Improving a Synechocystis-based Photoautotrophic Chassis Through Systematic Genome Mapping and Validation of Neutral Sites

Overview

Journal DNA Res

Publisher Oxford University Press

Specialties Genetics
Molecular Biology

Date 2015 Oct 23

PMID 26490728

Citations 19

Authors

Filipe Pinto

Catarina C Pacheco

Paulo Oliveira

Arnau Montagud

Andrew Landels

Narciso Couto

Phillip C Wright

Javier F Urchueguia

Paula Tamagnini

Affiliations

Soon will be listed here.

Abstract

The use of microorganisms as cell factories frequently requires extensive molecular manipulation. Therefore, the identification of genomic neutral sites for the stable integration of ectopic DNA is required to ensure a successful outcome. Here we describe the genome mapping and validation of five neutral sites in the chromosome of Synechocystis sp. PCC 6803, foreseeing the use of this cyanobacterium as a photoautotrophic chassis. To evaluate the neutrality of these loci, insertion/deletion mutants were produced, and to assess their functionality, a synthetic green fluorescent reporter module was introduced. The constructed integrative vectors include a BioBrick-compatible multiple cloning site insulated by transcription terminators, constituting robust cloning interfaces for synthetic biology approaches. Moreover, Synechocystis mutants (chassis) ready to receive purpose-built synthetic modules/circuits are also available. This work presents a systematic approach to map and validate chromosomal neutral sites in cyanobacteria, and that can be extended to other organisms.

Citing Articles

Efficient Multiplex Genome Editing of the Cyanobacterium Synechocystis sp. PCC6803 via CRISPR-Cas12a.

Du W, Meister L, van Grinsven T, Branco Dos Santos F Biotechnol Bioeng. 2024; 122(3):736-743.

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Elucidating light-induced changes in excitation energy transfer of photosystem I and II in whole cells of two model cyanobacteria.

Biswas S, Niedzwiedzki D, Pakrasi H Photosynth Res. 2024; 163(1):1.

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Current Metabolic Engineering Strategies for Photosynthetic Bioproduction in Cyanobacteria.

Satta A, Esquirol L, Ebert B Microorganisms. 2023; 11(2).

PMID: 36838420 PMC: 9964548. DOI: 10.3390/microorganisms11020455.

The Molecular Toolset and Techniques Required to Build Cyanobacterial Cell Factories.

Opel F, Axmann I, Klahn S Adv Biochem Eng Biotechnol. 2022; 183:65-103.

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References

Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A . Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene. 1994; 145(1):69-73. DOI: 10.1016/0378-1119(94)90324-7. View

Marchler-Bauer A, Anderson J, Chitsaz F, Derbyshire M, DeWeese-Scott C, Fong J . CDD: specific functional annotation with the Conserved Domain Database. Nucleic Acids Res. 2008; 37(Database issue):D205-10. PMC: 2686570. DOI: 10.1093/nar/gkn845. View

Taton A, Unglaub F, Wright N, Zeng W, Paz-Yepes J, Brahamsha B . Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria. Nucleic Acids Res. 2014; 42(17):e136. PMC: 4176158. DOI: 10.1093/nar/gku673. View

Aoki R, Goto T, Fujita Y . A heme oxygenase isoform is essential for aerobic growth in the cyanobacterium Synechocystis sp. PCC 6803: modes of differential operation of two isoforms/enzymes to adapt to low oxygen environments in cyanobacteria. Plant Cell Physiol. 2011; 52(10):1744-56. DOI: 10.1093/pcp/pcr108. View

Griese M, Lange C, Soppa J . Ploidy in cyanobacteria. FEMS Microbiol Lett. 2011; 323(2):124-31. DOI: 10.1111/j.1574-6968.2011.02368.x. View

Huckauf J, Nomura C, Forchhammer K, Hagemann M . Stress responses of Synechocystis sp. strain PCC 6803 mutants impaired in genes encoding putative alternative sigma factors. Microbiology (Reading). 2000; 146 ( Pt 11):2877-2889. DOI: 10.1099/00221287-146-11-2877. View

Fulda S, Mikkat S, Huang F, Huckauf J, Marin K, Norling B . Proteome analysis of salt stress response in the cyanobacterium Synechocystis sp. strain PCC 6803. Proteomics. 2006; 6(9):2733-45. DOI: 10.1002/pmic.200500538. View

Aoki S, Kondo T, Ishiura M . Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol. 1995; 177(19):5606-11. PMC: 177371. DOI: 10.1128/jb.177.19.5606-5611.1995. View

Ohkawa H, Price G, Badger M, Ogawa T . Mutation of ndh genes leads to inhibition of CO(2) uptake rather than HCO(3)(-) uptake in Synechocystis sp. strain PCC 6803. J Bacteriol. 2000; 182(9):2591-6. PMC: 111325. DOI: 10.1128/JB.182.9.2591-2596.2000. View

10.

Berla B, Pakrasi H . Upregulation of plasmid genes during stationary phase in Synechocystis sp. strain PCC 6803, a cyanobacterium. Appl Environ Microbiol. 2012; 78(15):5448-51. PMC: 3416404. DOI: 10.1128/AEM.01174-12. View

11.

Trautmann D, Voss B, Wilde A, Al-Babili S, Hess W . Microevolution in cyanobacteria: re-sequencing a motile substrain of Synechocystis sp. PCC 6803. DNA Res. 2012; 19(6):435-48. PMC: 3514855. DOI: 10.1093/dnares/dss024. View

12.

Eriksson J, Salih G, Ghebramedhin H, Jansson C . Deletion mutagenesis of the 5' psbA2 region in Synechocystis 6803: identification of a putative cis element involved in photoregulation. Mol Cell Biol Res Commun. 2000; 3(5):292-8. DOI: 10.1006/mcbr.2000.0227. View

13.

Sergeyenko T, Los D . Identification of secreted proteins of the cyanobacterium Synechocystis sp. strain PCC 6803. FEMS Microbiol Lett. 2000; 193(2):213-6. DOI: 10.1111/j.1574-6968.2000.tb09426.x. View

14.

Bensadoun A, Weinstein D . Assay of proteins in the presence of interfering materials. Anal Biochem. 1976; 70(1):241-50. DOI: 10.1016/s0003-2697(76)80064-4. View

15.

Ishii A, Hihara Y . An AbrB-like transcriptional regulator, Sll0822, is essential for the activation of nitrogen-regulated genes in Synechocystis sp. PCC 6803. Plant Physiol. 2008; 148(1):660-70. PMC: 2528100. DOI: 10.1104/pp.108.123505. View

16.

Tamagnini P, Troshina O, Oxelfelt F, Salema R, Lindblad P . Hydrogenases in Nostoc sp. Strain PCC 73102, a Strain Lacking a Bidirectional Enzyme. Appl Environ Microbiol. 1997; 63(5):1801-7. PMC: 1389151. DOI: 10.1128/aem.63.5.1801-1807.1997. View

17.

Git A, Dvinge H, Salmon-Divon M, Osborne M, Kutter C, Hadfield J . Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression. RNA. 2010; 16(5):991-1006. PMC: 2856892. DOI: 10.1261/rna.1947110. View

18.

Koksharova O, Wolk C . Genetic tools for cyanobacteria. Appl Microbiol Biotechnol. 2002; 58(2):123-37. DOI: 10.1007/s00253-001-0864-9. View

19.

Burnap R, Qian M, Shen J, Inoue Y, Sherman L . Role of disulfide linkage and putative intermolecular binding residues in the stability and binding of the extrinsic manganese-stabilizing protein to the photosystem II reaction center. Biochemistry. 1994; 33(46):13712-8. DOI: 10.1021/bi00250a023. View

20.

Zhou H, Hu H, Lai M . Non-coding RNAs and their epigenetic regulatory mechanisms. Biol Cell. 2010; 102(12):645-55. DOI: 10.1042/BC20100029. View