Exclusive Production of Gentamicin C1a from Micromonospora purpurea by Metabolic Engineering.
ABSTRACT: Gentamicin C1a is an important precursor to the synthesis of etimicin, a potent antibiotic. Wild type Micromonospora purpurea Gb1008 produces gentamicin C1a, besides four other gentamicin C components: C1, C2, C2a, and C2b. While the previously reported engineered strain M. purpurea GK1101 can produce relatively high titers of C1a by blocking the genK pathway, a small amount of undesirable C2b is still being synthesized in cells. Gene genL (orf6255) is reported to be responsible for converting C1a to C2b and C2 to C1 in Micromonospora echinospora ATCC15835. In this work, we identify the genL that is also responsible for the same methylation in Micromonospora purpurea. Based on M. purpurea GK1101, we construct a new strain with genL inactivated and show that no C2b is produced in this strain. Therefore, we successfully engineer a strain of M. purpurea that solely produces gentamicin C1a. This strain can potentially be used in the industrial production of C1a for the synthesis of etimicin.
Project description:Gentamicin C complex from Micromonospora echinospora remains a globally important antibiotic, and there is revived interest in the semisynthesis of analogs that might show improved therapeutic properties. The complex consists of five components differing in their methylation pattern at one or more sites in the molecule. We show here, using specific gene deletion and chemical complementation, that the gentamicin pathway up to the branch point is defined by the selectivity of the methyltransferases GenN, GenD1, and GenK. Unexpectedly, they comprise a methylation network in which early intermediates are ectopically modified. Using whole-genome sequence, we have also discovered the terminal 6'-N-methyltransfer required to produce gentamicin C2b from C1a or gentamicin C1 from C2, an example of an essential biosynthetic enzyme being located not in the biosynthetic gene cluster but far removed on the chromosome. These findings fully account for the methylation pattern in gentamicins and open the way to production of individual gentamicins by fermentation, as starting materials for semisynthesis.
Project description:Isepamicin is a weakly toxic but highly active aminoglycoside antibiotic derivative of gentamicin B. Gentamicin B is a naturally occurring minor component isolated from Micromonospora echinospora. 2'-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2'-OH-containing gentamicin B is produced as a minor component. However, the biosynthetic pathway of gentamicin B remains unclear. Considering that gentamicin B shares a unique C2' hydroxyl group with kanamycin A, we aimed to design a new biosynthetic pathway of gentamicin B by combining twelve steps of gentamicin biosynthesis and two steps of kanamycin biosynthesis.We blocked the biosynthetic pathway of byproducts and generated a strain overproducing JI-20A, which was used as a precursor of gentamicin B biosynthesis, by disrupting genK and genP. The amount of JI-20A produced in M. echinospora ?K?P reached 911 ?g/ml, which was 14-fold higher than that of M. echinospora ?P. The enzymes KanJ and KanK necessary to convert 2'-NH2 into 2'-OH from the kanamycin biosynthetic pathway were heterologously expressed in M. echinospora ?K?P to transform JI-20A into gentamicin B. The strain with kanJK under PermE* could produce 80 ?g/ml of gentamicin B, which was tenfold higher than that of the wild-type strain. To enhance gentamicin B production, we employed different promoters and gene integration combinations. When a PhrdB promoter was used and kanJ and kanK were integrated in the genome through gene replacement, gentamicin B was generated as the major product with a maximum yield of 880 ?g/ml.We constructed a new biosynthetic pathway of high-level gentamicin B production; in this pathway, most byproducts were removed. This method also provided novel insights into the biosynthesis of secondary metabolites via the combinatorial biosynthesis.
Project description:BACKGROUND:New semi-synthetic aminoglycoside antibiotics generally use chemical modifications to avoid inactivity from pathogens. One of the most used modifications is 3',4'-di-deoxygenation, which imitates the structure of gentamicin. However, the mechanism of di-deoxygenation has not been clearly elucidated. RESULTS:Here, we report that the bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3',4'-di-deoxygenation via reduction and transamination activities. Following disruption of genB4 in wild-type M. echinospora, its products accumulated in 6'-deamino-6'-oxoverdamicin (1), verdamicin C2a (2), and its epimer, verdamicin C2 (3). Following disruption of genB4 in M. echinospora ?genK, its products accumulated in sisomicin (4) and 6'-N-methylsisomicin (5, G-52). Following in vitro catalytic reactions, GenB4 transformed sisomicin (4) to gentamicin C1a (9) and transformed verdamicin C2a (2) and its epimer, verdamicin C2 (3), to gentamicin C2a (11) and gentamicin C2 (12), respectively. CONCLUSION:This finding indicated that in addition to its transamination activity, GenB4 exhibits specific 4',5' double-bond reducing activity and is responsible for the last step of gentamicin 3',4'-di-deoxygenation. Taken together, we propose three new intermediates that may refine and supplement the specific biosynthetic pathway of gentamicin C components and lay the foundation for the complete elucidation of di-deoxygenation mechanisms.
Project description:In pharmaceutical industry, isepamicin is mainly manufactured from gentamicin B, which is produced by Micromonospora echinospora as a minor component of the gentamicin complex. Improvement of gentamicin B production through metabolic engineering is therefore important to satisfy the increasing demand for isepamicin. We hypothesized that gentamicin B was generated from gentamicin JI-20A via deamination of the C2' amino group. Using kanJ and kanK as the gene probes, we identified the putative deamination-related genes, genR and genS, through genome mining of the gentamicin B producing strain M. echinospora CCTCC M 2018898. Interestingly, genR and genS constitute a gene cassette located approximately 28.7 kb away from the gentamicin gene cluster. Gene knockout of genR and genS almost abolished the production of gentamicin B in the mutant strain, suggesting that these two genes, which are responsible for the last steps in gentamicin B biosynthesis, constitute the missing part of the known gentamicin biosynthetic pathway. Based on these finding, we successfully constructed a gentamicin B high-yielding strain (798 mg/L), in which an overexpression cassette of genR and genS was introduced. Our work fills the missing piece to solve the puzzle of gentamicin B biosynthesis and may inspire future metabolic engineering efforts to generate gentamycin B high-yielding strains that could eventually satisfy the need for industrial manufacturing of isepamicin.
Project description:Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster in Micromonospora echinospora has enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A(2), the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes from M. echinospora and other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain of Streptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression of gtmG together with gtmB-gtmA-gacH led to production of 2'-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that adds N-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression of gtmM in a 2'-N-acetylparomamine-producing recombinant S. venezuelae strain generated paromamine. Expression of gtmE in an engineered paromamine-producing strain of S. venezuelae successfully generated gentamicin A(2), indicating that GtmE is another glycosyltransferase that attaches d-xylose to paromamine. These results represent in vivo evidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.
Project description: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.
Project description:A systematic approach was developed to investigate the stability of gentamicin sulfate (GS) and GS/poly (lactic-co-glycolic acid) (PLGA) coatings on hydroxyapatite surfaces. The influence of environmental factors (light, humidity, oxidation and heat) upon degradation of the drug in the coatings was investigated using liquid chromatography with evaporative light scattering detection and mass spectrometry. GS coated rods were found to be stable across the range of environments assessed, with only an oxidizing atmosphere resulting in significant changes to the gentamicin composition. In contrast, rods coated with GS/PLGA were more sensitive to storage conditions with compositional changes being detected after storage at 60 °C, 75% relative humidity or exposure to light. The effect of γ-irradiation on the coated rods was also investigated and found to have no significant effect. Finally, liquid chromatography-mass spectrometry analysis revealed that known gentamines C1, C1a and C2 were the major degradants formed. Forced degradation of gentamicin coatings did not produce any unexpected degradants or impurities.
Project description:Curcumin is a polyphenolic nutraceutical that acts on multiple biological targets, including protein kinase C (PKC). PKC is a family of serine/threonine kinases central to intracellular signal transduction. We have recently shown that curcumin selectively inhibits PKC?, but not PKC?, in CHO-K1 cells [Pany, S. (2016) Biochemistry 55, 2135-2143]. To understand which domain(s) of PKC? is responsible for curcumin binding and inhibitory activity, we made several domain-swapped mutants in which the C1 (combination of C1A and C1B) and C2 domains are swapped between PKC? and PKC?. Phorbol ester-induced membrane translocation studies using confocal microscopy and immunoblotting revealed that curcumin inhibited phorbol ester-induced membrane translocation of PKC? mutants, in which the ?C1 domain was replaced with ?C1, but not the PKC? mutant in which ?C1 was replaced with the ?C1 domain, suggesting that ?C1 is a determinant for curcumin's inhibitory effect. In addition, curcumin inhibited membrane translocation of PKC? mutants, in which the ?C1A and ?C1B domains were replaced with the ?C1A and ?C1B domains, respectively, indicating the role of both ?C1A and ?C1B domains in curcumin's inhibitory effects. Phorbol 13-acetate inhibited the binding of curcumin to ?C1A and ?C1B with IC50 values of 6.27 and 4.47 ?M, respectively. Molecular docking and molecular dynamics studies also supported the higher affinity of curcumin for ?C1B than for ?C1A. The C2 domain-swapped mutants were inactive in phorbol ester-induced membrane translocation. These results indicate that curcumin binds to the C1 domain of PKC? and highlight the importance of this domain in achieving PKC isoform selectivity.
Project description:Single cell RNA sequencing of the mouse colonic mesenchyme. Mesenchymal/lamina propria cells were isolated from the middle and distal colon of wild type mice in two biological replicates. For each biological replicate the colons of n = 2 mice were pooled. The vast majority of intestinal epithelial cells were depleted by Ethylenediaminetetraacetic acid (EDTA) treatment of the tissue samples and mesenchymal/lamina propria cells were isolated after enzymatic treatment with collagenase XI and dispase. Single cell RNA sequencing was performed with the Drop-seq protocol. N =5 Drop-seq collections (samples) were processed in total, two from the first biological replicate and three from the second. Overall design: Two biological replicates (C1 and C2): one with two Drop-seq collections (samples C1A, C1B) and one with three Drop-seq collections (samples C2A, C2B, C2C)
Project description:The gentamicin drug product is a complex mixture of numerous components, many of which have not individually undergone safety and efficacy assessments. This is in contrast to the majority of medicines that require rigorous characterizations of trace impurities and are dosed as single components. In gentamicin, four components, known as gentamicin congeners C1, C1a, C2, and C2a, comprise the majority of the mixture. A liquid chromatography-mass spectroscopy analysis revealed that the relative abundances of each gentamicin congener in commercial formulations can vary up to 1.9-fold depending on the commercial source of the gentamicin. To determine if the gentamicin used for antibiotic susceptibility testing (AST) would be predictive of the microbiological activity of the product used to dose patients, the relative abundances of the four congeners contained on commercial AST disks were measured. It was found that the congener abundances on the commercial AST disks varied up to 4.1-fold. After purification of the four gentamicin congeners, similar potencies against bacterial strains lacking aminoglycoside-modifying enzymes (AMEs) were observed. However, the potency of the congeners against strains harboring a common AME differed up to 128-fold. Nephrotoxicity of the individual gentamicin congeners also differed significantly in cell-based and repeat-dose rat nephrotoxicity studies. Variations in the composition of commercial gentamicin products combined with toxicity differences between gentamicin congeners suggest that some gentamicin formulations may be more nephrotoxic. Our results also raise the concern that gentamicin susceptibility test results may not be predictive of patient outcomes and could lead to unexpected clinical treatment failures.