title: Characterization of new parts for synthetic biology applications in bacteria and yeast supported by automated methods
Creators: name: Zoll, Thomas
Publisher: name: UNSPECIFIED
Year: 2023

description: The recently presented IPCC report once again urges us to intensify efforts to build a sustainable economy. The UN also emphasizes that most of the economy still operates in a linear model and urgently needs to be reformed. Biotechnological applications can offer a valuable contribution, as they can replace energyintensive processes in the chemical industry. In addition, they have the charm that the growth of the used organisms is not dependent on crude oil sources. Further, they can use inexpensive energy sources from industrial by-products as energy sources. The oil yeast Yarrowia lipolytica in particular is emerging as a valuable workhorse for industry and science. Due to the natural accumulation of lipids, the yeast represents an interesting production host for industrial applications. To date, unfortunately, only a limited repertoire of metabolic engineering approaches or SynBio circuits is available for yeast. This thesis aims, among other things, to expand the portfolio of SynBio parts for this yeast. To this end, chapter 3 presents ways to further exploit the organism. Thus, a broad-hostrange plasmid (section 3.3.1) was developed, which allows rapid testing of constructs in three organisms. This plasmid could be used to test a tetracycline aptamer in Y. lipolytica (section 3.3.2). The aptamer was previously developed for Saccharomyces cerevisiae and could be successfully used for translation inactivation. Y. lipolytica was successfully modified with a monomer of the aptamer. Unfortunately, growth assays revealedthat the strain carrying the aptamer was severely impaired in fitness. Thus, a clear conclusion about the functionality was not possible. An expansion of the repertoire of Y. lipolytica and other organisms was pursued by establishing artificial landing pads (section 3.3.3). The computer-generated DNA sequences were designed to cause as little offtarget effects as possible when using Cas9 in the organisms Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica. To this end, up to 25 artificial protospacers were lined up in a sequence up to 900 bp long. These were successfully integrated in the genome of the organisms. Experiments with CRISPRi and CRISPRa served as proof-of-concept. By addressing different protospacers, different expression levels should be achieved depending on the distance between protospacer and reporter gene. Fine-graded activation of expression was demonstrated in S. cerevisiae. The use of CRISPRa increased the basal activity of the minimal promoter by 3.4-fold. In Y. lipolytica, there was no clear gradient as in S. cerevisiae. The effect of CRISPRi in B. subtilis was dependent on the promoter used. Automation methods were developed to support the molecular biology and microbiology work (chapter 4). Due to a clearly visible edge effect when using the incubator of the CompuGene Robotics platform, a method was developed to randomize the samples in the microtiter plates (section 4.4). Here it was shown that this method cannot eliminate the edge effect, but it can improve comparability between samples. In addition, a method was developed to use the liquid handler of the automation platform to pick colonies from agar plates (section 4.5).