4.7 GenomeeReducing Efforts and the Immact of Streamlining 674.7.3 Complex Streamlining Efforts Based on Growth Properties
In a study focusing on cell growth in minimal medium, long, scarless deletions of
E. coli W3110 were constructed (Figure 4.4) [47]. The long-term goal of the work
is to create streamlined-genome strains, which are suitable platforms for meta-
bolic engineering. To identify deletable chromosomal segments, the genome
sequences of E. coli K-12 MG1655 and Buchnera sp. APS were used for compara-
tive genomics, and genes unique for E. coli were selected. Essential genes
reported in the PEC database (http://www.shigen.nig.ac.jp/ecoli/pec/index.jsp)
were excluded from the deletion list. The annotations of the remaining genes were
surveyed in databases to judge their importance for efficient growth in M9 mini-
mal medium, and regions with more than 10 continuous deletable genes were
chosen for deletion. Genomic deletions were made by using λ-Red-mediated
recombination and the negative selection marker sacB. Individual deletion strains
were checked for growth in M9 minimal medium, and only the well-growing con-
structs were chosen for further use. By combining the individual deletions, a top-
performer strain (designated minimum genome factory 01 (MGF-01)) with 1 Mb
total genome reduction was obtained. MGF-01 grew as fast as the parental W3110
strain and reached higher optical density and higher number of colony-forming
units (CFUs) in stationary phase in minimal medium. This higher- density growth
property emerged by superpositioning the individual deletions and might be
caused by the lower level accumulation of growth-inhibiting acetate, presumably
due to the elevated expression of glyoxylate shunt-related genes aceA and aceB.
This more efficient metabolism could also be the reason MGF-01 with an
l- threonine-producing unit integrated into the genome produced 2.4-fold higher
amount of l-threonine than the parental strain carrying the same unit.
The genome of MGF-01 was further reduced via step-by-step accumulation of
additional deletions made in W3110 (Figure 4.4) [98]. Noncore regions were
chosen for deletions. Starting with 37 individual deletions, strains with normal
phenotypes were selected, and 10 of them were added to MGF-01 in subsequent
cycles, generating MGF-02. Analysis of the growth phenotype of MGF-02
revealed that deletion of gcvA encoding a positive regulator of the glycine cleav-
age system enhanced initial growth in minimal medium. To further optimize the
strain, two intrinsic mutations of parental MG1655, ilvG and rph-1 (causing
valine sensitivity and partial pyrimidine starvation, respectively), were fixed both
in MGF-01 and MGF-02, creating DGF-362 and DGF-348, respectively. Starting
from DGF-348, further deletions were added by keeping only those without
growth-reducing synergistic effects. The proVXW carrying region, deleted ini-
tially, was reintroduced into the genome to fix sensitivity to high osmolarity.
Eventually, the strain with the smallest genome (DGF-298) possessed a 2.98 Mb
chromosome and was free from all IS elements. DGF-298 grew better in M9
minimal medium than parental W3110 and also had higher cell yield in a simple
medium (CSL) in fermentation. Transcriptome analysis showed that a heat-
shock chaperone (IbpAB) and a protease for abnormal proteins (Lon) are down-
regulated in DGF strains. The authors concluded that downregulation of the
genes encoding chaperones and proteases is one of the factors that improve the
fitness of DGF strains.