RESUMO
Miniature bioreactors under parallel fed-batch operations are not only useful screening tools for bioprocess development but also provide a suitable basis for eventual scale-up. In this study, three feeding strategies were investigated: besides the established intermittent feeding by a liquid handler, an optimized microfluidic device and a new enzymatic release system were applied for parallel fed-batch cultivation of Escherichia coli HMS174(DE3) and BL21(DE3) strains in stirred-tank bioreactors on a 10 mL scale. Lower fluctuation in dissolved oxygen (DO) and higher optical densities were measured in fed-batch processes applying the microfluidic device or the enzymatic glucose/fructose release system (conversion of intermittently added sucrose by an invertase), but no difference in dry cell weights (DCW) were observed. With all three feeding strategies high cell densities were realized on a milliliter scale with final optical density measured at 600 nm (OD600 ) of 114-133 and final DCW concentrations of 69-70 g L(-1) . The effect of feeding strategies on the expression of two heterologous proteins was investigated. Whereas no impact was observed on the expression of the spider silk protein eADF4(C16), the fluorescence of enhanced green fluorescence protein (eGFP) was reproducibly lower, if an intermittent glucose feed was applied. Thus, the impact of feeding strategy on expression is strongly dependent on the E. coli strain and/or expressed protein. As a completely continuous feed supply is difficult to realize in miniature bioreactors, the enzymatic release approach from this study can be easily applied in all microfluidic system to reduce fluctuations of glucose supply and DO concentrations.
Assuntos
Reatores Biológicos/microbiologia , Biotecnologia/métodos , Técnicas de Cultura de Células/métodos , Técnicas Analíticas Microfluídicas/métodos , Proteínas Recombinantes , Animais , Contagem de Células , Escherichia coli/metabolismo , Fibroínas/análise , Fibroínas/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismoRESUMO
This study presents a comparative reaction engineering analysis of metabolically engineered sucrose-utilizing Escherichia coli derived from E. coli K12 MG1655 for the anaerobic production of succinic acid. Production capacities of 16 different recombinant strains were evaluated in 48 parallel fed-batch-operated milliliter-scale stirred tank bioreactors (10 mL) with continuous CO2 sparging. The effects of recombinant sucrose-utilization systems (csc-operon or scr-operon), enhancements of anaplerotic reactions (pck, ppc, maeA, maeB or heterologous pyc) and gene deletions (ldhA, adhE, ack-pta and ptsG) were studied with respect to the overall process performances of the respective recombinant strains. Both sucrose-utilization systems enabled the production of succinic acid from sucrose in E. coli K12 MG1655. Maximum succinate production was observed by overexpressing the pyruvate carboxylase from Corynebacterium glutamicum resulting in a succinate concentration of 26.8 g L⻹ after 48 h and a cell-specific productivity of 0.14 g g⻹ h⻹. Further experiments in a fed-batch-operated laboratory-scale stirred tank bioreactor (2 L) showed that micro-aerobic conditions preceding the anaerobic phase enhance succinic acid production of E. coli K12 MG1655-derived strains. The work demonstrates the importance of parallel approaches within the scope of applied metabolic engineering studies.