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Intact Genomics, Collaborators Develop Workflow for Natural Products Discovery


NEW YORK (GenomeWeb) – Intact Genomics and its collaborators have developed a high-throughput pipeline for identifying secondary metabolites produced by filamentous fungi.

Detailed in a paper published this week in Nature Chemical Biology, the method combines the St. Louis-based company's capabilities in large insert DNA cloning and manipulation and bacterial artificial chromosome technology with mass spectrometry to enable better identification and production of fungal secondary metabolites, which are of great interest in drug discovery research.

Fungi secondary metabolites are a potentially rich source of natural products that could prove useful in a variety of areas including therapeutics and pesticides, said Chengcang Wu, Intact's founder and CEO and an author on the Nature Chemical Biology study. However, isolating and identifying these metabolites has proved challenging.

One difficulty, Wu said, is the fact that many of these organisms are difficult or impossible to culture. Additionally, he noted, even in the case of organisms that can be cultured, they will not typically produce their full repertoire of secondary metabolites under standard lab culture conditions.

These factors have led researchers to explore the possibility of capturing the genes responsible for ultimately producing various secondary metabolites and expressing them in hosts that can be cultured.

This, though, comes with difficulties as well. As opposed to proteins, for which researchers can, in theory, isolate the specific gene responsible for their production, secondary metabolites are generated "most of the time by multiple gene pathways or gene clusters," Wu said. "And if you capture just part [of a target gene cluster] you will never see the compound."

These gene clusters can total 50Kb or more in sequence length, which presents challenges for commonly used genomic techniques like PCR or next-generation sequencing, Wu noted. Such large sequences are also poorly compatible with vectors like cosmids and fosmids, which, the study authors noted, "are often not large enough to encode complete secondary metabolite BGCs [biosynthetic gene clusters]."

Wu and his collaborators address these issues by using the company's fungal artificial chromosome (FAC) technology, which is able to capture large intact sequences of fungal DNA which can then be analyzed via PCR-based library screening or sequencing to determine if it is likely to contain a secondary metabolite-producing BGCs. The company developed much of this technology working under a $1.8 million Phase II Small Business Innovation Research grant from the National Institutes of Health that ran from 2014 to 2016, Wu said.

Once isolated and identified, promising BGCs can then be expressed in a culturable host (the fungi Aspergillus nidulans in the case of the Nature Chemical Biology study), and metabolites generated by these BGCs can then be identified using mass spectrometry.

This mass spec-based identification also comes with complications, however, due to the challenge of distinguishing between metabolites produced by the BGC of interest and those produced by the host expressing the cluster. Wu and his co-authors noted that a previous study they undertook looking at FAC-encoded gene clusters failed to identify novel metabolites "because of the lack of an effective mechanism for sorting through the [roughly] 5,000 LC-mass spectrometry signals detected from each FAC-transformed strain."

Working with Northwestern University researcher Neil Kelleher, first author on the paper and an expert in various areas of mass spectrometry including top-down proteomics, the team devised a system by which they looked at the abundance of a given compound across controls and FAC-transformed organisms to distinguish likely BGC-produced compounds from those made by the host endogenously. In this way, they winnowed the number of potential compounds down from on the order of thousands per strain to, typically, one or a few, the authors noted.

This still left the possibility, though, that these apparently BGC-produced compounds were actually host metabolites whose production was upregulated due to the insertion of the BGC of interest. To account for this possibility, Wu and his colleagues deleted key genes from the BGCs, reinserted them into the host and looked to see whether the metabolite was still present.

In this way, the researchers looked at 56 uncharacterized BGCs from three different fungal species, identifying 15 compounds, 14 of which they were unable to match to previously identified secondary metabolites.

Wu suggested that the workflow "has huge potential for drug discovery." He said that Intact has patents covering the FAC workflow as well as provisional patents covering workflows enabling similar unbiased extraction of large genomic elements from bacteria and samples including soil and water metagenomes.

"Ninety nine percent of those metagenomes are not culturable under lab conditions," he said, adding that in addition to drug discovery the approach could be useful for identifying new compounds for agricultural and other applications. "Crop protection, for instance," he said. "Every big [agriculture] company is looking right now for the next-generation RoundUp [Monsanto's glyphosate-based herbicide]."

Wu also said that Intact has ongoing collaborations involving the FAC-based metabolite discovery workflow with several large firms involved in drug development and crop science.