Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae.

Ana Molina-Márquez,Marta Vila,Federico García-Maroto,Emilio Fernández,Rocío Rengel,Rosa León,Javier Vigara

doi:10.3390/ijms21030718

Abstract

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3′-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

Highlights

Microalgae are a heterogeneous group of photosynthetic microorganisms highly attractive for the production of many different interesting metabolites
We have demonstrated that screening of the transformants with increasing amounts of the antibiotic paromomycin provides a simple method for the selection of clones with the highest level of expression of APHVIII gene, and of the gene of interest, among the obtained transformants
We found that the increase in the transformation efficiency with the new Phyco69 plasmid is due to the fact that in Phyco69 the APHVIII gene is in the same reading frame than the initiation codon, which is immediately before the RbcS2 intron

Summary

Introduction

Microalgae are a heterogeneous group of photosynthetic microorganisms highly attractive for the production of many different interesting metabolites. The high cost of microalgal culture systems and the low productivity of many of these compounds have hampered their commercial applications. Competition with synthetic products or with those obtained from other sources makes the production of microalgal-based compounds non-viable from the economical point of view [2,3,4,5,6]. More attention is being paid to the genetic engineering of microalgae as a potential tool to achieve economically feasible production of bulk materials and to enhance the productivity of the high added value ones [7,8,9]. Low nuclear expression of transgenes in microalgae has hampered the efficient and stable engineering of metabolic pathways and has limited the use of these microorganisms for the commercial production of recombinant proteins. Nuclear expression provides the recombinant proteins with desirable characteristics, which cannot be acquired if they are expressed in the chloroplasts, such as post-translational modifications or targeting of the protein products to different organelles [15,16]

Methods

Results

Conclusion