Synthetic yeast based cell factories for vanillin-glucoside production

The yeast Saccharomyces cerevisiae is well a characterized microorganism and widely used as eukaryotic model organism as well as a key cell factory for bioproduction of various products. The latter comprise a large variety of scientifically and industrially relevant products such as low-value bulk chemicals and biofuels, food additives, high-value chemicals and recombinant proteins. Despite the recent achievements in the fields of systems biology and metabolic engineering together with availability of broad genetic engineering toolbox, the full potential of S. cerevisiae as a cell factory is not yet exploited. This will require additional insights into functionality of the production system and improved genetic engineering strategies for efficient cell factory design. The aim of this project was to develop novel genetic engineering tools that allow for rapid and efficient assembly of metabolic pathways and controlled expression/overexpression of genes of interest. De novo biosynthetic pathway for vanillin-β-glucoside production was employed as a model system for several case studies in this project.
In order to construct yeast cell factories fulfilling current demands of industrial biotechnology, methods allowing for the introduction of large and complex metabolic pathways need to be added to the existing repertoire. To reduce the number of gene engineering steps required for cell factory construction, a new set of integrative “EasyClone” vectors have been developed in this study. This platform enables simultaneous integration of multiple genes with an option of recycling selection markers. Moreover, EasyClone vectors combine the advantage of efficient uracil-excision reaction based cloning that allows integration of one or two genes per plasmid and Cre-LoxP mediated marker recycling system. As a proof of concept, it was demonstrated that using EasyClone system it is possible to simultaneously integrate three DNA fragments carrying genes encoding for either yellow, cyan or red fluorescent proteins. In addition, all genetic markers were successfully removed using Cre-mediated recombination without compromising production levels of all three fluorescent proteins.
Assembly of multi-enzyme pathways into yeast does not guarantee high production levels per se. Moreover, pathway engineering requires precise control over the genes of interest. In this work, a novel gene amplification system was designed for fast, controlled and efficient gene overexpression in a manner that is based on targeted integration of multiple gene copies into defined loci in the yeast genome. For a proof of concept two genes encoding red and cyan fluorescent proteins were successfully amplified up to ten copies using the developed method. Linear correlation between gene copy number and mean fluorescence intensity for both reporter proteins was observed. The system was compared to multi-copy plasmids based systems and parameters such as expression stability and homogeneity were assessed. Moreover, the gene amplification method was further applied for balancing vanillin-β-glucoside production in S. cerevisiae. It was previously demonstrated that de novo biosynthetic pathway is not capable to efficiently convert its precursor metabolite into vanillin-β-glucoside, which resulted in significant accumulation of several intermediates. Here, the gene amplification system was used to systematically overexpress individual genes or gene combinations of the biosynthetic pathway. Using this strategy, metabolic bottlenecks were identified and the production yield of vanillin-β-glucoside was 6-fold improved.
Several S. cerevisiae strains are commonly used by the yeast community. Among those, the S288c and CEN.PK strain backgrounds have been most frequently applied for metabolic engineering experiments. As a result, these strains have been subjected to extensive comparison studies with respect to genotype and phenotype differences. In this study, it was investigated how strain genetic background affects heterologous production of a given product. For that reason vanillin-β-glucoside biosynthetic pathway was identically reconstructed in S288c and CEN.PK strains. Comparison of two producer strains revealed that genetic background has a large impact on the vanillin-β-glucoside yield.
In summary, this work contributes with novel insights, genetic engineering tools and methodologies for improved yeast cell factory construction and metabolic engineering strategies.