Zedler Julie A Z, Gangl Doris, Guerra Tiago, Santos Edgar, Verdelho Vitor V, Robinson Colin
Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK.
A4F-Algae For the Future, S.A., Campus do Lumiar-Edifício E-R/C, Estrada do Paço do Lumiar, 1648-038, Lisbon, Portugal.
Appl Microbiol Biotechnol. 2016 Aug;100(16):7061-70. doi: 10.1007/s00253-016-7430-y. Epub 2016 Mar 12.
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.
微藻已成为生产重组蛋白和高价值产品的潜在强大平台。莱茵衣藻是一种潜在的重要宿主物种，因为已为这种单细胞绿藻开发了一系列遗传工具。叶绿体基因组转化相对于核转化具有重要优势，现在已有多种重组蛋白在莱茵衣藻菌株的叶绿体中表达。这通常在细胞壁缺陷型突变体中进行，这些突变体更易于转化。然而，仅有一项研究报道了莱茵衣藻中试规模生长的数据，而细胞壁缺陷型菌株的生长情况则完全没有报道。在此，我们报告了转基因、细胞壁缺陷型莱茵衣藻菌株的首次中试规模生长研究。表达细胞色素P450（CYP79A1）或双功能二萜合酶（顺式枞香醇合酶，TPS4）的菌株在磷酸三乙酸盐培养基中混合营养条件下培养7天。这些菌株在3-4天内达到了0.3 g/L的干细胞重量，并且在整个放大培养过程中重组蛋白表达水平稳定。尽管具有细胞壁缺陷型表型，但这些菌株总体上表现出较强的适应性，不过在光养条件下生长较差。数据表明，细胞壁缺陷型菌株可能非常易于转化，并且适合在混合营养生长模式下进行商业规模的操作。
