Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2015 Jan 14:5:774.
doi: 10.3389/fmicb.2014.00774. eCollection 2014.

Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi

Ralph A Cacho  1 , Yi Tang  2 , Yit-Heng Chooi  3
Affiliations
Review

Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi

Ralph A Cacho et al. Front Microbiol. .

Abstract

Genomics has revolutionized the research on fungal secondary metabolite (SM) biosynthesis. To elucidate the molecular and enzymatic mechanisms underlying the biosynthesis of a specific SM compound, the important first step is often to find the genes that responsible for its synthesis. The accessibility to fungal genome sequences allows the bypass of the cumbersome traditional library construction and screening approach. The advance in next-generation sequencing (NGS) technologies have further improved the speed and reduced the cost of microbial genome sequencing in the past few years, which has accelerated the research in this field. Here, we will present an example work flow for identifying the gene cluster encoding the biosynthesis of SMs of interest using an NGS approach. We will also review the different strategies that can be employed to pinpoint the targeted gene clusters rapidly by giving several examples stemming from our work.

Keywords: filamentous fungi; gene clusters; genome mining; next generation sequencing; secondary metabolites.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Next-generation sequencing (NGS) workflow for targeted secondary metabolite (SM) gene cluster discovery.
FIGURE 2
FIGURE 2
Comparative genomics and knowledge-based prediction strategies for targeted SM gene cluster discovery. New biosynthetic knowledge and insights into gene-to-molecular structure relationship would be useful for target SM gene cluster predictions and knowledge-based genome mining for discovery of novel SM compounds.
FIGURE 3
FIGURE 3
Comparative genomics approach for discovery of SM biosynthetic gene cluster. Comparison of the PKS genes in Penicillium aethiopicum and P. chrysogenum was utilized in searching for the griseofulvin and viridicatumtoxin gene cluster (A). In order to narrow down possible candidates for the tryptoquialanine gene cluster in P. aethiopicum, NRPS genes that are non-orthologous to P. chrysogenum NRPS genes but are orthologous to the NRPS genes of the tryptoquivaline producer A. clavatus were found (B). The echinocandin NRPS gene was found in Emericella rugulosa was found by searching for a hexamodule NRPS gene in E. rugulosa that is non-orthologous to A. nidulans NRPS genes (C).
FIGURE 4
FIGURE 4
Griseofulvin biosynthetic gene clusterin P. aethiopicum. Shown are the roles of the genes in the biogenesis of the compound. The non-reducing polyketide synthase (NR-PKS) gene gsfA is found via comparison of PKS genes in P. aethiopicum and P. chrysogenum. Two structural features, O-methylation and chlorination (in red text), were used as criteria to find the candidate gene clusters.
FIGURE 5
FIGURE 5
Viridicatumtoxin biosynthetic gene cluster in P. aethiopicum. As with the case for the griseofulvin, the NR-PKS gene vrtA is non-orthologous to PKS genes in P. chrysogenum. The presence of the prenyltransferase gene vrtC and the O-methyltransferase gene vrtF (in red text) led to the identification of the gene cluster.
FIGURE 6
FIGURE 6
Tryptoquialanine biosynthetic gene cluster in P. aethiopicum. The presence of acyltransferase gene tqaD and the anthranilic acid-activating A domain of TqaA (in red text) were clues to identification of the cluster.
FIGURE 7
FIGURE 7
Echinocandin B biosynthetic gene cluster in E. rugulosa. The gene cluster was found in two loci (ecd and hty). The ecd cluster contained a six module NRPS gene ecdA and an acyl-CoA ligase gene ecdI (in red text), indicating that the gene cluster is involved in the biosynthesis of a lipo-hexapeptide SM. The separate homotyrosine biosynthesis hty gene cluster at a different locus was located by searching for an isopropylmalate synthase (IPMS) homolog (in red text).
See this image and copyright information in PMC

References

    1. Adefarati A. A., Giacobbe R. A., Hensens O. D., Tkacz J. S. (1991). Biosynthesis of L-671,329, an echinocandin-type antibiotic produced by Zalerion arboricola: origins of some of the unusual amino acids and the dimethylmyristic acid side chain. J. Am. Chem. Soc. 113 3542–3545 10.1021/ja00009a048 - DOI
    1. Ahuja M., Chiang Y. M., Chang S. L., Praseuth M. B., Entwistle R., Sanchez J. F., et al. (2012). Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans. J. Am. Chem. Soc. 134 8212–8221 10.1021/ja3016395 - DOI - PMC - PubMed
    1. Ali H., Ries M. I., Lankhorst P. P., Van Der Hoeven R. A., Schouten O. L., Noga M., et al. (2014). A non-canonical NRPS is involved in the synthesis of fungisporin and related hydrophobic cyclic tetrapeptides in Penicillium chrysogenum. PLoS ONE 9:e98212 10.1371/journal.pone.0098212 - DOI - PMC - PubMed
    1. Ames B. D., Walsh C. T. (2010). Anthranilate-activating modules from fungal nonribosomal peptide assembly lines. Biochemistry 49 3351–3365 10.1021/bi100198y - DOI - PMC - PubMed
    1. Awakawa T., Yokota K., Funa N., Doi F., Mori N., Watanabe H., et al. (2009). Physically discrete beta-lactamase-type thioesterase catalyzes product release in atrochrysone synthesis by iterative type I polyketide synthase. Chem. Biol. 16 613–623 10.1016/j.chembiol.2009.04.004 - DOI - PubMed