A. LAMBERT, BRI L. MOREL, BRI M. BÉLAND, BRI N. LASNIER, BRI L. BÉLANGER, BRI C.B. MIGUEZ., BRI
Title
Development of a high-cell density recombinant protein expression system with Methylobacterium extorquensfed-batch fermentation
Abstract Text
Methylobacterium extorquensATCC 55366 was cultivated in a fed-batch fermentation mode using methanol as a sole carbon and energy source and a completely minimal culture medium. Green fluorescent protein (GFP) was used as a model for the production of heterologous protein. An adaptative control process was developed to regulate the methanol feed rate according to the cell growth rate with the objective of maintaining the methanol concentration in the fermenter at a non-toxic level. The methanol concentration was monitored with an on-line semiconductor gas sensor in the culture broth while the cell growth rate was estimated with the oxygen consumption rate from the gas analyzer. Several fed-batch fermentations were run in a 20-L stirred tank fermenter (V init=10 L ) using different clones of M. extorquens. The same growth profile was achieved in all fermentations demonstrating the robustness of the process. The maximum growth rate (µmax) was 0.18 h –1 with an overall yield (YXS) of 0.3 g cell dry weight (CDW) /g methanol. Using two different minimal media, we were able to reach a cell biomass of 60 g (CDW)/L after 60-70 h of fermentation without oxygen enrichment.
Two different expression vectors with two different promoters were used to express GFP. The clone PmxaF-GFP-pCM110 containing the vector pCM110 with native methanol dehydrogenase promoter (P mxaF) produced approximately 60 fold more GFP than the clone Plac-GFP-pRK310 containing the vector pRK310 with the heterogeneous promoter Plac. Using the described high-cell density fermentation process, we obtained high levels (up to 4 g/L) of GFP with the clone PmxaF-GFP-pCM110. The specific GFP production with this clone was 70-80 mg GFP/ g CDW. Due to the presence of methanol as a carbon source, the promoter PmxaF was induced and expression of GFP occurred continuously throughout the fermentation. However, this situation did not seem to affect the yield of production of the cell biomass and/or the recombinant protein GFP. These results suggest that the M. extorquensATCC 55366 could be an interesting candidate for overexpression of recombinant proteins at the industrial level.
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