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Parallelized gene cluster editing illuminates mechanisms of epoxyketone proteasome inhibitor biosynthesis
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Huang, Chuan, Zabala, Daniel, de los Santos, Emmanuel, Song, Lijiang, Corre, Christophe, Alkhalaf, Lona M. and Challis, Gregory L. (2023) Parallelized gene cluster editing illuminates mechanisms of epoxyketone proteasome inhibitor biosynthesis. Nucleic Acids Research, 51 (3). pp. 1488-1499. doi:10.1093/nar/gkad009 ISSN 1362-4962.
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Official URL: https://doi.org/10.1093/nar/gkad009
Abstract
Advances in DNA sequencing technology and bioinformatics have revealed the enormous potential of microbes to produce structurally complex specialized metabolites with diverse uses in medicine and agriculture. However, these molecules typically require structural modification to optimize them for application, which can be difficult using synthetic chemistry. Bioengineering offers a complementary approach to structural modification but is often hampered by genetic intractability and requires a thorough understanding of biosynthetic gene function. Expression of specialized metabolite biosynthetic gene clusters (BGCs) in heterologous hosts can surmount these problems. However, current approaches to BGC cloning and manipulation are inefficient, lack fidelity, and can be prohibitively expensive. Here, we report a yeast-based platform that exploits transformation-associated recombination (TAR) for high efficiency capture and parallelized manipulation of BGCs. As a proof of concept, we clone, heterologously express and genetically analyze BGCs for the structurally related nonribosomal peptides eponemycin and TMC-86A, clarifying remaining ambiguities in the biosynthesis of these important proteasome inhibitors. Our results show that the eponemycin BGC also directs the production of TMC-86A and reveal contrasting mechanisms for initiating the assembly of these two metabolites. Moreover, our data shed light on the mechanisms for biosynthesis and incorporation of 4,5-dehydro-l-leucine (dhL), an unusual nonproteinogenic amino acid incorporated into both TMC-86A and eponemycin.
| Item Type: | Journal Article | |||||||||||||||
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| Subjects: | Q Science > QH Natural history > QH426 Genetics Q Science > QP Physiology |
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| Divisions: | Faculty of Science, Engineering and Medicine > Science > Chemistry | |||||||||||||||
| SWORD Depositor: | Library Publications Router | |||||||||||||||
| Library of Congress Subject Headings (LCSH): | Propylene oxide , Biosynthesis -- Research, Nucleotide sequence, DNA | |||||||||||||||
| Journal or Publication Title: | Nucleic Acids Research | |||||||||||||||
| Publisher: | Oxford University Press (OUP) | |||||||||||||||
| ISSN: | 1362-4962 | |||||||||||||||
| Official Date: | February 2023 | |||||||||||||||
| Dates: |
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| Volume: | 51 | |||||||||||||||
| Number: | 3 | |||||||||||||||
| Page Range: | pp. 1488-1499 | |||||||||||||||
| DOI: | 10.1093/nar/gkad009 | |||||||||||||||
| Status: | Peer Reviewed | |||||||||||||||
| Publication Status: | Published | |||||||||||||||
| Access rights to Published version: | Open Access (Creative Commons) | |||||||||||||||
| Date of first compliant deposit: | 27 February 2023 | |||||||||||||||
| Date of first compliant Open Access: | 27 February 2023 | |||||||||||||||
| RIOXX Funder/Project Grant: |
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