The untapped chemical diversity in nature holds tremendous promise of biological and pharmacological relevance. Indeed, as highlighted in this RFA, roughly 75% of antibacterial and anticancer drugs are natural products or inspired by natural products (NPs). However, discovery of secondary metabolite NPs has historically been a laborious and costly process involving producer species that are often difficult to impossible to cultivate. The recent explosion in genome sequence data has additionally revealed that only a fraction of the secondary metabolites from even well-studied species have actually been discovered, due to the clusters being transcriptionally silent under laboratory conditions. Efforts to activate these cryptic or silent gene clusters have been laborious and not scalable to high-throughput discovery. We propose to address this bottleneck in NP discovery by altogether bypassing native, uncultivable hosts and instead developing yeast as a super-host capable of expressing a large variety of NP gene clusters. We propose to achieve this objective through four specific aims.
Aim 1. Tools for NP DNA design and synthesis. We will use de novo DNA synthesis and develop new synthetic biology tools and genomic technologies to allow heterologous expression of diverse NP pathways in yeast. We will develop this host and its tools while performing heterologous expression of ~600 natural product gene clusters mined from 10 filamentous fungi of diverse ecological origin.
Aim 2. Tools to improve yeast as a host for heterologous transcription of fungal NP gene clusters. We will characterize promoter libraries and engineer a fungal spliceosome in S. cerevisiae to allow proper intron splicing.
Aim 3. Tools to improve yeast as a host for heterologous translation of fungal NP gene clusters. We will identify yeast strains that serve as improved hosts for heterologous protein expression by screening genome-wide yeast and fungal gene libraries and performing directed in-lab evolution, coupled with a high-throughput readout for protein expression.
Aim 4. Tools to improve yeast as a host for heterologous NP production. We will use the library and in-lab evolution screening developed in Aim 3, here applied to a screen for improved NP production. We will also develop tools that users can use to improve expression their own NP pathways of interest. Our proposal addresses all aspects of the genome to NP process, starting with genomic sequence and ending with NPs. The strategy outlined here will allow discovery of an unprecedented number of new NPs.

Public Health Relevance

Natural products are exceptionally valuable therapeutic compounds: roughly 75% of antibacterial and anticancer drugs are natural products or inspired by natural products. However, only a small fraction of these compounds have been discovered because the producer species do not grow under laboratory conditions. Our project will provide access to a large variety of entirely novel natural products that will impact human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01GM110706-05
Application #
9495703
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Sledjeski, Darren D
Project Start
2014-06-01
Project End
2019-04-30
Budget Start
2018-05-01
Budget End
2019-04-30
Support Year
5
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Medema, Marnix H (2018) Computational Genomics of Specialized Metabolism: from Natural Product Discovery to Microbiome Ecology. mSystems 3:
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Roy, Kevin R; Smith, Justin D; Vonesch, Sibylle C et al. (2018) Multiplexed precision genome editing with trackable genomic barcodes in yeast. Nat Biotechnol 36:512-520
Hang, Leibniz; Tang, Man-Cheng; Harvey, Colin J B et al. (2017) Reversible Product Release and Recapture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase Domain. Angew Chem Int Ed Engl 56:9556-9560
Vandova, Gergana A; O'Brien, Robert V; Lowry, Brian et al. (2017) Heterologous expression of diverse propionyl-CoA carboxylases affects polyketide production in Escherichia coli. J Antibiot (Tokyo) 70:859-863
Smith, Justin D; Schlecht, Ulrich; Xu, Weihong et al. (2017) A method for high-throughput production of sequence-verified DNA libraries and strain collections. Mol Syst Biol 13:913
Billingsley, John M; DeNicola, Anthony B; Barber, Joyann S et al. (2017) Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae. Metab Eng 44:117-125
Celaj, Albi; Schlecht, Ulrich; Smith, Justin D et al. (2017) Quantitative analysis of protein interaction network dynamics in yeast. Mol Syst Biol 13:934
Tang, Man-Cheng; Zou, Yi; Watanabe, Kenji et al. (2017) Oxidative Cyclization in Natural Product Biosynthesis. Chem Rev 117:5226-5333
Billingsley, John M; DeNicola, Anthony B; Tang, Yi (2016) Technology development for natural product biosynthesis in Saccharomyces cerevisiae. Curr Opin Biotechnol 42:74-83

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