Advancing chloroplast synthetic biology through high-throughput plastome engineering of Chlamydomonas reinhardtii

Abstract
Chloroplast synthetic biology holds promise for developing improved crops through improving the function of plastids. However, chloroplast engineering efforts face limitations due to the scarcity of genetic tools and the low throughput of plant-based systems. To address these challenges, we here established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology. We developed an automation workflow that enables the generation, handling, and analysis of thousands of transplastomic strains in parallel, expanded the repertoire of selection markers for chloroplast transformation, established new reporter genes, and characterized over 140 regulatory parts, including native and synthetic promoters, UTRs, and intercistronic expression elements. We integrated the system within the Phytobrick cloning standard and demonstrate several applications, including a library-based approach to develop synthetic promoter designs in plastids. Finally, we provide a proof-of-concept for prototyping novel traits in plastids by introducing a chloroplast-based synthetic photorespiration pathway and demonstrating a twofold increase in biomass production. Overall, our study advances chloroplast engineering, and provides a promising platform to rapidly prototype chloroplast manipulations before their transfer into higher plants and crops.

Description
In this study René Inckemann, Tanguy Chotel and colleagues in the lab of Tobias Erb developed a complete expression toolkit for the chloroplast in Chlamydomonas, taking a synthetic biology approach based on a Modular Cloning (MoClo) framework. Prior to this work, no systematic approach had been taken to understand heterologous expression in the Chlamydomonas chloroplast. René and Tanguy developed a high throughput approach to generate and characterize transplastomic lines that were generated by particle bombardment with huge number of synthetic and naturally occurring expression parts. I contributed to this study in the development of fluorescent protein reporters for the chloroplast (mScarlet-I, mCherry, mVenus and mCerulean). We also used vectors developed by René in our recent expression of the Chlorella pyrenoid linker protein (CsLinker) in Chlamydomonas, which you can read about here.

Cite: Inckemann, R. et al. (2024). "Advancing chloroplast synthetic biology through high-throughput plastome engineering of Chlamydomonas reinhardtii" bioRxiv. (2024) https://www.biorxiv.org/content/10.1101/2024.05.08.593163v2