Manipulating the Chlamydomonas chloroplast genome

Abstract

The chloroplast offers an ideal system in which to address fundamental questions about genome structure and function on the one hand, and to develop a sophisticated production platform for high value products on the other. The Chlamydomonas chloroplast genome is one of the most well-characterised and simple system and we have taken advantage of this to carry out extensive engineering. We have established a synthetic biology workflow that allows us rapidly to delete and insert genes without leaving scars, and are using this to determine what would constitute a viable minimal genome as well as to refactor gene clusters. We have also been able to recode chloroplast genes, reducing the number of codons required. This can form the basis of attempts to introduce non-canonical amino acids by reassigning the unused codons. In parallel we have established approaches to optimize metabolic engineering of the chloroplast, taking advantage of the high biosynthetic potential of the organelle and establishing ways to regulate production in a tuneable fashion.