Heterologous expression, biosynthesis, and mutagenesis of type II lantibiotics from Bacillus licheniformis in Escherichia coli

Lichenicidin is a class II two-component lantibiotic produced by Bacillus licheniformis. It is composed of the two peptides Bliα and Bliβ, which act synergistically against various Gram-positive bacteria. The lichenicidin gene cluster was successfully expressed in Escherichia coli, thus constituting the first report to our knowledge of a full reconstitution of a lantibiotic biosynthetic pathway in?vivo by a Gram-negative host. This system was further exploited to characterize and assign the function of proteins encoded in the biosynthetic gene cluster in the maturation of lichenicidin peptides. Moreover, a trans complementation system was developed for expression of Bliα and Bliβ variants in?vivo. This contribution will spur future studies in the heterologous expression and engineering of lantibiotics.     

Cloning and characterization of an environmental DNA-derived gene cluster that encodes the biosynthesis of the antitumor substance BE-54017

Soil is predicted to contain thousands of unique bacterial species per gram. Soil DNA libraries represent large reservoirs of biosynthetic diversity from which diverse secondary metabolite gene clusters can be recovered and studied. The screening of an archived soil DNA library using primers designed to target oxytryptophan dimerization genes allowed us to identify and functionally characterize the first indolotryptoline biosynthetic gene cluster. The recovery and heterologous expression of an environmental DNA-derived gene cluster encoding the biosynthesis of the antitumor substance BE-54017 is reported here. Transposon mutagenesis identified two monooxygenases, AbeX1 and AbeX2, as being responsible for the transformation of an indolocarbazole precursor into the indolotryptoline core of BE-54017.     

High-throughput quantification of lincomycin traces in fermentation broth of genetically modified Streptomyces spp. Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography with UV detection

A new separation and quantification method using ultra-performance liquid chromatography (UPLC) with UV detection was developed for detection of lincomycin traces in fermentation broth of different Streptomyces spp. A similar high-performance liquid chromatography (HPLC) protocol was simultaneously developed for comparison purposes. Both methods were validated and showed a linear range of detector response for quantification of lincomycin in concentration from 3.125 to 1000.0 microgml(-1) with correlation coefficient 0.999 and recoveries ranging from 81.5 to 89.85% with precision < or =5%. Compared with the HPLC, the UPLC method offered high sample throughput and about 10 times lower consumption of solvents. The developed assays were used for determination of lincomycin production in genetically manipulated production strain Streptomyces lincolnensis and for determination of lincomycin production after heterologous expression of lincomycin biosynthetic gene cluster in non-producing strain Streptomyces coelicolor.     

Identification and analysis of the core biosynthetic machinery of tubulysin, a potent cytotoxin with potential anticancer activity

Myxobacteria are well known for their biosynthetic potential, especially for the production of cytotoxic compounds with potential anticancer activities. The tubulysins are currently in preclinical development. They are produced in very low quantities, and genetic manipulation of producing strains has never been accomplished. We report the development of a mariner-based transposon mutagenesis system for Angiococcus disciformis An d48. Extracts from a library of 1200 mutants were analyzed for the presence of tubulysin by a microscopic cell nucleus fragmentation bioassay. The transposition sites of four tubulysin-negative mutants were identified by vector recovery, which led to the identification and the sequencing of the corresponding core biosynthetic gene locus. Sequence analysis of more than 80,000 bp reveals an unusual multimodular hybrid polyketide synthase/peptide synthetase assembly line with a variety of unprecedented features.     

Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.     

Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity.

BACKGROUND: Combinatorial biosynthesis techniques using polyketide synthases (PKSs) in heterologous host organisms have enabled the production of macrolide aglycone libraries in which many positions of the macrolactone ring have been manipulated. However, the deoxysugar moieties of macrolides, absent in previous libraries, play a critical role in contributing to the antimicrobial properties exhibited by compounds such as erythromycin. Since the glycosidic components of polyketides dramatically alter their molecular binding properties, it would be useful to develop general expression hosts and vectors for synthesis and attachment of deoxysugars to expand the nature and size of such polyketide libraries. RESULTS: A set of nine deoxysugar biosynthetic and auxiliary genes from the picromycin/methymycin (pik) cluster was integrated in the chromosome of Streptomyces lividans to create a host which synthesizes TDP-D-desosamine. The pik desosaminyl transferase was also included so that when the strain was transformed with a previously constructed library of expression plasmids encoding genetically modified PKSs that produce different macrolactones, the resulting strains produced desosaminylated derivatives. Although conversion of the macrolactones was generally low, bioassays revealed that, unlike their aglycone precursors, these novel macrolides possessed antibiotic activity. CONCLUSIONS: Based on the structural differences among the compounds that were glycosylated it appears that the desosaminyl transferase from the pik gene cluster is quite tolerant of changes in the macrolactone substrate. Since others have demonstrated tolerance towards modifications in the sugar substituent, one can imagine employing this approach to alter both polyketide and deoxysugar pathways to produce 'unnatural' natural product libraries.     

Biosynthetic pathway for high structural diversity of a common dilactone core in antimycin production

We herein report comparative analysis of two versions of the biosynthetic gene clusters of antimycins, a natural product family possessing up to 44 distinct entities. The biosynthetic pathway of antimycins is amenable to the high structural variation of the substrates, supported by successes in heterologous expression of the ant cluster and in fluorine incorporation. The latter facilitated the investigation of the structure-activity relationship into the usually invariable 3-formamidosalicylic acid moiety of the molecules.     

Characterization of the maduropeptin biosynthetic gene cluster from Actinomadura madurae ATCC 39144 supporting a unifying paradigm for enediyne biosynthesis

The biosynthetic gene cluster for the enediyne antitumor antibiotic maduropeptin (MDP) from Actinomadura madurae ATCC 39144 was cloned and sequenced. Cloning of the mdp gene cluster was confirmed by heterologous complementation of enediyne polyketide synthase (PKS) mutants from the C-1027 producer Streptomyces globisporus and the neocarzinostatin producer Streptomyces carzinostaticus using the MDP enediyne PKS and associated genes. Furthermore, MDP was produced, and its apoprotein was isolated and N-terminal sequenced; the encoding gene, mdpA, was found to reside within the cluster. The biosynthesis of MDP is highlighted by two iterative type I PKSs--the enediyne PKS and a 6-methylsalicylic acid PKS; generation of (S)-3-(2-chloro-3-hydroxy-4-methoxyphenyl)-3-hydroxypropionic acid derived from L-alpha-tyrosine; a unique type of enediyne apoprotein; and a convergent biosynthetic approach to the final MDP chromophore. The results demonstrate a platform for engineering new enediynes by combinatorial biosynthesis and establish a unified paradigm for the biosynthesis of enediyne polyketides.     

Cloning, sequencing, analysis, and heterologous expression of the fredericamycin biosynthetic gene cluster from Streptomyces griseus

Fredericamycin (FDM) A, a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique chiral spiro carbon center, exhibits potent cytotoxicity and has been studied as a new type of anticancer drug lead because of its novel molecular architecture. The fdm gene cluster was localized to 33-kb DNA segment of Streptomyces griseus ATCC 49344, and its involvement in FDM A biosynthesis was proven by gene inactivation, complementation, and heterologous expression experiments. The fdm cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS) and tailoring enzymes as well as several regulatory and resistance proteins. The FDM PKS features a KSalpha subunit with heretofore unseen tandem cysteines at its active site, a KSbeta subunit that is distinct phylogenetically from KSbeta of hexa-, octa-, or decaketide PKSs, and a dedicated phosphopantetheinyl transferase. Further study of the FDM PKS could provide new insight into how a type II PKS controls chain length in aromatic polyketide biosynthesis. The availability of the fdm genes, in vivo characterization of the fdm cluster in S. griseus, and heterologous expression of the fdm cluster in Streptomyces albus set the stage to investigate FDM A biosynthesis and engineer the FDM biosynthetic machinery for the production of novel FDM A analogues.     

Biosynthesis of the Structurally Unique Polycyclopropanated Polyketide–Nucleoside Hybrid Jawsamycin (FR‐900848)

The biosynthetic gene cluster of antifungal agent jawsamycin (FR‐900848) has been identified by heterologous expression. A series of gene inactivations and in vitro and in vivo analysis of key enzymes in the biosynthetic pathway established their functions. A novel mechanism involving a radical S‐adenosyl methionine (SAM) cyclopropanase collaborating with an iterative polyketide synthase is proposed for the construction of the unique polycyclopropanated backbone. Our reconstitution system sets the stage for studying the catalytic mechanism of this intriguing contiguous cyclopropanation.     

An artificial pathway to 3,4-dihydroxybenzoic acid allows generation of new aminocoumarin antibiotic recognized by catechol transporters of E. coli

An artificial operon was synthesized, consisting of the genes for chorismate pyruvate-lyase of E. coli and for 4-hydroxybenzoate 3-hydroxylase of Corynebacterium cyclohexanicum. This operon, directing the biosynthesis of 3,4-dihdroxybenzoate, was expressed in the heterologous expression host Streptomyces coelicolor M512, together with a modified biosynthetic gene cluster for the aminocoumarin antibiotic clorobiocin. The resulting strain produced a clorobiocin derivative containing a 3,4-dihdroxybenzoyl moiety. Its structure was confirmed by MS and NMR analysis, and it was found to be a potent inhibitor of the gyrases from Escherichia coli and Staphylococcus aureus. Bioassays against different E. coli mutants suggested that this compound is actively imported by catechol siderophore transporters in the cell envelope. This study provides an example of the structure of a natural product that can be rationally modified by synthetic biology.     

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.     

Heterologous Reconstitution of Ikarugamycin Biosynthesis in E. coli

Polycyclic tetramate macrolactams (PTMs) are a family of biomedically promising natural products with challenging molecular frameworks. Despite these interesting properties, so far only relatively little is known about the biosynthetic origin of PTMs, in particular concerning the mechanism by which their ring systems are formed. Herein we present the first insights into these processes by using the biosynthesis of ikarugamycin as an example. This has been facilitated by the first heterologous expression of a PTM biosynthetic gene cluster in Escherichia coli. With this approach it will not only become possible to mechanistically investigate already known PTM biosynthetic pathways in more detail in the future, but also to interrogate cryptic PTM biosynthetic pathways chemically and biochemically.     

Refactoring the Concise Biosynthetic Pathway of Cyanogramide Unveils Spirooxindole Formation Catalyzed by a P450 Enzyme

Cyanogramide ( 1 ) from the marine actinomycete Actinoalloteichus cyanogriseus WH1‐2216‐6 features a unique spirooxindole skeleton and exhibits significant bioactivity to efficiently reverse drug resistance in tumor cells. The biosynthetic gene cluster of 1 in A. cyanogriseus WH1‐2216‐6 was identified and refactored by promoter engineering for heterologous expression in Streptomyces coelicolor YF11, thereby enabling the production of 1 and five new derivatives. Interesting, four of them, including 1 , were identified as enantiomeric mixtures in different ratios. The functions of tailoring enzymes, including two methyltransferases (CyaEF), and three cytochrome P450 monooxygenases (CyaGHI) were confirmed by gene inactivation and feeding experiments, leading to the elucidation of a concise biosynthetic pathway for 1 . Notably, CyaH was biochemically verified to catalyze the formation of the spirooxindole skeleton in 1 through an unusual carbocation‐mediated semipinacol‐type rearrangement reaction.     

The complete gene cluster of the antitumor agent gilvocarcin V and its implication for the biosynthesis of the gilvocarcins

Gilvocarcin V, an antitumor agent produced by the bacterium Streptomyces griseoflavus G? 3592, is the most studied representative of the distinct family of benzo[d]naphtho[1,2-b]pyran-6-one aryl C-glycoside antibiotics, which show excellent antitumor activity and a remarkably low toxicity. Its biosynthesis contains many intriguing steps, including an oxidative rearrangement, the C-glycosylation, and the generation of a vinyl side chain. These steps all contribute to structural elements of the drug, which are essential for its biological activity, but only poorly understood. Herein we report the cloning and characterization of the gilvocarcin (gil) gene cluster from S. griseoflavus G? 3592, and its heterologous expression in a foreign host (S. lividans). This is the first reported gene cluster encoding the biosynthesis of a benzo[d]naphtho[1,2-b]pyran-6-one aryl C-glycoside antibiotic, which not only provides insights regarding the biosynthesis of gilvocarcin V but also lays the foundation for the detailed studies of its intriguing biosynthetic steps and possibly for the generation of gilvocarcin analogues with improved biological activities through combinatorial biosynthesis.     

Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster

Fosfomycin is a clinically utilized, highly effective antibiotic, which is active against methicillin- and vancomycin-resistant pathogens. Here we report the cloning and characterization of a complete fosfomycin biosynthetic cluster from Streptomyces fradiae and heterologous production of fosfomycin in S. lividans. Sequence analysis coupled with gene deletion and disruption revealed that the minimal cluster consists of fom1-4, fomA-D. A LuxR-type activator that was apparently required for heterologous fosfomycin production was also discovered approximately 13 kb away from the cluster and was named fomR. The genes fomE and fomF, previously thought to be involved in fosfomycin biosynthesis, were shown not to be essential by gene disruption. This work provides new insights into fosfomycin biosynthesis and opens the door for fosfomycin overproduction and creation of new analogs via biomolecular pathway engineering.     

Polyketide β-branching in bryostatin biosynthesis: identification of surrogate acetyl-ACP donors for BryR, an HMG-ACP synthase

In vitro analysis of natural product biosynthetic gene?products isolated from unculturable symbiotic bacteria is necessary to probe the functionalities of these enzymes. Herein, we report the biochemical characterization of BryR, the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) homolog implicated in β-branching at C13 and C21 of the core ring system from the bryostatin metabolic pathway (Bry). We confirmed the activity of BryR using two complementary methods, radio-SDS PAGE, and Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS). The activity of BryR depended on pairing of the native acetoacetyl-BryM3 acceptor acyl carrier protein (ACP) with an appropriate donor acetyl-ACP from a heterologous HMGS cassette. Additionally, the ability of BryR to discriminate between various ACPs was assessed using a surface plasmon resonance (SPR)-based protein-protein binding assay. Our data suggest that specificity for a protein-bound acyl group is a distinguishing feature between HMGS homologs found in PKS or PKS/NRPS biosynthetic pathways and those of primary metabolism. These findings reveal an important example of molecular recognition between protein components that are essential for biosynthetic fidelity in natural product assembly and modification.