An improved ARS2‐derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas

Abstract

The model alga Chlamydomonas reinhardtii has been used to pioneer genetic engineering techniques for high-value protein and biofuel production from algae. To date, most studies of transgenic Chlamydomonas have utilized the chloroplast genome due to its ease of engineering, with a sizeable suite of reporters and well-characterized expression constructs. The advanced manipulation of algal nuclear genomes has been hampered by limited strong expression cassettes, and a lack of high-throughput reporters. We have improved upon an endogenous reporter gene - the ARS2 gene encoding an arylsulfatase enzyme - that was first cloned and characterized decades ago but has not been used extensively. The new construct, derived from ARS2 cDNA, expresses significantly higher levels of reporter protein and transforms more efficiently, allowing qualitative and quantitative screening using a rapid, inexpensive 96-well assay. The improved arylsulfatase expression cassette was used to screen a new transgene promoter from the ARG7 gene, and found that the ARG7 promoter can express the ARS2 reporter as strongly as the HSP70-RBCS2 chimeric promoter that currently ranks as the best available promoter, thus adding to the list of useful nuclear promoters. This enhanced arylsulfatase reporter construct improves the efficiency and ease of genetic engineering within the Chlamydomonas nuclear genome, with potential application to other algal strains.

Identification and Metabolite Profiling of Chemical Activators of Lipid Accumulation in Green Algae

“…Statistical significance was accepted at P , 0.05. Primary screening data were analyzed using HCS-Analyzer, an open-source application for high-content screening (Specht et al, 2015), and chemoinformatics analysis was done using the Bioconductor Chemmine R package (Schaffer, 2003) or Tibco Spotfire Lead Discovery. Structural rendering of the compounds was done using the ChemDraw Professional 14 suite (PerkinElmer).…”

Synthetic promoters capable of driving robust nuclear gene expression in the green alga Chlamydomonas reinhardtii

“…Synthetic promoters were cloned with the RBCS2 5'UTR, which was amplified with appropriate primers to allow 15 bp overhangs with the synthetic promoters as well the digested backbone (Table S8), resulting in the constructs in Figure 1b. To rearrange sap11 with the hygromycin cassette downstream of the mCherry cassette, each half of pBR4 was amplified with appropriate primers for USER cloning into the HCR1, a modified pBlueScript II (Agilent, Santa Clara, CA), as previously described (Specht et al, 2015) (Table S8). The rearranged construct was then digested with NdeI and XbaI to remove ar1 and replace it with sap11 which was PCR amplified and SliCE cloned into the rearranged pBR4.…”

Genetic tools and techniques for Chlamydomonas reinhardtii

“…Potential reasons are biased codon usage ), epigenetic transgene silencing (Cerutti et al 1997), positional effects and chromatin structure (Specht et al 2014;Strenkert et al 2011), aberrant processing, lack of suitable regulatory sequences, or potentially further currently unknown mechanisms (Fuhrmann et al 1999). Efficient transgene expression strongly relies on non-coding, cis-acting elements (Barnes et al 2005;Lumbreras et al 1998;Sizova et al 2001), and several elements promoting stable transgene expression have been identified.…”

“…For this reason, we selected this hybrid system to develop a versatile, modular vector tool for nuclear encoded recombinant protein production (Lauersen et al 2013;Lauersen et al 2015). A 1425-bp promoter/5′-UTR region upstream of the endogenous ARG7 gene was recently demonstrated to convey promoter activity of similar strength as the hybrid HSP70A-RBCS2 promoter in the context of an ARS2 reporter assay (Specht et al 2014), therefore representing an interesting alternative. Promoter trapping (Haring and Beck 1997) has also been performed and led to the identification of several efficient regulatory promoter sequences (Vila et al 2012).…”