Developing the Chlamydomonas Reinhardtii Plastid As a Platform for Directed Evolution of Photosynthetic Proteins

Although plants are essential to human life and industry, their photosynthetic properties are not perfectly efficient. Much effort is being invested to improve photosynthesis, especially crop species. These efforts are often hampered by the plants’ genomic intractability, large space requirements and long generation time. We propose Chlamydomonas reinhardtii, the “green yeast,” as a new platform for high throughput engineering of photosynthetic proteins.

C reinhardtii is a haploid, unicellular alga that has already been developed as a model organism for study of photosynthesis because of its close homology to land plants. Proteins from plants functionally complement the knockout of their algal homologs. The plastome is readily transformed, allowing photosynthetic proteins to be expressed in their native chloroplast environment. Additionally, homologous recombination in the plastome allows protein variants to be readily compared because identical genetic context eliminates the confounding effects traditionally seen with random genomic integration. Because of the alga’s small size and rapid growth, protein variants can be assayed on a scale not feasible in plants. When a plant protein is used to complement an algal knockout, the phenotype will be determined by the plant protein so that the characteristics of that protein can be selected directly. In this way, high throughput directed evolution of plant proteins can be realistically achieved. When improved protein variants are found and characterized in the algal system, they will be validated in land plants.

We present initial progress to create a Golden Gate system for integration and expression of plant genes from the C reinhardtii plastome. The protein Rubisco Activase has been chosen for proof of concept because its lack of thermal stability is recognized as a key limitation for heat tolerance in C4 crops such as maize.