Abstract
BackgroundShake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom.ResultsComparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (kLa ≈ 80 h−1) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and β-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response.ConclusionsIn moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in RAM (up to the maximum) was not enough to avoid the classical oxygen limitation that happens in OM shake flasks. However, RAM presents a decrease of oxygen limitation, resulting in a favorable effect on biomass growth and volumetric rPLA2 production. While under OM a higher recombinant protein yield was obtained.
Highlights
Shake flasks are widely used during the development of bioprocesses for recombinant proteins
We report for the first time the production of a recombinant protein and culture behavior at different agitation rates in a novel mixing system for shake flasks by acoustic resonance (RAM) that reduce oxygen transfer limitations, using E. coli BL21 (DE3) producing recombinant phospholipase A2
In this work, we performed a comparison, based on the same initial kLa, between resonant acoustic mixing (RAM) and orbital mixing (OM) in terms of production of a recombinant protein in E. coli (7.5g and 200 rpm in 250-mL conventional Erlenmeyer glass flasks with 20% of filling volume)
Summary
Shake flasks are widely used during the development of bioprocesses for recombinant proteins. As for oxygen transfer, in shake flasks, only “one big bubble” transfers oxygen to the liquid, whereas in other systems (like stirred tank bioreactors), small bubbles enhance the air–liquid transfer area and the oxygen transfer rate, favoring the delivery of oxygen to the microorganisms [2, 5,6,7] These mass transfer variations have a relevant impact on the growth of Escherichia coli because oxygen participates as a nutrient during aerobic growth, with the main function to act as the final electron acceptor of the respiratory chain, through which the energy required for cell growth and maintenance is generated [8].
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