Erratum: The dabABC operon is a marker of C4-alkylated monobactam biosynthesis and responsible for (2S,3R)-diaminobutyrate production.

[This corrects the article DOI: 10.1016/j.isci.2024.109202.].

The dabABC operon is a marker of C4-alkylated monobactam biosynthesis and responsible for (2S,3R)-diaminobutyrate production.

Non-ribosomal peptide synthetases (NRPSs) assemble metabolites of medicinal and commercial value. Both serine and threonine figure prominently in these processes and separately can be converted to the additional NRPS building blocks 2,3-diaminopropionate (Dap) and 2,3-diaminobutyrate (Dab). Here we bring extensive bioinformatics, in vivo and in vitro experimentation to compose a unified view of the biosynthesis of these widely distributed non-canonical amino acids that both derive by pyridoxal-mediated ß-elimination of the activated O-phosphorylated substrates followed by ß-addition of an amine donor. By examining monobactam biosynthesis in Pseudomonas and in Burkholderia species where it is silent, we show that (2S,3R)-Dab synthesis depends on an l-threonine kinase (DabA), a ß-replacement reaction with l-aspartate (DabB) and an argininosuccinate lyase-like protein (DabC). The growing clinical importance of monobactams to both withstand Ambler Class B metallo-ß-lactamases and retain their antibiotic activity make reprogrammed precursor and NRPS synthesis of modified monobactams a feasible and attractive goal.

Synthesis of functionalized 2,3-diaminopropionates and their potential for directed monobactam biosynthesis.

The N-sulfonated monobactams harbor considerable potential to combat emerging bacterial infections that are problematic to treat due to their metallo-ß-lactamase mediated resistance against conventional ß-lactam antibiotics. Herein, we report a divergent synthesis of C3-substituted 2,3-diaminopropionates featuring an array of small functional groups and examine their potential as alternative precursors during monobactam biosynthesis in a mutant strain (ΔsulG) of Pseudomonas acidophila that is deficient in the supply of this native precursor. In vitro assays revealed high diastereoselectivity, as well as a substrate tolerance by the terminal adenylation domain of the non-ribosomal peptide synthetase (NRPS) SulM toward the majority of synthetic analogs. Chemical complementation of this mutant yielded a fluorinated, bioactive monobactam through fermentation as confirmed by a combination of spectrometric data and microbiological assays. This study demonstrates site-specific functionalization of a clinically important natural product and sets in place a platform for further strain improvements and engineered NRPS-biosynthesis of non-native congeners.

Colorimetric Determination of Adenylation Domain Activity in Nonribosomal Peptide Synthetases by Using Chrome Azurol S.

Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurol S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630 nm, visualized by a prominent color change from blue to orange.

Early Oxidative Transformations During the Biosynthesis of Terrein and Related Natural Products.

The mycotoxin terrein is derived from the C10 -precursor 6-hydroxymellein (6-HM) via an oxidative ring contraction. Although the corresponding biosynthetic gene cluster (BGC) has been identified, details of the enzymatic oxidative transformations are lacking. Combining heterologous expression and in vitro studies we show that the flavin-dependent monooxygenase (FMO) TerC catalyzes the initial oxidative decarboxylation of 6-HM. The reactive intermediate is further hydroxylated by the second FMO TerD to yield a highly oxygenated aromatic species, but further reconstitution of the pathway was hampered. A related BGC was identified in the marine-derived Roussoella sp. DLM33 and confirmed by heterologous expression. These studies demonstrate that the biosynthetic pathways of terrein and related (polychlorinated) congeners diverge after oxidative decarboxylation of the lactone precursor that is catalyzed by a conserved FMO and further indicate that early dehydration of the side chain is an essential step.

Co-Oxidative Transformation of Piperine to Piperonal and 3,4-Methylenedioxycinnamaldehyde by a Lipoxygenase from Pleurotus sapidus.

The valuable aroma compound piperonal with its vanilla-like olfactory properties is of high interest for the fragrance and flavor industry. A lipoxygenase (LOXPsa 1) of the basidiomycete Pleurotus sapidus was identified to convert piperine, the abundant pungent principle of black pepper (Piper nigrum), to piperonal and a second volatile product, 3,4-methylenedioxycinnamaldehyde, with a vanilla-like odor through an alkene cleavage. The reaction principle was co-oxidation, as proven by its dependence on the presence of linoleic or α-linolenic acid, common substrates of lipoxygenases. Optimization of the reaction conditions (substrate concentrations, reaction temperature and time) led to a 24-fold and 15-fold increase of the piperonal and 3,4-methylenedioxycinnamaldehyde concentration using the recombinant enzyme. Monokaryotic strains showed different concentrations of and ratios between the two reaction products.

Biosynthesis of 6-Hydroxymellein Requires a Collaborating Polyketide Synthase-like Enzyme.

The polyketide synthase (PKS)-like protein TerB, consisting of inactive dehydratase, inactive C-methyltransferase, and functional ketoreductase domains collaborates with the iterative non reducing PKS TerA to produce 6-hydroxymellein, a key pathway intermediate during the biosynthesis of various fungal natural products. The catalytically inactive dehydratase domain of TerB appears to mediate productive interactions with TerA, demonstrating a new mode of trans-interaction between iterative PKS components.

The same but different: multiple functions of the fungal flavin dependent monooxygenase SorD from Penicillium chrysogenum.

Sorbicillinoids are a large family of fungal secondary metabolites with a diverse range of structures and numerous bioactivites, some of which have pharmaceutical potential. The flavin-dependent monooxygenase SorD from Penicillium chrysogenum (PcSorD) utilizes sorbicillinol to catalyze a broad scope of reactions: formation of oxosorbicillinol and epoxysorbicillinol; intermolecular Diels-Alder and Michael-addition dimerization reactions; and dimerization of a sorbicillinol derivative with oxosorbicillinol. PcSorD shares only 18.3% sequence identity with SorD from Trichoderma reesei (TrSorD) and yet unexpectedly catalyzes many of the same reactions, however, the formation of oxosorbicillinol and bisvertinolone by PcSorD extends the range of reactions catalyzed by a single enzyme. Phylogenetic analysis indicates that PcSorD and TrSorD bind the flavin cofactor covalently but via different residues and point mutations confirm these residues are essential for activity.

Diels-Alder Reactions During the Biosynthesis of Sorbicillinoids.

The sorbicillinoids are a class of biologically active and structurally diverse fungal polyketides arising from sorbicillin. Through co-expression of sorA, sorB, sorC, and sorD from Trichoderma reesei QM6a, the biosynthetic pathway to epoxysorbicillinol and dimeric sorbicillinoids, which resemble Diels-Alder-like and Michael-addition-like products, was reconstituted in Aspergillus oryzae NSAR1. Expression and feeding experiments demonstrated the crucial requirement of the flavin-dependent monooxygenase SorD for the formation of dimeric sorbicillinoids, hybrid sorbicillinoids, and epoxysorbicillinol in vivo. In contrast to prior reports, SorD catalyses neither the oxidation of 2',3'-dihydrosorbicillin to sorbicillin nor the oxidation of sorbicillinol to oxosorbicillinol. This is the first report that both the intermolecular Diels-Alder and Michael dimerization reactions, as well as the epoxidation of sorbicillinol are catalysed in vivo by SorD.