50 4 Rational Efforts to Streamline the Escherichia coli Genome
living cells possess the intrinsic ability for physiological and genetic adaptation,
unwanted genotypic and phenotypic alterations may arise when challenged by
artificial genetic constructs [3–5].
Conveniently, recent advances in genome manipulation and synthetic DNA
construction techniques [6–11] as well as our rapidly expanding knowledge of
the wealth of genome sequences [12] make genome-scale engineering possible,
and, consequently, elimination of the disadvantageous features of the host cell
can be attempted. Rationally redesigned, streamlined, and semisynthetic cus-
tom-made genomes could then replace naturally evolved gene sets, leading to an
effective domestication of the microbial world [3, 11, 13–16].
In this chapter we will discuss the concept of the streamlined bacterial chassis,
argue that E. coli is a primary choice for a versatile host, and review the tools and
approaches of genome reduction. Next, results of E. coli genome streamlining
and selected applications of the reduced-genome strains will be presented.
Finally, future directions, gaps in our knowledge to be filled in, and perspectives
of genome streamlining will be briefly discussed.4.2 The Concept of a Streamlined Chassis
Natural cells are complex biological systems reflecting a long evolutionary
history. The intrinsic functional robustness of natural cells, due to intertwining
networks, functional redundancies, and feedback regulatory mechanisms make
them resilient to synthetic reprogramming [17]. Moreover, their genomes are
riddled with remnants of past adaptation events that may be irrelevant at present
[18]. In addition, well-defined laboratory or industrial settings can be rather dif-
ferent from complex and changing natural environments [19, 20], rendering the
existing genomic capabilities partially dispensable. Even if a number of empiri-
cally selected or purposefully introduced modifications shaped the genomes of
some widely used experimental or industrial organisms, they still are unneces-
sarily complex and heterogeneous biological systems with a vast number of com-
ponents and network interactions, largely unsuitable for precise and rational
engineering.
Developing simple cells that provide only the very basic cellular machinery for
maintaining and expressing designed constructs in a predefined range of condi-
tions would thus greatly facilitate predictable engineering. Such a biological
“chassis” could be used as a starting point to add new modules and build more
complex systems adjusted to special needs [21]. Moreover, creating a more ame-
nable and embraceable system would facilitate our understanding of general bio-
logical phenomena, such as transcriptome complexity, energy metabolism, and
robustness [22, 23]. (It should be noted that a somewhat different interpretation
of the chassis restricts it to a DNA-less cellular container, into which synthetic
genomes could be transplanted [21]. We use here the term for a self-sustaining
cellular system, complete with a simple genome.)
What are the desired features of a cellular chassis? First, it should have signifi-
cantly reduced complexity. By eliminating unnecessary components, predict-
ability of reprogramming could be enhanced. Second, the chassis should be