Synthetic biology tools for yeast cell factories

Research output: Book/ReportPh.D. thesis

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Abstract

Budding yeast, Saccharomyces cerevisiae, has been used for millennia for production of wine, beer and bread. Nowadays yeast is also used as a cell factory chasis for synthesizing a wide range of chemical products with many applications. Metabolic engineering designs and implements modifications to cellular metabolism for increased carbon flux towards the desired metabolites resulting in more efficient cell factories with higher titers, rates and yields. Synthetic biology circuits have proved useful for metabolic engineering efforts. Such circuits have often embedded biosensors that sense concentrations of metabolites, proteins or physiological conditions, and trigger expression of reporter or actuator genes. These circuits are useful to assess many modifications in cell factories in parallel, therefore reducing metabolic engineering projects’ turnaround time.

The work presented in this thesis focuses mainly on synthetic biology tools development for yeast cell factories, specifically in the development of biosensor systems for muconic acid and insoluble protein expression. Muconic acid is a precursor for plastics such as polyethylene terephthalate and nylon. Insoluble protein is used as a proxy for misfolded protein, as misfolded protein tends to form insoluble aggregates. Poor expression and misfolding have been observed to be a bottleneck in some cell factories, including the cell factories producing polyketides, a diverse family of compounds with applications such as pharmaceuticals, and fine and bulk chemicals.

In the first study a sensor-selector system for muconic acid is developed. The system is based on coupling a muconic acid-responsive allosteric transcription factor to the expression of an antibiotic marker. The study first demonstrates that growth under selection increases with higher amounts of fed muconic acid, and that the sensor-selector is orthogonal to muconic acid production. Next, culture conditions are optimized to reduce activation of sensor-selector in non- or low-producing cells by adjusting pH of the medium, followed by demonstration that the selection system can be used to select high muconic acid producers in a pooled library. Finally, we tested two antibiotic-resistant clones from our library in bioreactors, observing muconic acid titers of ~2g/L.

The second study describes the construction and optimization of a promoter-reporter for insoluble protein expression in yeast. In this study, proteins upregulated upon expression of insoluble protein are assessed by proteomics. Next, promoter-reporters are designed and built using promoters of genes encoding proteins with the highest response to insoluble protein expression. Furthermore, the performance of the best promoter-reporters from the screen is optimized by promoter engineering and by optimizing culture conditions. Finally, the best promoter-reporter was applied for detection of expression of misfolding-prone proteins, and furthermore demonstrated useful for sorting of cells expressing soluble protein diluted in a mixed population with cells expressing insoluble protein.

In the third study, protein fusions of misfolding-prone proteins and solubility-enhancing tags are constructed in order to try to improve expression and activity of three target proteins. This study focuses on two misfolding-prone mutants of the yellow fluorescence protein (YFPm3 and YFPm4) and the polyketide synthase PKS12. We observed that fusion to solubility-enhancing tags can enhance YFPm3 and PKS12 fluorescence and activity, respectively. Although three solubility-enhancing tags had a positive effect for both YFPm3 and PKS12, most of the tags had different effects for both targets.

Altogether, these three studies describe the development and application of synthetic biology tools that permit the screening of yeast strains with improved metabolite production and protein expression, and the optimization of expression of misfolding-prone proteins, ultimately providing tools and workflows of relevance to the development of a multitude of yeast cell factories.
Original languageEnglish
Number of pages157
Publication statusPublished - 2020

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