In the last decades, the industrial or white biotechnology which uses micro-organisms and enzymes for the sustainable production of industrial relevant compounds is on the rise. One
group of such interesting compounds are flavonoids, plant secondary metabolites with promising bioactive properties for treatments against viral and bacterial infections, cancer
and inflammation. As such, these molecules attain huge attention for usage in the human health sector making their secured and defined supply essential. Currently, the industrial
production of these molecules has some inherent drawbacks like low yields using plant extraction and multiple reaction steps, harsh reaction conditions and the difficulty of chiral
centers in chemical synthesis. As such, production of these specialized plant metabolites in microbial cell factories can be a valuable alternative. However, developing suitable
microbial strains with profitable product titers for an industrial environment is challenging, especially due to the difficulty of tuning all steps in a (heterologous) production pathway
and the native metabolism. In this respect, tremendous efforts to enhance the strain development process in the field of metabolic engineering and synthetic biology have been
made. Nevertheless, this still remains a cost – and labor-intensive undertaking, not the least in the attractive eukaryotic host Saccharomyces cerevisiae. To this end, this doctoral
research aimed to develop novel tools to facilitate the alteration of gene expression at the transcriptional and translational level, as such speeding up the construction of yeast cell
factories which was applied here on a naringenin production strain as proof of concept.
The use of characterized, modular regulatory parts and the standardized sharing of biological data plays an indispensable role to transform the synthetic biology field to a mature engineering field. In this respect, the obscure demarcation of yeast’s transcriptional and translational control elements slows down this transformation process in eukaryotes.
As such, novel biological parts on the one hand influencing transcription, i.e. semi-synthetic core promoters, and on the other hand affecting translation, i.e. 5’UTRs with a predictive
outcome on gene expression, were developed. The yeast core promoter is known to be the main determinant of transcription levels, making it an interesting target for modifying
biosynthetic pathways. Additionally, minimal core promoters with equal or better activities
as the cumbersome native yeast promoters could immensely facilitate the assembly of transcription units. Therefore, the well-characterized TEF1 promoter was truncated to
elucidate the minimal length needed for functional gene expression. This minimal sequence served as template for the creation of a core promoter library leading to short, functional
semi-synthetic core promoters which were equally or twice as strong as commonly long yeast promoters. Besides modulating transcription, altering a gene’s translation initiation
rate has proven to work well as a tool to predictably modify gene expression, especially in prokaryotes. Therefore, a similar forward engineering approach was set up in S. cerevisiae by developing a partial least square (PLS) regression model linking 13 5’UTR features with
protein levels. This model was used for the de novo design of 5’UTRs with a predictive outcome on gene expression in different genetic contexts. In vivo testing of these 5’UTR sequences showed a good general applicability of the model since adequate coefficients of determination (R²) were obtained in all experiments. As such, this data-driven algorithm
expands the small toolbox of existing methods for the novel design of biological parts in yeast.
Besides monocistronic regulation, previous studies have shown the possibility of eukaryotic pathway balancing through multicistronic expression. However, this is still a mainly
unexplored tool in S. cerevisiae. To this end, a thorough evaluation of this technique was performed by the usage of T2A peptides enabling ribosome skipping at the end of a coding
sequence and proven to be efficient in different yeast species. Typically, their multiple use in long pathways is hindered because of the risk of unwanted homologous recombination.
To allow this, five T2A sequences were developed differing as much as possible in their nucleotide sequence and evaluated for their effectiveness as a tool for pathway optimization. The T2A peptides, with the exception of one T2A having some lower
reliability, effectively led to spliced proteins. Finally, their performance as real regulatory elements in a polycistronic pathway was tested in the genome for bi-, tri-, and quadcistronic constructs. While all constructs were stably integrated in the genome and for bi and tricistronic expression acceptable protein levels was observed, a complete lack of
expression was noticed for the last positioned protein in the quadcistronic transcription unit. To this end, the usage of multicistronic pathways in baker’s yeast is preferably limited
to bi- and tricistronic expression units.
To show the ability of S. cerevisiae as an industrial host for the biosynthesis of specialty metabolites, the S288c wild-type yeast was transformed into a cell factory for naringenin
production. To do so, cutting-edge synthetic biology tools such as CRISPR/Cas9 and the versatile genetic assembly system (VEGAS) were used. Yeast’s native metabolism was
rewired to enhance the supply of flavonoid precursors phenylalanine, tyrosine and malonylCoA. First, the improvement of the phenylalanine and tyrosine pool was assessed indirectly by measuring p-coumaric acid after introduction of its pathway. Next, the augmented
cytosolic malonyl-CoA pool was evaluated by analyzing naringenin titers by introducing the last three genes of the pathway and feeding the strains with p-coumaric acid. Finally, both approaches were combined in the strains with the most promising metabolic backgrounds to produce naringenin de novo from glucose with maximal productivity. Acceptable titers
up to 4.0 mg/l were obtained. However, to reach a full profitable production strain further optimization will be needed. As only the native precursor pools were modified and no finetuning of the naringenin pathway itself was performed yet, the latter looks the most obvious way to be working on as a future perspective to increase final product titers. To this end,
our developed design tool for 5’UTRs was tested for the predictive expression of the Rhodobacter capsulatustal1 gene, converting tyrosine to p-coumaric acid. The initial results
were promising for further pathway optimization in that way that p-coumaric acid titers were proportional with the predicted protein abundance.
In general, several tools were developed and evaluated during this Ph.D. research which could facilitate future development and optimization of S. cerevisiae cell factories. More
specifically, short semi-synthetic core promoters and a forward engineering approach to alter a gene’s translation were constructed. Also the capacity of 2A peptides as a tool for
multicistronic expression in yeast was investigated. Additionally, since the main goal of industrial biotechnology is to set up green production processes for economically relevant
compounds, a naringenin producing yeast strain was created. Overall, this Ph.D. dissertation showed the potential of S. cerevisiae as an interesting host for secondary metabolite
production and contributed to the expansion of the yeast synthetic biology toolbox enabling the reduction of strain development times for future bioprocesses.