6 research outputs found
Mutations in the Proteolipid Subunits of the Vacuolar H<sup>+</sup>‑ATPase Provide Resistance to Indolotryptoline Natural Products
Indolotryptoline natural products
represent a small family of structurally
unique chromopyrrolic acid-derived antiproliferative agents. Like
many prospective anticancer agents before them, the exploration of
their potential clinical utility has been hindered by the limited
information known about their mechanism of action. To study the mode
of action of two closely related indolotryptolines (BE-54017, cladoniamide
A), we selected for drug resistant mutants using a multidrug resistance-suppressed
(MDR-sup) <i>Schizosaccharomyces pombe</i> strain. As fission
yeast maintains many of the basic cancer-relevant cellular processes
present in human cells, it represents an appealing model to use in
determining the potential molecular target of antiproliferative natural
products through resistant mutant screening. Full genome sequencing
of resistant mutants identified mutations in the c and c′ subunits
of the proteolipid substructure of the vacuolar H<sup>+</sup>-ATPase
complex (V-ATPase). This collection of resistance-conferring mutations
maps to a site that is distant from the nucleotide-binding sites of
V-ATPase and distinct from sites found to confer resistance to known
V-ATPase inhibitors. Acid vacuole staining, cross-resistance studies,
and direct c/c′ subunit mutagenesis all suggest that indolotryptolines
are likely a structurally novel class of V-ATPase inhibitors. This
work demonstrates the general utility of resistant mutant selection
using MDR-sup <i>S. pombe</i> as a rapid and potentially
systematic approach for studying the modes of action of cytotoxic
natural products
Targeted Metagenomics: Finding Rare Tryptophan Dimer Natural Products in the Environment
Natural
product discovery from environmental genomes (metagenomics)
has largely been limited to the screening of existing environmental
DNA (eDNA) libraries. Here, we have coupled a chemical-biogeographic
survey of chromopyrrolic acid synthase (CPAS) gene diversity with
targeted eDNA library production to more efficiently access rare tryptophan
dimer (TD) biosynthetic gene clusters. A combination of traditional
and synthetic biology-based heterologous expression efforts using
eDNA-derived gene clusters led to the production of hydroxysporine
(<b>1</b>) and reductasporine (<b>2</b>), two bioactive
TDs. As suggested by our phylogenetic analysis of CPAS genes, identified
in our survey of crude eDNA extracts, reductasporine (<b>2</b>) contains an unprecedented TD core structure: a pyrrolinium indolocarbazole
core that is likely key to its unusual bioactivity profile. This work
demonstrates the potential for the discovery of structurally rare
and biologically interesting natural products using targeted metagenomics,
where environmental samples are prescreened to identify the most phylogenetically
unique gene sequences and molecules associated with these genes are
accessed through targeted metagenomic library construction and heterologous
expression
Discovery and Synthetic Refactoring of Tryptophan Dimer Gene Clusters from the Environment
Here we investigate bacterial tryptophan
dimer (TD) biosynthesis
by probing environmental DNA (eDNA) libraries for chromopyrrolic acid
(CPA) synthase genes. Functional and bioinformatics analyses of TD
clusters indicate that CPA synthase gene sequences diverge in concert
with the functional output of their respective clusters, making this
gene a powerful tool for guiding the discovery of novel TDs from the
environment. Twelve unprecedented TD biosynthetic gene clusters that
can be arranged into five groups (A–E) based on their ability
to generate distinct TD core substructures were recovered from eDNA
libraries. Four of these groups contain clusters from both cultured
and culture independent studies, while the remaining group consists
entirely of eDNA-derived clusters. The complete synthetic refactoring
of a representative gene cluster from the latter eDNA specific group
led to the characterization of the erdasporines, cytotoxins with a
novel carboxy-indolocarbazole TD substructure. Analysis of CPA synthase
genes in crude eDNA suggests the presence of additional TD gene clusters
in soil environments
A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology
Centralized
facilities for genetic engineering, or “biofoundries”,
offer the potential to design organisms to address emerging needs
in medicine, agriculture, industry, and defense. The field has seen
rapid advances in technology, but it is difficult to gauge current
capabilities or identify gaps across projects. To this end, our foundry
was assessed via a timed “pressure test”, in which 3
months were given to build organisms to produce 10 molecules unknown
to us in advance. By applying a diversity of new approaches, we produced
the desired molecule or a closely related one for six out of 10 targets
during the performance period and made advances toward production
of the others as well. Specifically, we increased the titers of 1-hexadecanol,
pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne
warhead underlying powerful antimicrobials, established a cell-free
system for monoterpene production, produced an intermediate toward
vincristine biosynthesis, and encoded 7802 individually retrievable
pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran
and barbamide were designed and constructed, but toxicity or analytical
tools inhibited further progress. In sum, we constructed 1.2 Mb DNA,
built 215 strains spanning five species (<i>Saccharomyces cerevisiae</i>, <i>Escherichia coli</i>, <i>Streptomyces albidoflavus</i>, <i>Streptomyces coelicolor</i>, and <i>Streptomyces
albovinaceus</i>), established two cell-free systems, and performed
690 assays developed in-house for the molecules
A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology
Centralized
facilities for genetic engineering, or “biofoundries”,
offer the potential to design organisms to address emerging needs
in medicine, agriculture, industry, and defense. The field has seen
rapid advances in technology, but it is difficult to gauge current
capabilities or identify gaps across projects. To this end, our foundry
was assessed via a timed “pressure test”, in which 3
months were given to build organisms to produce 10 molecules unknown
to us in advance. By applying a diversity of new approaches, we produced
the desired molecule or a closely related one for six out of 10 targets
during the performance period and made advances toward production
of the others as well. Specifically, we increased the titers of 1-hexadecanol,
pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne
warhead underlying powerful antimicrobials, established a cell-free
system for monoterpene production, produced an intermediate toward
vincristine biosynthesis, and encoded 7802 individually retrievable
pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran
and barbamide were designed and constructed, but toxicity or analytical
tools inhibited further progress. In sum, we constructed 1.2 Mb DNA,
built 215 strains spanning five species (<i>Saccharomyces cerevisiae</i>, <i>Escherichia coli</i>, <i>Streptomyces albidoflavus</i>, <i>Streptomyces coelicolor</i>, and <i>Streptomyces
albovinaceus</i>), established two cell-free systems, and performed
690 assays developed in-house for the molecules
A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology
Centralized
facilities for genetic engineering, or “biofoundries”,
offer the potential to design organisms to address emerging needs
in medicine, agriculture, industry, and defense. The field has seen
rapid advances in technology, but it is difficult to gauge current
capabilities or identify gaps across projects. To this end, our foundry
was assessed via a timed “pressure test”, in which 3
months were given to build organisms to produce 10 molecules unknown
to us in advance. By applying a diversity of new approaches, we produced
the desired molecule or a closely related one for six out of 10 targets
during the performance period and made advances toward production
of the others as well. Specifically, we increased the titers of 1-hexadecanol,
pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne
warhead underlying powerful antimicrobials, established a cell-free
system for monoterpene production, produced an intermediate toward
vincristine biosynthesis, and encoded 7802 individually retrievable
pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran
and barbamide were designed and constructed, but toxicity or analytical
tools inhibited further progress. In sum, we constructed 1.2 Mb DNA,
built 215 strains spanning five species (<i>Saccharomyces cerevisiae</i>, <i>Escherichia coli</i>, <i>Streptomyces albidoflavus</i>, <i>Streptomyces coelicolor</i>, and <i>Streptomyces
albovinaceus</i>), established two cell-free systems, and performed
690 assays developed in-house for the molecules