Expanding CRISPR-based toolbox for enhanced production of β-carotene in S. cerevisiae

Abstract

β-carotene is a natural pigment with antioxidant properties, widely used in food and animal feed production, pharmaceutical and beauty industries. The market demand for β-carotene is constantly rising which motivates development of new production processes such as biosynthesis in microbial cell factories, a sustainable alternative for the chemical synthesis. However, β-carotene biosynthesis in the model host organism – Saccharomyces cerevisiae leads to an imbalance in metabolic pathways (e.g. ergosterol biosynthesis), altered morphology (e.g. composition of the cellular membrane), activation of stress response (i.e. pleiotropic drug resistance transporters) and consequently hampered cell growth and instability of the production pathway genes. This thesis explores the CRISPR systems as novel molecular tools for the optimization of β-carotene biosynthesis in S. cerevisiae and mitigation of the related production burden. The protocol for multiplex manipulation of the yeast genome using the CRISPR-Cas12a system in combination with crRNA arrays was proposed to enable fast construction of carotenoids-producing strains. The nuclease deficient CRISPR-dCas12a effector was then implemented for specific downregulation of gene expression, so-called CRISPR interference. Development of CRISPRi system was supported by a systematic study of the key design features for efficient dCas12a mediated gene downregulation, such as the amount and position of the nuclear location signal, fusion to an additional repression domain, position of the target and architecture of the gRNA. The optimized dCas12a system was successfully applied for multiplex downregulation of β-carotene production. To mitigate the burden caused by the production of β-carotene in S. cerevisiae, the CRISPRi-dCas12a system was used to develop a feedback control system (FCS). In principle, the FCS temporarily downregulates β-carotene production when the production burden occurs so that the cell can restore its fitness. To achieve dynamic regulation, gRNA was expressed from ERG3 or PDR5 promoters. According to the conducted RNA sequencing, these promoters drive expression of genes which are highly upregulated upon carotenogenesis. The carotenoids producing strains with and without the feedback control system were then compared in terms of growth, carotenoids production and genetic stability of the expression cassettes encoding genes responsible for β-carotene biosynthesis. Although the FCS was shown to effectively repress β-carotene production, further optimization of the system is required to provide the host organism with the synthetic regulatory system which would exhibit improved growth, genetic stability and consequently higher product yield. Finally, CRISPR-Cas13a and -Cas13b systems were explored for specific mRNA transcript knockdown in S. cerevisiae. However, activation of Cas13 led to the collateral effect (unspecific cleavage of RNA present in the cell) which was shown to reversibly block cellular growth but did not lead to the cells death. Based on these learnings, CRISPR-Cas13a system was proposed to be used as a molecular tool to control growth of S. cerevisiae.