Biotechnology of microalgae is a fast-growing field and several species have become targets for transgenic manipulation. Microalgae provide low-cost and scalable production platforms for manufacturing recombinant proteins and other high value products. However, the exploitation of microalgae as expression systems is restricted by the low yield of recombinant proteins and the limited availability of tools for the genetic manipulation of commercially important species. This thesis explores transgene instability and gene autoregulation as causes for low recombinant protein accumulation in the chloroplast of Chlamydomonas reinhardtii and describes the isolation of a mutant phytoene desaturase (PDS) gene which confers resistance to the herbicide norflurazon for future use as a selection marker for the marine microalga Dunaliella tertiolecta. Recombination between short dispersed DNA repeats (SDR) found in the chloroplast genome of C. reinhardtii was identified as a cause of transgene instability. The genes coding for β-glucuronidase (GUS) and peridinin-chlorophyll binding protein (PCP) were inserted in the chloroplast genome next to the atpB 3' UTR by homologous recombination. Recombination of a 30bp SDR located within the 3' UTR of atpB was identified as the cause of transgene excision in the transplastomic lines. Such transgene instability was tackled by replacing the 3' UTR of atpB with the rbcL 3' UTR from D. tertiolecta. Using this 3'UTR sequence from a different species produced a photosynthetic strain and prevented excision of the transgene by SDR recombination in all transfomants. Very low levels of recombinant GUS and PCP accumulated in chloroplast transformants when using the psbD 5' regulatory region to drive their expression. To address low levels of accumulation caused by regulatory pathways that inhibit transgene expression, I have engineered the chloroplast genome of a non-photosynthetic atpB mutant of C. reinhardtii by replacing the endogenous psbD promoter and 5'UTR with the promoter and 5'UTR of psbA. The engineered strain was subsequently transformed with the wildtype atpB and two different reporter genes driven by the psbD regulatory regions: gusA and kat, which code for GUS and the fluorescent protein Katushka respectively. Analysis of the transformants showed that accumulation of recombinant proteins in our engineered strain was 10 to 20 fold higher than in the nonengineered cells. Most of the selectable markers used in plants and algae are inefficient in Dunaliella, which is naturally resistant to many of the antibiotics used for the selection of transformants. Norflurazon inhibits PDS, an essential enzyme for carotenoid biosynthesis. Using forward genetics I have isolated, sequenced and characterised mutant PDS alleles conferring norflurazon resistance in D. tertiolecta. Independent mutations in pds, leading to substitutions R265C, S472L, S472F and L502F, all result in high resistance to norflurazon but differ in sensitivity to other bleaching herbicides. By mapping the four amino acid substitutions on 3D models of D. tertiolecta PDS I determined that R265C, S472L, S472F and L502F, cluster together in proximity to a Rossman-like domain and to aminoacids F128 and V469, previously reported to confer norflurazon resistance. This suggests that the mode of action of norflurazon is by competition with flavin adenine dinucleotide (FAD) for its binding site. A unique aspect of the R265C substitution is its negative cross-resistance to diflufenican and beflutamid which could be advantageous for its use as a positive/negative selection marker for transformation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:727988 |
| Date | January 2017 |
| Creators | Herrera Rodriguez, Leopoldo |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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