Our goal is to develop the yeast host and tools for natural product (NP) expression, using novel genomic and synthetic biology technologies. We will showcase these tools through heterologous expression of 600 natural product gene clusters from 10 filamentous fungi of diverse ecological origin. Our project will address all aspects of the genome to natural product process, including high-throughput and cost-effective gene cluster DNA synthesis, gene transcription and protein expression in yeast, through natural product biosynthesis. Our team will have three classes of deliverables: (1) tools and strains, (2) compounds, and (3) biosynthetic knowledge. This project is a collaboration between the Stanford Genome Technology Center, the Stanford ChEM-H Metabolic Chemistry Analysis Center, and the University of California, Los Angeles. Find out more about our team members here.
Our approach comprises four major aims:
Aim 1. Tools for NP DNA design and synthesis.
We will use de novo DNA synthesis to produce pathways optimized for yeast expression in a high-throughput, low-cost manner. We will refactor these gene cluster sequences for optimal expression in S. cerevisiae, using codon optimization, mRNA secondary structure optimization, and translational arrest sequences. We have developed novel methods for facile DNA assembly and error correction of synthetic oligonucleotides. We will focus on natural product gene clusters from 10 different fungal species whose genomes have been sequenced, and which are prolific producers of NPs.
Aim 2. Tools to improve yeast as a host for heterologous transcription of fungal NP gene clusters.
After introducing synthetic NP gene clusters into hosts we must confirm their proper transcription. In native hosts, these clusters are often transcriptionally silent under laboratory conditions. We will express these gene clusters under a series of yeast promoters, test and/or develop alternative promoters as necessary, and characterize them for the community in terms of NP gene transcription. We will maintain a database of promoter use and successes and failures in NP strains, characterizing them for the community in terms of NP gene transcription.
Fungal gene expression, similar to that in higher eukaryotes, requires accurate splicing of introns by the spliceosome. To ensure functional expression of our fungal genes in S. cerevisiae, we must identify and remove introns during cloning. Direct synthesis of genomic DNA for processing by a compatible splicing machinery in yeast is desirable.
Aim 3. Tools to improve yeast as a host for heterologous translation of fungal NP gene clusters.
Once biosynthetic NP gene clusters have been transcribed in the yeast host, the heterologous proteins must be translated. Our group has extensive experience expressing heterologous proteins in S. cerevisiae. However, heterologous expression can be limiting, especially for fungal enzymes larger than 10kb, and improving NP production in yeast will require improved protein expression. We will develop tools to identify genes in yeast that enhance or impair overall protein biosynthesis for heterologous biosynthetic pathways. We also aim to identify additional genetic changes to yeast that improve heterologous protein expression in a general manner.
We will apply our extensive expertise in yeast genome-wide library construction and characterization to identify genes that improve expression of heterologous proteins to improve yeast as a super-host for natura product expression.
Aim 4. Tools to improve yeast as a host for heterologous NP production.
We will discover genetic changes that are beneficial for NP production in general by screenings the yeast and fungal gene libraries. In this aim, our readout is a genetic selection based on the endogenous yeast transcriptional response to NPs, in which metabolite-responsive promoters are placed upstream of selectable marker genes. In this manner we couple the presence of NP metabolites to growth, allowing for simple and high-throughput readouts to identify improved host strains. In addition to general host improvement, we will develop tools that will aid productivity of specific biosynthetic pathways in yeast.
We will perform structural class prediction using our existing yeast functional genomic pipeline to compare profiles from the newly discovered biosynthetic pathways to previously characterized compounds. Finally, we will isolate and characterize novel NPs using LC/MS and NMR approaches.