High yield plasmid DNA production under oxygen limitation using microaerobically induced replication

With the aim of increasing plasmid DNA (pDNA) production under oxygen limitation, a self-inducible replication system was created. An extra copy of the gene coding for rnaII, which is a positive control molecule for pMB1-derived replicons, was placed under control of the lac or trp promoters and cloned in plasmid pUC18. The modified plasmid pUC18-Ptrc rnaII resulted in a strong overexpression of rnaII which in turn triggered the plasmid copy number in more than the double of that of pUC18. Based on this, a microaerobically-inducible plasmid was created by inserting an extra copy of rnaII under control of the microaerobic promoter from the Vitreoscilla hemoglobin (Pvgb). Such plasmid was tested in fed-batch cultures of the strain W3110 recA- in which dissolved oxygen was depleted for nearly 6 h. Upon oxygen depletion, rnaII was efficiently induced and pDNA titer increased steadily for pUC18- Pvgb rnaII, reaching nearly 400 mg/L. In contrast, only 200 mg/L of the unmodified pUC18 were obtained. In order to improve cellular performance under oxygen limitations, engineered strains expressing the Vitreoscilla hemoglobin encoded in the chromosome, were created. The vgb gene was inserted in BL21 and W3110 strains and the performance of both strains were compared in biphasic aerobic-oxygen limited cultures. Interesting differences were observed in the kinetic behavior, metabolic fluxes distribution and gene expression levels when the vgb gene was expressed in BL21 or W3110 recA- vgb+, therefore, this strain was used for production of the inducible plasmid. The amount of pUC18 produced by W3110 recA- vgb+ under oxygen limitation doubled that of W3110 recA-. However, when pUC18-Pvgb rnaII was used, the engineered strain produced only 20 mg/L. Moreover, the size of the obtained plasmid was strongly shortened. Plasmid sequencing revealed that an important fraction of the origin of replication was lost. These results demonstrate the feasibility of microaerobically-induced pDNA production, and that the performance of genetic constructions depend on the strain used. Furthermore, unexpected changes in plasmid fidelity can arise when using genetically modified strains

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

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

Engineering E. coli for improved microaerobic pDNA production.

peer reviewedEscherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA(-), which, together with W3110recA(-)vgb(+), was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production

Evaluation of microbial globin promoters for oxygen-limited processes using Escherichia coli

Abstract Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTRmax) of 7 and 11 mmol L−1 h−1. Different FbFP fluorescence intensities were observed and the OTRmax affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor, the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli

Characterization of Endogenous and Reduced Promoters for Oxygen-Limited Processes Using Escherichia coli

Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the <i>Vitreoscilla</i> hemoglobin promoter (P_<i>vgb</i>) and a set of microaerobic endogenous promoters in <i>Escherichia coli</i>. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a nonoxygen dependent marker protein, was used as a reporter. Fluorescence and fluorescence emission rates under oxygen-limited conditions were the highest when FbFP was under transcriptional control of P_<i>adhE</i>, P_<i>pfl</i> and P_<i>vgb</i>. The lengths of the <i>E. coli</i> endogenous promoters were shortened by 60%, maintaining their key regulatory elements. This resulted in improved promoter activity in most cases, particularly for P_<i>adhE</i>, P_<i>pfl</i> and P_<i>narK</i>. Selected promoters were also evaluated using an engineered <i>E. coli</i> strain expressing <i>Vitreoscilla</i> hemoglobin (VHb). The presence of the VHb resulted in a better repression using these promoters under aerobic conditions, and increased the specific growth and fluorescence emission rates under oxygen-limited conditions. These results are useful for the selection of promoters for specific applications and for the design of modified artificial promoters