Chloroplast genetic engineering in the microalga Chlamydomonas reinhardtii: molecular tools and applications.
Doctoral thesis, UCL (University College London).
Microalgae such as the unicellular chlorophycean Chlamydomonas reinhardtii represent attractive biological systems for industrial biotechnology programmes aimed at producing novel metabolites such as biofuels and nutraceuticals, or high value therapeutic proteins. Of particular interest is the genetic engineering of the chloroplast genome (=plastome) since this organelle is the site of numerous important biosynthetic pathways. Furthermore, the targeted introduction and high level expression of multiple foreign genes in the plastome is (in principle) feasible. However, current progress in the genetic manipulation of the C. reinhardtii plastome is hindered by a lack of advanced molecular tools, and a limited understanding of factors allowing optimised expression of foreign genes. Consequently, there have been only a few reports of successful metabolic engineering of the chloroplast, or researchers achieving protein production levels of more than a few percent of total soluble protein. In this thesis, I describe: i) efforts to manipulate the isoprenoid biosynthesis pathway through the introduction of various novel enzymes; ii) my research on the development of new expression vectors, a new recipient strain and a new selectable marker for chloroplast transformation in C. reinhardtii. In the first part of the work, biotechnological applications of chloroplast metabolic engineering were explored by introducing genes encoding enzymes involved in isoprenoid biosynthesis into the plastome and analysing the carotenoid profile of the transgenic lines. The enzymes chosen were the archaeal phytoene synthase from Sulfolobus and the β-carotene ketolase from the chlorophyte algae Muriella zofingiensis and Haematococcus pluvialis. Transgenic lines were generated successfully for all the constructs and molecular analysis confirmed the correct integration, homoplasmy and the transcriptions of the foreign genes. HPLC analysis failed to detect any changes in carotenoid levels or any novel ketocarotenoids produced in the ketolase transformants. In addition, no ketolase protein could be detected by Western blot analysis, suggesting that no active enzyme was accumulating in the lines. Overall, these findings illustrate the challenges of manipulating chloroplast metabolism to create designer algae and suggested that new molecular tools are needed. In the second part of the work, a series of chloroplast expression vectors were constructed that differ in the endogenous promoter/5’ untranslated region (5’UTR) used to drive expression of the foreign gene. Each vector also carries the essential photosynthetic gene psbH, allowing this gene to be used as a selectable marker in which \DeltapsbH recipient cells are rescued to photoautotrophic growth. Five promoter/5’UTR elements were tested with the aim of identifying which gave the highest expression level, and also which were functional in E. coli thereby allowing the plasmids to be used as dual E. coli/C. reinhardtii vectors. Expression of the zeocin-resistance gene BLE in E. coli and a synthetic gene for human growth hormone in C. reinhardtii revealed that all vectors support some level of expression in both the bacterium and the chloroplast, but that the pSSapI vector containing the promoter/5’UTR from psaA is the most suitable as a dual expression vector. DNA delivery into the chloroplast compartment is typically achieved using microparticle bombardment, although agitation of a cell/DNA suspension with glass beads has previously been reported as a simpler alternative method. Since this latter method requires the prior removal of the cell wall, either by enzymatic digestion or mutation, a new \DeltapsbH recipient strain (TN72) was created using a cell wall-deficient (cw15) mutant. In addition, the disruption of psbH in TN72 was designed to avoid the occurrence of ‘marker only’ transformants in which a functional copy of psbH integrates into the plastome, but the foreign gene-of-interest does not. Testing of the new strain showed that transformant lines could be generated quickly and easily using the glass bead method. Sophisticated engineering of the C. reinhardtii plastome is currently limited by a lack of different selectable markers. The TN72/glass bead system was therefore used to test whether the bacterial gene (cat) encoding chloramphenicol acetyltransferase could be developed as a new marker. Transgenic lines were successfully generated and molecular analysis showed that all contained the cat gene construct, thereby validating the design of the new recipient host. Unfortunately, enhanced resistance to chloramphenicol was not observed in the lines, possibly because of a secondary effect of chloramphenicol on mitochondrial ribosome function.
|Title:||Chloroplast genetic engineering in the microalga Chlamydomonas reinhardtii: molecular tools and applications|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
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