An exogenous chloroplast genome for complex sequence manipulation in algae

Abstract

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.

Figure 1.

Cloning of the C. reinhardtii chloroplast genome. (A) Flow diagram of genome assembly and maintenance in yeast and bacteria. Each arrow represents a transformation step to assemble or modify the genome in yeast or to transfer the genome from yeast to bacteria. Arrows with a gray ‘X’ indicate transformations that did not yield the target genome in the desired cloning host. In all cases, green boxes indicate PSII-encoding genes, red boxes indicate yeast selection markers, blue boxes indicate the hybrid vector elements from pTrp-10, purple boxes indicate bacterial F-factor replication elements, and gray boxes indicate the large, inverted repeats. Numbers adjacent to each assembly fragment correspond to the pSC vector from which they were liberated. The solid triangles indicate the unique AsiSI restriction site and the open triangles indicate the RsrII restriction sites. (B and C) Analysis of the cloned chloroplast genome. (B) pCr03 was digested with AsiSI and analyzed by pulsed-field gel electrophoresis on a 1% agarose gel in 0.5× TBE. λ indicates the lambda ladder (NEB). (C) Sequence coverage of pCr03 from 454 FLX Titanium pyrosequencing. Features are identical to those in panel (A). Scale bar indicates fold coverage.

Figure 2.

Exogenous chloroplast genome transformation strategy. Color of the chloroplast indicates whether it is able to carry out photosynthesis (green) or not (white). Green boxes indicate PSII-encoding genes, red boxes indicate yeast selection markers, blue boxes indicate the hybrid vector elements from pTrp-10, and purple boxes indicate bacterial F-factor replication elements. Unique markers for screening are labeled M1–M4.

Figure 3.

Characterization of the cloned C. reinhardtii chloroplast genome in vivo. (A) A nested set representing the presence of increasing numbers of markers in primary transformants of pCr03 into a psbD knockout strain as determined by PCR (Table 2; primers used as follows: M1, 11 606 and 11 607; M2, 5512 and 5513; M3, 11 456 and 11 457; and M4, 14 067 and 14 068.). The broken circle shows the subset of transformants with M1, M2, M3 and M4 that gave rise to the same genotype upon rescreening. (B–E) Southern blot analysis of EcoRI (B, C and E) or NdeI (D) digests (see ‘Materials and Methods’ section). Probes were specific for sequences adjacent to integration sites for M1 (B), M2 (C), M3 (D) and M4 (E). All samples are arranged as follows: Lane L, 1 kb DNA ladder (Invitrogen; Carlsbad, CA); lane 1, wild-type; lane 2, purified pCr03; and lane 3, a representative algae clone containing all unique markers. A single band in lane 3 indicates homoplasmic integration of the marker, while two bands indicate heteroplasmy with the wild-type locus.

Figure 4.

Gene replacement in an exogenous chloroplast genome. (A) Quantum efficiency of PSII in strains with a single PSII gene replacement. Values are reported as the average and standard deviation of three analytical replicates of a representative transformant. (B) The two steps of gene replacement. The endogenous locus (green box) is first displaced by a cassette containing one positive and one negative selection marker (red box) using recombination in yeast. The displacement cassette is then replaced with a new sequence (green box with ‘Asterisk’) targeted by the same flanking regions. (C) Flow diagram of genome assembly and maintenance in yeast and bacteria. Each arrow represents a transformation step to assemble or modify the genome in yeast or to transfer the genome from yeast to bacteria. In all cases, green boxes indicate PSII-encoding genes, red boxes indicate yeast selection markers, blue boxes indicate the hybrid vector elements from pTrp-34, purple boxes indicate bacterial F-factor replication elements, and gray boxes indicate the large, inverted repeats. Numbers adjacent to each assembly fragment correspond to the pSC vector from which they were liberated. The black triangle in pCr05 indicates the unique AsiSI restriction site. (D) DNA isolated from bacteria was digested with AsiSI and analyzed by pulsed-field gel electrophoresis on a 1% agarose gel in 0.5× TBE. λ indicates the lambda ladder.