Establishing a standardised DNA assembly and biosensor-based screening pipeline for natural product pathway engineering in Saccharomyces cerevisiae

Abstract

Many pharmaceutical drugs currently available on the market are natural products, which are extracted from biological sources (microorganisms or plants). It can prove challenging to maintain a constant supply of these drugs, considering that they are often derived from microorganisms that are difficult to culture or plants, where availability can be strongly impacted by demand for alternative crops or severe weather conditions. Synthetic biology presents a promising and more sustainable option, through the engineering of easily cultured and genetically tractable microorganisms such as Saccharomyces cerevisiae to enable their production of pharmaceutical compounds. However, the process of heterologous expression of a bacteria-derived natural product pathway in S. cerevisiae can be challenging. Many variables need to be adjusted, such as codon optimisation, gene expression, pathway flux, enzyme variants utilised and the activity of different enzymes within the pathway. In the process of optimisation and screening, a major rate-limiting step is the assembly of diverse pathway variants, which can be time consuming and costly.

The aim of this project was to utilise synthetic biology tools and techniques to engineer yeast strains to produce different therapeutic compounds. This involves the establishment of a pipeline to be able to 1) assemble many different variants of large pathways, and 2) rapidly screen for the production of a bioactive compound and identify producer or high producer strains. In this body of work, I describe the development of YeastFab, a toolkit of standardised and modular biological yeast ‘parts’ that can be used to hierarchically assemble large natural product pathways that encode for production of therapeutic drugs within the span of a week. To date, hundreds of promoters and terminators have been standardised into the YeastFab format and have been further characterised to determine their activities. This assembly strategy is subsequently applied in the context of a collaborative project, funded by the Bill and Melinda Gates Foundation, for heterologous expression of exemplar pathways from different classes of natural products (NRPS, PKS, RiPPs, nucleosides, alkaloids and flavonoids). I have primarily focussed on the heterologous production of viomycin, rebeccamycin and naringenin. Different variants of each pathway have been assembled and screened using a bioassay to detect antibiotic production or using a biosensor specific to the compound of interest. As a proof of principle, I have developed a high throughput assay, utilising an Escherichia coli transcription factor-based biosensor to quantify the amount of naringenin produced by each yeast strain, demonstrating comparable quantification to mass spectrometry measurements. Further improvements of naringenin yield are being carried out through chassis optimisation by genome engineering techniques such as SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxPsyn-mediated Evolution), with the use of the E. coli biosensor to sift through the population of SCRaMbLEd strains for high producers.

In conclusion, I have developed and demonstrated a pipeline for rapid assembly and screening of natural products biosynthetic pathways in yeast, which can facilitate the development of novel processes for manufacture of many therapeutic compounds.