A comprehensive study of glucose and oxygen gradients in a scaled-down model of recombinant HuGM-CSF production in thermoinduced <i>Escherichia coli</i> fed-batch cultures

The effect of gradients of elevated glucose and low dissolved oxygen in the addition zone of fed-batch E. coli thermoinduced recombinant high cell density cultures can be evaluated through two-compartment scale-down models. Here, glucose was fed in the inlet of a plug flow bioreactor (PFB) connected to a stirred tank bioreactor (STB). E. coli cells diminished growth from 48.2 ± 2.2 g/L in the stage of RP production if compared to control (STB) with STB-PFB experiments, when residence time inside the PFB was 25 s (34.1 ± 3.5 g/L) and 40 s (25.6 ± 5.1 g/L), respectively. The recombinant granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) production decreased from 34 ± 7% of RP in inclusion bodies (IB) in control cultures to 21 ± 8%, and 7 ± 4% during the thermoinduction production phase when increasing residence time inside the PFB to 25 s and 40 s, respectively. This, along with the accumulation of acetic and formic acid (up to 4 g/L), indicates metabolic redirection of central carbon routes through metabolic flow and mixed acid fermentation. Special care must be taken when producing a recombinant protein in heat-induced E. coli, because the yield and productivity of the protein decreases as the size of the bioreactors increases, especially if they are carried at high cell density. Thermoinduced recombinant E. coli grew less in a two-compartment scale-down model.Heat-inducible E. coli cultures at a large scale significantly decrease recombinant protein production.The accumulation of acetic and formic acid increases when E. coli is exposed to glucose and oxygen gradients.The axial flow pattern inside the PFB mimics glucose and dissolved oxygen gradients at the industrial scale. Thermoinduced recombinant E. coli grew less in a two-compartment scale-down model. Heat-inducible E. coli cultures at a large scale significantly decrease recombinant protein production. The accumulation of acetic and formic acid increases when E. coli is exposed to glucose and oxygen gradients. The axial flow pattern inside the PFB mimics glucose and dissolved oxygen gradients at the industrial scale.</p

The metabolic switch can be activated in a recombinant strain of Streptomyces lividans by a low oxygen transfer rate in shake flasks

Abstract Background In Streptomyces, understanding the switch from primary to secondary metabolism is important for maximizing the production of secondary metabolites such as antibiotics, as well as for optimizing recombinant glycoprotein production. Differences in Streptomyces lividans bacterial aggregation as well as recombinant glycoprotein production and O-mannosylation have been reported due to modifications in the shake flask design. We hypothetized that such differences are related to the metabolic switch that occurs under oxygen-limiting conditions in the cultures. Results Shake flask design was found to affect undecylprodigiosin (RED, a marker of secondary metabolism) production; the RED yield was 12 and 385 times greater in conventional normal Erlenmeyer flasks (NF) than in baffled flasks (BF) and coiled flasks (CF), respectively. In addition, oxygen transfer rates (OTR) and carbon dioxide transfer rates were almost 15 times greater in cultures in CF and BF as compared with those in NF. Based on these data, we obtained respiration quotients (RQ) consistent with aerobic metabolism for CF and BF, but an RQ suggestive of anaerobic metabolism for NF. Conclusion Although the metabolic switch is usually related to limitations in phosphate and nitrogen in Streptomyces sp., our results reveal that it can also be activated by low OTR, dramatically affecting recombinant glycoprotein production and O-mannosylation and increasing RED synthesis in the process