Our Science

Terra Bioforge has developed a new generation of tools to overcome limitations and pain points in natural product discovery and manufacturing that are 10-100x faster than existing methods.

BIGDNATM Tools for Manipulating BGCs

Natural product biosynthesis is encoded in large, complex regions of DNA known as Biosynthetic Gene Clusters (BGCs).  We have overcome the historical challenges of manipulating large contiguous BGCs to seamlessly integrate natural product development with modern approaches in synthetic biology. 

  • Our DNAtrapTM captures intact BGCs up to 150kb, irrespective of GC content or repetitive sequences
  • Effectively captures BGCs from complex genomic DNA (gDNA) or metagenomic library substrates
  • De-Risked: DNAtrapTM has been used to clone >130 intact BGCs

 

Engineering Expression of BGCs

In both native and heterologous microbial hosts natural product production is an innately regulated process, oftentimes limiting detection, analysis, and optimization of these valuable molecules. Our team has pioneered novel methodologies to predictably activate BGCs for both natural product screening and titer optimization workflows.

  • Our patented BGCexpressTM inducible expression technology enables broad control over transcriptional activation and titer
    • Induction increases natural product titers at least 2-3x facilitating functional bioactivity screening
  • BGCrefactorTM in vitro tool for quick and precise refactoring of BGCs
    • Multiplexed, marker-free refactoring workflow for
      optimizing entire natural product biosynthesis

 

Metabologenomics for Natural Product BGC Identification

Identifying putative BGCs in digital databases is routine, and so is broadly detecting natural products from microbial extracts. One of the major challenges that limits scaling discovery and optimization of these valuable molecules has been linking BGCs to their respective natural products.

To overcome this, we have developed patented bioinformatics and machine learning tools that link microbial metabolites with their biosynthetic pathways.

  • Prospect Fungi is a proprietary analysis tool for in silico prediction and annotation of fungal BGCs
    • An AI platform that grows and learns from known BGC:Natural Product relationships
  • HAYSTACK-MS: Finding new drugs via targeted cloning
    • Solves needle-in-the-haystack problem of finding target compounds in complex metabolite mixtures
    • Rapidly identifies and produces natural products by heterologous expression

 

Hosts for Heterologous Expression

When it comes to optimal heterologous expression of natural products, host selection matters. Precursor metabolites, cofactors, among other host-specific variables can greatly influence the successful production of specific molecules. Our access to a suite of optimal heterologous hosts provides the greatest opportunity for natural product expression.

  • CleanChassisTM strains have been engineered to reduce competing metabolite production
    • Streptomyces, Bacillus, Aspergillus, and Gram Negative bacteria
  • For every BGC of interest, our growing portfolio of CleanChassisTM strains represents an opportunity to accelerate optimal expression

 

For additional information on Terra Bioforge technical capabilities, check out our conference posters on  New Tools for Targeted Cloning and Over Expression of Biosynthetic Gene Clusters and Expanding Functional Access to Fungal Natural Products with Metabologenomics and Heterologous Expression.

Publications

A Survey of Didemnin Depsipeptide Production in Tistrella. Stankey RJ, Johnson D, Duggan BM, Mead DA, La Clair JJ. Marine Drugs. 2023; 21(2):56. https://doi.org/10.3390/md21020056

Correlative metabologenomics of 110 fungi reveals metabolite/gene cluster pairs. Caesar LK, Butun FA, Robey MT, Ayon NJ, Gupta R, Dainko D, Bok JW, Nickles G, Stankey RJ, Johnson D, Mead DA, Cank KB, Earp CE, Raja HA, Oberlies NH, Keller NP, Kelleher NK. Nature Chemical Biology. 2023.

Nanoscaled discovery of a shunt rifamycin from Salinispora arenicola using a three-colour GFP-tagged Staphylococcus aureus macrophage infection assay. Nhan T. Pham, Joana Alves, Fiona A. Sargison, Reiko Cullum, Jan Wildenhain, William Fenical, Mark S. Butler, David A. Mead, Brendan M. Duggan, J. Ross Fitzgerald, James J. La Clair, Manfred Auer. bioRxiv 2022.12.04.519019; doi: https://doi.org/10.1101/2022.12.04.519019

An interpreted atlas of biosynthetic gene clusters from 1,000 fungal genomes. Robey MT, Caesar LK, Drott MT, Keller NP, Kelleher NL. Proc Natl Acad Sci U S A. 2021 May 11;118(19):e2020230118. doi: 10.1073/pnas.2020230118. PMID: 33941694; PMCID: PMC8126772.

Discovery of the Biosynthetic Machinery for Stravidins, Biotin Antimetabolites. Montaser R, Kelleher NL. ACS Chem Biol. 2020 May 15;15(5):1134-1140. doi:10.1021/acschembio.9b00890. Epub 2020 Jan 9. PMID: 31887014; PMCID: PMC7230017.

Discovery of Novel Biosynthetic Gene Cluster Diversity from a Soil Metagenomic Library. Santana-Pereira ALR, Sandoval-Powers M, Monsma S, Zhou J, Santos SR, Mead DA, Liles MR. Front Microbiol. 2020 Dec 7;11:585398. doi: 10.3389/fmicb.2020.585398

Chloramphenicol Derivatives with Antibacterial Activity Identified by Functional Metagenomics. Nasrin S, Ganji S, Kakirde KS, Jacob MR, Wang M, Ravu RR, Cobine PA, Khan IA, Wu CC, Mead DA, Li XC, Liles MR. J Nat Prod. 2018 Jun 22;81(6):1321-1332.

A scalable platform to identify fungal secondary metabolites and their gene clusters. Clevenger KD, Bok JW, Ye R, Miley GP, Verdan MH, Velk T, Chen C, Yang K, Robey MT, Gao P, Lamprecht M, Thomas PM, Islam MN, Palmer JM, Wu CC, Keller NP, Kelleher NL. Nat Chem Biol. 2017 Aug;13(8):895-901. doi: 10.1038/nchembio.2408. Epub 2017 Jun 12. PMID: 28604695; PMCID: PMC5577364.

Challenges and Opportunities in the Discovery of Secondary Metabolites Using a Functional Metagenomics Approach. Pereira A, Liles MR, In: Functional Metagenomics: Tools and Applications, edited by Charles T, Liles MR, and Sessitch A. pp. 119-138. (2017) Berlin Heidelberg: Springer-Verlag.

Meeting Report for Synthetic Biology for Natural Products 2017: The Interface of (Meta)Genomics, Machine Learning, and Natural Product Discovery. Smanski MJ, Mead D, Gustafsson C, Thomas MG. ACS Synth Biol. 2017 May 19;6(5):737-743.

Metabologenomics: Correlation of Microbial Gene Clusters with Metabolites Drives Discovery of a Nonribosomal Peptide with an Unusual Amino Acid Monomer. Goering AW, McClure RA, Doroghazi JR, Albright JC, Haverland NA, Zhang Y, Ju KS, Thomson RJ, Metcalf WW, Kelleher NL. ACS Cent Sci. 2016 Feb 24;2(2):99-108. doi: 10.1021/acscentsci.5b00331. Epub 2016 Jan 20. PMID: 27163034; PMCID: PMC4827660.

Fungal artificial chromosomes for mining of the fungal secondary metabolome. BMC Genomics. Bok JW, Ye R, Clevenger KD, Mead D, Wagner M, Krerowicz A, Albright JC, Goering AW, Thomas PM, Kelleher NL, Keller NP, Wu CC.  2015 Apr 29;16(1):343. doi: 10.1186/s12864-015-1561-x. PMID: 25925221; PMCID: PMC4413528.

Bacillusin A, an Antibacterial Macrodiolide from Bacillus amyloliquefaciens AP183. Ravu RR, Jacob MR, Chen X, Wang M, Nasrin S, Kloepper JW, Liles MR, Mead DA, Khan IA, Li XC. J Nat Prod. 2015 Apr 24;78(4):924-8.

Draft genome sequence of Bacillus amyloliquefaciens AP183 with antibacterial activity against methicillin-resistant Staphylococcus aureus. Nasrin S, Hossain MJ, and Liles MR (2015) Genome Announcements, 3(2). pii: e00162-15.

A roadmap for natural product discovery based on large-scale genomics and metabolomics. Doroghazi JR, Albright JC, Goering AW, Ju KS, Haines RR, Tchalukov KA, Labeda DP, Kelleher NL, Metcalf WW.  Nat Chem Biol. 2014 Nov;10(11):963-8. doi: 10.1038/nchembio.1659. Epub 2014 Sep 28. PMID: 25262415; PMCID: PMC4201863.

Biological agents for control of disease in aquaculture. Carrias A, Ran C, Terhune J, and Liles MR (2012), pp. 353-393. In: Infectious Diseases in Aquaculture, S. Austin (Ed.). London: Woodhead Publishing.

Recovery of as-yet-uncultured soil Acidobacteria on dilute solid media. George IF, Hartmann M, Liles MR, and Agathos SN. (2011) Applied & Environmental Microbiology, 77(22):8184-8188.

Polyketide synthase pathways identified from a metagenomic library are derived from soil Acidobacteria. Parsley LC, Goode AM, Becklund K, George I, Linneman J, Lopanik NB, Goodman RM, and Liles MR. (2011) FEMS Microbiology Ecology, 78(1):176-187.

Gram-negative shuttle BAC vector for heterologous expression of metagenomic libraries. Kakirde KS, Wild J, Godiska R, Mead D, Wiggins AG, Szybalski W, and Liles MR. (2011) Gene, 475(2):57-62.

Size Does Matter: Application-driven Approaches for Soil Metagenomics. Kakirde KS, Parsley LC, and Liles MR. (2010) Soil Biology and Biochemistry, 42(11):1911-1923.

Isolation and cloning of high molecular weight metagenomic DNA from soil microorganisms.  Liles M R, Williamson LL, Rodbumrer J, Parsley L, Torsvik V, Goodman RM, and Handelsman J. (2009) Cold Spring Harbor Protocols, doi:10.1101/pdb.prot5271.

Recovery, purification, and cloning of high molecular weight genomic DNA from soil microorganisms. Liles MR, Williamson LL, Rodbumrer J, Torsvik V, Goodman RM, and Handelsman J. (2008) Applied and Environmental Microbiology, 74:3302-3305.

Isolation of high molecular weight genomic DNA from soil bacteria for genomic library construction.  Liles MR, Williamson LL, Goodman RM, and Handelsman J.  (2004) In: Molecular Methods in Environmental Microbiology, de Bruijn FJ, Head IM, Akkermans AD, van Elsas JD. (Eds.), pp. 839-852.

Cloning the metagenome: Culture-independent access to the diversity and functions of the uncultivated microbial world. Handelsman J, Liles MR, Mann D, Riesenfeld C, and Goodman RM. (2002) Methods in Microbiology, 33:241-255.

Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA. Gillespie DE, Brady SF, Bettermann AD, Cianciotto NP, Liles MR, Rondon MR, Clardy J, Goodman RM, Handelsman J. (2002). Applied and Environmental Microbiology, 68:4301-4306.

Cloning the soil metagenome: A strategy for accessing the genetic and functional diversity of uncultured microorganisms. Rondon MR, August PR, Bettermann AD, Brady SF, Grossman TH, Liles MR, Loiacono KA, Lynch BA, MacNeil IA, Minor C, Tiong CL, Gilman M, Osbourne MS, Clardy J, Handelsman J, and Goodman RM  (2000) Applied and Environmental Microbiology, 66 (6): 2541-2547.

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