Expression of genes in cyanobacteria: adaptation of endogenous plasmids as platforms for high-level gene expression in Synechococcus sp. PCC 7002.

Synechococcus sp. PCC 7002 is an ideal model cyanobacterium for functional genomics and biotechnological applications through metabolic engineering. A gene expression system that takes advantage of its multiple, endogenous plasmids has been constructed in this cyanobacterium. The method involves the integration of foreign DNA cassettes with selectable markers into neutral sites that can be located on any of the several endogenous plasmids of this organism. We have exploited the natural transformability and powerful homologous recombination capacity of this organism by using linear DNA fragments for transformation. This approach overcomes barriers that have made the introduction and expression of foreign genes problematic in the past. Foremost among these is the natural restriction endonuclease barrier that can cleave transforming circular plasmid DNAs before they can be replicated in the cell. We describe herein the general methodology for expressing foreign and homologous genes in Synechococcus sp. PCC 7002, a comparison of several commonly used promoters, and provide examples of how this approach has successfully been used in complementation analyses and overproduction of proteins with affinity tags.

[1]  D. Bryant,et al.  Biogenesis of Phycobiliproteins , 2008, Journal of Biological Chemistry.

[2]  D. Bryant,et al.  A Novel Nitrate/Nitrite Permease in the Marine CyanobacteriumSynechococcus sp. Strain PCC 7002 , 1999, Journal of bacteriology.

[3]  D. Bryant,et al.  Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation. , 2004, Methods in molecular biology.

[4]  H. Hayashi,et al.  A high temperature-sensitive mutant of Synechococcus sp. PCC 7002 with modifications in the endogenous plasmid, pAQ1. , 2002, Plant & cell physiology.

[5]  D. Bryant,et al.  CpcM Posttranslationally Methylates Asparagine-71/72 of Phycobiliprotein Beta Subunits in Synechococcus sp. Strain PCC 7002 and Synechocystis sp. Strain PCC 6803 , 2008, Journal of bacteriology.

[6]  S. Stevens,et al.  Chromosomal transformation in the cyanobacterium Agmenellum quadruplicatum , 1990, Journal of bacteriology.

[7]  J. Markley,et al.  Cloning, sequencing and transcriptional studies of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120. , 1994, Microbiology.

[8]  S. Slater,et al.  Application of the Synechococcus nirA Promoter To Establish an Inducible Expression System for Engineering the Synechocystis Tocopherol Pathway , 2005, Applied and Environmental Microbiology.

[9]  C. Krebs,et al.  Biogenesis of Iron-Sulfur Clusters in Photosystem I , 2008, Journal of Biological Chemistry.

[10]  D. Bryant,et al.  Identification and Characterization of a New Class of Bilin Lyase , 2006, Journal of Biological Chemistry.

[11]  D. Bryant,et al.  Synechoxanthin, an aromatic C40 xanthophyll that is a major carotenoid in the cyanobacterium Synechococcus sp. PCC 7002. , 2008, Journal of natural products.

[12]  J. Myers,et al.  THE PRODUCTION OF HYDROGEN PEROXIDE BY BLUE‐GREEN ALGAE: A SURVEY 1 , 1973 .

[13]  H. Miyasaka,et al.  Nucleotide sequence of plasmid pAQ1 of marine cyanobacterium Synechococcus sp. PCC7002. , 1998, DNA research : an international journal for rapid publication of reports on genes and genomes.

[14]  Donald A. Bryant,et al.  Characterization of a Synechococcus sp. strain PCC 7002 mutant lacking Photosystem I. Protein assembly and energy distribution in the absence of the Photosystem I reaction center core complex , 1995, Photosynthesis Research.

[15]  T. Roberts,et al.  The blue-green alga agmenellum quadruplicatum contains covalently closed DNA circles , 1976, Cell.

[16]  R. Porter,et al.  Transformation in cyanobacteria. , 1986, Critical reviews in microbiology.

[17]  Jindong Zhao,et al.  Assembly of Photosystem I , 2002, The Journal of Biological Chemistry.

[18]  Julia A Maresca,et al.  Identification of a fourth family of lycopene cyclases in photosynthetic bacteria , 2007, Proceedings of the National Academy of Sciences.

[19]  S. Stevens,et al.  Photoheterotrophic growth of Agmenellum quadruplicatum PR-6 , 1986, Journal of bacteriology.

[20]  D. Bryant,et al.  Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis , 2004, Archives of Microbiology.

[21]  S. Stevens,et al.  Plasmid transformation in Agmenellum quadruplicatum PR-6: construction of biphasic plasmids and characterization of their transformation properties , 1983, Journal of bacteriology.

[22]  D. Bryant,et al.  The Biosynthetic Pathway for Myxol-2′ Fucoside (Myxoxanthophyll) in the Cyanobacterium Synechococcus sp. Strain PCC 7002 , 2009, Journal of bacteriology.

[23]  D. Bryant,et al.  The Biosynthetic Pathway for Synechoxanthin, an Aromatic Carotenoid Synthesized by the Euryhaline, Unicellular Cyanobacterium Synechococcus sp. Strain PCC 7002 , 2008, Journal of bacteriology.

[24]  D. Bryant,et al.  The cpcE and cpcF genes of Synechococcus sp. PCC 7002. Construction and phenotypic characterization of interposon mutants. , 1992, The Journal of biological chemistry.

[25]  M. Ikeuchi,et al.  2,2'-beta-hydroxylase (CrtG) is involved in carotenogenesis of both nostoxanthin and 2-hydroxymyxol 2'-fucoside in Thermosynechococcus elongatus strain BP-1. , 2008, Plant & cell physiology.

[26]  X. Chen,et al.  Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002 , 1993, Journal of bacteriology.

[27]  Hong-liang Wang,et al.  Alterations in Global Patterns of Gene Expression in Synechocystis sp. PCC 6803 in Response to Inorganic Carbon Limitation and the Inactivation of ndhR, a LysR Family Regulator* , 2004, Journal of Biological Chemistry.

[28]  D. Bryant,et al.  Prokaryotic photosynthesis and phototrophy illuminated. , 2006, Trends in microbiology.

[29]  S. Golden,et al.  Specialized techniques for site-directed mutagenesis in cyanobacteria. , 2007, Methods in molecular biology.

[30]  C. Wolk,et al.  A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. , 1988, Gene.

[31]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[32]  R. Haselkorn,et al.  Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions , 2001, Proceedings of the National Academy of Sciences of the United States of America.