.
Figueira
M M
MM
Bourque
D
D
Morel
L
L
Béland
M
M
Laramée
L
L
Groleau
D
D
Míguez
C B
CB
D001426:Bacterial Proteins / Q000235:genetics*; D015169:Colony Count, Microbial; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D020601:Methylobacterium extorquens / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D008828:Microbiological Techniques; D010100:Oxygen / Q000494:pharmacology; D010957:Plasmids
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D010100:Oxygen