58 4 Rational Efforts to Streamline the Escherichia coli Genome
they can be a nuisance in synthetic biology constructions. Deleting them elimi-
nates barriers to genome engineering procedures that involve the introduction of
genetic material into a heterologous host.4.5.2.4 Genes of Unknown and Exotic Functions
A significant portion of the genome codes for genes with unknown function
(~20%) [31]. Not excluding the possibility of discovering new and important
functions, deletion of these genes might be attempted with high confidence.
Similarly, metabolic and transport genes associated with substrates not com-
monly used are primary targets for removal. It might be intuitively argued that
metabolic genes not needed under a particular condition are not expressed;
hence deletion of them provides little gain in the economical use of resources.
However, in fact, it was shown that, under conditions of declining carbon source
quality, cells switch into a scavenging mode and express a variety of transport
and metabolic genes to prepare for any substrate availability [62]. Thus, even if
actually not used, exotic transport and metabolic genes can pose a metabolic
burden on the cell.4.5.2.5 Repeat Sequences
The largest repeat sequences of E. coli, rearrangement hot spot (Rhs) elements,
are about 8 kb in length on average and collectively constitute about 1% of the
genome [18]. Although widespread in E. coli strains, their function is poorly
understood [63]. Rhs elements carry dispensable genes responsible for polysac-
charide synthesis and export and for genes with unknown functions and might
promote RecA-dependent rearrangements of the chromosome and are thus
undesired for synthetic biology applications.4.5.2.6 Virulence Factors and Surface Structures
The commonly used E. coli K-12 MG1655 strain is non-pathogenic due to the
lack of a type-III secretion system and haemolysin expression, in addition to an
impaired O-antigen synthesis [18]. Nevertheless, the strain harbors a number
of virulence‐associated factors, like flagella, fimbriae, siderophores and a
cryptic haemolysin. There is a theoretical chance that safe strains acquire
mutations or horizontally transferred additional virulence factors that
transform them into a pathogen. It is a cause for concern that a double point
mutation change in the gene coding for histon-like protein HUα can turn K-12
into an invasive strain [64]. Deletion of the genes associated with virulence
therefore makes the cells safer. Elimination of surface structures might bring
about other gains as well. For instance, the flagellar apparatus, not needed in a
fermentor, consumes an estimated 1–2% of the total cellular energy. In addi-
tion, flagella break off and regrow constantly, and these proteins, shed in the
environment, constitute a net loss for the cell [65]. Deletion of flagellar and
chemotaxis gene clusters might thus result in energy savings. Elimination of
other surface structures (fimbriae, curli, lipopolysaccharide outer core, colanic
acid capsule) could further improve the cellular economy and also reduce the
propensity of the cell for biofilm formation [66].