4 research outputs found
Mechanistic Insights into the Bifunctional Non-Heme Iron Oxygenase Carbapenem Synthase by Active Site Saturation Mutagenesis
The
carbapenem class of β-lactam antibiotics is known for
its remarkable potency, antibacterial spectrum, and resistance to
β-lactamase-mediated inactivation. While the biosynthesis of
structurally “complex” carbapenems, such as thienamycin,
share initial biochemical steps with carbapenem-3-carboxylate (“simple”
carbapenem), the requisite inversion at C5 and formation of the characteristic
α,β-unsaturated carboxylate are different in origin between
the two groups. Here, we consider carbapenem synthase, a mechanistically
distinct bifunctional non-heme iron α-ketoglutarate-dependent
enzyme responsible for the terminal reactions, C5 epimerization and
desaturation, in simple carbapenem production. Interestingly, this
enzyme accepts two stereoisomeric substrates and transforms each to
a common active antibiotic. Owing both to enzyme and product instability,
resorting to saturation mutagenesis of active site and selected second-sphere
residues gave clearly differing profiles of CarC tolerance to structural
modification. Guided by a crystal structure and the mutational data, <i>in silico</i> docking was used to suggest the positioning of
each disastereomeric substrate in the active site. The two orientations
relative to the reactive iron-oxo center are manifest in the two distinct
reactions, C5-epimerization and C2/3-desaturation. These observations
favor a two-step reaction scheme involving two complete oxidative
cycles as opposed to a single catalytic cycle in which an active site
tyrosine, Tyr67, after hydrogen donation to achieve bicyclic ring
inversion, is further hypothesized to serve as a radical carrier
A High-Throughput Screen for the Engineered Production of β-Lactam Antibiotics
High-throughput screens and selections have had profound
impact
on our ability to engineer proteins possessing new, desired properties.
These methods are especially useful when applied to the modification
of existing enzymes to create natural and unnatural products. In an
advance upon existing methods we developed a high-throughput, genetically
regulated screen for the <i>in vivo</i> production of β-lactam
antibiotics using a green fluorescent protein (gfp) reporter. This
assay proved reliable and sensitive and presents a dynamic range under
which a wide array of β-lactam architectural subclasses can
be detected. Moreover, the graded response elicited in this assay
can be used to rank mutant activity. The utility of this development
was demonstrated <i>in vivo</i> and then applied to the
first experimental investigation of a putative catalytic residue in
carbapenem synthase (CarC). Information gained about the mutability
of this residue defines one parameter for enzymatic activity and sets
boundaries for future mechanistic and engineering efforts
Engineering Terpene Biosynthesis in <i>Streptomyces</i> for Production of the Advanced Biofuel Precursor Bisabolene
The
past decade has witnessed a large influx of research toward
the creation of sustainable, biologically derived fuels. While significant
effort has been exerted to improve production capacity in common hosts,
such as <i>Escherichia coli</i> or <i>Saccharomyces cerevisiae</i>, studies concerning alternate microbes comparatively lag.
In an effort to expand the breadth of characterized hosts for fuel
production, we map the terpene biosynthetic pathway in a model actinobacterium, <i>Streptomyces venezuelae</i>,
and further alter secondary metabolism to afford the advanced biofuel
precursor bisabolene. Leveraging information gained from study of
the native isoprenoid pathway, we were able to increase bisabolene
titer nearly 5-fold over the base production strain, more than 2 orders
of magnitude greater than the combined terpene yield in the wild-type
host. We also explored production on carbon sources of varying complexity
to, notably, define this host as one able to perform consolidated
bioprocessing
Production of Odd-Carbon Dicarboxylic Acids in <i>Escherichia coli</i> Using an Engineered Biotin–Fatty Acid Biosynthetic Pathway
Dicarboxylic acids are commodity
chemicals used in the production
of plastics, polyesters, nylons, fragrances, and medications. Bio-based
routes to dicarboxylic acids are gaining attention due to environmental
concerns about petroleum-based production of these compounds. Some
industrial applications require dicarboxylic acids with specific carbon
chain lengths, including odd-carbon species. Biosynthetic pathways
involving cytochrome P450-catalyzed oxidation of fatty acids in yeast
and bacteria have been reported, but these systems produce almost
exclusively even-carbon species. Here we report a novel pathway to
odd-carbon dicarboxylic acids directly from glucose in <i>Escherichia
coli</i> by employing an engineered pathway combining enzymes
from biotin and fatty acid synthesis. Optimization of the pathway
will lead to industrial strains for the production of valuable odd-carbon
diacids