Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit.

Kyle J Lauersen,Jan H Mussgnug,Olaf Kruse

doi:10.1007/s00253-014-6354-7

Kyle J Lauersen, Jan H Mussgnug + Show 1 more

Open Access

https://doi.org/10.1007/s00253-014-6354-7

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Abstract

We present a versatile vector toolkit for nuclear transgene expression in the model green microalga Chlamydomonas reinhardtii. The vector was designed in a modular fashion which allows quick replacement of regulatory elements and genes of interest. The current toolkit comprises two antibiotic resistance markers (paromomycin and hygromycin B), five codon-optimized light emission reporters, including the Gaussia princeps luciferase, as well as bright cyan, green, yellow, and red fluorescent protein variants. The system has demonstrated robust functional flexibility with signal options to target the protein of interest to the cytoplasm, the nucleus, cellular microbodies, the chloroplast, mitochondria, or via the endoplasmic reticulum-Golgi apparatus secretory pathway into the culture medium. Successful fluorescent reporter protein fusion to C. reinhardtii Rubisco small subunit 1 was accomplished with this system. Localization of the fluorescently tagged protein was observed in the chloroplast pyrenoid via live cell fluorescence microscopy, the first report of heterologous protein localization to this cellular structure. The functionalities of the vector toolkit, the individual modular elements, as well as several combinations thereof are demonstrated in this manuscript. Due to its strategic design, this vector system can quickly be adapted to individual tasks and should therefore be of great use to address specific scientific questions requiring nuclear recombinant protein expression in C. reinhardtii.

Highlights

Microalgae are a heterogeneous group of organisms, including photosynthetic proand eukaryotes which are sources of biotechnologically relevant products (Hallmann2007; Wijffels et al 2013)
The heat shock 70A-Rubisco small subunit 2 fusion promoter with Rubisco small subunit intron 1 and RBCS2 3’ untranslated region (3’ UTR) were chosen as regulatory elements for both cassettes, because these elements resulted in efficient transgene expression in previous work
Vector pSI103 was constructed by combining the gene expression cassette of the Sh ble resistance gene (Lumbreras et al 1998), containing the RBCS2 promoter, with the HSP70A promoter, RBCS2 intron 1 (HSP70A-RBCS2-i1), and the aphVIII gene to confer resistance to paromomycin (Sizova et al 2001)

Summary

Introduction

Microalgae are a heterogeneous group of organisms, including photosynthetic proand eukaryotes which are sources of biotechnologically relevant products (Hallmann2007; Wijffels et al 2013). Microalgae are a heterogeneous group of organisms, including photosynthetic proand eukaryotes which are sources of biotechnologically relevant products Growth and inexpensive cultivation with sunlight energy in simple mineral salt solutions make microalgae attractive as sustainable sources of a vast array of natural bio-products with many application potentials. Genetic manipulation has been discussed for several decades as the means to achieve some of the ambitious goals of microalgal technologies, including the competitive production of lipids for sustainable solarbased liquid fuels, or the synthesis of complex chemicals which do not occur naturally, by metabolic engineering (Vieler et al 2012; Bogen et al 2013; Liu et al.2013)

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