Good News for Nuclear Transgene Expression in Chlamydomonas

Abstract

Chlamydomonas reinhardtii is a well-established model system for basic research questions ranging from photosynthesis and organelle biogenesis, to the biology of cilia and basal bodies, to channelrhodopsins and photoreceptors. More recently, Chlamydomonas has also been recognized as a suitable host for the production of high-value chemicals and high-value recombinant proteins. However, basic and applied research have suffered from the inefficient expression of nuclear transgenes. The combined efforts of the Chlamydomonas community over the past decades have provided insights into the mechanisms underlying this phenomenon and have resulted in mutant strains defective in some silencing mechanisms. Moreover, many insights have been gained into the parameters that affect nuclear transgene expression, like promoters, introns, codon usage, or terminators. Here I critically review these insights and try to integrate them into design suggestions for the construction of nuclear transgenes that are to be expressed at high levels.

Overexpression of sedoheptulose-1,7-bisphosphatase enhances photosynthesis inChlamydomonas reinhardtiiand has no effect on the abundance of other Calvin-Benson cycle enzymes

“…We chose to 178 use the genomic version of the gene including all seven exons and six introns to adapt it to the MoClo 179 syntax (Weber et al, 2011;Patron et al, 2015). For this, we followed the protocol suggested previously 180 (Schroda, 2019), which required two PCR amplifications to alter sequences around the start and stop 181 codons and to remove an internal BpiI recognition site ( Figure 1A). Using the Chlamydomonas MoClo 182 toolkit (Crozet et al, 2018), the domesticated SBP1 gene was equipped with the strong constitutive 183…”

“…It is likely that the 323 screening of more transformants would have allowed recovering lines with even higher expression 324 levels. Furthermore, a SBP1 gene re-synthesized with optimal codon usage and the three RBCS2 introns 325 probably would have allowed higher expression levels (Barahimipour et al, 2015;Schroda, 2019). By 326 combining the MoClo strategy with Chlamydomonas as a model, a complete cycle of the iterative 327 process of construct design and assembly, transformation, screening, and phenotype test can be 328 achieved in as little as 6 weeks.…”

A recombineering pipeline to clone large and complex genes in Chlamydomonas

“…PCR-based cloning from gDNA presents its own challenges and limitations that are particularly problematic when working with Chlamydomonas nuclear genes, which generally have a high GC content (68% in coding regions), contain one or more introns and can include complex repeating regions (Merchant et al, 2007). On the other hand, cloning from complementary DNA can result in low or no expression of target genes most likely due to lack of introns and lack of regulatory elements (Lumbreras et al, 1998;Schroda, 2019). Some of the challenges associated with PCR-based cloning can be circumvented via gene synthesis, however in many cases the need to include introns, high GC content and gene complexity results in synthesis failure or is prohibitively expensive.…”

“…Improved Chlamydomonas target gene and foreign gene (collectively transgenes) expression (e.g., GFP) have been achieved through strain optimization (Neupert et al, 2009), the development of systems with linked transgene and antibiotic resistance gene expression (Rasala et al, 2012;Onishi and Pringle, 2016) and an advanced understanding of transgene silencing (reviewed in Schroda, 2019). Furthermore, release of the Chlamydomonas Golden Gate based Modular Cloning kit has provided a cloning framework and selection of genetic elements to enable labs to rapidly assemble and test transgene constructs (Crozet et al, 2018).…”

“…Furthermore, release of the Chlamydomonas Golden Gate based Modular Cloning kit has provided a cloning framework and selection of genetic elements to enable labs to rapidly assemble and test transgene constructs (Crozet et al, 2018). Independent of background strain and expression system, it is now clear that inserting or maintaining introns, correct codon usage and promoter sequence are all critical for robust transgene expression (Barahimipour et al, 2015;López-Paz et al, 2017;Baier et al, 2018;Weiner et al, 2018;Schroda, 2019). These considerations have made the cloning of Chlamydomonas target genes directly from gDNA the community standard for mutant complementation and fluorescent protein tagging.…”