Regulation of nucleus-encoded trans-acting factors allows orthogonal fine-tuning of multiple transgenes in the chloroplast of Chlamydomonas reinhardtii.

The green microalga Chlamydomonas reinhardtii is a promising host organism for the production of valuable compounds. Engineering the Chlamydomonas chloroplast genome offers several advantages over the nuclear genome, including targeted gene insertion, lack of silencing mechanisms, potentially higher protein production due to multiple genome copies and natural substrate abundance for metabolic engineering. Tuneable expression systems can be used to minimize competition between heterologous production and host cell viability. However, complex gene regulation and a lack of tight regulatory elements make this a challenge in the Chlamydomonas chloroplast. In this work, we develop two synthetic tuneable systems to control the expression of genes on the chloroplast genome, taking advantage of the properties of the vitamin B-responsive METE promoter and a modified thiamine (vitamin B) riboswitch, along with nucleus-encoded chloroplast-targeted regulatory proteins NAC2 and MRL1. We demonstrate the capacity of these systems for robust, fine-tuned control of several chloroplast transgenes, by addition of nanomolar levels of vitamins. The two systems have been combined in a single strain engineered to avoid effects on photosynthesis and are orthogonal to each other. They were then used to manipulate the production of an industrially relevant diterpenoid, casbene, by introducing and tuning expression of the coding sequence for casbene synthase, as well as regulating the metabolite flux towards casbene precursors, highlighting the utility of these systems for informing metabolic engineering approaches.