The Development Of Microalgae As A Bioreactor System For The Production Of Recombinant Proteins

摘要: Dunaliella, a genus of unicellular, biflagellate green algae, is one the most studied microalgae for mass culture and commercial importance as source natural -carotene. Dunaliella species have desirable properties halotolerance photoautotrophy that makes their large-scale simple cheap using resources unsuitable conventional agriculture. The ease cost-effectiveness target increased production compounds by metabolic engineering or exploitation biological factories synthesis novel high-value compounds. However, lack efficient genetic transformation systems has been major limitation in manipulation these microalgae. In chapter four we describe development nuclear system tertiolecta. gene encoding phleomycin-binding protein from Streptoalloteichus hindustanus, was chosen selectable marker this retains activity at high salt concentrations. To drive expression marker, two highly expressed tertiolecta RbcS genes associated 5' 3' regulatory regions were isolated characterised (chapter three). cassettes containing promoter terminator flanking ble antibiotic resistance constructed. These tested Chlamydomonas reinhardtii cells found to heterologous system. This study also demonstrated truncation both D. RbcS1 RbcS2 significantly increases C. cells. determine if foreign DNA could stably integrate into genome, methods: microprojectile bombardment, glass bead-mediated transformation, PEG-mediated electroporation number parameters varied. Southern blot analysis revealed plasmid transiently entered following but rapidly degraded. Following electroporation, transformed line recovered. first demonstration stable alga. Chloroplast becoming favoured method recombinant proteins plants, levels are often higher than those achieved transforming nucleus. chloroplast genome not genetically characterised, thus there no existing sequences intergenic be used vectors chloroplast. Therefore, aimed isolate characterise promoters matching terminators capable driving transgene expression, would suitable insertion sites vector construct five). complete sequence psbB rbcL including well coding psbA cloned sequenced. In addition, useful introduced mutations confer herbicide 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU). Two homologous constructs based on mutated developed bombardment. A including: size gold particle, distance plates point discharge, plating onto membranes filter paper, helium pressure, addition an osmoticum medium recovery time. Although transformants recovered study, recombination should prove protocol. The other component investigate use proteins. Transformation reinhardtii, related developed. six, examined human proteins, -lactalbumin IGF-1 reinhardtii. Plasmids upstream cDNAs glass-bead mediated transformation. Transgenic lines generated shown contain transgenes PCR hybridisation. RT- northern hybridisation subsequently demonstrate transcriptionally active. transcripts however, only detected RT-PCR indicating transcribed low levels. Accumulation demonstrated, suggesting although transcribed, they either translated below sensitivity western any produced Previous studies indicated codon usage vital translation protein. Codon modification IGF-I lead accumulation. This reports successful Therefore it now feasible can bioreactor (psbB rbcL) corresponding cassette