Title: Engineering yeasts for in situ production of fungal tetracyclines
Description: Synthetic biology consists of the design and construction of customized cell-based systems, and metabolic engineering is its co-discipline that aims to engineer these cells into biological factories for the production of drugs, chemical commodities and fuels. Together, these two disciplines continue to provide various innovative solutions to current problems of humanity in the areas of medicine, agriculture and energy. In this dissertation, we use synthetic biology and metabolic engineering approaches to explore the potential of engineered live yeasts as therapeutic platforms for treating inflammatory bowel disease (IBD). The vast majority of microbial-based therapeutics at the moment have focused on bacteria instead of yeasts, and all of these engineered live bacterial platforms use either proteins or peptides as therapeutic agents of choice. This dissertation seeks to enhance yeast’s beneficial properties to humans by genetically engineering them to produce TAN-1612, a small molecule tetracycline with therapeutic potential.

We choose tetracyclines as our small molecule therapeutic agent because these compounds are one of the most impactful natural products that humanity has benefited from due to its significant antimicrobial and anti-inflammatory properties. We genetically engineer strains of baker’s yeast Saccharomyces cerevisiae and the probiotic yeast Saccharomyces cerevisiae var boulardii to produce in situ the fungal tetracycline TAN-1612, a natural product with anti-inflammatory properties (instead of anti-microbial so as to not disturb the gut microbiome), and to study the molecular mechanisms involved in their potential beneficial effects for IBD. Our engineered live yeast therapeutics would provide an effective, safe, and cheap alternative to treating IBD and other gastrointestinal tract disorders compared to the currently available but costly and laborious therapies. In Chapter 1, we review key milestones in the fields of synthetic biology and metabolic engineering that have enabled and inspired the generation of both engineered live microbial-based systems and small molecules as the therapeutic agents for the potential treatment of a wide array of human diseases such IBD, cancer, and pathogenic infections.

In Chapter 2, we develop synthetic biology and metabolic engineering approaches for designing, building, and testing of the biosynthetic pathway of TAN-1612 in genetically engineered yeasts such as S. cerevisiae and S. boulardii. These approaches enable the production of TAN-1612 in yeasts with titers as high as ~61 mg/L which represent a 100-fold improvement from previous reported yeast strains. These engineering approaches hold great potential to advance the heterologous biosynthesis of other small molecule therapeutics in yeasts. In Chapter 3, we explore the role of TAN-1612 as an anti-inflammatory agent, inhibitor of tetracycline inactivating enzymes, and inducer of gene expression with the goal of identifying its best therapeutic or biological application that can be leveraged for the development of engineered live yeast-based systems for the in situ treatment of IBD. Advances in DNA synthesis and sequencing technologies have spurred the high-throughput construction of microbial strains for numerous applications in synthetic biology and metabolic engineering.

Breakthrough technologies in our abilities to screen and select target molecule biosynthesis, however, are needed in order to realize the potential of both of these disciplines for drug discovery and production. Current state-of-the-art methods such as liquid/gas chromatography – mass spectrometry (LC/GC – MS) are applicable to screen or select a variety of target molecules but their throughput remains low (~102 samples/day). Other screening or selection methods available are highly dependent on the molecule of interest and generally inapplicable to other compounds. Therefore, in Chapter 4 we propose that the Fluorescence Polarization (FP) assay can be readily adapted as a general, medium-throughput (~104 samples/day) screen for synthetic biology and metabolic engineering applications. As a proof-of-principle, we develop an FP assay to detect the immunosuppressant polyketide FK506, use this assay to detect FK506 biosynthesized from Streptomyces tsukubaensis cultures in microtiter plates, and finally apply this FP assay to generate S. tsukubaensis strains with increased production of FK506.

Lastly, we outline the experimental steps necessary to adapt the FP to screen different classes of natural products beyond polyketides such as FK506 or tetracyclines. The fungal polyketide TAN-1612 is an attractive chemical scaffold for the generation of novel tetracycline-based therapeutics using metabolic engineering approaches. Libraries of > 108 are usually required to generate chemical compound diversity to identify drugs with significant therapeutic activities. Compared to screening methods, genetic selections allow the detection of target molecules at a much higher throughput (> 108 samples/day) because the population of cells can be cultured and assayed in one-pot manner. Thus, in Chapter 5 we establish the yeast three hybrid (Y3H) assay as a general, high-throughput selection technology to detect tetracycline derivatives. We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating producer and non-producer yeast strains of TAN-1612. The Y3H assay can be used for the heterologous biosynthesis of tetracycline analogues in yeasts especially because the Y3H would enable the production and detection of these derivatives in the same yeast cell.