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.
Department of Natural Resource Sciences, Microbiology Unit, McGill University, 21111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9.
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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