PUB - Publikationen an der Universität Bielefeld

**SUMMARY**
In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycistronic mRNAs inChlamydomonas reinhardtiinatively carrying multiple co‐expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expressionin vivoto drive successful co‐expression of several terpene synthases with the selection markeraphVIII. Bicistronic transgene design resulted in significantly increased (E)‐α‐bisabolene production of 7.95 mg L⁻¹from a single open reading frame, 18.1× fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high‐level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to theaphVIIImarker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co‐expression from the algal nuclear genome.

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**Significance Statement**
The designed expression concepts enable reliable and robust transgene expression from the nuclear genome ofChlamydomonas reinhardtiiand will promote the use of this microalga as a green cell factory. This work is highly valuable to the greater synthetic biology research community and may have implications for other green microalgae as well.