Title

Effect of the oxygen transfer rate on oxygen-limited production of plasmid DNA by Escherichia coli

Conference Dates

March 4-8, 2018

Abstract

Oxygen limitation can increase the pDNA yield in cultures of Escherichia coli. Nevertheless, such effect has not been studied systematically. Namely, only cultures at low DOT have been performed, excluding important factors like the oxygen transfer rate (OTR). Moreover, to the best of our knowledge, there is no information regarding the impact of oxygen availability on the topology of the plasmid. The supercoiling of DNA requires energy and it is hypothesized that oxygen availability will affect the produced isoforms. In the present study, we performed fully aerobic and oxygen-limited cultures of E. coli bearing a high copy number plasmid. Cultures at OTRmax values of 10, 14, 30, 45 (for oxygen-limited cultures) and 110 mmol L-1 h-1 (for aerobic cultures) were performed in microtiter plates with DOT, pH, biomass (measured as scattered light) and NADH fluorescence online monitoring. To further investigate the impact of oxygen limitation on pDNA topology, an E. coli strain constitutively expressing the Vitreoscilla hemoglobin (VHb) was used. VHb is known to improve aerobic respiration and consequently ATP generation at low oxygen availability. Our results show that the pDNA yields on biomass (YpDNA/X) were inversely proportional to the OTRmax for both strains, and increased more than two-fold in cultures at the lowest OTRmax, compared to aerobic cultures. Expression of VHb resulted in lower YpDNA/X, compared to cultures of the parent strain. The strain expressing the VHb displayed higher specific growth rates at OTRmax of 10, 14 and 30 mmol L-1 h-1, compared to the parent strain. However, at OTRmax of 45 and 110 mmol L-1 h-1, the growth rate of the parent strain was higher. In general, the specific NADH fluorescence was lower in cultures of the engineered strain, which can be associated to a more oxidized intracellular state, in agreement with the proposed effect of VHb on the cellular metabolism. The pDNA supercoiled fraction (SCF) was maximum in cultures at OTRmax of 30 mmol L-1 h-1, reaching 92.9 % for the wild type strain and 98.7 % for the strain expressing VHb, while no linearized pDNA was detected. This condition was replicated in a 1 L stirred tank bioreactor (STB) for W3110 recA-, due to the higher productivity of this strain. The performance of cultures in the STB was very similar to that of cultures in the MTP concerning accumulated fermentative by-products, cell growth and pDNA production and SCF.

Altogether, these results show the existence of an optimal OTRmax for oxygen-limited production of plasmid DNA. Furthermore, we demonstrate that studies in microtiter plates are excellent to predict culture performance of STB and to scale-up plasmid DNA production cultures.

This document is currently not available here.

Share

COinS