title: Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide.
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.
authors.affiliation: The San Diego Center for Algae Biotechnology and Division of Molecular Biology, University of California San Diego, San Diego, California, United States of America.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Rasala
authors.forename: Philip A
authors.initials: PA
authors.lastname: Lee
authors.forename: Zhouxin
authors.initials: Z
authors.lastname: Shen
authors.forename: Steven P
authors.initials: SP
authors.lastname: Briggs
authors.forename: Michael
authors.initials: M
authors.lastname: Mendez
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Mayfield
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014995:Xylosidases