The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae.

Ana Molina-Márquez,Ana Borrero,Rosa León,Javier Vigara,Marta Vila

doi:10.3390/metabo9030049

Abstract

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

Highlights

Microalgae have attracted considerable interest for the production of a wide range of compounds of applied interest due to their easy growth, their ability to fix atmospheric CO2, and the valuable metabolites that some species can produce, which include pigments, food supplements, vitamins, antioxidants, polysaccharides, lipids, and other bioactive products [1,2,3]
A synthetic CRTI gene with the codon usage adapted to C. reinhardtii was designed and synthesized by Genescript Co (Piscataway, NJ, USA)
We demonstrated that the expression of the foreign CRTIop, which is not affected by the and microalgae [34]

Summary

Introduction

Microalgae have attracted considerable interest for the production of a wide range of compounds of applied interest due to their easy growth, their ability to fix atmospheric CO2 , and the valuable metabolites that some species can produce, which include pigments, food supplements, vitamins, antioxidants, polysaccharides, lipids, and other bioactive products [1,2,3]. In the last years, there has been an increasing interest in microalgae as a potential source of biofuels [4,5]. Despite the expectations generated, the production of biofuels and other useful compounds from microalgae will not be economically feasible unless the cost of microalgae cultivation and harvesting is lowered and the productivity increased. Genetic manipulation and synthetic biology show great promise to further boost the productivity of microalgae-based compounds [6,7,8,9]. One important issue is the fact that the selection of microalgal transformants depends mainly on the use of antibiotics as selective agents [10]. Antibiotic resistance genes continue to be the most commonly used selectable markers for the genetic manipulation of Metabolites 2019, 9, 49; doi:10.3390/metabo9030049 www.mdpi.com/journal/metabolites

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Results

Conclusion