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

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.

Recent Advances on Key Enzymes of Microbial Origin in the Lycopene Biosynthesis Pathway

“…79 By contrast, the four reactions from phytoene to lycopene are catalyzed by a single enzyme in bacteria and fungi, the so-called bacterial-type phytoene desaturase (encoded by the crtI gene). 80 Bacterial-type PDS with different catalytic specificity can convert phytoene via two-to six-step dehydrogenation reactions to generate different dehydrogenation products. 81 The native phytoene dehydrogenase is a 3-step dehydrogenase that catalyzes the conversion of phytoene to neurosporene in Rhodobacter sphaeroides.…”

“…In cyanobacteria and some plants, the production of lycopene from phytoene requires four different enzymes including phytoene desaturase, resulting in the formation of lycopene, the first colored compound in the carotenoid biosynthesis pathway . By contrast, the four reactions from phytoene to lycopene are catalyzed by a single enzyme in bacteria and fungi, the so-called bacterial-type phytoene desaturase (encoded by the crtI gene) . Bacterial-type PDS with different catalytic specificity can convert phytoene via two- to six-step dehydrogenation reactions to generate different dehydrogenation products .…”

Microalgal lutein biosynthesis: Recent trends and challenges to enhance the lutein content in microalgal cell factories

“…Finally, neurosporene is converted to lycopene by z-carotene desaturase (ZDS), z-carotene isomerase (Z-iso), and carotenoid isomerase (CRTISO) (Cunningham and Gantt, 1998;Liang et al, 2019). This process differs in cyanobacteria, where a single phytoene desaturase (CrtI) enzyme catalyses the phytoene into lycopene (Molina-Maŕquez et al, 2019). Lycopene is usually the bright color carotenoid found in vegetables such as tomatoes.…”

“…In photosynthetic microorganisms, lycopene is further converted into carotene by cyclization of one or both lycopene ends which is a crucial branching point for carotenoids synthesis. Finally, a-carotene is formed by b-cyclase (LCY-b) and ϵ-cyclase (LCY-e) that is responsible for b and ϵ rings formation at both ends of lycopene and is mainly found in plants and microalgae (Molina-Maŕquez et al, 2019). On the other hand, b-carotene is formed by the action of LCY-b alone, which catalyzed the synthesis of two b-rings at both ends of lycopene.…”