Bicistronic expression of nuclear transgenes in Chlamydomonas reinhardtii

Abstract

SUMMARYIn 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 in Chlamydomonas reinhardtii natively 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 expression in vivo to drive successful co‐expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)‐α‐bisabolene production of 7.95 mg L−1 from 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 the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co‐expression from the algal nuclear genome.

Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae

“…In light of these findings, the observations described by Onishi and Pringle (51) and Jacobebbinghaus et al (54) are consistent with some contribution from leaky ribosome scanning to bicistronic gene expression in their systems. The bicistronic transgene constructs analyzed in Jacobebbinghaus et al contained multiple ATG 3-mers within ORF 1 reporter and fusion genes, features which we expect would inhibit ORF 2 translation.…”

“…However, our in vivo mutational analyses of endogenous and synthetic bicistronic loci demonstrated that ORF 2 translation was directly influenced by sequence manipulations at the ORF 1 initiation site (Figures 5 and 6), contradicting the IRES hypothesis. We conclude that the inter-ORF sequences do not contain IRES-like elements, but rather 5' UTR length, Kozak-like strength, and the frequency of alternative translation initiation sites upstream of ORF 2 determine the amount of leaky scanning and ability of the ribosome to reach ORF 2, which may explain the limited success in achieving bicistronic gene expression in previous studies (51,54).…”

“…Recently, Jacobebbinghaus et al (54) integrated a series of transgenic constructs expressing bicistronic mRNAs in Chla. reinhardtii, and they observed that the different inter-ORF sequences alone were sufficient to produce dramatic differences in expression of both ORF 1 and ORF 2, as judged by fluorescence activity and transformation efficiency, respectively (54).…”

“…Recently, Jacobebbinghaus et al (54) integrated a series of transgenic constructs expressing bicistronic mRNAs in Chla. reinhardtii, and they observed that the different inter-ORF sequences alone were sufficient to produce dramatic differences in expression of both ORF 1 and ORF 2, as judged by fluorescence activity and transformation efficiency, respectively (54). The authors suggested that specific inter-ORF sequences might produce differences in mRNA secondary structure and folding energy that could influence downstream ORF translation.…”

“…In our previous analysis, prediction software did not uncover any evidence of known IRES within polycistronic loci (27), and experimentally tested IRES from other organisms have historically performed poorly in green algae (51,56). Onishi and Pringle (51) and Jacobebbinghaus et al (54) both reported improved transformation efficiency (presumably via increased aphVIII expression) from truncated versions of inter-ORF sequences that were far too small to contain complex IRES structural elements. Our own observations also argue against IRES-like features in green algae bicistronic genes.…”