MassIVE MSV000098583

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GNPS - A simple and versatile cell-free expression method for producing secondary metabolites

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Description

Secondary metabolites are a major source of natural products with industrially relevant bioactivities. Lysate-based cell-free expression (CFE) is an emerging platform for accelerating the discovery and engineering of these natural products. While Escherichia coli cell extracts are widely used for CFE, Streptomyces extracts are believed to offer a more biochemically compatible environment for natural product synthesis. However, current Streptomyces-based CFE systems remain underdeveloped, with protocols that are either strain-specific or not readily scalable. To address these limitations and enable broader access to cell-free natural product biosynthesis, we present a generalizable and simple set of reaction conditions that support high-yield protein expression (180–230?µg/mL) in lysates derived from Streptomyces venezuelae NRRL B-65422 and Streptomyces lividans TK24. Like Escherichia coli-based systems, these extracts enable iterative and pathway-level biosynthesis, as demonstrated by the production of the polyketide flaviolin and the cyclic dipeptide albonoursin. Notably, the S. lividans lysate outperforms E. coli systems by also supporting the expression and catalytic activity of a (~250?kDa) type I polyketide synthase (T1PKS), producing its corresponding ethyl ketone product, 2-methyl-3-pentanone, without the need for precursor or post-translational modification supplements. To our knowledge, this represents the first demonstration coupling both expression and catalysis of a megasynthase in a Streptomyces-based system, and of a T1PKS in any bacterial extract. By addressing key challenges in the generalizability and scalability of prior Streptomyces CFE, we establish a protocol that enables parallelized evaluation of diverse lysate systems and provide a foundation for high-throughput T1PKS engineering in vitro. The work (proposal:https://doi.org/10.46936/10.25585/60008760) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231. [doi:10.25345/C5DV1D18H] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: cell-free expression ; natural products ; biosynthesis ; DatasetType:Metabolomics

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Principal Investigators:
(in alphabetical order) 		Hiroshi Otani, LBL, United States
Submitting User: bpbowen
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GNPS content goes here (MSV000098583 [task=38cdc54c33ae41c3b5175d12974c13c1])
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Distinct condition labels are counted across all files submitted in the "Metadata" category having a "Condition" column in this dataset.

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Number of distinct biological replicates across all analyses (original submission and reanalyses) associated with this dataset.

Distinct replicate labels are counted across all files submitted in the "Metadata" category having a "BioReplicate" or "Replicate" column in this dataset.

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Number of distinct technical replicates across all analyses (original submission and reanalyses) associated with this dataset.

The technical replicate count is defined as the maximum number of times any one distinct combination of condition and biological replicate was analyzed across all files submitted in the "Metadata" category. In the case of fractionated experiments, only the first fraction is considered.

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Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

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Number of distinct protein accessions reported across all analyses (original submission and reanalyses) associated with this dataset.

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Number of distinct unmodified peptide sequences reported across all analyses (original submission and reanalyses) associated with this dataset.

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Number of distinct peptide sequences (including modified variants or peptidoforms) reported across all analyses (original submission and reanalyses) associated with this dataset.

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Total number of peptide-spectrum matches (i.e. spectrum identifications) reported across all analyses (original submission and reanalyses) associated with this dataset.

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Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

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Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
This dataset may not contain all raw spectra data as originally deposited in PRIDE. It has been imported to MassIVE for reanalysis purposes, so its spectra data here may consist solely of processed peak lists suitable for reanalysis with most software.