György PÓSFAI
scientific adviser
deputy director-general

picture
Gabriella BALIKÓ research associate
Zsuzsanna GYÖRFY research associate
Edit TÍMÁR research associate
Kinga UMENHOFFER research associate
Viktor VERNYIK junior research associate
Ildikó KARCAGI scientific administrator

GENOME ENGINEERING


Microbial genome engineering protocols

Current gene synthesis and assembly methods, combined with high-throughput multiplex genome modification protocols, open up new possibilities in microbial genome construction. However, in order to manipulate and assemble whole genomes at will, further technical improvements are needed. In the past, we have developed a set of simple tools and protocols for homologous recombination-based, precise manipulation of bacterial genomes. Currently we are adapting these methods to high-throughput protocols both for random deletion-generation and for more specific, multiplex genome modifications.


Minimizing the genomes of E. coli strains

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 (mostly the strain-specific, horizontally transmitted genomic “islands”) as possible to develop core-genome strains of E. coli K-12 MG1655 with robust metabolic performance. 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.

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). Compared with the parental wild-type strain, some multideletional strains display beneficial changes: improvements in growth rate, physiological uniformity, and genome stability. Currently we are pursuing additional genome reduction and optimization involving K-12 and other strains.

The genome reduction/optimization work is done in collaboration with F.R. Blattner (Scarab Genomics LLC, Madison, USA).

Genome architecture and evolution

Genomes are more than their gene content. When designing artificial microbial genomes (as anticipated to happen in the near future), architectural principles, such as the domain structure, or the copy number, orientation, and genomic position of genomic elements, have to be taken into account. Our genome modification protocols and reduced-genome strains provide excellent tools and platforms to construct genome variations designed to uncover these principles. We are currently studying the effect of the copy number and position of ribosomal operons, expression of gene cassettes inserted in various positions/domains, and effect of the number and position of mobile genetic elements on genomic plasticity.

Selected publications

Pósfai, G., Kolisnychenko, V., Bereczki, Z. and Blattner, F.R. (1999). Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucleic Acids Res. 27: 4409-4415.

Perna, N.T., Plunkett, G., Burland, V., Mau, B., Glasner, J.D., Rose, D.J., Mayhew, G.F., Evans, P.S., Gregor, J., Kirkpatrick, H.A., Pósfai, G., Hackett, J,, Klink, S., Boutin, A,, Shao, Y., Miller. L., Grotbeck, E.J., Davis, N.W., Lim, A., Dimalanta, E.T., Potamousis, K.D., Apodaca, J., Anantharaman, T.S., Lin, J., Yen, G., Schwartz, D.C., Welch, R.A., Blattner, F.R. (2001). Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409: 529-533.

Kolisnychenko, V., Fehér, T., Herring, C.D., Plunkett, G. III, Blattner, F.R. and Pósfai, G. (2002). Engineering a reduced E. coli genome. Genome Research 12: 640-647.

Pósfai, G., Plunkett, G3rd., Fehér, T., Frisch, D., Keil, G., Umenhoffer, K., Kolisnychenko, V., Stahl, B., Arruda, M., Sharma, S.S., Burland, V., Harcum, S.W., Blattner, F.R. (2006). Emergent properties of reduced-genome Escherichia coli. Science 312: 1044-1046.

Fehér, T., Papp, B., Pál, C. and Pósfai, G. (2007). Systematic genome reductions: Theoretical and experimental aspects. Chem. Rev. 107: 3498-3513.

Umenhoffer K, Fehér T, Balikó G, Ayaydin F, Pósfai J, Blattner FR, Pósfai G (2010). Reduced evolvability of Escherichia coli MDS42, an IS-less cellular chassis for molecular and synthetic biology applications. Microb Cell Fact. 9:38.

Csörgő, B., Fehér, T., Timár, E., Blattner, F.R. and Pósfai, G. (2012). Low-mutation-rate, reduced-genome Escherichia coli: An improved host for faithful maintenance of engineered genetic constructs. Microb. Cell Fact. 11:11.

Fehér, T., Bogos, B., Méhi, O., Fekete, G., Csörgő, B., Kovács, K., Pósfai, G., Papp, B., Hurst, L.D., Pál, C. (2012). Competition between Transposable Elements and Mutator Genes in Bacteria. Mol. Biol. Evol. 29:3153-3159.

Fehér, T., Burland, V., Pósfai, G. (2012). In the fast lane: Large-scale bacterial genome engineering. J. Biotechnol. 160: 72-79.

Lázár V., Singh G.P. , Spohn R., Nagy I. , Horváth B., Hrtyan M. , Busa-Fekete R. , Bogos B., Méhi O., Csörgő B., Pósfai G., Fekete G., Szappanos B., Kégl B., Papp B., Pal C. (2013) Bacterial evolution of antibiotic hypersensitivity. Mol. Syst. Biol. 9:700

Nyerges A., Csörgő B., Nagy I., Latinovics D., Szamecz B., Posfai G., Pal C. (2014). Conditional DNA repair mutants enable highly precise genome engineering. Nucleic Acids Research 1–10 doi:10.1093/nar/gku105

Pál, C., Papp, B., Pósfai, G. (2014). The dawn of evolutionary genome engineering. Nature Reviews Genetics doi:10.1038/nrg3746

Gyorfy, Z., Draskovits, G., Vernyik, V., Blattner, F.R., Gaal, T., Posfai, G. (2015). Engineered ribosomal RNA operon copy-number variants of E. coli reveal the evolutionary trade-offs shaping rRNA operon number. Nucleic Acids Research doi: 10.1093/nar/gkv040

Nyerges, Á., Csörgő, B., Nagy, I., Bálint, B., Bihari, P., Lázár, V., Apjok, G., Umenhoffer, K., Bogos, B., Pósfai, G., Pál, C. (2016). A highly precise and portable genome engineering method allows comparison of mutational effects acrossbacterial species. Proc Natl Acad Sci U S A. 2016 Feb 16. pii: 201520040.

Karcagi, I., Draskovits, G., Umenhoffer, K., Fekete, G., Kovács, K., Méhi, O., Balikó, G, Szappanos, B., Györfy, Z., Fehér, T, Bogos, B., Blattner, F.R., Pál, C., Pósfai, G., Papp, B. (2016). Indispensability of horizontally transferred genes and its impact on bacterial genome streamlining. Molecular Biology and Evolution 2016 Jan 14. pii: msw009.