70 4 Rational Efforts to Streamline the Escherichia coli Genome
4.8.4 Codon Reassignment
Incorporation of unnatural amino acids (uaas) in proteins in living cells would
enable evolution of novel protein functions [109]. Relatively rarely used stop
codons, coupled with orthogonal tRNA/synthetase pairs, can be exploited to
genetically introduce uaas. A major limitation of using a stop codon to encode
uaas is the low efficiency of incorporation due to competition of the suppressor
tRNA with endogenous release factors (RF1 and RF2 in prokaryotes). UAG is the
least used stop codon in E. coli (present in 7% of the genes) and is recognized by
RF1, but not by RF2. To achieve full reassignment of UAG, the reportedly essen-
tial RF1 must be removed from the system. It was shown that, after modifying
the activity of RF2, the gene encoding RF1 (prfA) can be deleted from the E. coli
genome. MDS42 was used as parental strain, because the deletion of nearly 700
genes may alleviate the termination load imposed on RF2. Besides the demon-
strated successes for multisite incorporations of uaas for protein research and
laboratory evolution, the RF1 knockout strains can also be valuable for investi-
gating the evolution of the genetic code [110].
Due to the degenerate nature of the genetic code, reassigning sense codons to
encode uaas is also conceivable, once the specific codons are successfully elimi-
nated from the genome. In a proof-of-concept work, the synonymous re-coding
of certain Ser, Leu, or Ala codons was attempted in a 20 kbp-long essential operon
of E. coli MDS42 [16]. Eight different re-coding schemes were tested, some of
which resulted in the exchange of 373 codons in a single step. Measuring the
efficiency of various codon exchanges permitted the definition of allowed and
disallowed synonymous re-coding schemes to be applied in future codon reas-
signment projects.
A similar project, on the long run, aimed at the re-coding of the complete
E. coli MDS42 genome to eliminate all 62 214 instances of seven different codons
[111]. In this endeavor, re-coding would take place by the stepwise exchange of
50 kbp-long segments of the chromosome. Testing the complementing ability of
the synthetic recoded DNA segments one by one, 99.5% of the recoded genes
were found to complement their wild-type counterparts without the need of fur-
ther optimization. The use of the MDS42 strain in such re-coding enterprises
warrants reduced synthesis costs and improved genome stability.4.8.5 Genome Architecture
As reduced-genome bacteria have altered positions and perturbed local context
of certain chromosomal segments, these strains could be useful for studying
genome architectural effects. A comparative protein occupancy profile of
MG1655 and MDS42 was analyzed using microarray-based chromatin immuno-
precipitation [112]. This work identified both highly transcribed and transcrip-
tionally silent extended protein occupancy domains, hiEPODs and tsEPODs,
respectively. It was suggested that the binding of tsEPODs by nucleoid proteins
(HU, Fis, H-NS, and IHF) establishes them as chromosomal organizing centers.
MDS42 lacks a large fraction of tsEPODs, but the remaining ones are similarly
located as in parental MG1655, supporting a dynamic role of the organizing
centers in the formation of a higher-order chromosome structure.