Engineering the Chloroplast Genome with Synthetic Biology Tools for Microcompartment Construction

Photosynthetic efficiency of C3 plants suffers from the slow catalytic rate and the reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) with O₂ instead of CO₂, leading to the costly process of photorespiration. Cyanobacteria are able to utilize a form of Rubisco exhibiting a higher catalytic rate by encapsulating the enzyme within a microcompartment known as a carboxysome. Engineering the cyanobacterial CO₂ concentrating mechanism (CCM) into chloroplasts is an approach to enhance photosynthesis in important C3 plants such as soybean, poplar, and Brassica species. Microcompartments may also be used to compartmentalize other biochemical reactions to confer new capabilities on transgenic plants. Achieving such a goal requires improving the technology for expressing multiple proteins from chloroplast operons. We have been developing strategies and vectors for expressing as many as 12 cyanobacterial genes from the chloroplast genome. Our strategy uses parts flanked by two restriction sites on each side where the distal site is a Type-II rare cutter (8bp recognition) and proximal site is TypeIIS (S for shifted cleavage). Usage of Type-IIS restriction enzyme, which cuts outside the recognition site and produces non-palindromic overhang, brings several unique advantages to traditional BioBrick strategy while maintaining its features. First, more than two parts can be inserted in a vector backbone in a single round of cloning. Second, usage of LguI allows to make a ‘scar-less’ 5’UTR-CDS junction or an in-frame joining of protein domains with 1-substitution (as opposed to two in the latest Biobrick standards). Third, the flexibility in the overhang sequence creates advantages similar to Golden Gate cloning. The strategy allows designing of case-specific approaches which can assemble even more number of parts simultaneously. And lastly, it reduces the domestication requirement for parts as it utilizes longer recognition sites for 3 out of 4 of the enzymes.