Making use of synthetic biology toolbox for industrial biotechnology applications: The case of enzyme production

Microorganisms have long been used in industrial biotechnology. However, since yield and diversity are limited by the natural capacity of the microorganism, it cannot adequately respond to the increasing demand. At this point, recombinant DNA technology offers alternative solutions. Compared to recombinant DNA technology methods, synthetic biology, which enables use of standardized parts and protocols, has been widely used in industrial biotechnology. The focus of synthetic biology is not only the integrated design of DNA fragments such as the promoter, coding region, and metabolic chemical reactions such as the pathway, but also the design of the microorganism itself, namely "host design". These designs aim to eliminate the challenges in the over-production of natural products, heterologous production, keeping the product stable for a longer time or utilization of alternative carbon sources in production. This thesis includes synthetic biology applications for strain development studies in industrially important filamentous fungi and yeast. In the first chapter, one of the well-studied filamentous fungi, Aspergillus brasiliensis, was used as the host and rendered protease-deficient. This strain ensured that the activity of natural or heterologous enzymes remained stable without degradation. Furthermore, promoter engineering studies were carried out on carbon catabolite repression. Glucoamylase promoter was selected and base addition/deletion was performed on the sequence to remove carbon catabolite repression. 3 different promoter variants were obtained and the variant that continued to exhibit activity in the presence of xylose was then used as promoter for heterologous protein expression in the protease-deficient strain. As a conclusion, heterologous protein production was carried out in the presence of an alternative lignocellulosic carbon source, enzyme activity was maintained during fermentation and simultaneous utilization of different carbon sources was achieved in this strain. In the second chapter, it was aimed to develop a CRISPR/Cas9 system to be used in gene disruption studies in Saccharomyces cerevisiae. In this context, beta-isopropylmalate dehydrogenase gene, LEU2, was targeted, related sgRNA was designed and a CRISPR plasmid was constructed using Golden Gate assembly. As a result, construction and utilization of a functional CRISPR/Cas9 system in yeast was confirmed.