Lab website: http://group.szbk.u-szeged.hu/sysbiol/

GENOME ENGINEERING

Using approaches of synthetic biology, we are focusing on the rational large-scale remodeling of the genome of Escherichia coli K-12. Our goal is to construct an improved, minimal genome E. coli to serve both as a reduced-complexity model organism, and as a programmable cellular chassis for biotechnological applications. In the process of gradually streamlining the genome, we are investigating the effect of genome architecture and gene content on the adaptation and evolution of the cells.

Towards a minimal Escherichia coli cell

E. coli K-12 is a model organism of basic research, the workhorse of molecular biology, and the platform of choice for the production of DNA, metabolites and many proteins of therapeutic or commercial interest. In spite of being one of the best understood model organisms, roughly one third of its ~4300 genes have no experimentally verified functions assigned to them. Moreover, as E. coli evolved in the intestinal tracts of animals, it has many genes that are not relevant to practical applications and some that may be detrimental. One of the objectives of this research is to eliminate as many of these unnecessary/unknown genes as possible to develop core-genome strains of E. coli K-12 MG1655 with robust metabolic performance, to which genetic modules carrying out specific practical applications can be added. By streamlining the genome, simpler and better characterized cells could be developed. Moreover, due to the removal of unnecessary products, the energy and metabolic resources of the cell could be redirected to useful biomolecule production.

Systems biology and comparative genomics methods are applied to identify dispensable genomic regions. Genomes of E. coli strains are now recognized to be mosaics in which a backbone of conserved genes in conserved order is interspersed with strain-specific horizontally transmitted “islands”. The islands contain genes supporting niche-specific adaptation as well as transposable elements. Using scarless genomic reduction of the sequenced K-12 strain MG1655, we physically realize strains whose genome consists of the backbone elements common to most E. coli strains.

In published studies, we have described the deletion of up to 43 genomic segments of MG1655, resulting in a >15% reduction of the genome (Kolisnychenko et al., 2002; Pósfai et al., 2006). This work progressed to the construction of multideletional strains with >70 deletions (>20% genome reduction). Regions deleted include large K-islands, prophages, phage remnants, restriction-modification genes, flagellar and chemotaxis related genes, genes with unknown functions, and transposable elements.

Strain characteristics important for laboratory applications (growth rate, biomass production, electroporation efficiency, plasmid yield and quality) were not adversely affected by the deletions. In fact, compared with the parental wild-type strain, some multideletional strains display a number of beneficial changes: increase/improvement in growth rate, physiological uniformity, electroporability, plasmid maintenance and genome stability. Currently we are pursuing large-scale characterization of the multideletional strains, including the matching of experimental data with metabolic models, as well as the analysis of newly emerging phenotypic traits.

The genome reduction/optimization work is done in collaboration with F.R. Blattner (Scarab Genomics LLC, Madison, USA). Systems biology analyses are done in collaboration with B. Papp and C. Pál (BRC).

The role of mobile genetic elements in adaptation and evolution

Insertion sequence (IS) elements in E. coli have traditionally been considered mostly “selfish” DNA, i.e., evolutionarily neutral. Having a unique tool, a multideletional strain stripped of its mobile genetic elements, we are investigating the potential role of ISs in adaptation to stressful conditions. Measuring mutation rates under various conditions, we find evidence of stress-induced transposition of ISs, resulting in genomic rearrangements and inactivation of protein overexpressing plasmid clones. Comparing isogenic strains with or without IS elements, their individual contribution to the adaptation of the cell can be investigated.