An exogenous chloroplast genome for complex sequence manipulation in algae

Bryan M. O'Neill 1 Kari L. Mikkelson 1 Noel M. Gutierrez 1 Jennifer L. Cunningham 1 Kari L. Wolff 1 Shawn J. Szyjka 1 Christopher B. Yohn 1 Kevin E. Redding 0 Michael J. Mendez 1 0 Department Chemistry & Biochemistry, Arizona State University , Tempe, AZ 85287-1604, USA 1 Sapphire Energy, Inc., San Diego, CA , 92121 We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome. - A promise of synthetic biology is the ability to rationally alter metabolic processes in ways that would be impossible, or at least prohibitively difficult, through traditional approaches like chemical mutagenesis, breeding, or expression of even a few heterologous genes. While the field remains ill-defined, it generally includes leveraging advanced methods for synthesis and cloning of DNA molecules to obtain novel sequences with desired functional properties (1,2). Some of synthetic biologys most notable achievements involve the assembly of genetic material into large DNA molecules that resemble chromosomal fragments or even whole genomes (3,4). Such large contigs may then be used to deliver all desired sequences into a target host in a single transformation step (5). However, as exogenous DNA molecules grow in size, so do the challenges of designing and maintaining correct sequences (6,7). Chloroplast genomes present a unique opportunity for the field of synthetic biology. In a single, relatively small molecule, they encode the most important genes of photosynthesis, natures principle method for converting sunlight into chemical energy and the progenitor of countless metabolites, and only a few other coding regions that support gene expression in the organelle (8). Most chloroplast genomes range between 150- and 205-kb, and many genomes representing diverse taxa have been sequenced and are publicly available (8). In addition, chloroplast transformation is a well-established technology in both plants and algae (9,10). Thus, these naturally minimized, manipulable genomes, which are of great interest for metabolic engineering for foods, fuels and myriad bio-products, are an ideally suited target for synthetic biology. Photosynthesis is among the best-understood processes in biology. Studies conducted during the last few decades have revealed the architecture and mechanism of action for every component in the photosynthetic apparatus, including Photosystem II (PSII), a multi-subunit complex responsible for utilizing light energy in oxidoreduction reactions to extract electrons from water and produce oxygen (11). At the core of the PSII complex are four highly conserved proteins: D1, D2, CP43 and CP47. Photochemistry takes place in the D1/D2 heterodimer while CP43 and CP47 bind additional *To whom correspondence should be addressed. Tel: +1 858 768 4766; Fax: +1 888 501 8353; Email: , pigments to increase the absorption cross-section of the complex (12). These subunits interact extensively with one another via polypeptide sequences that are among the most conserved in all of biology (13). D1, CP47, CP43 and D2 are encoded by psbA, psbB, psbC and psbD, respectively, which exist at disparate locations in the chloroplast genome of all photosynthetic eukaryotes (8). We sought to utilize the power of synthetic biology methods to directly manipulate the core genetics of photosynthesis by cloning a chloroplast genome from algae ex vivo using a yeast-bacteria hybrid system (14). This cloning system exploits yeast for its ability to stably maintain large DNA molecules and to support homologous recombination for sequence assembly and modification, and exploits bacteria for its ability to produce large quantities of specific DNA molecules, which are required for biolistic chloroplast transformation (14,15). Here, we demonstrate exogenous assembly and modification of the entire Chlamydomonas reinhardtii chloroplast genome, followed by transformation into algae cells and simultaneous alteration of at least six independent sites, including some that encode the core subunits of PSII. MATERIALS AND METHODS Primers used in this study are listed in Supplementary Table S1. Hybrid vector elements from pED-R2D2-ADE/URA [including a yeast centromere, yeast autonomously replication sequence, yeast selection marker (TRP1), bacterial replication origins (P1 rep and P1 lytic), and bacterial selection marker (Kanr) (14)] were combined with algae-specific sequences, enabling maintenance of the exogenous algae chloroplast genome in yeast and bacteria. The combination of these DNA sequences is described here. The vector pDOCI was first generated to manipulate pED-R2D2-ADE/URA. Two portions of pED-R2D2ADE/URA were amplified using PCR primer pairs (462 and 465, and 469 and 473) that anneal to sites surrounding the region encompassing TEL, ADE2 and URA3, assembled into a single DNA fragment by PCR assembly using a single primer pair (462 and 473), digested with NotI and ligated to a NotI-digested variant of pUC19, forming pDOCI. Portions of the C. reinhardtii chloroplast genome were then PCR-amplified using two primer pairs specific for adjacent regions near the psbD locus (791 and 792, and 793 and 794), digested with NotI and I-SceI and three-way ligated to I-SceI-digested pDOCI to form pDOCI-10. Next, a sequence conferring kanamycin resistance in algae (16) was cloned into pDOCI-10 using SnaBI to form pDOCI-10-Kan. The hybrid vectors for cloning chloroplast DNA, pTRP-10 and pTRP-10-Kan, were constructed using recombination in yeast. pDOCI-10 and pDOCI-10-Kan were digested with PacI and AscI to liberate cassettes that introduce chloroplast genome-specific elements into the hybrid cloning vector. This cassette was transformed along with pEDR2D2-ADE/URA into yeast. Transformants that correctly integrated each cassette were isolated based on growth on CSM-Trp agar media containing 5-fluoroorotic acid (5-FOA) and by red color. Plasmid DNA was isolated from yeast clon (...truncated)