Exploring genetic engineering strategies to enable heterologous monoterpenoid production in model microalgae, Chlamydomonas reinhardtii and Phaeodactylum tricornutum

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This thesis focuses on next generation engineering strategies for 𝘊𝘩𝘭𝘢𝘮𝘺𝘥𝘰𝘮𝘰𝘯𝘢𝘴 𝘳𝘦𝘪𝘯𝘩𝘢𝘳𝘥𝘵𝘪𝘪 and 𝘗𝘩𝘢𝘦𝘰𝘥𝘢𝘤𝘵𝘺𝘭𝘶𝘮 𝘵𝘳𝘪𝘤𝘰𝘳𝘯𝘶𝘵𝘶𝘮, exploring aspects at the genomic and phenotypic level, to understand the biochemical implications and potential of heterologous monoterpenoid production in microalgae. 𝗖𝗵𝗮𝗽𝘁𝗲𝗿 𝟭 outlines the ecological, and biotechnological relevance of microalgae, in the context of genetic engineering strategies for heterologous monoterpenoid production. 𝗖𝗵𝗮𝗽𝘁𝗲𝗿 𝟮 investigates different strategies for delivering CRISPR-Cas9 ribonucleoprotein (RNP) into 𝘊. 𝘳𝘦𝘪𝘯𝘩𝘢𝘳𝘥𝘵𝘪𝘪 for targeted genome editing. This study highlighted major bottlenecks in CRISPR-Cas9 genome editing in this species, specifically low delivery efficiencies and unreliable endogenous markers. 𝗖𝗵𝗮𝗽𝘁𝗲𝗿 𝟯 explores extrachromosomal expression (EE) and randomly integrated chromosomal expression (RICE) strategies to genetically engineer 𝘗. 𝘵𝘳𝘪𝘤𝘰𝘳𝘯𝘶𝘵𝘶𝘮 to express 𝘊𝘢𝘵𝘩𝘢𝘳𝘢𝘯𝘵𝘩𝘶𝘴 𝘳𝘰𝘴𝘦𝘶𝘴 geraniol synthase (GES) for production of the monoterpenoid, geraniol. We identified superior RICE geraniol-yielding strains by developing a high-throughput phenotyping analysis and used long-read whole genome sequencing to interrogate the genomes of highly expressing cell lines. This revealed precise integration locations and unexpectedly large concatenated arrangements. We also demonstrated that exogenous DNA designed for EE does not inadvertently integrate into the nuclear genome. 𝗖𝗵𝗮𝗽𝘁𝗲𝗿 𝟰 investigates CRISPR-Cas9 mediated targeted integration (TGI) for geraniol production in 𝘗. 𝘵𝘳𝘪𝘤𝘰𝘳𝘯𝘶𝘵𝘶𝘮 in the genomic loci identified in Chapter 3. We showed that CRISPR-Cas9 RNP delivery is still inefficient in this species and that the recently described endogenous marker gene uridine-5’-monophosphate synthase (𝘜𝘔𝘗𝘚) is unreliable in 𝘗. 𝘵𝘳𝘪𝘤𝘰𝘳𝘯𝘶𝘵𝘶𝘮, due to the highly mutagenic effect of 5-fluoroorotic acid, the selectable agent required to screen 𝘜𝘔𝘗𝘚 knock-out mutants. 𝗖𝗵𝗮𝗽𝘁𝗲𝗿 𝟱 explores metabolic engineering approaches for increasing heterologous geraniol production in 𝘗. 𝘵𝘳𝘪𝘤𝘰𝘳𝘯𝘶𝘵𝘶𝘮. We fused two genes encoding adjacent enzymes in geraniol biosynthesis pathway, 𝘎𝘌𝘚 and 𝘈𝘣𝘪𝘦𝘴 𝘨𝘳𝘢𝘯𝘥𝘪𝘪 geranyl pyrophosphate synthase, and showed that this approach decreased geraniol production, while constitutive expression of 𝘎𝘌𝘚 using a strong promoter resulted in a three times increased geraniol production. We used these strains to demonstrate that heterologous geraniol production in 𝘗. 𝘵𝘳𝘪𝘤𝘰𝘳𝘯𝘶𝘵𝘶𝘮 did not perturb the native biosynthesis of major sterols and pigments. 𝗖𝗵𝗮𝗽𝘁𝗲𝗿 𝟲 discusses why these findings are important for (1) providing insight as to why CRISPR-Cas9-based editing is still difficult to achieve in microalgae (2) improving 𝘗. 𝘵𝘳𝘪𝘤𝘰𝘳𝘯𝘶𝘵𝘶𝘮’𝘴 status for heterologous terpenoid production with regard to its metabolic flexibility and capacity for high geraniol accumulation (3) characterising both well-established and new genetic engineering tools, including uncovering putative safe harbour loci for TGI required for developing more complex synthetic biology approaches in 𝘗. 𝘵𝘳𝘪𝘤𝘰𝘳𝘯𝘶𝘵𝘶𝘮.
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