Abstract
As a key focus of synthetic biology, building a minimal artificial cell has given rise to many discussions. A synthetic minimal cell will provide an appropriate chassis to integrate functional synthetic parts, devices and systems with functions that cannot generally be found in nature. The design and construction of a functional minimal genome is a key step while building such a cell/chassis since all the cell functions can be traced back to the genome. Kinds of approaches, based on bioinformatics and molecular biology, have been developed and proceeded to derive essential genes and minimal gene sets for the synthetic minimal genome. Experiments about streamlining genomes of model bacteria revealed genome reduction led to unanticipated beneficial properties, such as high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Recent achievements in chemical synthesis technology for large DNA segments together with the rapid development of the wholegenome sequencing, have transferred synthesis of genes to assembly of the whole genomes based on oligonucleotides, and thus created strong preconditions for synthesis of artificial minimal genome. Here in this article, we review briefly the history and current state of research in this field and summarize the main methods for making a minimal genome. We also discuss the impacts of minimized genome on metabolism and regulation of artificial cell.
Keywords: synthetic biology, minimal genome, essential gene, minimal cell
References
- Akerley B.J., Rubin E.J., Novick V.L., Amaya K., Judson N., Mekalanos J.J. A genome-scale analysis for identification of genes required for growth or survival of Haemophilus influenzae. Proc Natl Acad Sci U S A. 2002;99:966–971. doi: 10.1073/pnas.012602299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ara K., Ozaki K., Nakamura K., Yamane K., Sekiguchi J., Ogasawara N. Bacillus minimum genome factory: effective utilization of microbial genome information. Biotechnol Appl Biochem. 2007;46:169–178. doi: 10.1042/BA20060111. [DOI] [PubMed] [Google Scholar]
- Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2,2006.0008. [DOI] [PMC free article] [PubMed]
- Becker S.A., Palsson B.O. Genome-scale reconstruction of the metabolic network in Staphylococcus aureus N315: an initial draft to the two-dimensional annotation. BMC Microbiol. 2005;5:12. doi: 10.1186/1471-2180-5-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Benner S.A., Sismour A.M. Synthetic biology. Nat Rev Genet. 2005;438:533–543. doi: 10.1038/nrg1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cello J., Paul A.V., Wimmer E. Chemical synthesis of poliovirus cDNA: generation of infections virus in the absence of natural template. Science. 2002;297:1016–1018. doi: 10.1126/science.1072266. [DOI] [PubMed] [Google Scholar]
- Charlebois R.L., Doolittle W.F. Computing prokaryotic gene ubiquity: rescuing the core from extinction. Genome Res. 2004;14:2469–2477. doi: 10.1101/gr.3024704. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Christian N., May P., Kempa S., Handorf T., Ebenhoh O. An integrative approach towards completing genome-scale metabolic networks. Mol BioSyst. 2009;5:1889–1903. doi: 10.1039/b915913b. [DOI] [PubMed] [Google Scholar]
- Covert M.W., Palsson B.O. Constraints-based models: Regulation of gene expression reduces the steady-state solution space. J Theor Biol. 2003;221:309–325. doi: 10.1006/jtbi.2003.3071. [DOI] [PubMed] [Google Scholar]
- de Berardinis V., Vallenet D., Castelli V., Besnard M., Pinet A., Cruaud C., Samair S., Lechaplais C., Gyapay G., Richez C., et al. A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1. Mol Syst Biol. 2008;4:174. doi: 10.1038/msb.2008.10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Diez M.S., Lam C.M., Leprince A., dos Santos V.A. (Re-)construction, characterization and modeling of systems for synthetic biology. Biotechnol J. 2009;4:1382–1391. doi: 10.1002/biot.200900173. [DOI] [PubMed] [Google Scholar]
- Duarte N.C., Herrgard M.J., Palsson B.O. Reconstruction and validation of Saccharomyces cerevisiae iND750, a fully compartmentalized genome-scale metabolic model. Genome Res. 2004;14:1298–1309. doi: 10.1101/gr.2250904. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fang G., Rocha E., Danchin A. How essential are nonessential genes? Mol Biol Evol. 2005;22:2147–2156. doi: 10.1093/molbev/msi211. [DOI] [PubMed] [Google Scholar]
- Fehér T., Papp B., Pál C., Pósfai G. Systematic genome reductions: theoretical and experimental approaches. Chem Rev. 2007;107:3498–3513. doi: 10.1021/cr0683111. [DOI] [PubMed] [Google Scholar]
- Foley P.L., Shuler M.L. Considerations for the design and construction of a synthetic platform cell for biotechnological applications. Biotechnol Bioeng. 2010;105:26–36. doi: 10.1002/bit.22575. [DOI] [PubMed] [Google Scholar]
- Forsyth R.A., Haselbeck R.J., Ohlsen K.L., Yamamoto R.T., Xu H., Trawick J.D., Wall D., Wang L., Brown-Driver V., Froelich J.M., et al. A genome-wide strategy for the identification of essential genes in Staphylococcus aureus. Mol Microbiol. 2002;43:1387–1400. doi: 10.1046/j.1365-2958.2002.02832.x. [DOI] [PubMed] [Google Scholar]
- Fraser C.M., Gocayne J.D., White O., Adams M.D., Clayton R.A., Fleischmann R.D., Bult C.J., Kerlavage A.R., Sutton G., Kelley J.M., et al. The minimal gene complement of Mycoplasma genitalium. Science. 1995;270:397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
- French C.T., Lao P., Loraine A.E., Matthews B.T., Yu H., Dybvig K. Large-scale transposon mutagenesis of Mycoplasma pulmonis. Mol Microbiol. 2008;69:67–76. doi: 10.1111/j.1365-2958.2008.06262.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gallagher L.A., Ramage E., Jacobs M.A., Kaul R., Brittnacher M., Manoil C. A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate. Proc Natl Acad Sci U S A. 2006;104:1009–1014. doi: 10.1073/pnas.0606713104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gerdes S.Y., Scholle M.D., Campbell J.W., Balazsi G., Ravasz E., Daugherty M.D., Somera A.L., Kyrpides N.C., Anderson I., Gelfand M.S., et al. Experimental determination and system level analysis of essential genes in Escherichia coli MG1655. J Bacteriol. 2003;185:5673–5684. doi: 10.1128/JB.185.19.5673-5684.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gibson D.G., Benders G.A., Andrews-Pfannkoch C., Denisova E. A., Baden-Tillson H., Zaveri J., Stockwell T.B., Brownley A., Thomas D.W., Algire M.A., et al. Complete chemical synthesis, assembly, and cloning of a mycoplasma genitalium genome. Science. 2008;319:1215–1220. doi: 10.1126/science.1151721. [DOI] [PubMed] [Google Scholar]
- Gibson D.G., Benders G.A., Axelrod K.C., Zaveri J., Algire M.A., Moodie M., Montague M.G., Venter J.C., Smith H.O., Ill C.A. H. One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci U S A. 2008;105:20404–20409. doi: 10.1073/pnas.0811011106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Giga-Hama Y., Tohda H., Takegawa K., Kumagai H. Schizosaccharomyces pombe minimum genome factory. Biotechnol Appl Biochem. 2007;46:147–155. doi: 10.1042/BA20060106. [DOI] [PubMed] [Google Scholar]
- Gil R., Sabater-Munoz B., Latorre A., Silva F.J., Moya A. Extreme genome reduction in Buchnera spp.: Toward the minimal genome needed for symbiotic life. Proc Natl Acad Sci U S A. 2002;99:4454–4458. doi: 10.1073/pnas.062067299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gil R., Silva F.J., Pereto J., Moya A. Determination of the core of a minimal bacterial gene set. Microbiol Mol Biol Rev. 2004;68:518–537. doi: 10.1128/MMBR.68.3.518-537.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Glass J.I., Assad-Garcia N., Alperovich N., Yooseph S., Lewis M. R., Maruf M., III C.A.H., Smith H.O., Venter J.C. Essential genes of a minimal bacterium. Proc Natl Acad Sci U S A. 2006;103:425–430. doi: 10.1073/pnas.0510013103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herring C.D., Glasner J.D., Blattner F.R. Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene. 2003;311:153–163. doi: 10.1016/S0378-1119(03)00585-7. [DOI] [PubMed] [Google Scholar]
- Hobom B. Surgery of genes — at the doorstep of synthetic biology. Med Klin. 1980;75:14–21. [PubMed] [Google Scholar]
- Holzhütter S., Holzhütter H.-G. Computational design of reduced metabolic networks. ChemBioChem. 2004;5:1401–1422. doi: 10.1002/cbic.200400128. [DOI] [PubMed] [Google Scholar]
- Hutchison C.A., Peterson S.N., Gill S.R., Cline R.T., White O., Fraser C.M., Smith H.O., Venter J.C. Global transposon mutagenesis and a minimal mycoplasma genome. Science. 1999;286:2165–2169. doi: 10.1126/science.286.5447.2165. [DOI] [PubMed] [Google Scholar]
- Itaya M. An estimation of minimal genome size required for life. FEBS Letters. 1995;362:257–260. doi: 10.1016/0014-5793(95)00233-Y. [DOI] [PubMed] [Google Scholar]
- Jacobs M.A., Alwood A., Thaipisuttikul I., Spencer D., Haugen E., Ernst S., Will O., Kaul R., Raymond C., Levy R., et al. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2003;100:14339–14344. doi: 10.1073/pnas.2036282100. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ji Y., Zhang B., Van S.F., Horn, Warren P., Woodnutt G., Burnham M.K.R., Rosenberg M. Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science. 2001;293:2266–2269. doi: 10.1126/science.1063566. [DOI] [PubMed] [Google Scholar]
- Judson N., Mekalanos J.J. TnAraOut, A transposon-based approach to identify and characterize essential bacterial genes. Nat Biotechnol. 2000;18:740–745. doi: 10.1038/77305. [DOI] [PubMed] [Google Scholar]
- Kitney R.I. Synthetic biology — engineering biologically-based devices and systems. In: Jarm T., Kramar P., Zupanic A., editors. 11th Mediterranean Conference on Medical and Biological Engineering and Computing 2007, Vols 1 and 2. Berlin: Springer-Verlag Berlin; 2007. pp. 1138–1139. [Google Scholar]
- Knuth K., Niesalla H., Hueck C.J., Fuchs T.M. Large-scale identification of essential Salmonella genes by trapping lethal insertions. Mol Microbiol. 2004;51:1729–1744. doi: 10.1046/j.1365-2958.2003.03944.x. [DOI] [PubMed] [Google Scholar]
- Kobayashi K., Ehrlich S.D., Albertini A., Amati G., Andersen K.K., Arnaud M., Asai K., Ashikaga S., Aymerich S., Bessieres P., et al. Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A. 2003;100:4678–4683. doi: 10.1073/pnas.0730515100. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koonin E.V. How many genes can make a cell: the minimalgene-set concept. Annu Rev Genomics Hum Genet. 2000;01:99–116. doi: 10.1146/annurev.genom.1.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koonin E.V. Comparative genomics, minimal gene-sets and the last universal common ancestor. Nat Rev Microbiol. 2003;1:127–136. doi: 10.1038/nrmicro751. [DOI] [PubMed] [Google Scholar]
- Liberati N.T., Urbach J.M., Miyata S., Lee D.G., Drenkard E., Wu G., Villanueva J., Wei T., Ausubel F.M. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc Natl Acad Sci U S A. 2006;103:2833–2838. doi: 10.1073/pnas.0511100103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liolios K., Mavromatis K., Tavernarakis N., Kyrpides N.C. The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucl Acids Res. 2008;36:D475–479. doi: 10.1093/nar/gkm884. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Medini D., Donati C., Tettelin H., Masignani V., Rappuoli R. The microbial pan-genome. Curr Opin Genet Dev. 2005;15:589–594. doi: 10.1016/j.gde.2005.09.006. [DOI] [PubMed] [Google Scholar]
- Murakami K., Tao E., Ito Y., Sugiyama M., Kaneko Y., Harashima S., Sumiya T., Nakamura A., Nishizawa M. Large scale deletions in the Saccharomyces cerevisiae genome create strains with altered regulation of carbon metabolism. Appl Microbiol Biotechnol. 2007;75:589–597. doi: 10.1007/s00253-007-0859-2. [DOI] [PubMed] [Google Scholar]
- Mushegian A.R., Koonin E.V. A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proc Natl Acad Sci U S A. 1996;93:10268–10273. doi: 10.1073/pnas.93.19.10268. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Posfai G., III G.P., Feher T., Frisch D., Keit G.M., Umenhoffer K., Kolisnychenko V., Stahl B., Sharma S.S., Arruda M.d., et al. Emergent properties of reduced-genome Escherichia coli. Science. 2006;312:1044–1046. doi: 10.1126/science.1126439. [DOI] [PubMed] [Google Scholar]
- Price N.D., Reed J.L., Palsson B.O. Genome-scale models of microbial cells: evaluating the consequences of constraints. Nat Rev Micro. 2004;2:886–897. doi: 10.1038/nrmicro1023. [DOI] [PubMed] [Google Scholar]
- Puchalka J., Oberhardt M.A., Godinho M., Bielecka A., Regenhardt D., Timmis K.N., Papin J.A., Martins dos Santos V.A. Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology. PLoS Comput Biol. 2008;4:e1000210. doi: 10.1371/journal.pcbi.1000210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rawls R.L. ’synthetic biology’ makes its debut. Chem Eng News. 2000;78:49–53. [Google Scholar]
- Reed J.L., Vo T.D., Schilling C.H., Palsson B.O. An expanded genome-scale model of Escherichia coli K-12 (iJR904 GSM/GPR) Genome Biol. 2003;4:12. doi: 10.1186/gb-2003-4-9-r54. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Salama N.R., Shepherd B., Falkow S. Global transposon mutagenesis and essential gene analysis of Helicobacter pylori. J Bacteriol. 2004;186:7926–7935. doi: 10.1128/JB.186.23.7926-7935.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sassetti C.M., Boyd D.H., Rubin E.J. Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol. 2003;48:77–84. doi: 10.1046/j.1365-2958.2003.03425.x. [DOI] [PubMed] [Google Scholar]
- Smith H.O., Hutchison C.A., Pfannkoch C., Venter J.C. Generating a synthetic genome by whole genome assembly: ϕ×174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci U S A. 2003;100:15440–15445. doi: 10.1073/pnas.2237126100. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Song J.-H., Ko K.S., Lee J.-Y., Baek J.Y., Oh W.S., Yoon H.S., Jeong J.-Y., Chun J. Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis. Mol Cells. 2005;19:365–374. [PubMed] [Google Scholar]
- Sung B.H., Lee J.H., Kim S.C. Escherichia coli genome engineering and minimization for the construction of a bioengine. In: Lee S.Y., editor. Systems Biology and Biotechnology of Escherichia coli. Springer: Daejeon; 2009. pp. 19–40. [Google Scholar]
- Suzuki N., Nonaka H., Tsuge Y., Okayama S., Inui M., Yukawa H. Multiple large segment deletion method for Corynebacterium glutamicum. Appl Microbiol Biotechnol. 2005;69:151–161. doi: 10.1007/s00253-005-1976-4. [DOI] [PubMed] [Google Scholar]
- Szathmáry E. Life: In search of the simplest cell. Nature. 2005;433:469–470. doi: 10.1038/433469a. [DOI] [PubMed] [Google Scholar]
- Tatusov R.L., Fedorova N.D., Jackson J.D., Jacobs A.R., Kiryutin B., Koonin E.V., Krylov D.M., Mazumder R., Mekhedov S.L., Nikolskaya A.N., et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 2003;4:14. doi: 10.1186/1471-2105-4-41. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thanassi J.A., Hartman-Neumann S.L., Dougherty T.J., Dougherty B.A., Pucci M.J. Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae. Nucleic Acids Res. 2002;30:3152–3162. doi: 10.1093/nar/gkf418. [DOI] [PMC free article] [PubMed] [Google Scholar]
- The Royal Academy of Engineering . Synthetic Biology: scope, applications and implications. London: The Royal Academy of Engineering; 2009. [Google Scholar]
- Wann E.R. Unlocking the secrets of another minimal genome. Trends Microbiol. 2000;8:544–545. doi: 10.1016/S0966-842X(00)01895-3. [DOI] [PubMed] [Google Scholar]
- Yu B.J., Sung B.H., Koob M.D., Lee C.H., Lee J.H., Lee W.S., Kim M.S., Kim S.C. Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system. Nat Biotech. 2002;20:1018–1023. doi: 10.1038/nbt740. [DOI] [PubMed] [Google Scholar]
- Zhang Y., Thiele I., Weekes D., Li Z., Jaroszewski L., Ginalski K., Deacon A.M., Wooley J., Lesley S.A., Wilson I.A., et al. Three-dimensional structural view of the central metabolic network of Thermotoga maritima. Science. 2009;325:1544–1549. doi: 10.1126/science.1174671. [DOI] [PMC free article] [PubMed] [Google Scholar]