A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.biosynthetic enzymes | mycobacteria | metal transport S mall-molecule secondary metabolites are produced by microbes as chemical weapons to combat competing organisms or as communication signals to control complex processes such as virulence, morphological differentiation, biofilm formation, and metal acquisition (1-3). One of the most important classes of secondary metabolites are nonribosomal peptides, which are typically biosynthesized by modular nonribosomal peptide synthetases (NRPSs) in an assembly-line manner (4). Two NRPS-encoding gene clusters (mbt and Rv0096-0101) have been identified from the genome of Mycobacterium tuberculosis, the leading causative agent of tuberculosis that currently infects one-third of the world's population. Although the cluster of mbt has been characterized to biosynthesize mycobactin siderophores that form mycobactin-Fe(III) complexes for iron sequestration (5), the role of Rv0096-0101 remains obscure despite various biological studies that have indicated the production of a virulence factor by this gene cluster (6-14). For example, using transposon-site hybridization, Rv0098 to Rv0101 were predicted to be required for M. tuberculosis survival in a mouse model of infection (10). Consistently, a transposon insertion of Rv0097 attenuated M. tuberculosis growth and survival in mice (7).An in silico homology search has revealed that gene clusters homologous to Rv0096-0101 are conserved in pathogenic mycobacteria, such as M. bovis, M. leprae, M. marinum, M. ulcerans, and M. abscessus (Fig. 1), but absent in nonpathogenic mycobacteria such as M. smegmatis, providing further indication of the virulenceassociated nature of the locus product in mycobacteria. Interestingly, in addition to the genus of Mycobacterium, related operons are found in the phylum of Actinobacteria across genera including Streptomyces, Kutzneria, Nocardia, and Rhodococcus (Fig. 1), suggesting a widespread presence of this cluster. Further bioinformatic analysis has shown that five genes (Rv0097-0101) are conserved across all identified gene cluste...
1-Alkenes are important platform chemicals that are almost exclusively produced from fossil hydrocarbons. Bioproduction of 1-alkenes can mitigate our dependence on declining petrochemical resources, thereby representing an important step in the field of green chemistry. Here, we report the discovery of a new family of membrane-bound desaturase-like enzymes that convert medium-chain fatty acids (10−16 carbons) into the corresponding 1-alkenes through oxidative decarboxylation. We further show that these desaturase-like enzymes could be efficient in transforming lauric acid to 1-undecene in E. coli compared to the existing 1-alkene biosynthetic enzymes. This work expands the enzyme inventory for the transformation of fatty acid precursors to hydrocarbons and promotes the industrial production of mediumchain 1-alkenes through microbial fermentation.
The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We here provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, of which several members are critical for the virulence of pathogenic mycobacteria.
P. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida. We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.
30Despite intensive study for 50 years, the biochemical and genetic links between lysine 31 metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish 32 these biochemical links, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq), a 33 genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple 34 novel enzymes in both L-and D-lysine metabolism. We first describe three pathway enzymes 35 that catabolize L-2-aminoadipate (L-2AA) to 2-ketoglutarate (2KG), connecting D-lysine to the 36 TCA cycle. One of these enzymes, PP_5260, contains a DUF1338 domain, a family with no 37 previously described biological function. Our work also identified the recently described CoA 38 independent route of L-lysine degradation that metabolizes to succinate. We expanded on 39 previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-40 oxoacid selectivity. Proteomics of select pathway enzymes revealed that expression of catabolic 41 genes is highly sensitive to particular pathway metabolites, implying intensive local and global 42 regulation. This work demonstrates the utility of RB-TnSeq for discovering novel metabolic 43 pathways in even well-studied bacteria, as well as a powerful tool for validating previous 44 research. 45 Importance 46 P. putida lysine metabolism can produce multiple commodity chemicals, conferring great 47 biotechnological value. Despite much research, connecting lysine catabolism to central 48 metabolism in P. putida remained undefined. Herein we use Random Barcode Transposon 49Sequencing to fill in the gaps of lysine metabolism in P. putida. We describe a route of 2-50 oxoadipate (2OA) catabolism in bacteria, which utilizes DUF1338 containing protein PP_5260. 51Despite its prevalence in many domains of life, DUF1338 containing proteins had no known 52 biochemical function. We demonstrate PP_5260 is a metalloenzyme which catalyzes an unusual 53 2OA to D-2HG decarboxylation. Our screen also identified a recently described novel glutarate 54 metabolic pathway. We validate previous results, and expand the understanding of glutarate 55hydroxylase CsiD by showing can it use either 2OA or 2KG as a cosubstrate. Our work 56 demonstrates biological novelty can be rapidly identified using unbiased experimental genetics, 57and that RB-TnSeq can be used to rapidly validate previous results. 58 Introduction 59Pseudomonas putida is an ubiquitous saprophytic soil bacterium and is a model organism 60for bioremediation (1). Interest in utilizing P. putida KT2440 as a chassis organism for metabolic 61 engineering has recently surged due to the existence of well-established genetic tools and its 62 robust metabolism of aromatic compounds that resemble lignin hydrolysis products (2-4). As 63 lignin valorization remains essential for the economic feasibility of cellulosic bioproducts, a 64 nuanced and predictable understanding of P. putida metabolism is highly desirable (5). 65Although its aromatic metabolism has garnered mu...
The electron-rich isonitrile is an important functionality in bioactive natural products,but its biosynthesis has been restricted to the IsnA family of isonitrile synthases.W eh erein providet he first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, aputative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus,w as shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides ar evised scheme for the biosynthesis of au nique class of isonitrile lipopeptides,o f which several members are critical for the virulence of pathogenic mycobacteria.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.
The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We here provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, members of which are critical for the virulence of pathogenic mycobacteria.[a]
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