The shikimate pathway: gateway to metabolic diversity.

Covering: 1997 to 2023The shikimate pathway is the metabolic process responsible for the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Seven metabolic steps convert phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) into shikimate and ultimately chorismate, which serves as the branch point for dedicated aromatic amino acid biosynthesis. Bacteria, fungi, algae, and plants (yet not animals) biosynthesize chorismate and exploit its intermediates in their specialized metabolism. This review highlights the metabolic diversity derived from intermediates of the shikimate pathway along the seven steps from PEP and E4P to chorismate, as well as additional sections on compounds derived from prephenate, anthranilate and the synonymous aminoshikimate pathway. We discuss the genomic basis and biochemical support leading to shikimate-derived antibiotics, lipids, pigments, cofactors, and other metabolites across the tree of life.

Enzymatic assembly of the salinosporamide γ-lactam-ß-lactone anticancer warhead.

The marine microbial natural product salinosporamide A (marizomib) is a potent proteasome inhibitor currently in clinical trials for the treatment of brain cancer. Salinosporamide A is characterized by a complex and densely functionalized γ-lactam-ß-lactone bicyclic warhead, the assembly of which has long remained a biosynthetic mystery. Here, we report an enzymatic route to the salinosporamide core catalyzed by a standalone ketosynthase (KS), SalC. Chemoenzymatic synthesis of carrier protein-tethered substrates, as well as intact proteomics, allowed us to probe the reactivity of SalC and understand its role as an intramolecular aldolase/ß-lactone synthase with roles in both transacylation and bond-forming reactions. Additionally, we present the 2.85-Å SalC crystal structure that, combined with site-directed mutagenesis, allowed us to propose a bicyclization reaction mechanism. This work challenges our current understanding of the role of KS enzymes and establishes a basis for future efforts toward streamlined production of a clinically relevant chemotherapeutic.

Genome mining methods to discover bioactive natural products.

Covering: 2016 to 2021With genetic information available for hundreds of thousands of organisms in publicly accessible databases, scientists have an unprecedented opportunity to meticulously survey the diversity and inner workings of life. The natural product research community has harnessed this breadth of sequence information to mine microbes, plants, and animals for biosynthetic enzymes capable of producing bioactive compounds. Several orthogonal genome mining strategies have been developed in recent years to target specific chemical features or biological properties of bioactive molecules using biosynthetic, resistance, or transporter proteins. These "biosynthetic hooks" allow researchers to query for biosynthetic gene clusters with a high probability of encoding previously undiscovered, bioactive compounds. This review highlights recent case studies that feature orthogonal approaches that exploit genomic information to specifically discover bioactive natural products and their gene clusters.

Discovery and engineering of the antibody response against a prominent skin commensal.

The ubiquitous skin colonist Staphylococcus epidermidis elicits a CD8 + T cell response pre-emptively, in the absence of an infection 1 . However, the scope and purpose of this anti-commensal immune program are not well defined, limiting our ability to harness it therapeutically. Here, we show that this colonist also induces a potent, durable, and specific antibody response that is conserved in humans and non-human primates. A series of S. epidermidis cell-wall mutants revealed that the cell surface protein Aap is a predominant target. By colonizing mice with a strain of S. epidermidis in which the parallel ß-helix domain of Aap is replaced by tetanus toxin fragment C, we elicit a potent neutralizing antibody response that protects mice against a lethal challenge. A similar strain of S. epidermidis expressing an Aap-SpyCatcher chimera can be conjugated with recombinant immunogens; the resulting labeled commensal elicits high titers of antibody under conditions of physiologic colonization, including a robust IgA response in the nasal mucosa. Thus, immunity to a common skin colonist involves a coordinated T and B cell response, the latter of which can be redirected against pathogens as a novel form of topical vaccination.

Refactoring the Cryptic Streptophenazine Biosynthetic Gene Cluster Unites Phenazine, Polyketide, and Nonribosomal Peptide Biochemistry.

The disconnect between the genomic prediction of secondary metabolite biosynthetic potential and the observed laboratory production profile of microorganisms is well documented. While heterologous expression of biosynthetic gene clusters (BGCs) is often seen as a potential solution to bridge this gap, it is not immune to many challenges including impaired regulation, the inability to recruit essential building blocks, and transcriptional and/or translational silence of the biosynthetic genes. Here we report the discovery, cloning, refactoring, and heterologous expression of a cryptic hybrid phenazine-type BGC (spz) from the marine actinomycete Streptomyces sp. CNB-091. Overexpression of the engineered spz pathway resulted in increased production and chemical diversity of phenazine natural products belonging to the streptophenazine family, including bioactive members containing an unprecedented N-formylglycine attachment. An atypical discrete adenylation enzyme in the spz cluster is required to introduce the formylglycine moiety and represents a phylogenetically distinct class of adenylation proteins.

Maternal transfer and sublethal immune system effects of brevetoxin exposure in nesting loggerhead sea turtles (Caretta caretta) from western Florida.

Blooms of Karenia brevis (also called red tides) occur almost annually in the Gulf of Mexico. The health effects of the neurotoxins (i.e., brevetoxins) produced by this toxic dinoflagellate on marine turtles are poorly understood. Florida's Gulf Coast represents an important foraging and nesting area for a number of marine turtle species. Most studies investigating brevetoxin exposure in marine turtles thus far focus on dead and/or stranded individuals and rarely examine the effects in apparently "healthy" free-ranging individuals. From May-July 2014, one year after the last red tide bloom, we collected blood from nesting loggerhead sea turtles (Caretta caretta) on Casey Key, Florida USA. These organisms show both strong nesting and foraging site fidelity. The plasma was analyzed for brevetoxin concentrations in addition to a number of health and immune-related parameters in an effort to establish sublethal effects of this toxin. Lastly, from July-September 2014, we collected unhatched eggs and liver and yolk sacs from dead-in-nest hatchlings from nests laid by the sampled females and tested these samples for brevetoxin concentrations to determine maternal transfer and effects on reproductive success. Using a competitive enzyme-linked immunosorbent assay (ELISA), all plasma samples from nesting females tested positive for brevetoxin (reported as ng brevetoxin-3[PbTx-3] equivalents [eq]/mL) exposure (2.1-26.7ng PbTx-3eq/mL). Additionally, 100% of livers (1.4-13.3ng PbTx-3eq/mL) and yolk sacs (1.7-6.6ng PbTx-3eq/mL) from dead-in-nest hatchlings and 70% of eggs (<1.0-24.4ng PbTx-3eq/mL) tested positive for brevetoxin exposure with the ELISA. We found that plasma brevetoxin concentrations determined by an ELISA in nesting females positively correlated with gamma-globulins, indicating a potential for immunomodulation as a result of brevetoxin exposure. While the sample sizes were small, we also found that plasma brevetoxin concentrations determined by an ELISA in nesting females significantly correlated with liver brevetoxin concentrations of dead-in-nest hatchlings and that brevetoxins could be related to a decreased reproductive success in this species. This study suggests that brevetoxins can still elicit negative effects on marine life long after a bloom has dissipated. These results improve our understanding of maternal transfer and sublethal effects of brevetoxin exposure in marine turtles.