Abstracts Category : Other

Genetic engineering tools for transforming the nucleusand chloroplast of microalgae

by Rodriguez Leopoldo Herrera

Biotechnology of microalgae is a fast-growing fieldand several species have become targets for transgenicmanipulation. Microalgae provide low-cost and scalable productionplatforms for manufacturing recombinant proteins and other highvalue products. However, the exploitation of microalgae asexpression systems is restricted by the low yield of recombinantproteins and the limited availability of tools for the geneticmanipulation of commercially important species. This thesisexplores transgene instability and gene autoregulation as causesfor low recombinant protein accumulation in the chloroplast ofChlamydomonas reinhardtii and describes the isolation of a mutantphytoene desaturase (PDS) gene which confers resistance to theherbicide norflurazon for future use as a selection marker for themarine microalga Dunaliella tertiolecta. Recombination betweenshort dispersed DNA repeats (SDR) found in the chloroplast genomeof C. reinhardtii was identified as a cause of transgeneinstability. The genes coding for -glucuronidase (GUS) andperidinin-chlorophyll binding protein (PCP) were inserted in thechloroplast genome next to the atpB 3 UTR by homologousrecombination. Recombination of a 30bp SDR located within the 3UTR of atpB was identified as the cause of transgene excision inthe transplastomic lines. Such transgene instability was tackled byreplacing the 3 UTR of atpB with the rbcL 3 UTR from D.tertiolecta. Using this 3UTR sequence from a different speciesproduced a photosynthetic strain and prevented excision of thetransgene by SDR recombination in all transfomants. Very low levelsof recombinant GUS and PCP accumulated in chloroplast transformantswhen using the psbD 5 regulatory region to drive their expression.To address low levels of accumulation caused by regulatory pathwaysthat inhibit transgene expression, I have engineered thechloroplast genome of a non-photosynthetic atpB mutant of C.reinhardtii by replacing the endogenous psbD promoter and 5UTRwith the promoter and 5UTR of psbA. The engineered strain wassubsequently transformed with the wildtype atpB and two differentreporter genes driven by the psbD regulatory regions: gusA and kat,which code for GUS and the fluorescent protein Katushkarespectively. Analysis of the transformants showed thataccumulation of recombinant proteins in our engineered strain was10 to 20 fold higher than in the nonengineered cells. Most of theselectable markers used in plants and algae are inefficient inDunaliella, which is naturally resistant to many of the antibioticsused for the selection of transformants. Norflurazon inhibits PDS,an essential enzyme for carotenoid biosynthesis. Using forwardgenetics I have isolated, sequenced and characterised mutant PDSalleles conferring norflurazon resistance in D. tertiolecta.Independent mutations in pds, leading to substitutions R265C,S472L, S472F and L502F, all result in high resistance tonorflurazon but differ in sensitivity to other bleachingherbicides. By mapping the four amino acidAdvisors/Committee Members: DAY, ANIL A, Curtis, Robin, Day, Anil.