Gábor Draskovits, Tamás Fehér , György Pósfai
Genome Engineering Group, Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
▌ Genes (innermost circle)
▌▌ Deletions
▌ K-islands
▌ Rhs-sequence
▌ IS-elements (outermost circle)
Genome reduction of E. coli K-12
Using approaches of synthetic biology, we are focusing on the rational large-scale remodeling of the genome of E. coli K-12 (Pósfai et al., 2006, Science). A cell with a streamlined, semisynthetic genome could serve as an improved model organism, and as a programmable cellular chassis for industrial applications. It was hypothesized that deletion of the mobile genetic elements and other unneeded genomic islands would result in a cell displaying lower complexity, higher genetic stability, and lower energy consumption. Lower complexity is evident, higher genetic stability has been previously demonstrated (Umenhoffer et al., 2010, Microb Cell Fact; Csörgő et al., 2012, Microb Cell Fact). Regarding cellular energetics, we hypothesized that due to the elimination of unnecessary, energy- consuming processes (e.g., lack of flagella synthesis), streamlined-genome strains use resources more economically than wild-type cells. This might be manifested in enhanced growth properties or in higher recombinant protein production. To accurately quantitate the energy consumption of the cellular machinery, we measured the maintenance energy (energy needed to maintain the cell at zero growth) requirement of the various cells in a series of controlled steady-state growth experiments in a chemostat.
Maintenance energy requirement and genetic adaptation of
streamlined-genome Escherichia coli
Escherichia coli K-12 MDS69
Deleted genes:
-Potential virulence factors -Mobile genetic elements -Unnecessary genes
-Genes of unknown function
Deletion map of MDS69, a fast-growing member of the MDS series obtained by deleting 69 genomic segments of wt MG1655.
E. coli K-12 MG1655
4 639 221 bp 4434 genes
MDS42
3 975 907 bp -14.28%
-704 genes
MDS69
3 699 230 bp -20.25%
-947genes
Genome reduction
Multiple Deletion Strains (MDS) used in this study. The number after the MDS marks the number of deletions.
MDS12
4 263 041 bp -8.1%
-423 genes
Our tool of investigation: the chemostat
Fresh medium influx
Stirring
Air efflux
- Aim: continuous growth (division) of bacterial cells
- Method: constant influx of growth medium, with carbon source (glucose) limitation
- Result: constant, reproducible, controlled cellular environment and physiological state
Dilution (D):
the proportion of the culture volume exchanged within a unit time (1h)
↓
In a chemostat, under steady- state conditions:
D = growth rate (μ) Efflux of used
medium and bacteria
Bacterial culture
Fermentor, operating in chemostat mode
Influx of sterile air
Calculating the energy requirement of growth:
q = μ/YG + m (Pirt, 1968) where
q: energy uptake rate μ: growth rate
YG: maximal biomass yield (at infinitely high growth rate)
m = maintenance energy or NGAM
(non-growth associated maintenance coefficient)
Calculating maintenance energy
0 0,1 0,2 0,3 0,4 0,5 0,6
growth rate (1/h)
glucose uptake rate mmol / (h*g)
Calculating maintenance energy (in this work)
y = 0.05 x + 0.25
0 0,2 0,4 0,6 0,8
0 1 2 3 4 5 6 7 8 9 10 11
1/D (h)
1/Yield (g/g)
Reorganized formula:
1/Y = 1/YG + m/μ
where Y: biomass yield
by plotting 1/Y vs. 1/μ = 1/D, we obtain a line with a slope of m (NGAM).
Rationale: measuring the glucose uptake rate per unit biomass at various growth rates, and extrapolation to obtain the glucose (energy) requirement at zero growth rate.
Measuring maintenance energy
Conclusions
Contrary to expectations, elimination of unnecessary, energy- consuming processes did not seem to result in significant reduction of the maintenance energy (with the possible exception of MDS12). We currently hypothesize that the potential gain was off-set by the loss of adaptation capabilities and by some structural disturbances of the chromosome, resulting in suboptimal working of the transcriptional machinery (preliminary data).
Paralleling the streamlining process, a tendency of reduced genetic adaptability was observed. Wild-type cells, displaying a certain initial maintenance energy requirement in the chemostat, quickly develop genetic changes that result in a phenotypically heterogeneous population and in a decrease of energy consumption. In contrast, the initial maintenance energy requirement of the streamlined-genome cells (especially those with more deletions) remains basically unchanged for a hundred generations, and the cells remain phenotypically and genetically uniform. Whole-genome sequencing of the adapted wild-type cells is underway to elucidate the genetic basis for this difference in the adaptation capabilities of the wild-type and streamlined- genome strains.
The slopes of the trendlines (red boxes) reflect the maintenance energy of the strains.
No significant differences were observed.
Biomass yield was measured in a glucose-limited chemostat at different dilutions (growth rates), following a 100-generation adaptation period at a dilution rate of 0.5 to the chemostat condition.
Maintenance energy requirements of MDS strains and wt E. coli, measured after an adaptation period
Maintenance energy of the wt strain, measured with and without adaptation to the chemostat
A 50-generation adaptation of the wt strain in the chemostat at a dilution rate of 0.1 results in approximately 40 % lower maintenance energy [~ 35 mg glucose/ (g dry weight * h)], compared to the original strain [~ 60 mg glucose/ (g dry weight * h)].
y = 0,0605x + 2,512
R² = 0,8939 y = 0,0374x + 2,3438
R² = 0,6292 y = 0,0337x + 2,3347 R² = 0,5176
1,6 1,8 2 2,2 2,4 2,6 2,8 3 3,2 3,4 3,6 3,8
0 1 2 3 4 5 6 7 8 9 10 11 12 13
1/ Yield (g dry biomass / g glucose)
1/Dilution rate
MG1655 unadapted MG1655 adapted
MG1655 adapted, new inoculation
0,14 0,18 0,22 0,26 0,30 0,34 0,38 0,42 0,46
0,00 20,00 40,00 60,00 80,00 100,00 120,00 140,00
Yield (dry biomass g / glucose g)
Generation
wt MG1655 (adaptation)
adapted MG1655 (re-inoculated) wt MG1655 (parallel adaptation) MDS12 (8% genome reduction) MDS42 (14% genome reduction)
Changes in biomass yield during adaptation to chemostat conditions
Wt E. coli MG1655 produced a significantly elevated biomass yield after 40-60 generations of growth at a dilution rate of 0.1. Re-inoculated wt cells continue to produce elevated biomass, indicating a genetic basis for adaptation. Spreading on a plate, wt adapted cells show phenotypically heterogeneous colonies. In contrast, the yields of MDS strains remain constant after 100 generation, and cells remain phenotypically homogeneous. MDS cells have a lower capacity for genetic adaptation.
References
1. Pósfai G, Plunkett G, III., Fehér T, Frisch D, Keil GM, Umenhoffer K, Kolisnychenko V, Stahl B, Sharma SS, de Arruda M et al:
Emergent properties of reduced-genome Escherichia coli.
Science 2006, 312(5776):1044-1046.
2. Umenhoffer K, Fehér T, Balikó G, Ayaydin F, Pósfai J, Blattner FR, Pósfai G:
Reduced evolvability of Escherichia coli MDS42, an IS-less cellular chassis for molecular and synthetic biology applications.
Microb Cell Fact 2010, 9:38.
3. Csörgő B, Fehér T, Tímár E, Blattner FR, Pósfai G:
Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs.
Microb Cell Fact. 2012 Jan 20;11:11.
4. Pirt, SJ:
The Maintenance Energy of Bacteria in Growing Cultures.
Proc R Soc Lond B Biol Sci. 1965,163:224
Pictures: Atomic force microscope, Attila Gergely Végh, Institute of Biophysics, BRC HAS
wt MG1655 E. coli cells MDS42 E. coli cells
