title: High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.
abstract: Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n, wherein n = 10 or 20]. The yields of the (SP)n-fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP)nproteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)nglycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)nglycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins.
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Frederiksberg C, Copenhagen, Denmark.
authors.forename: Erick Miguel
authors.initials: EM
authors.lastname: Ramos-Martinez
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Frederiksberg C, Copenhagen, Denmark.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Fimognari
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Frederiksberg C, Copenhagen, Denmark.
authors.forename: Yumiko
authors.identifier: http://orcid.org/0000-0002-9405-5197
authors.initials: Y
authors.lastname: Sakuragi
mesh_terms: D001709:Biotechnology; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media; D017930:Genes, Reporter; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D003470:Culture Media; D006023:Glycoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D045726:Metalloproteases
keywords: 
  C. reinhardtii
  ; glycomodule; protein secretion; signal sequence; yellow fluorescent protein