Enhancing microaerobic plasmid DNA production by chromosomal expression of Vitreoscilla hemoglobin in E. coli

Abstract

• VHb reduced overflow metabolism. • VHb improved pDNA production under oxygen limitations. • VHb improved pDNA yields even at lower RNAII/RNAI ratios. • Higher lactate production may be caused by stronger NAD + demand. Oxygen availability and overflow metabolism are often limiting factors in high cell-density cultures. In this study, Vitreoscilla hemoglobin (VHb) was expressed in the chromosome of E. coli as a strategy to improve plasmid DNA (pDNA) production in biphasic fed-batch cultures. During the aerobic batch phase, the strain expressing VHb accumulated 28 % less acetate and 19 % more pDNA than the wild type strain. The culture was shifted from aerobic to microaerobic conditions during the fed-batch phase. pDNA yields increased consistently in the VHb-expressing strain during the whole culture, while decreased progressively for the wild type under microaerobic conditions. Unexpectedly, the ratio of positive/negative replication control molecules (RNAII/RNAI) were lower for the VHb-expressing than for the wild type strain. However, the final pDNA titer was ca. 74 % higher for the former. Flux balance analysis suggests that VHb presence increases the fluxes in anaplerotic pathways. The higher lactate production observed in VHb-expressing cells may be triggered by an increased demand of NAD + in glycolysis under microaerobic conditions.