Heterogeneous oxygen availability affects the titer and topology but not the fidelity of plasmid DNA produced by Escherichia coli

Abstract

BackgroundDissolved oxygen tension (DOT) is hardly constant and homogenously distributed in a bioreactor, which can have a negative impact in the metabolism and product synthesis. However, the effects of DOT on plasmid DNA (pDNA) production and quality have not been thoroughly investigated. In the present study, the effects of aerobic (DOT ≥30% air sat.), microaerobic (constant DOT = 3% air sat.) and oscillatory DOT (from 0 to 100% air sat.) conditions on pDNA production, quality and host performance were characterized.ResultsMicroaerobic conditions had little effect on pDNA production, supercoiled fraction and sequence fidelity. By contrast, oscillatory DOT caused a 22% decrease in pDNA production compared with aerobic cultures. Although in aerobic cultures the pDNA supercoiled fraction was 98%, it decreased to 80% under heterogeneous DOT conditions. The different oxygen availabilities had no effect on the fidelity of the produced pDNA. The estimated metabolic fluxes indicated substantial differences at the level of the pentose phosphate pathway and TCA cycle under different conditions. Cyclic changes in fermentative pathway fluxes, as well as fast shifts in the fluxes through cytochromes, were also estimated. Model-based genetic modifications that can potentially improve the process performance are suggested.ConclusionsDOT heterogeneities strongly affected cell performance, pDNA production and topology. This should be considered when operating or scaling-up a bioreactor with deficient mixing. Constant microaerobic conditions affected the bacterial metabolism but not the amount or quality of pDNA. Therefore, pDNA production in microaerobic cultures may be an alternative for bioreactor operation at higher oxygen transfer rates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0378-x) contains supplementary material, which is available to authorized users.

Strain engineering to reduce acetate accumulation during microaerobic growth conditions in Escherichia coli

“…These conditions resulted in a rapid increase in acetate accumulation. During the shift from aerobic to oxygen limited conditions, the TCA cycle activity decreases while the flux through the pflB gene product increases . Consistent with these results, higher levels of acetate accumulated under oxygen limited conditions when compared to aerobic conditions in bioreactor cultures …”

“…There is a steady increase in acetate accumulation during the first 8 hours of the fermentation and then acetate decreased for a brief period of time until dO 2 reaches 0%. The molecular basis for this decrease in acetate is unknown and is not consistent with previous studies where the acetate levels increased continuously from aerobic to oxygen limited conditions . During hours 13–15, the dO 2 is near zero in the presence of excess glucose.…”

“…A metabolic flux model in W3110 grown under oxygen limited conditions predicted a 5.5‐fold higher flux towards conversion of formate to CO 2 when compared to formate transport to extracellular medium . Under oxygen limited conditions, the flux towards accumulation of acetate is higher than accumulation of formate and other mixed acids in the extracellular medium . Consistent with these results, extracellular formate, succinate and lacate levels were very low compared to acetate under oxygen limited conditions .…”

“…Oxygen limited conditions have been utilized for the production of several products including reduced chemicals, polymers, and biopharmaceuticals . However, a well‐known disadvantage with oxygen limitation is that there is a higher rate of acetate accumulation when compared to aerobic conditions, which can subsequently affect biomass and product yields. Thus there is a strong driver to understand the molecular mechanisms involved in acetate accumulation and engineer biopharmaceutical production strains that prevent acetate accumulation under oxygen limitation.…”

Comprehensive analysis of metabolic sensitivity of 1,4‐butanediol producing Escherichia coli toward substrate and oxygen availability

“…Such effects can be simulated under lab scale conditions following the scale-down approach with compartmented bioreactor systems, providing frequent oscillation in the environmental conditions while parts of the culture pass the additional compartments. 22 Depending on the microbial system and the redox state of substrate and product, microbial production processes can be distinguished. Such studies provided detailed insight into microbial production of for example valinomycin in E. coli, 19 L-lysine in Corynebacterium glutamicum, 20 preproinsulin in E. coli 21 or even the efficiency of plasmid DNA production in general.…”