Optimizing nuclear transgene expression and genome editing for high-value compound production in the green microalga Chlamydomonas reinhardtii

Heterologous production of valuable compounds like terpenoids is essential to overcome low production in their native hosts and to avoid harsh extraction methods, which produce large amounts of toxic waste. Various organisms were genetically engineered to express various terpene synthases, however, photosynthetic model organisms like the green alga Chlamydomonas reinhardtii only recently gained interest as sustainable production chassis. This eukaryotic microalga inherits multiple beneficial features for fundamental research as well as industrial applications. Naturally, this organism grows relatively fast with a doubling time of 6-8 hours and also provides a sufficient pool of precursors needed for valuable production mediated by a strong carbon flux towards the methylerythritol phosphate pathway. Since it has a relatively long history in research, many strains and mutants are available, not only for the investigation of fundamental questions, but also for genetic engineering of this host. However, nuclear transgene expression rates remain rather low compared to heterotrophic cultivated bacteria or yeast, displaying a major limitation for the use of the alga in biotechnology. Especially, the complex eukaryotic genome structure and transgene silencing mechanisms interfere with the production of valuable products in this host. Eukaryotic gene expression involves numerous protein factors and regulators, and expression systems applied to prokaryotic production systems, like expression from plasmids as well as polycistronic expression, are not transferable to C. reinhardtii. Development of various tools for cloning expression cassettes, establishing a variety of strong promoters, and using expression-enhancing techniques like codon optimization and intron spreading supports elevating transgene expression rates and therefore production. Additional establishment of targeted genome editing makes these approaches more feasible, however, present transgene silencing and lack of understanding of regulatory expression mechanisms suppress the application of C. reinhardtii in biotechnology. Addressing these limitations, intergenic regions of recently discovered polycistronic loci were tested for biotechnological application of nuclear gene expression in C. reinhardtii. Simultaneous expression of terpene synthases was demonstrated with the potent selection marker aphVIII. By applying this transgene structure, terpenoid production of E-(α)-bisabolene could be enhanced by 3.2-fold compared to monocistronic expressed constructs, and the time-dependent transgene silencing mechanism was suppressed. In a systematic study, the importance of the flanking regions were identified to facilitate bicistronic expression and the aphVIII was demonstrated as a main contributor to bicistronic transgene expression. The investigations could not reveal the mode of action behind bicistronic transgene expression, however, the results suggest mRNA secondary structure as an important key player. Moreover, the interaction between efficient homology directed repair and the length of homologous regions for this repair mechanism was shown. In this work, homology directed repair was improved for CRISPR/Cas9 mediated double strand breaks, developing a guideline of homology arm lengths. This enhanced tool was used to target endogenous high expressed native loci, to investigate their capacity for predictable high gene expression via reporter protein accumulation. Promising candidate loci were tested for terpenoid production and could increase the mean valencene production by 1.7-fold compared to the random insertion transformation method. Additionally, the application of this method was highlighted for in vivo endogenous expression monitoring via fusion of fluorescence reporters to endogenous genes. Despite the revealed changes in target loci mRNA amounts, physiological changes could not be identified. In a third approach to enhance transgene expression levels, known and putative protein factors involved in transgene silencing and time-dependent silencing were disrupted. While performing single, double, and triple knock-outs of these factors, enhanced expression of the applied reporter construct and terpenoid production was confirmed. Simultaneously, the additive effects of different protein factors were determined, and the long-term silencing could be prevented. Additionally, the proportion of high expressing transformants towards the transformant population was drastically enlarged, allowing easier screening. In order to preserve valuable selection markers for subsequent transformations, the most promising strain was recreated using the newly developed SpecR split intein tool for targeting two loci. Concluding all findings of this thesis, multiple novel tools were established for genetic engineering, including bicistronic expression, targeted integration, and development of a domesticated strain for elevated gene expression levels and enhanced valuable terpenoid production. A combination of these tools with already existing engineering tools for C. reinhardtii is the key to powerful production in sustainable biotechnology. This work provides guidelines for enhanced transgene expression, presents strains for advanced genetic engineering approaches, and gains insights into various expression regulation mechanisms in this alga.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2025

Author dc:creator

• Jacobebbinghaus, Nick

Repository record source_url

https://pub.uni-bielefeld.de/record/3005669

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:3005669