Critical analysis of polycyclic tetramate macrolactam biosynthetic gene cluster phylogeny and functional diversity.

Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggest their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity. To address the origins of this complexity and understand its influence on PTM discovery, we engaged in a combination of bioinformatics to systematically classify PTM BGCs and PTM-targeted metabolomics to compare the products of select BGC types. By comparing groups of producers and BGC mutants, we exposed knowledge gaps that complicate bioinformatics-driven product predictions. In sum, we provide new insights into the evolution of PTM BGCs while systematically accounting for the PTMs discovered thus far. The combined computational and metabologenomic findings presented here should prove useful for guiding future discovery.IMPORTANCEPolycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically important bacteria, including Streptomyces and Lysobacter spp. Their molecular products are typically bioactive, having substantial agricultural and therapeutic interest. Leveraging bacterial genomics for the discovery of new related molecules is thus desirable, but drawing accurate structural predictions from bioinformatics alone remains challenging. This difficulty stems from a combination of previously underappreciated biosynthetic complexity and remaining knowledge gaps, compounded by a stream of yet-uncharacterized PTM biosynthetic loci gleaned from recently sequenced bacterial genomes. We engaged in the following study to create a useful framework for cataloging historic PTM clusters, identifying new cluster variations, and tracing evolutionary paths for these molecules. Our data suggest new PTM chemistry remains discoverable in nature. However, our metabolomic and mutational analyses emphasize the practical limitations of genomics-based discovery by exposing hidden complexity.

Characterization of Levan Fructan Produced by a Gluconobacter japonicus Strain Isolated from a Sugarcane Processing Facility.

During raw sugarcane processing, a significant portion of lost sucrose is attributable to microbial degradation. Sucrose consumption by many bacteria is also linked to the production of exopolysaccharides (EPS) such as dextrans and fructans. These resulting EPS cause operational challenges during raw sugar manufacturing. Here, we report the characterization of EPS from a fructan-forming Gluconobacter japonicus bacterium that we previously isolated from a Louisiana sugarcane factory. The genome sequencing revealed the presence of two encoded levansucrase genes, lsrA and lsrB. One levansucrase, LsrB, was detected in the secreted protein fraction of G. japonicus LASM12 by QTOF LC-MS. The spotting assays indicated that G. japonicus produces EPS using sucrose and raffinose as substrates. The G. japonicus EPS correlated with levan fructan commercial standards by 1H-NMR, and with the characteristic carbohydrate fingerprint region for FTIR spectra, confirming that the G. japonicus EPS is levan fructan. The glycosyl composition and glycosyl linkage analysis revealed a linear ß-2,6-fructofuranosyl polysaccharide with occasional (5.7%) ß-2,1-fructofuranosyl branches. The gel permeation chromatography of the levan fructan EPS showed two main peaks at 4.5 kDa and 8 kDa and a very minor peak at 500 kDa. G. japonicus was identified as a producer of levan fructan. These findings will be useful for future studies aimed at evaluating the impact of levan fructans on sugar crop processing, which have been historically underestimated in industry.

Draft genomes of 17 bacterial isolates from Louisiana raw sugarcane factory juices and biofilms.

We report the draft genomes for 17 bacterial isolates belonging to the genera Gluconobacter, Leuconostoc, and Pantoea that were obtained from Louisiana raw sugarcane factory juice and biofilm samples.

Microbiome Analysis of Sugarcane Juices and Biofilms from Louisiana Raw Sugar Factories.

During postharvest processing of sugarcane for raw sugar, microbial activity results in sucrose loss and undesirable exopolysaccharide (EPS) production. Historically, culture-based approaches have focused on the bacterium Leuconostoc mesenteroides as the main contributor to both processes. However, recent studies have shown that diverse microbes are present in sugarcane factories and may also contribute to sugarcane juice deterioration. In the present study, high-throughput amplicon-based sequence profiling was applied to gain a more comprehensive view of the microbial community in Louisiana raw sugar factories. Microbial profiling of the bacterial and fungal microbiomes by 16S V4 and ITS1 sequences, respectively, identified 417 bacterial amplicon sequence variants (ASVs) and 793 fungal ASVs. While Leuconostoc was indeed the most abundant bacterial genus overall (40.9% of 16S sequences), multiple samples were dominated by other taxa such as Weissella and Lactobacillus, underscoring the microbial diversity present in sugarcane factories. Furthermore, flask cultures inoculated with the same samples demonstrated differences in the rate of sucrose consumption, as well as the production of exopolysaccharides and other organic acids, which may result from the observed differences in microbial composition. IMPORTANCE Amplicon-based sequencing was utilized to address long-ignored gaps in microbiological knowledge about the diversity of microbes present in processing streams at Louisiana sugarcane raw sugar factories. These results support an emerging model where diverse organisms contribute to sugarcane juice degradation, help to contextualize microbial contamination problems faced by raw sugar factories, and will guide future studies on biocontrol measures to mitigate sucrose losses and operational challenges due to exopolysaccharide production.

Recovery of Aconitic Acid from Sweet Sorghum Plant Extract Using a Solvent Mixture, and Its Potential Use as a Nematicide.

Trans-aconitic acid (TAA) is naturally present in sweet sorghum juice and syrup, and it has been promoted as a potential biocontrol agent for nematodes. Therefore, we developed a process for the extraction of aconitic acid from sweet sorghum syrup. The process economics were evaluated, and the extract was tested for its capability to suppress the motility of the nematodes Caenorhabditis elegans and Meloidogyne incognita. Aconitic acid could be efficiently extracted from sweet sorghum syrup using acetone:butanol:ethanol mixtures, and it could be recovered from this solvent with a sodium carbonate solution, with an overall extraction and recovery efficiency of 86%. The estimated production cost was USD 16.64/kg of extract and this was highly dependent on the solvent cost, as the solvent was not recycled but was resold for recovery at a fraction of the cost. The extract was effective in reducing the motility of the parasitic M. incognita and causing over 78% mortality of the nematode when 2 mg/mL of TAA extract was added. However, this positive result could not conclusively be linked solely to TAA. An unidentified component (or components) in the acetone:butanol:ethanol sweet sorghum extract appears to be an effective nematode inhibitor, and it may merit further investigation. The impact of aconitic acid on C. elegans appeared to be entirely controlled by pH.

Discovery of unusual dimeric piperazyl cyclopeptides encoded by a Lentzea flaviverrucosa DSM 44664 biosynthetic supercluster.

Rare actinomycetes represent an underexploited source of new bioactive compounds. Here, we report the use of a targeted metabologenomic approach to identify piperazyl compounds in the rare actinomycete Lentzea flaviverrucosa DSM 44664. These efforts to identify molecules that incorporate piperazate building blocks resulted in the discovery and structural elucidation of two dimeric biaryl-cyclohexapeptides, petrichorins A and B. Petrichorin B is a symmetric homodimer similar to the known compound chloptosin, but petrichorin A is unique among known piperazyl cyclopeptides because it is an asymmetric heterodimer. Due to the structural complexity of petrichorin A, solving its structure required a combination of several standard chemical methods plus in silico modeling, strain mutagenesis, and solving the structure of its biosynthetic intermediate petrichorin C for confident assignment. Furthermore, we found that the piperazyl cyclopeptides comprising each half of the petrichorin A heterodimer are made via two distinct nonribosomal peptide synthetase (NRPS) assembly lines, and the responsible NRPS enzymes are encoded within a contiguous biosynthetic supercluster on the L. flaviverrucosa chromosome. Requiring promiscuous cytochrome p450 crosslinking events for asymmetric and symmetric biaryl production, petrichorins A and B exhibited potent in vitro activity against A2780 human ovarian cancer, HT1080 fibrosarcoma, PC3 human prostate cancer, and Jurkat human T lymphocyte cell lines with IC50 values at low nM levels. Cyclic piperazyl peptides and their crosslinked derivatives are interesting drug leads, and our findings highlight the potential for heterodimeric bicyclic peptides such as petrichorin A for inclusion in future pharmaceutical design and discovery programs.

Multifunctional P450 Monooxygenase CftA Diversifies the Clifednamide Pool through Tandem C-H Bond Activations.

Polycyclic tetramate macrolactams (PTMs) are a class of structurally complex hybrid polyketide-nonribosomal peptide (PK-NRP) natural products produced by diverse bacteria. Several PTMs display pharmaceutically interesting bioactivities, and the early stages of PTM biosynthesis involving polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymology are well studied. However, the timing and mechanisms of post PKS-NRPS oxidations by P450 monooxygenases encoded in PTM biosynthetic gene clusters (BGCs) remain poorly characterized. Here we demonstrate that CftA, encoded in clifednamide-type PTM BGCs, is a multifunctional P450 monooxygenase capable of converting the C29-C30 ethyl side chain of ikarugamycin to either a C29-C30 methyl ketone or a C29-C30 hydroxymethyl ketone through C-H bond activation, resulting in the formation of clifednamide A or clifednamide C, respectively. We also report the complete structure of clifednamide C solved via multidimensional NMR (COSY, HSQC, HMBC, NOESY, and TOCSY) using material purified from an engineered Streptomyces strain optimized for production. Finally, the in vitro reconstitution of recombinant CftA catalytic activity revealed the oxidation cascade for sequential conversion of ikarugamycin to clifednamide A and clifednamide C. Our findings confirm prior genetics-based predictions on the origins of clifednamide complexity via P450s encoded in PTM BGCs and place CftA into a growing group of multifunctional P450s that tailor PTM natural products through late-stage regioselective C-H bond activation.

A comparative metabologenomic approach reveals mechanistic insights into Streptomyces antibiotic crypticity.

Streptomyces genomes harbor numerous, biosynthetic gene clusters (BGCs) encoding for drug-like compounds. While some of these BGCs readily yield expected products, many do not. Biosynthetic crypticity represents a significant hurdle to drug discovery, and the biological mechanisms that underpin it remain poorly understood. Polycyclic tetramate macrolactam (PTM) antibiotic production is widespread within the Streptomyces genus, and examples of active and cryptic PTM BGCs are known. To reveal further insights into the causes of biosynthetic crypticity, we employed a PTM-targeted comparative metabologenomics approach to analyze a panel of S. griseus clade strains that included both poor and robust PTM producers. By comparing the genomes and PTM production profiles of these strains, we systematically mapped the PTM promoter architecture within the group, revealed that these promoters are directly activated via the global regulator AdpA, and discovered that small promoter insertion-deletion lesions (indels) differentiate weaker PTM producers from stronger ones. We also revealed an unexpected link between robust PTM expression and griseorhodin pigment coproduction, with weaker S. griseus-clade PTM producers being unable to produce the latter compound. This study highlights promoter indels and biosynthetic interactions as important, genetically encoded factors that impact BGC outputs, providing mechanistic insights that will undoubtedly extend to other Streptomyces BGCs. We highlight comparative metabologenomics as a powerful approach to expose genomic features that differentiate strong, antibiotic producers from weaker ones. This should prove useful for rational discovery efforts and is orthogonal to current engineering and molecular signaling approaches now standard in the field.

Draft Genome Sequences of Two Polycyclic Tetramate Macrolactam Producers, Streptomyces sp. Strains JV180 and SP18CM02.

Here, we report the draft genome sequences of two related Streptomyces sp. strains, JV180 and SP18CM02. Despite their isolation from soils in Connecticut and Missouri (USA), respectively, they are strikingly similar in gene content. Both belong to the Streptomyces griseus clade and harbor several secondary metabolite biosynthetic gene clusters.

Bioinformatic and Functional Evaluation of Actinobacterial Piperazate Metabolism.

Piperazate (Piz) is a nonproteinogenic amino acid noted for its unusual N-N bond motif. Piz is a proline mimic that imparts conformational rigidity to peptides. Consequently, piperazyl molecules are often bioactive and desirable for therapeutic exploration. The in vitro characterization of Kutzneria enzymes KtzI and KtzT recently led to a biosynthetic pathway for Piz. However, Piz anabolism in vivo has remained completely uncharacterized. Herein, we describe the systematic interrogation of actinobacterial Piz metabolism using a combination of bioinformatics, genetics, and select biochemistry. Following studies in Streptomyces flaveolus, Streptomyces lividans, and several environmental Streptomyces isolates, our data suggest that KtzI-type enzymes are conditionally dispensable for Piz production. We also demonstrate the feasibility of Piz monomer production using engineered actinobacteria for the first time. Finally, we show that some actinobacteria employ fused KtzI-KtzT chimeric enzymes to produce Piz. Our findings have implications for future piperazyl drug discovery, pathway engineering, and fine chemical bioproduction.

Draft Genome Sequence of Streptomyces sp. Strain JV178, a Producer of Clifednamide-Type Polycyclic Tetramate Macrolactams.

Here, we report the draft genome sequence of Streptomyces sp. JV178, a strain originating from Connecticut (USA) garden soil. This strain produces the polycyclic tetramate macrolactam compounds clifednamides A and B. The draft genome contains 10.65 Mb, 9,045 predicted protein coding sequences, and several natural product biosynthetic loci.

Native and Engineered Clifednamide Biosynthesis in Multiple Streptomyces spp.

Polycyclic tetramate macrolactam (PTM) natural products are produced by actinomycetes and other bacteria. PTMs are often bioactive, and the simplicity of their biosynthetic clusters make them attractive for bioengineering. Clifednamide-type PTMs from Streptomyces sp. strain JV178 contain a distinctive ketone group, suggesting the existence of a novel PTM oxidizing enzyme. Here, we report the new cytochrome P450 enzyme (CftA) is required for clifednamide production. Genome mining was used to identify several new clifednamide producers, some having improved clifednamide yields. Using a parallel synthetic biology approach, CftA isozymes were used to engineer the ikarugamycin pathway of Streptomyces sp. strain NRRL F-2890 to yield clifednamides. Further, we observed that strong CftA expression leads to the production of a new PTM, clifednamide C. We demonstrate the utility of both genome mining and synthetic biology to rapidly increase clifednamide production.