Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Tunable Leaky Ribosomal Scanning Governs Translation of Polycistronic Genes in Green Algae

Authors:

Marco Dueñas* (marco.duenas@berkeley.edu), Jeffrey L. Moseley, Rory J. Craig, Sean D. Gallaher, Sabeeha S. Merchant

Institutions:

University of California–Berkeley

Goals

Facile gene targeting in the nuclear genome of Auxenochlorella protothecoides, a unicellular, freshwater Trebouxiophyte, make this a useful reference organism for discovery and a platform for synthetic biology. Researchers aim to expand the molecular genetic toolkit with additional neutral integration sites, transformation markers, regulatory sequences, and reporter genes, along with improving transformation efficiency and developing RNP-mediated gene-editing methods for genome modification. Researchers are employing systems analyses and metabolic modeling approaches to inform engineering of the Calvin Benson cycle for improved photosynthetic carbon fixation, and to identify signaling pathways and regulators responsible for controlling fatty acid and triacylglycerol biosynthesis. Genome modifications predicted from these analyses to increase lipid productivity will be combined with strain engineering to produce cyclopropane fatty acids. Non-photochemical quenching and a regulatory circuit for maintaining photosynthesis under Cu-limitation, both of which are absent in A. protothecoides, will be introduced to improve photosynthetic resilience.

Abstract

Polycistronic genes encode two or more polypeptides on a single mRNA molecule. In prokaryotic, mitochondrial, and plastid genomes, open reading frames (ORFs) are usually clustered via function, and transcription and translation of polycistronic transcripts are coupled. In eukaryotes, canonical gene expression is generally considered exclusively monocistronic since the cap-dependent scanning mechanism of translation initiation limits recognition of multiple ORFs. However, advances in sequencing technology have revealed cases of polycistronic mRNAs in various plants, animals, and fungi. In the process of updating genome annotations for two green algae, Chlamydomonas reinhardtii and Chromochloris zofingensis, long-read Iso-seq sequencing revealed an abundance of polycistronic genes, though the mechanism controlling the synthesis of proteins remained elusive (Gallaher et al 2021). Auxenochlorella protothecoides, an oleaginous Trebouxiophyte that is evolutionarily divergent from both C. reinhardtii and C. zofingensis, is a valuable reference organism for molecular genetics and biotechnology. While assembling and annotating the Auxenochlorella nuclear genome researchers discovered a new set of conserved polycistronic genes. The team also updated the inventory of polycistronic genes in the genome of C. reinhardtii, using available Ribo-seq data to strengthen the assignments, and established high confidence polycistronic gene datasets from both C. reinhardtii and A. protothecoides for investigation. Examination of the structural features of bicistronic genes revealed preferences for short 5’ untranslated regions (UTRs), weak Kozak sequences for the first ORF, and bias against alternative ATG translation start codons in all regions upstream of the second ORF. Auxenochlorella endogenous polycistronic loci were cloned and integrated at a neutral locus by homologous recombination, and the second ORF was swapped with a Venus fluorescent reporter gene. Manipulation of the ORF 1 Kozak sequence altered the expression of the Venus reporter with a weaker Kozak sequence conferred greater expression and a stronger Kozak sequence decreasing expression. Removal of the ORF 1 start codon substantially increased ORF 2 expression. A synthetic polycistronic dual reporter showed inversely adjustable activity of green fluorescent protein (GFP) expressed from ORF 1 and luciferase (LUC) from ORF2, depending on the ORF 1 Kozak strength. The results demonstrate that expression of multiple ORFs in green algal polycistronic transcripts occurs by means of alternative translation initiation and are consistent with leaky ribosome scanning as the most probable mechanism. The design logic behind these polycistronic genes will be implemented in future metabolic engineering projects to co-express and precisely control the stoichiometry of proteins produced by transgenes. In addition, researchers will undertake functional analysis of enigmatic endogenous polycistronic loci.

References

Gallaher, S. D., et al. 2021. “Widespread Polycistronic Gene Expression in Green Algae,” Proceedings of the National Academy of Sciences 118, e2017714118.

Funding Information

This work was supported by the DOE Office of Science, BER program under award no. DE-SC0023027.