Green microalgae are an important source of natural products such as β-carotene, and have recently become objects of intense study for producing biodiesel and valuable recombinant proteins. Application of chloroplast engineering in microalgae is limited by the availability of tools for genetically engineering the chloroplast of commercially important species. The phytoene desaturase gene of a previously isolated norflurazon tolerant mutant of Chlamydomonas reinhardtii was isolated and sequenced. A thymine to guanine transversion in exon 2 changes codon 131 resulting in a F131V mutation that is located in the NADP binding site domain on the primary structure. This mutation clusters with three conserved amino acids, whose substitution confers norflurazon tolerance in other species, in a pocket on a 3-D structure of the protein. The pocket identifies the target site of norflurazon. The side pocket is at the opening of a tunnel leading to the enzyme's NADP binding site. The mutant gene was cloned and used as marker for glass-bead mediated nuclear transformation of C. reinhardtii using direct selection with 5 μM norflurazon. Integration was by illegitimate recombination and transformants were able to grow in media containing 150 μM norflurazon. Transformants exhibited cross tolerance to fluridone, flurtamone, and diflufenican but were more sensitive to beflubutamid than wildtype. This allows mutant pds gene to act as a dual negative/positive selectable marker that is conditional on the herbicide used. The F131V mutation was introduced into a synthetic gene encoding a Dunaliella salina phytoene desaturase that contained codons used frequently in C. reinhardtii chloroplast genes. The 1.8 kbp CpPDS1 gene was assembled from 74 oligonucleotides by overlap PCR. The coding sequence was inserted into a Dunaliella tertiolecta chloroplast targeting vector that integrated the CpPDS1 sequence into the ycf3-trnL-rbcL region of the plastome. The resulting vector was transformed into D. salina and D. tertiolecta chloroplasts using particle bombardment with plasmid coated gold microprojectiles. Norflurazon tolerant colonies were isolated and the D. salina and D. tertiolecta clones were shown to contain a pds gene integrated in the plastome using PCR analyses. Transformation of the CpPDS1 gene into C. reinhardtii chloroplasts by rescue of an atpB mutation only gave rise to herbicide tolerant colonies if the presequence was removed. Industrial production of algae in large volumes is limited by the availability of light to drive algal growth. This problem was addressed by expressing fluorescent protein Katushka in the chloroplast of C. reinhardtii which converts yellow light to red light. The Katushka gene was transformed into chloroplasts using vector pB10, which was constructed to rescue a deletion in the chloroplast atpB gene in the mutant CC373 strain. The Katushka coding sequence was codon-optimised for expression in chloroplasts and expressed from three different promoter and 5' UTRs (atpA, atpB and psbD). C. reinhardtii wild type cells were able to grow under either blue or red LED lights but grew best when both were present. Wild type cell grew poorly in yellow LED lighting. Cells expressing Katushka in the chloroplast exhibited enhanced autotrophic growth in yellow light and under conditions where yellow light was present and red light was limiting. The improvement in growth was related to the levels of Katushka fluorescence detected in chloroplast transformants, which was highest for the atpA promoter and UTR.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764276 |
| Date | January 2014 |
| Creators | Valencia Suarez, Julio Enrique |
| Contributors | Robson, Geoffrey ; Day, Anil |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/development-of-tools-for-biotechnology-of-microalgae(b94627d0-c6c0-4055-bbaf-06e9cb9c565e).html |
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