Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins

Ashley E Sproles,Francis J Fields,Stephen P Mayfield,Anthony Berndt

doi:10.1007/s00253-022-11790-9

Abstract

The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas, that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products.Key points• A protein expression vector using double-antibiotic resistance genes was designed• Double antibiotic selection causes fewer colonies with more positive for phenotype• Coupling the new vector with FACS improves microalgal screening efficiency > 60%

Highlights

Microalgae, which include photosynthetic eukaryotes and cyanobacteria, hold great potential as a source of commercial bioproducts, and are currently gaining popularity in biotechnology applications
A FLAG-tagged, modified green fluorescent protein (GFP), mClover, derived from the pOpt series of vectors, is used as the gene-of-interest to act as a reporter since it is one of the brightest and most photostable GFP variants codon optimized for use in C. reinhardtii (Lam et al 2012; Lauersen et al 2015)
The higher relative fluorescence units (RFU) of the hyg + zeo-screened transformants only after fluorescence activated cell sorting (FACS) enrichment shows that the double antibiotic screen more efficiently isolates clones with higher expression of mClover

Summary

Introduction

Microalgae, which include photosynthetic eukaryotes and cyanobacteria, hold great potential as a source of commercial bioproducts, and are currently gaining popularity in biotechnology applications. Algae can be grown on non-arable land in open ponds or in bioreactors using nonpotable water, and have the potential to directly sequester CO2 from power plant flue gases to reduce carbon emissions (Khoo et al 2013; Cheah et al 2014). Their biological features allow cultures to quickly reach high densities, producing larger quantities of biomass per area than plants, and can use resources that do not compete with food crops (Dismukes et al 2008; Herrero and Ibáñez 2015; MorenoGarcia et al 2017). Investigations into potential co-products has identified many high-value naturally derived compounds, including proteins, oils, carbohydrates, pigments, and small molecules, that can be sold in a variety of markets, such as specialty chemicals, nutritional supplements, feeds and foods, cosmetics, and pharmaceuticals (León-Bañares et al 2004; Borowitzka 2013; Chew et al 2017)

Methods

Results

Conclusion