+ Resolve Article
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter

+ Translate
+ Subscribe to Site Feed
GeoScience Most Shared ContentMost Shared Content

Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering

, : Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering. Chembiochem 13(13): 1946-1952

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce unnatural derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.Reconstructing the path: We report the direct cloning and genetic engineering of a biosynthetic gene cluster based on Red/ET recombineering, combined with suitable heterologous expression, that led to the identification of two new syringolin members and production of all of the syringolins in E. coli.


PMID: 22851214

DOI: 10.1002/cbic.201200310

Other references

Mccarthy J.F.; Jacobson D.N.; Shugart L.R.; Jimenez B.D., 1989: Pre exposure to 3 methylcholanthrene increases benzo a pyrene adducts on dna of bluegill sunfish. Marine Environmental Research 28(1-4): 323-328

Wagner, D.; Hufeland, C.W., 1987: The epidemiology of Christoph Wilhelm Hufeland (1762-1836). A contribution to the history of pre-bacteriologic epidemiology. Zeitschrift für die Gesamte Hygiene und Ihre Grenzgebiete 33(10): 477-479

Anonymous, 1973: Session 3A. Cereal diseases. The following papers are noted:a. Lescar, L.; Bouchet, F.; Faivre-Dupaigre, R. (Inst. Tech. Cereales et Fourrages, Paris). Fungicidal control of winter wheat foliage diseases in France (97-104, 1 diag., 3 graphs, 4 tab.).b. Frost, A.J.P.; Brown, E...

Hoekstra, S.S.; Harkes, P.A.; Verpoorte, R.; Libbenga, K.R., 1990: Effect of auxin on cytodifferentiation and production of quinoline alkaloids in compact globular structures of Cinchona ledgeriana. Fine cell suspension cultures of Cinchona ledgeriana produce only very low amounts of quinoline alkaloids. These cultures formed self-propagating compact globular structures (CGS) on medium containing 2,4-D and BAP. These CGS could be induced to p...

Fischer, M.S.; Schilling, N.; Schmidt, M.; Haarhaus, D.; Witte, H., 2002: Basic limb kinematics of small therian mammals. A comparative study of quantitative kinematic data of fore- and hindlimb movements of eight different mammalian species leads to the recognition of basic principles in the locomotion of small therians. The description of kinematics comprises fore-...

Jones, L.; Williams, M., 1980: Economic impact of agricultural production in Texas: part 1, major production regions (TAEX Districts) Texas Agricultural EXtension Service. Data contained in this handbook rank the value and economic impact of agricultural production during 1976 through 1978 in Texas, and on Area Economic Growth Program regions within the state as defined by the Texas Agricultural Extension Service....

Rodoman G.V.; Kostrova M.V.; Salikov A.V., 1991: Immune status in wound complications after herniotomy. Sovetskaya Meditsina (4): 58-60

Wösten, H.A.; Richter, M.; Willey, J.M., 1999: Structural proteins involved in emergence of microbial aerial hyphae. After a submerged feeding mycelium has been established, both filamentous fungi and filamentous bacteria may leave the hydrophilic environment to form spore bearing aerial structures. These are hydrophobic and are characterized by rodlet-decorated...

Peck, Aj, 1979: How saltland can be reclaimed. Journal of agriculture Western Australia0(3): 72-73

Voy, A., 1953: Linear growth and phases of post-embryonal development in the female phasma (Clonopsis gallica Charp.). Comptes Rendus Hebdomadaires des Seances de L'academie des Sciences 236(25): 2447-2449