66 4 Rational Efforts to Streamline the Escherichia coli Genome
of a heterologous toxic protein. Versions of MDS42, rendering it less recombi-
nogenic (recA−), suitable for blue-white selection cloning (lacZΔM15), or
expressing inducible T7 polymerase are also available for common applications.
Continuing the MDS series, reduced-genome strains with up to 69 deletions
were created by removing further putatively horizontally transferred regions
[100].The final member of the series, MDS69 lost 965 genes, 20.3% of the
genome.
A novel, rapid streamlining workflow, the MDS series based on genome
shuffling and CRISPR/Cas-assisted MAGE was developed to improve the stabil-
ity of the E. coli BL21(DE3), a host frequently used for high-level recombinant
protein production [101]. All 9 resident prophages were deleted and all 50 active
IS elements were removed or inactivated. The DE3 prophage carrying an induc-
ible T7 RNA polymerase gene was exchanged with a tightly controlled T7 RNA
polymerase cassette. Additional strain variants with inactivated error-prone
DNA polymerases were also constructed. The streamlined BL21(DE3)-K-12
hybrid strains retained the favorable characteristics of BL21(DE3), displayed
increased genomic and plasmid stability, and allowed elevated electroporation
efficiencies [95].4.7.2 Genome Reduction Based on Gene Essentiality
In another attempt to reduce the genome of E. coli MG1655, a series of
medium-scale and large-scale markerless deletions were constructed using linear
targeting, DNA/λ-Red-mediated recombination, and sacB-based counterselec-
tion methods (Figure 4.4) [97]. Deletion targets were selected by excluding essen-
tial genes and maximizing the potentially deletable chromosomal segments.
First, many nonessential regions were removed from the chromosome. Next, by
combining consecutive deletions, a series of mutants were constructed, lacking
up to 29.7% (16 combined deletions) of the chromosome. Mutants with individ-
ual deletions grew like the wild-type strain. The mutants with an increasing
number of combined deletions, however, grew increasingly slower than the
parental strain in rich medium. The mutant with the largest number of deletions
(16) grew much slower than the parental strain (45.4 min vs. 26.2 min doubling
time) and showed aberrant nucleoid morphology, as well as altered cell shape and
size. It was concluded that the additive effect of large deletions can sometimes
not be predicted, but the deletion of nonessential chromosome regions may be
valuable for elucidating cellular processes governed by multiple systems.
The interspersed nature of essential genes within bacterial chromosomes
would normally require genome reduction projects to execute numerous
short deletions targeting the flanked nonessential segments. To accelerate
this process, the MEGA (see above) technique replaces long chromosomal
stretches with short DNA cassettes comprising solely the essential genes of
the targeted segment [94]. As a proof of principle, three regions ranging from
80 to 205 kbp were deleted this way in the E. coli chromosome with each tar-
get containing two to eight essential genes. The authors envisioned the step-
wise, complete replacement of the E. coli genome with the gene set essential
to sustain life.