title: Pilot-scale cultivation of wall-deficient transgenic Chlamydomonas reinhardtii strains expressing recombinant proteins in the chloroplast.
abstract: Microalgae have emerged as potentially powerful platforms for the production of recombinant proteins and high-value products. Chlamydomonas reinhardtii is a potentially important host species due to the range of genetic tools that have been developed for this unicellular green alga. Transformation of the chloroplast genome offers important advantages over nuclear transformation, and a wide range of recombinant proteins have now been expressed in the chloroplasts of C. reinhardtii strains. This is often done in cell wall-deficient mutants that are easier to transform. However, only a single study has reported growth data for C. reinhardtii grown at pilot scale, and the growth of cell wall-deficient strains has not been reported at all. Here, we report the first pilot-scale growth study for transgenic, cell wall-deficient C. reinhardtii strains. Strains expressing a cytochrome P450 (CYP79A1) or bifunctional diterpene synthase (cis-abienol synthase, TPS4) were grown for 7 days under mixotrophic conditions in a Tris-acetate-phosphate medium. The strains reached dry cell weights of 0.3 g/L within 3-4 days with stable expression levels of the recombinant proteins during the whole upscaling process. The strains proved to be generally robust, despite the cell wall-deficient phenotype, but grew poorly under phototrophic conditions. The data indicate that cell wall-deficient strains may be highly amenable for transformation and suitable for commercial-scale operations under mixotrophic growth regimes.
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK.
authors.forename: Julie A Z
authors.initials: JA
authors.lastname: Zedler
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK.
authors.forename: Doris
authors.initials: D
authors.lastname: Gangl
authors.affiliation: A4F-Algae For the Future, S.A., Campus do Lumiar-Edifício E-R/C, Estrada do Paço do Lumiar, 1648-038, Lisbon, Portugal.
authors.forename: Tiago
authors.initials: T
authors.lastname: Guerra
authors.affiliation: A4F-Algae For the Future, S.A., Campus do Lumiar-Edifício E-R/C, Estrada do Paço do Lumiar, 1648-038, Lisbon, Portugal.
authors.forename: Edgar
authors.initials: E
authors.lastname: Santos
authors.affiliation: A4F-Algae For the Future, S.A., Campus do Lumiar-Edifício E-R/C, Estrada do Paço do Lumiar, 1648-038, Lisbon, Portugal.
authors.forename: Vitor V
authors.initials: VV
authors.lastname: Verdelho
authors.affiliation: Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK. c.robinson-504@kent.ac.uk.
authors.forename: Colin
authors.initials: C
authors.lastname: Robinson
mesh_terms: D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000254:growth & development; D002736:Chloroplasts / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018014:Gene Transfer Techniques; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase
keywords: CYP79A1; Cell wall-deficient; Chlamydomonas; Microalgae; Pilot scale; cis-abienol synthase