UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems

Abstract

In this investigation, we succeeded to generate Chlamydomonas mutants that bear dramatically enhanced ability for transgene expression. To yield these mutants, we utilized DNA methyltransferase deficient strain. These mutants must be useful as a plant cell factory. Chlamydomonas reinhardtii (hereafter Chlamydomonas) is a green freshwater microalga. It is a promising cell factory for the production of recombinant proteins because it rapidly grows in simple salt-based media. However, expression of transgenes integrated into the nuclear genome of Chlamydomonas is very poor, probably because of severe transcriptional silencing irrespective of the genomic position. In this study, we generated Chlamydomonas mutants by ultraviolet (UV)-mediated mutagenesis of maintenance-type DNA methyltransferase gene (MET1)-null mutants to overcome this disadvantage. We obtained several mutants with an enhanced ability to overexpress various transgenes irrespective of their integrated genomic positions. In addition, transformation efficiencies were significantly elevated. Our findings indicate that in addition to mechanisms involving MET1, transgene expression is regulated by a DNA methylation-independent transgene silencing system in Chlamydomonas. This is in agreement with the fact that DNA methylation occurs rarely in this organism. The generated mutants may be useful for the low-cost production of therapeutic proteins and eukaryotic enzymes based on their rapid growth in simple salt-based media.

Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii

“…Such an enhancer could function to recruit the elements of the transcription machinery by either direct transcription factor binding or acting on the epigenetic level, making the insertion site more accessible to regulatory protein binding. It was previously reported that knock out of the met1 gene leads to a globally reduced DNA methylation status, and consequently to higher transgene expression from the nuclear genome of C. reinhardtii ( 13 , 49 ). The strain UVM11, which also exhibits improved nuclear transgene expression capacities, reportedly has an reduced Histone 3 occupancy, increased Histone 4 acetylation and an reduced H3 Lysine 9 monomethylation status at the gene of interest site ( 15 ).…”

“…Although genetic manipulation is well established in C. reinhardtii , the capacity for robust and reliable transgene expression from the nuclear genome, and consequently successful genetic engineering, is limited by its characteristic low transgene expression levels ( 8–11 ). Several attempts have been made to overcome this limitation by: cell line improvement ( 12 , 13 ), design of synthetic promoters ( 14 ), and transgene codon optimization ( 15 , 16 ). However, most reports of robust genetic engineering in C. reinhardtii involve the expression of only reporter proteins with relatively short coding sequences (CDS) ( 15 , 17 ).…”

Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii

“…When the met1 strain was further mutated by UV irradiation, we obtained met1(UVM)-47A and B mutants, which enable robust expression of nuclear transgenes regardless of the genomic position of the insertion, due to disruption of DNA methylation-dependent and -independent silencing systems. Moreover, met1(UVM) mutants were more capable of robustly expressing native, F o r P e e r R e v i e w fluorescent, or heterologous proteins than were other Chlamydomonas strains tested (Kurniasih et al 2016).…”

“…These strains have been used as background strains for protein subcellular localization studies, as host strains for protein secretion, and for the production of terpene squalene (Kajikawa et al 2015, Lauersen et al 2013. However, these UVM strains are still subjected to transgene silencing, probably because the gene silencing mechanisms have not been completely knocked out (Kong et al 2015, Kurniasih et al 2016).…”

Algevir: An Expression System for Microalgae Based on Viral Vectors

“…In the case of nuclear expression, genetic engineering faces two challenges: the improvement of yields and the genetic stability of the transformed clones (Rosales-Mendoza, 2016b). In an effort to overcome these obstacles, UV mutagenesis has been used to generate Chlamydomonas reinhardtii strains with improved transgene expression (showing yields up to 0.2% TSP; Neupert et al, 2009; Kurniasih et al, 2016). Secretion approaches have also been implemented in these mutant strains, leading to yields up to 10 mg of secreted reporter protein per liter of culture (Lauersen et al, 2013).…”