'21ENGBIO' Towards SYnthetic CHLOroPlastS (SYCHLOPS)

Bacteria and yeast with fully or partially synthetic genomes have been generated and these are proving to be useful platforms for engineering biology. For example, a well-designed synthetic genome can allow key genes to be swapped in and out or rearranged at will. In addition, synonymous codons and UAG stop codons have been reassigned to allow an expanded genetic code. A good illustration of this is the E. coli strain Syn61 in which all serine UCG and UCA codons were replaced along with all UAG stop codons. This tour de force allowed the re-assignment of these codons and the incorporation of two noncanonical amino acids into proteins made from introduced transgenes. However, yeast and bacterial genomes are relatively large and creating full or partially synthetic versions is challenging, time consuming and costly.

Plants contain three genomes, the nuclear, the chloroplast (plastid) and the mitochondrial. The nuclear genome is the largest and typically encodes in excess of 27,000 genes using from 130 to several thousand megabases depending on plant species. In contrast, the chloroplast genome is typically made up of just 150 thousand base pairs into which 120 genes are tightly packed. However, a leaf cell can contain in excess of 100 chloroplasts each with 100 or more copies of the chloroplast genome. Thus, despite representing less than 0.1% of the sequence complexity of the cell, chloroplasts can contribute over 10% of the DNA content. In part because of this, genes located on the chloroplast genome can produce much higher levels of protein than an equivalent single copy gene located on the nuclear genome (up to 300 fold higher). In addition, chloroplasts are excluded from pollen and the chloroplast DNA is only inherited from the pollinated and not the pollinating crop plant. This has made chloroplasts very attractive as "green factories" for producing novel high value proteins, metabolites and bio-polymers, where high levels of gene expression are required. In addition, research groups are interested in improving photosynthetic efficiency by manipulating key proteins such as re-engineering the CO2 fixing RuBisCO large subunit or replacing it with that from plants or algae adapted to different light environments. However, such experiments are currently beyond the capabilities of the existing plastid engineering technology. Chloroplasts encode the ribosomal and tRNAs necessary for supporting the protein synthesis of the coding sequences present on their genome (mostly related to housekeeping functions and photosynthesis), but the majority of proteins found in the chloroplast (including aminoacyl-tRNA synthetases and RNA pol subunits) are encoded on the nuclear genome and imported from the cytoplasm.

Direct transformation of the chloroplast genome was first achieved thirty years ago, but remains technically challenging due to the difficulty in introducing large DNA elements and the problem of ensuring transgenic plastid genomes fully replace the unmodified ones. Similar problems would also exist if attempts to replace large sections of the genome with fully synthetic sequence were to be made.
We have recently addressed two of these problems using a two-component gene drive system and plants engineered to contain a single giant chloroplast instead of >100 smaller ones. Utilising this system, we will construct and test a plastid engineering tool set for 1) the iterative introduction of large cassettes for the introduction of complex biochemical pathways and 2) for carrying out targeted genome rearrangements, ultimately allowing substantial sections of the plastid genome to be replaced with bespoke synthetic versions.

The SYCHLOPS tool kit will utilise an inducible two component gene drive system, giant chloroplasts, phage integrase and a prokaryote transposon system. This will allow efficient integration and selection of plastid transgene cassettes which can be added to in an iterative process. The transposon system will give two recombinase attP sites in the plastome, allowing the replacement of the intervening sequences with synthetic DNA (delivered directly to giant plastids) containing the complementary attB sites at their ends.

Transgenic plastids give high levels of gene expression, transgene containment (maternal inheritance), and direct access to key biochemical pathways. Moreover, with its genome of just 150,000 base pairs, the plastome is an attractive target for synthetic genome engineering. However, chloroplast transformation is inefficient due to three issues; 1) small size of the chloroplast as a biolistic target and limited size of DNA fragments that can be introduced this way, 2) poor selection of transformation events when the introduced DNA constitutes the minority of the chloroplast sequences in a cell, 3) obtaining plants where all wild type plastid genomes are eliminated. For these reasons, more complex genome-wide modifications/replacements are currently not possible. We have addressed these challenges using giant chloroplasts in cells misexpressing FtsZ and a two-component gene-drive system that drives integration and amplification of plastid transgenes. We generate transplastomic plants containing Tn5 transposon boarders and combinations of PhiC31 recombinase attachment (att) sites and we will cross these with plants expressing plastid imported inducible Tn5 transposase and PhiC31 recombinase. We will also improve our gene drive system by placing the plastid Cas9 under a chemically inducible promoter.