Development of synthetic biology tools and methods for plant systems

Plant synthetic biology is a fast-evolving field in which standardised tools and methods are developed to empower research and commercial bioproduction in plant systems. Nevertheless, in the whole synthetic biology landscape, plant systems lag compared to microbial and mammalian systems. Plant cell cultures are becoming a popular chassis for bioproduction, which can combine the metabolic diversity and capacity of the plants with the benefits of cell cultures as in microbial systems, such as fast growth, resource- and energy-efficient cultivation, and secure containment. My PhD was focused on the establishment of multiple enabling tools and methods to confer complex yet predictive genetic engineering in plant systems. These plant synthetic biology resources are being implemented to development cell-type-specific plant cell biofactories particularly competent in the biosynthesis of specific compounds. Firstly, I have developed Mobius Assembly, a user-friendly Golden Gate Assembly system for fast and easy generation of complex DNA constructs. Mobius Assembly toolkit was adapted for Plant Systems (MAPS). I devised a new category of compact binary vectors, which can be employed for the whole plant, plant cell culture, and plant protoplast transformations. I found that the combination of binary vectors and Agrobacterial strains determine the efficiency of plant cell culture transformation. Secondly, I developed a high-throughput, protoplast expression protocol. Using Mobius Assembly and the new protoplast expression analysis platform, I characterised a library of short promoters and terminators for their regulatory effect on reporter gene expression. The results highlighted the strong influence of terminators in gene expression depending on different promoters; this observation implies a synergistic interaction between terminators and promoters. I further made and optimised standardised parts of three commonly used chemically inducible gene expression systems in plant science and tested them for possible crosstalks when used in conjunction. Lastly, I employed MAPS to create a sequential plant expression system that can be used to activate multiple gene expression at differential levels and timings. As a pilot study, this sequential system was used to express master transcription factors for the differentiation of plant cell cultures into xylem vessels, a cell type that produces a high level of phenolic compounds and thus may serve as biofactories for production of anthocyanin and other phenolics.