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 P_mxaF produced approximately 100-fold more GFP than the clone PRK-GFP containing the vector pRK310 with the heterogeneous promoter P_lac. 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 (μ^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. [Copyright &y& Elsevier]