Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large‐scale processes

Abstract

We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.

DNA plasmid production in different host strains of Escherichia coli

“…Since signiWcant diVerences in supercoiling were observed between the strains, the fraction of supercoiled DNA appears also to be determined by the strain. This conclusion using a fed-batch process at two controlled growth rates is consistent with recent batch results using complex media, in which a wide range of percentage supercoiling was observed among diVerent strains [52].…”

“…Our results show that small-scale shake Xask experiments can aide in strain selection for DNA production, even though they operate exclusively at the maximum growth rate. Another recent study similarly compared 17 strains and three plasmids in shake Xasks using complex medium instead of deWned medium [52]. In this previous study for a 5.6 kb plasmid, MG1655 and BL21 yielded the most DNA at the small scale, but larger scale studies were not completed.…”

“…Studies have generally focused on only a very few strains such as DH5 [26,30] or DH5 [37]. Recently, a comprehensive study of 17 diVerent E. coli strains demonstrated signiWcant diVerences in DNA yield and supercoiling, although for this study complex medium was used in shake Xasks [52]. Currently, supercoiled DNA is thought to be the most eVective form at transferring gene expression [33], and the US FDA recommends a minimum speciWcation for supercoiled plasmid content (of greater than 80%) be established [47].…”

Deletion formation mutations in plasmid expression vectors are unfavored by runaway amplification conditions and differentially selected under kanamycin stress

“…Altered pMB1 copy number mutants arising by changes in the RNAI gene dosage, were also described (Moser and Campbell, 1983). Recently, a runaway-type replication as high as 2000 plasmid copies per cell was described to occur in pUC-based plasmids hosted in E. coli HB101, MG1655 and TG1 (Yau et al, 2008). In our case, the high amplification level of pCIneo at 42 • C might be related to the presence of a G → A transition frequently associated with temperature sensitive plasmid amplification (Minton et al, 1988;Lin-Chao et al, 1992) (Fig.…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…These observations are also found for BL21 host strains. In an extensive shake flask evaluation of plasmid specific yield and % supercoiling in 17 hosts with three different pUC origins, BL21 (DE3) was the poor producer due to the RecA+ gene product (Yau et al 2008). In contrast, Phue and co-workers reported high yields (1.9 g/L) of predominantly supercoiled plasmid in a 30ºC to 42ºC inducible process (Phue et al 2008).…”