MELICOMO - Metabolic Engineering mit lichtkontrollierten Modulen

For the first time in 2018, Zhao et al applied optogenetic modules to regulate biotechnological productions. These tools allowed to switch between growth- and production-phase using light, but focused only on the control of the heterologous biosynthesis. Until this thesis, optimizations of heterologous production were missing to completely acquire the biotechnology as optogenetic field of use. During this thesis, optogenetic switches were developed and applied in Saccharomyces cerevisiae, in context of metabolic engineering. With the combination of light controlled transcription and photosensitive degradation, a new ultrasensitive switch was generated. This synergistic optogenetic multistep control (SOMCo) allowed full control over the activity, even under low blue light intensities. Thanks to the comparison with the well-studied ADH1 promotor, new applications will be easy to discover. For the first time, the impact of light regulated PKA activity, on heterologous biosynthesis, was investigated. This was accomplished by fusing a psd module to the native adenylycyclase Cyr1 or different constructs, expressing the photosensitive adenylyl cyclase bPAC. Both approaches were characterized prior. All these different regulatory modules allowed to improve heterologous processes. With the help of different optogenetic constructs, improved production of β-Carotene, Cordycepin and Betulinic acid was achieved. A different part of this thesis dealt with the generation of a gibberellic acid producing S. cerevisiae strain. Therefore, 8 heterologous genes had to be implemented. To monitor the production, a new in vivo reporter, based on the GA dependent interaction of AtGAI and AtGID1A, was developed. To track this interaction, each component was fused with a different split-Venus sequence, which is able to reassemble with their fusion partners in close proximity. With the help of this newly developed „Gibberellic Acid Venus Reporter” (GAVR), different GA3 concentrations could be detected within the yeast media. Additionally, GAVR lead to the first promising results of successful GA4 production in yeast. Thanks to the development of new light controlled modules, during this thesis, the optogenetic toolbox was enriched with the SOMCo module, the novel bPAC constructs and the GA reporter GAVR. An increased production of β-Carotene from FPP, Betulinic acid from 2,3 Oxidosqualene and Cordycepin from 3’AMP could be reached. Additionally, the GA4 production was visualized. All this was made possible by photosensitive protein degradation, control of a central protein kinase by blue light and a fluorescent reporter for a specific product. The application of these modules allowed acquiring the biotechnology as optogenetic field of application.