Abstract
Microalgae have recently received attention as a potential low-cost host for the production of recombinant proteins and novel metabolites. However, a major obstacle to the development of algae as an industrial platform has been the poor expression of heterologous genes from the nuclear genome. Here we describe a nuclear expression strategy using the foot-and-mouth-disease-virus 2A self-cleavage peptide to transcriptionally fuse heterologous gene expression to antibiotic resistance in Chlamydomonas reinhardtii. We demonstrate that strains transformed with ble-2A-GFP are zeocin-resistant and accumulate high levels of GFP that is properly ‘cleaved’ at the FMDV 2A peptide resulting in monomeric, cytosolic GFP that is easily detectable by in-gel fluorescence analysis or fluorescent microscopy. Furthermore, we used our ble2A nuclear expression vector to engineer the heterologous expression of the industrial enzyme, xylanase. We demonstrate that linking xyn1 expression to ble2A expression on the same open reading frame led to a dramatic (∼100-fold) increase in xylanase activity in cells lysates compared to the unlinked construct. Finally, by inserting an endogenous secretion signal between the ble2A and xyn1 coding regions, we were able to target monomeric xylanase for secretion. The novel microalgae nuclear expression strategy described here enables the selection of transgenic lines that are efficiently expressing the heterologous gene-of-interest and should prove valuable for basic research as well as algal biotechnology.
Highlights
Microalgae are a diverse group of photosynthetic microorganisms with considerable biotechnological potential
Functional analysis of the 2A peptide in C. reinhardtii To determine whether the foot and mouth disease virus (FMDV) 2A ‘self-cleaving’ peptide could be expressed and is functional in C. reinhardtii, we constructed a nuclear transformation vector in which 2A was placed between the sh-ble bleomycin/zeocin-resistance gene [50] and nuclear codon-optimized GFP [35] (Figure 1B)
We show that FMDV 2A is properly processed by the microalga and that strains transformed with ble2A-GFP are zeocin resistant and accumulate monomeric, cytosolic GFP that is detectable by in-gel fluorescence analysis (Figure 2A), fluorescent microscopy (Figure 2B), immunoblotting and flow cytometry
Summary
Microalgae are a diverse group of photosynthetic microorganisms with considerable biotechnological potential. Microalgae have the potential to be a valuable source of bioenergy [4,5,6]. Transgenic microalgae have the potential to be low-cost bioreactors for commercially valuable recombinant proteins such as therapeutic proteins and industrial enzymes [11,12,13,14]. Genetic engineering of microalgae is still far behind other microorganisms. A major obstacle remains low transgene expression levels from the nuclear genome of many microalgae. We report the robust expression and secretion of a commercially valuable industrial enzyme, xylanase, from the nuclear genome of the microalga Chlamydomonas reinhardtii by linking the xylanase gene directly to an antibiotic resistance gene via the foot and mouth disease virus (FMDV) self cleaving 2A sequence
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