Arno Krieger (Universität Bielefeld)
Arno Krieger's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Universität Bielefeld in the year 2024 with the title:
Mitigating transgene silencing in C. reinhardtii – Examining synergisms of transgene silencing factors through genome editing
Krieger generated a series of C. reinhardtii strains using CRISPR/Cas9 mediated genome editing, each carrying different combinations of knock-out mutations with the aim to systematically investigate the function of various silencing factors and potential synergisms between them and to develop a strain which is capable of expressing transgenes on a high level.
The unicellular microalga Chlamydomonas reinhardtii gets growing attention as a viable production host for biotechnology. Its capacity for rapid growth under phototrophic conditions, coupled with a haploid genome and well-established genetic engineering protocols, positions it as a potent and sustainable alternative to heterotrophic hosts for the biotechnological synthesis of valuable compounds. C. reinhardtii offers a great variety of biosynthetic capacities and presents several unique features for commercial exploration. Advancements in metabolic engineering empowered C. reinhardtii to successfully synthesize a diverse array of recombinant proteins and high-value molecules, like algae-based pharmaceuticals, biofuels, and terpenoids.
However, efficient transgene silencing mechanisms pose a challenge for expressing transgenes in C. reinhardtii, limiting its biotechnological potential. In C. reinhardtii, transgene silencing occurs mainly at the chromatin level. This silencing mechanism involves a variety of epigenetic modifications, including histone deacetylation, methylation and phosphorylation and DNA methylation. Although several key factors of transgene silencing were identified, the mechanism and interactions between these factors remain poorly understood.
This thesis aims to prevent transgene silencing in C. reinhardtii by genome editing, to generate strains with robust transgene expression levels. Therefore, several key factors for epigenetic transgene silencing, including SRTA, HLM4, VIG1, Mut9, and DMC5, were knocked out in different combinations, exploiting the CRISPR/Cas9 system. To investigate the transgene expression capabilities of the mutant strains, all strains were transformed with a transgenic reporter gene construct, expressing a patchoulol synthase fused to the fluorescence reporter mVenus. The transgene expression level was analysed by quantification of the fluorescence signal and the patchoulol productivity.
This study demonstrated, that CRISPR/Cas9-based strain engineering is a powerful tool to enhance transgene expression in C. reinhardtii. It was shown that epigenetic transgene silencing mechanisms can be partially prevented by disruption of the underlying key factors. Moreover, it was demonstrated that the combination of these knockouts can synergize and partially yield additive effects in the repression of transgene silencing. Especially, the combination of the SRTA, DMC5 and VIG1 knockout exhibited robust transgene expression, exceeding the patchoulol productivity of its parental strain of up to 400%. Moreover, this project accomplished a collection of 28 knockout strains, offering a foundation to decipher the mechanisms of transgene silencing. Through comparative analysis of the transgene expression capabilities of these knockout strains, hypothetical interactions between several epigenetic modifications and silencing factors could be observed.
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Arno Krieger conducted this work in the working group Algenbiotechnologie and Bioenergie of Prof. Olaf Kruse under supervision from Dr. Alexander Einhaus.
