19 Search Results

Abstract Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometrymore » uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.« less

Abstract Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α‐diazoester functional group. Herein, we report the discovery of the azaserine ( aza ) biosynthetic gene cluster from Glycomyces harbinensis . Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)‐producing enzymes, implicating HYAA in α‐diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2‐electron oxidation of a hydrazonoacetyl intermediate is required for α‐diazoester formation, constituting amore » distinct logic for diazo biosynthesis. Uncovering this biological route for α‐diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene‐mediated biosynthetic transformations in vivo.« less

Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C–C bond formation. Here, in this study, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E. coli cells grown under aerobic conditions, as expected they are either reduced by the wild-type MG1655 strain or stabilized by a strain engineered for reduced aromatic aldehyde reduction (the E. coli RARE strain). Surprisingly, when these same aldehydes are supplemented to restingmore » cell preparations of either E. coli strain, under many conditions we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 h after their addition. Given that our newly engineered strain exhibits reduced oxidation and reduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. We applied the new strain to resting cell biocatalysis for two kinds of reactions – the reduction of 2-furoic acid to furfural and the condensation of 3-hydroxybenzaldehyde and glycine to form a non-standard β-hydroxy-α-amino acid. In each case, we observed substantial improvements in product titer 20 h after reaction initiation (9-fold and 10-fold, respectively). Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.« less

Abstract In the biosynthesis of the iron‐guanylylpyridinol (FeGP) cofactor, 6‐carboxymethyl‐5‐methyl‐4‐hydroxy‐2‐pyridinol ( 1 ) is 3‐methylated to form 2 , then 4‐guanylylated to form 3 , and converted into the full cofactor. HcgA‐G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S ‐adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis Δ hcgA strain was complemented with HcgA or precursors 1 , 2 or 3 . The results suggested that HcgA catalyzes the biosynthetic reactionmore » that forms 1 . We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the Δ hcgG system was recovered by HcgG but not by 3 , which indicated that HcgG catalyzes the reactions after the biosynthesis of 3 . The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.« less

Waste plastics represent major environmental and economic burdens due to their ubiquity, slow breakdown rates, and inadequacy of current recycling routes. Polyethylenes are particularly problematic, because they lack robust recycling approaches despite being the most abundant plastics in use today. We report a novel chemical and biological approach for the rapid conversion of polyethylenes into structurally complex and pharmacologically active compounds. We present conditions for aerobic, catalytic digestion of polyethylenes collected from post-consumer and oceanic waste streams, creating carboxylic diacids that can then be used as a carbon source by the fungus Aspergillus nidulans. As a proof of principle, wemore » have engineered strains of A. nidulans to synthesize the fungal secondary metabolites asperbenzaldehyde, citreoviridin, and mutilin when grown on these digestion products. This hybrid approach considerably expands the range of products to which polyethylenes can be upcycled.« less

Sustaining a robust metabolic network requires a balanced and fully functioning proteome. In addition to amino acids, many enzymes require cofactors (coenzymes and engrafted prosthetic groups) to function properly. Extensively validated resource allocation models, such as genome-scale models of metabolism and gene expression (ME-models), have the ability to compute an optimal proteome composition underlying a metabolic phenotype, including the provision of all required cofactors. Here we apply the ME-model for Escherichia coli K-12 MG1655 to computationally examine how environmental conditions change the proteome and its accompanying cofactor usage. We found that: (1) The cofactor requirements computed by the ME-model mostlymore » agree with the standard biomass objective function used in models of metabolism alone (M-models); (2) ME-model computations reveal non-intuitive variability in cofactor use under different growth conditions; (3) An analysis of ME-model predicted protein use in aerobic and anaerobic conditions suggests an enrichment in the use of peroxyl scavenging acids in the proteins used to sustain aerobic growth; (4) The ME-model could describe how limitation in key protein components affect the metabolic state of E . coli . Genome-scale models have thus reached a level of sophistication where they reveal intricate properties of functional proteomes and how they support different E . coli lifestyles.« less

Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of D-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their L-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of nocardicin biosynthesis mediates both the effectively complete L- to D-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures ofmore » the nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain.« less

Formycin A is a potent purine nucleoside antibiotic with a C-glycosidic linkage between the ribosyl moiety and the pyrazolopyrimidine base. In this work, a cosmid is identified from the Streptomyces kaniharaensis genome library that contains the for gene cluster responsible for the biosynthesis of formycin. Subsequent gene deletion experiments and in vitro characterization of the forBCH gene products established their catalytic functions in formycin biosynthesis. Results also demonstrated that PurH from de novo purine biosynthesis plays a key role in pyrazolopyrimidine formation during biosynthesis of formycin A. The participation of PurH in both pathways represents a good example of howmore » primary and secondary metabolism are interlinked.« less

Small molecules containing the N-nitroso group, such as the bacterial natural product streptozotocin, are prominent carcinogens and important cancer chemotherapeutics. Despite the considerable importance of this functional group to human health, enzymes dedicated to the assembly of the N-nitroso unit have not been identified. In this work, we show that SznF, a metalloenzyme from the biosynthesis of streptozotocin, catalyses an oxidative rearrangement of the guanidine group of N^ω-methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF reveal two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. This biosynthetic reaction, which hasmore » little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-haem-iron-dependent enzymes to include N–N bond formation. We find that biosynthetic gene clusters that encode SznF homologues are widely distributed among bacteria—including environmental organisms, plant symbionts and human pathogens—which suggests an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites.« less

Abstract Complex reduced polyketides represent the largest class of natural products that have applications in medicine, agriculture, and animal health. This structurally diverse class of compounds shares a common methodology of biosynthesis employing modular enzyme systems called polyketide synthases (PKSs). The modules are composed of enzymatic domains that share sequence and functional similarity across all known PKSs. We have used the nomenclature of synthetic biology to classify the enzymatic domains and modules as parts and devices, respectively, and have generated detailed lists of both. In addition, we describe the chassis (hosts) that are used to assemble, express, and engineer themore » parts and devices to produce polyketides. We describe a recently developed software tool to design PKS system and provide an example of its use. Finally, we provide perspectives of what needs to be accomplished to fully realize the potential that synthetic biology approaches bring to this class of molecules.« less