Engineering of Streptoalloteichus tenebrarius 2444 for Sustainable Production of Tobramycin.

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius. However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound's purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆aprK), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆aprK mutant. Fermentation by the engineered strain (∆aprK_1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆aprK). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

Cryptic phosphorylation in nucleoside natural product biosynthesis.

Kinases are annotated in many nucleoside biosynthetic gene clusters but generally are considered responsible only for self-resistance. Here, we report an unexpected 2'-phosphorylation of nucleoside biosynthetic intermediates in the nikkomycin and polyoxin pathways. This phosphorylation is a unique cryptic modification as it is introduced in the third of seven steps during aminohexuronic acid (AHA) nucleoside biosynthesis, retained throughout the pathway's duration, and is removed in the last step of the pathway. Bioinformatic analysis of reported nucleoside biosynthetic gene clusters indicates the presence of cryptic phosphorylation in other pathways and the importance of functional characterization of kinases in nucleoside biosynthetic pathways in general. This study also functionally characterized all of the enzymes responsible for AHA biosynthesis and revealed that AHA is constructed via a unique oxidative C-C bond cleavage reaction. The results indicate a divergent biosynthetic mechanism for three classes of antifungal nucleoside natural products.

Molecular Networking and Whole-Genome Analysis Aid Discovery of an Angucycline That Inactivates mTORC1/C2 and Induces Programmed Cell Death.

Rediscovery of known compounds and time consumed in identification, especially high molecular weight compounds with complex structure, have let down interest in drug discovery. In this study, whole-genome analysis of microbe and Global Natural Products Social (GNPS) molecular networking helped in initial understanding of possible compounds produced by the microbe. Genome data revealed 10 biosythethic gene clusters that encode for secondary metabolites with anticancer potential. NMR analysis of the pure compound revealed the presence of a four-ringed benz[a]anthracene, thus confirming angucycline; molecular networking further confirmed production of this class of compounds. The type II polyketide synthase gene identified in the microbial genome was matched with the urdamycin cluster by BLAST analysis. This information led to ease in identification of urdamycin E and a novel natural derivative, urdamycin V, purified from Streptomyces sp. OA293. Urdamycin E (Urd E) induced apoptosis and autophagy in cancer cell lines. Urd E exerted anticancer action through inactivation of the mTOR complex by preventing phosphorylation at Ser 2448 and Ser 2481 of mTORC1 and mTORC2, respectively. Significant reduction in phosphorylation of the major downstream regulators of both mTORC1 (p70s6k and 4e-bp1) and mTORC2 (Akt) were observed, thus further confirming complete inhibition of the mTOR pathway. Urd E presents itself as a novel mTOR inhibitor that employs a novel mechanism in mTOR pathway inhibition.

Minor components of aminoglycosides: recent advances in their biosynthesis and therapeutic potential.

Covering: up to 2019 There is significant demand for new aminoglycoside antibiotics due to the widespread emergence of multidrug-resistant Gram-negative bacteria and their high toxicity, but these are not easily accessible in nature because their biosynthetic gene clusters are less commonly found in actinomycetes than are other natural products. Mining minor aminoglycoside components whose pharmacological activity has not yet been assessed could be an alternative approach for the development of next-generation antibiotics for use in the post-antibiotic era. Here, we review the biosynthetic steps responsible for the structural diversity of aminoglycosides and highlight current developments regarding the use of natural minor and semi-synthetic aminoglycosides as promising therapeutic leads or candidates.

Recent advances in the biosynthesis of nucleoside antibiotics.

Nucleoside antibiotics are a diverse class of natural products with promising biomedical activities. These compounds contain a saccharide core and a nucleobase. Despite the large number of nucleoside antibiotics that have been reported, biosynthetic studies on these compounds have been limited compared with those on other types of natural products such as polyketides, peptides, and terpenoids. Due to recent advances in genome sequencing technology, the biosynthesis of nucleoside antibiotics has rapidly been clarified. This review covering 2009-2019 focuses on recent advances in the biosynthesis of nucleoside antibiotics.

Characterization of MtdV as a chorismate lyase essential to A201A biosynthesis and precursor-directed biosynthesis of new analogs.

The antimicrobial nucleoside antibiotic A201A is produced by the deep-sea derived Marinactinospora thermotolerans SCSIO 00652. Bioinformatics analysis revealed that mtdV, downstream within the A201A biosynthetic gene cluster, encodes a protein with low homology to a group of chorismate pyruvate-lyases. To explore the role of mtdV in A201A biosynthesis, mtdV was inactivated and HPLC analysis revealed that the resulting ΔmtdV mutant failed to produce A201A; production was partially restored by adding exogenous 4-hydroxybenzoic acid (4HB) to the fermentation. In vitro biochemical assays showed that MtdV catalyzes the conversion of chorismate into 4HB, thereby firmly demonstrating that MtdV is a chorismate lyase involved in A201A biosynthesis. In addition, supplementation of the ΔmtdV mutant with various 4HB analogs enabled production of seven new A201A analogs. Antimicrobial assays showed that the purified A201A analogs 3'-F-A201A and 3'-Cl-A201A were just as active as A201A against the test strains with MIC values of 1-8 µg mL-1.

Biosynthetic and Synthetic Strategies for Assembling Capuramycin-Type Antituberculosis Antibiotics.

Mycobacterium tuberculosis (Mtb) has recently surpassed HIV/AIDS as the leading cause of death by a single infectious agent. The standard therapeutic regimen against tuberculosis (TB) remains a long, expensive process involving a multidrug regimen, and the prominence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant (TDR) strains continues to impede treatment success. An underexplored class of natural products-the capuramycin-type nucleoside antibiotics-have been shown to have potent anti-TB activity by inhibiting bacterial translocase I, a ubiquitous and essential enzyme that functions in peptidoglycan biosynthesis. The present review discusses current literature concerning the biosynthesis and chemical synthesis of capuramycin and analogs, seeking to highlight the potential of the capuramycin scaffold as a favorable anti-TB therapeutic that warrants further development.

In Vivo Characterization of Phosphotransferase-Encoding Genes istP and forP as Interchangeable Launchers of the C3',4'-Dideoxygenation Biosynthetic Pathway of 1,4-Diaminocyclitol Antibiotics.

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'- dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ΔforP mutant ( or ΔistP mutant strain) successfully restored the biosynthesis of 3',4'- dideoxy fortimicins and istamycins , thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

Gentamicin B (GB), a valuable starting material for the preparation of the semisynthetic aminoglycoside antibiotic isepamicin, is produced in trace amounts by the wild-type Micromonospora echinospora. Though the biosynthetic pathway to GB has remained obscure for decades, we have now identified three hidden pathways to GB production via seven hitherto unknown intermediates in M. echinospora. The narrow substrate specificity of a key glycosyltransferase and the C6'-amination enzymes, in combination with the weak and unsynchronized gene expression of the 2'-deamination enzymes, limits GB production in M. echinospora. The crystal structure of the aminotransferase involved in C6'-amination explains its substrate specificity. Some of the new intermediates displayed similar premature termination codon readthrough activity but with reduced toxicity compared to the natural aminoglycoside G418. This work not only led to the discovery of unknown biosynthetic routes to GB, but also demonstrated the potential to mine new aminoglycosides from nature for drug discovery.

Landomycin biosynthesis and its regulation in Streptomyces.

This mini-review is centered on genetic aspects of biosynthesis of landomycins (La), a family of angucycline polyketides. From the very discovery in the 1990s, La were noted for unusual structure and potent anticancer properties. La are produced by a few actinobacteria that belong to genus Streptomyces. Biochemical logic behind the production of La aglycon and glycoside halves and effects of La on mammalian cells have been thoroughly reviewed in 2009-2012. Yet, the genetic diversity of La biosynthetic gene clusters (BGCs) and regulation of their production were not properly reviewed since discovery of La. Here, we aim to fill this gap by focusing on three interrelated topics. First, organization of known La BGCs is compared. Second, up-to-date scheme of biosynthetic pathway to landomycin A (LaA), the biggest (by molar weight) member of La family, is succinctly outlined. Third, we describe genetic and nutritional factors that influence La production and export. A summary of the practical utility of the gained knowledge and future directions to study La biosynthesis conclude this mini-review.

Discovery of 16-Demethylrifamycins by Removing the Predominant Polyketide Biosynthesis Pathway in Micromonospora sp. Strain TP-A0468.

A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms.IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains.

Members of the orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically requires oxidative chemistry. Correspondingly, the evd gene cluster encoding everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate-dependent oxygenases and one bifunctional enzyme with an N-terminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and that of the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.

Protein-protein interactions in polyketide synthase-nonribosomal peptide synthetase hybrid assembly lines.

Covering: up to early 2018 Polyketides and nonribosomal peptides are two major families of natural product with a broad range of biological activities. Polyketide synthases (PKSs) assemble small acetic acid-type acyl building blocks into polyketides through C-C bonds, and nonribosomal peptide synthetases (NRPSs) assemble amino acids into peptides through amide bonds. PKS-NRPS hybrid assembly lines build structurally complex polyketide-amino acid/peptide hybrid molecules that incorporate both acyl and aminoacyl building blocks into their products. Their combined functionalities expand the biological activities of these molecules by mixing their chemical properties. Protein-protein interactions are necessary within PKS-NRPS hybrid assembly lines to achieve accurate linkage between the PKS and NRPS systems. This review summarizes the current understanding of the roles and importance of the protein-protein interactions in various PKS-NRPS hybrid assembly lines.

Enzymology of Anthraquinone-γ-Pyrone Ring Formation in Complex Aromatic Polyketide Biosynthesis.

Aromatic-fused γ-pyrones are structural features of many bioactive natural products and valid scaffolds for medicinal chemistry. However, the enzymology of their formation has not been completely established. Now it is demonstrated that TxnO9, a CalC-like protein belonging to a START family, functions as an unexpected anthraquinone-γ-pyrone synthase involved in the biosynthesis of antitumor antibiotic trioxacarcin A (TXN-A). Structural analysis by NMR identified a likely substrate/product-binding mode and putative key active sites of TxnO9, which allowed an enzymatic mechanism to be proposed. Moreover, a subset of uncharacterized homologous proteins bearing an unexamined Lys-Thr dyad exhibit the same function. Therefore, the functional assignment and mechanistic investigation of this γ-pyrone synthase elucidated an undescribed step in TXN-A biosynthesis, and the discovery of this new branch of polyketide heterocyclases expands the functions of the START superfamily.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

The Immunosuppressant Brasilicardin: Determination of the Biosynthetic Gene Cluster in the Heterologous Host Amycolatopsis japonicum.

Nocardia terpenica IFM 0406 is the producer of the immunosuppressants brasilicardins A-D. Brasilicardin is a promising compound because of its unique mode of action and its higher potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin is so far hampered as Nocardia terpenica IFM 0406 synthesizes brasilicardin in only low amounts and represents a human pathogen (biosafety level 2 BSL2). In order to achieve a safe and high yield production of brasilicardin A (BraA), the authors heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum (A. japonicum::bcaAB01), which is fast growing, genetically accessible and closely related to N. terpenica IFM 0406. In A. japonicum::bcaAB01, four brasilicardin congeners, intermediates of the BraA biosynthesis, are produced. Investigation of the genes flanking the previously defined brasilicardin biosynthetic gene cluster revealed two novel genes (bra0, bra12), which are involved in brasilicardin biosynthesis: bra12 encodes a transcriptional activator of the brasilicardin gene cluster. bra0 codes for a dioxygenase involved in methoxylation of brasilicardin. Based on this finding the authors are able to revise the proposed brasilicardin biosynthesis.

Activation and mechanism of a cryptic oviedomycin gene cluster via the disruption of a global regulatory gene, adpA, in Streptomyces ansochromogenes.

Genome sequencing analysis has revealed at least 35 clusters of likely biosynthetic genes for secondary metabolites in Streptomyces ansochromogenes. Disruption of adpA encoding a global regulator (AdpA) resulted in the failure of nikkomycin production, whereas other antibacterial activities against Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis were observed with the fermentation broth of ΔadpA but not with that of the wild-type strain. Transcriptional analysis showed that a cryptic gene cluster (pks7), which shows high identity with an oviedomycin biosynthetic gene cluster (ovm), was activated in ΔadpA. The corresponding product of pks7 was characterized as oviedomycin by MS and NMR spectroscopy. To understand the molecular mechanism of ovm activation, the roles of six regulatory genes situated in the ovm cluster were investigated. Among them, proteins encoded by co-transcribed genes ovmZ and ovmW are positive regulators of ovm AdpA directly represses the transcription of ovmZ and ovmW Co-overexpression of ovmZ and ovmW can relieve the repression of AdpA on ovm transcription and effectively activate oviedomycin biosynthesis. The promoter of ovmOI-ovmH is identified as the direct target of OvmZ and OvmW. This is the first report that AdpA can simultaneously activate nikkomycin biosynthesis but repress oviedomycin biosynthesis in one strain. Our findings provide an effective strategy that is able to activate cryptic secondary metabolite gene clusters by genetic manipulation of global regulatory genes.

Biosynthetic pathways of aminoglycosides and their engineering.

Despite decades long clinical usage, aminoglycosides still remain a valuable pharmaceutical source for fighting Gram-negative bacterial pathogens, and their newly identified bioactivities are also renewing interest in this old class of antibiotics. As Nature's gift, some aminoglycosides possess natural defensive structural elements that can circumvent drug resistance mechanisms. Thus, a detailed understanding of aminoglycoside biosynthesis will enable us to apply Nature's biosynthetic strategy towards expanding structural diversity in order to produce novel and more robust aminoglycoside analogs. The engineered biosynthesis of novel aminoglycosides is required not only to develop effective therapeutics against the emerging 'superbugs' but also to reinvigorate antibiotic lead discovery in readiness for the emerging post-antibiotic era.

Deciphering the sugar biosynthetic pathway and tailoring steps of nucleoside antibiotic A201A unveils a GDP-l-galactose mutase.

Galactose, a monosaccharide capable of assuming two possible configurational isomers (d-/l-), can exist as a six-membered ring, galactopyranose (Galp), or as a five-membered ring, galactofuranose (Galf). UDP-galactopyranose mutase (UGM) mediates the conversion of pyranose to furanose thereby providing a precursor for d-Galf Moreover, UGM is critical to the virulence of numerous eukaryotic and prokaryotic human pathogens and thus represents an excellent antimicrobial drug target. However, the biosynthetic mechanism and relevant enzymes that drive l-Galf production have not yet been characterized. Herein we report that efforts to decipher the sugar biosynthetic pathway and tailoring steps en route to nucleoside antibiotic A201A led to the discovery of a GDP-l-galactose mutase, MtdL. Systematic inactivation of 18 of the 33 biosynthetic genes in the A201A cluster and elucidation of 10 congeners, coupled with feeding and in vitro biochemical experiments, enabled us to: (i) decipher the unique enzyme, GDP-l-galactose mutase associated with production of two unique d-mannose-derived sugars, and (ii) assign two glycosyltransferases, four methyltransferases, and one desaturase that regiospecifically tailor the A201A scaffold and display relaxed substrate specificities. Taken together, these data provide important insight into the origin of l-Galf-containing natural product biosynthetic pathways with likely ramifications in other organisms and possible antimicrobial drug targeting strategies.