Pyridoxal-5'-phosphate-dependent enzyme GenB3 Catalyzes C-3',4'-dideoxygenation in gentamicin biosynthesis.

BACKGROUND: The C-3',4'-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3'-phosphotransferase (APH(3')) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown. RESULTS: Here, we report that GenP catalyzes 3' phosphorylation of the gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that the pyridoxal-5'-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3',4'-dideoxy-4',5'-ene-6'-oxo products. The following C-6'-transamination and the GenB4-catalyzed reduction of 4',5'-olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP-dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis. CONCLUSIONS: This discovery solves a long-standing puzzle in gentamicin biosynthesis and enriches our knowledge of the chemistry of PLP-dependent enzymes. Interestingly, these results demonstrate that to evade APH(3') deactivation by pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3') to activate hydroxyls as leaving groups for the 3',4'-dideoxygenation in gentamicin biosynthesis.

Application of Genetic Engineering Approaches to Improve Bacterial Metabolite Production.

Genetic engineering is a powerful method to improve the fermentation yield of bacterial metabolites. Since many biosynthetic mechanisms of bacterial metabolites have been unveiled, genetic engineering approaches have been applied to various issues of biosynthetic pathways, such as transcription, translation, post-translational modification, enzymes, transporters, etc. In this article, natamycin, avermectins, gentamicins, piperidamycins, and ß-valienamine have been chosen as examples to review recent progress in improving their production by genetic engineering approaches. In these cases, not only yields of target products have been increased, but also yields of by-products have been decreased, and new products have been created.

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.

Crystal Structure of GenD2, an NAD-Dependent Oxidoreductase Involved in the Biosynthesis of Gentamicin.

Gentamicins are clinically relevant aminoglycoside antibiotics produced by several Micromonospora species. Gentamicins are highly methylated and functionalized molecules, and their biosynthesis include glycosyltransferases, dehydratase/oxidoreductases, aminotransferases, and methyltransferases. The biosynthesis of gentamicin A from gentamicin A2 involves three enzymatic steps that modify the hydroxyl group at position 3″ of the unusual garosamine sugar to provide its substitution for an amino group, followed by an N-methylation. The first of these reactions is catalyzed by GenD2, an oxidoreductase from the Gfo/Idh/MocA protein family, which reduces the hydroxyl at the C3″ of gentamicin A to produce 3''-dehydro-3''-oxo-gentamicin A2 (DOA2). In this work, we solved the structure of GenD2 in complex with NAD+. Although the structure of GenD2 has a similar fold to other members of the Gfo/Idh/MocA family, this enzyme has several new features, including a 3D-domain swapping of two ß-strands that are involved in a novel oligomerization interface for this protein family. In addition, the active site of this enzyme also has several specialties which are possibly involved in the substrate specificity, including a number of aromatic residues and a negatively charged region, which is complementary to the polycationic aminoglycoside-substrate. Therefore, docking simulations provided insights into the recognition of gentamicin A2 and into the catalytic mechanism of GenD2. This is the first report describing the structure of an oxidoreductase involved in aminoglycoside biosynthesis and could open perspectives into producing new aminoglycoside derivatives by protein engineering.

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.

Cobalamin-dependent radical S-adenosyl-l-methionine enzymes in natural product biosynthesis.

Covering: 2011 to 2018 This highlight summarizes the investigation of cobalamin (Cbl)- and radical S-adenosyl-l-methionine (SAM)-dependent enzymes found in natural product biosynthesis to date and suggests some possibilities for the future. Though some mechanistic aspects are apparently shared, the overall diversity of this family's functions and abilities is significant and may be tailored to the specific substrate and/or reaction being catalyzed. A little over a year ago, the first crystal structure of a Cbl- and radical SAM-dependent enzyme was solved, providing the first insight into what may be the shared scaffolding of these enzymes.

Methyltransferases of gentamicin biosynthesis.

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.

Improving gentamicin B and gentamicin C1a production by engineering the glycosyltransferases that transfer primary metabolites into secondary metabolites biosynthesis.

Gentamicin B and gentamicin C1a are the direct precursor for Isepamicin and Etimicin synthesis, respectively. Although producing strains have been improved for many years, both gentamicin B titer and gentamicin C1a titer in the fermentation are still low. Because all gentamicin components are biosynthesized using UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and UDP-xylose as precursors, we tried to explore strategies for development of strains capable of directing greater fluxes of these precursors into production of gentamicins. The glycosyltransferases KanM1 and GenM2, which are responsible for UDP-GlcNAc and UDP-xylose transfer, respectively, were overexpressed in gentamicin B producing strain Micromonospora echinospora JK4. It was found that gentamicin B could be improved by up to 54% with improvement of KanM1 and GenM2 expression during appropriately glucose feeding. To prove this strategy is widely usable, the KanM1 and GenM2 were also overexpressed in gentamicin C1a producing strain, titers of gentamicin C1a improved by 45% when compared with titers of the starting strain. These results demonstrated overexpression the glycosyltransferases that transfer primary metabolites into secondary metabolites is workable for improvement of gentamicins production.

Biosynthesis of 3″-demethyl-gentamicin C components by genN disruption strain of Micromonospora echinospora and test their antimicrobial activities in vitro.

Gentamicin consists primarily of four components, which have different patterns of methylation at C-6' position. The methyl groups have a significant impact on gentamicin antimicrobial activity. Sequence analysis predicted that GenN was a methyltransferase in the gentamicin biosynthetic pathway. To study the function of genN, it was disrupted in Micromonospora echinospora. The genN disruption strains produced 3″-N-demethyl-gentamicin C complex instead of the gentamicin C complex. In this study, 3″-N-demethyl gentamicin C1a was purified from the broth of disruption strain, and its structure was elucidated using MS and NMR. Besides 3″-N-demethyl products corresponding to gentamicin C1a, C2, and C2a, two 3″-N-demethyl products corresponding to gentamicin C1 were detected, which were concluded as C-6' epimers originating from decreased substrate specificity of 6'-N methyltransferase. To explore the effects of 3″-N-methyl on gentamicin antimicrobial activity, antimicrobial activity of these demethyl gentamicin analogues were tested in vitro. 3″-N-Demethyl gentamicin components have identical activity with corresponding components of gentamicin. The results of bioassays showed that the 3″-N-methyl group has little impact on gentamicin activity. However, these highly bioactive compounds afforded a unique opportunity for creating new and high potent aminoglycoside antibiotics.

[Aminoglycoside gentamicin research: fundamental progress and new application prospects].

As an important aminoglycosides antibiotic, gentamicin has been used clinically over decades. With the development in modern biological technology, the mechanisms of gentamicin action and resistance, its biosynthesis and structural modification were studied in great depth. Meanwhile, its emerging novel bioactivities and potential applications are also under extensive exploration. Here we summarize the latest progresses and prospects towards the future development of gentamicin for more efficient and effective uses.

Delineating the biosynthesis of gentamicin x2, the common precursor of the gentamicin C antibiotic complex.

Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.

Genetic engineering combined with random mutagenesis to enhance G418 production in Micromonospora echinospora.

G418, produced by fermentation of Micromonospora echinospora, is an aminoglycoside antibiotic commonly used in genetic selection and maintenance of eukaryotic cells. Besides G418, M. echinospora produces many G418 analogs. As a result, the G418 product always contains impurities such as gentamicin C1, C1a, C2, C2a, gentamicin A and gentamicin X2. These impurities are less potent but more toxic than G418, but the purification of G418 is difficult because it has similar properties to its impurities. G418 is an intermediate in the gentamicin biosynthesis pathway. From G418 the pathway proceeds via successive dehydrogenation and aminotransferation at the C-6' position to generate the gentamicin C complex, but genes responsible for these steps are still obscure. Through disruption of gacJ, which is deduced to encode a C-6' dehydrogenase, the biosynthetic impurities gentamicin C1, C1a, C2 and C2a were all removed, and G418 became the main product of the gacJ disruption strain. These results demonstrated that gacJ is in charge of conversion of the 6'-OH of G418 into 6'-NH2. Disruption of gacJ not only eliminates the impurities seen in the original strain but also improves G418 titers by 15-fold. G418 production was further improved by 26.6 % through traditional random mutagenesis. Through the use of combined traditional and recombinant genetic techniques, we produced a strain from which most impurities were removed and G418 production was improved by 19 fold.

Specificity and promiscuity at the branch point in gentamicin biosynthesis.

Gentamicin C complex is a mixture of aminoglycoside antibiotics used to treat severe Gram-negative bacterial infections. We report here key features of the late-stage biosynthesis of gentamicins. We show that the intermediate gentamicin X2, a known substrate for C-methylation at C-6' to form G418 catalyzed by the radical SAM-dependent enzyme GenK, may instead undergo oxidation at C-6' to form an aldehyde, catalyzed by the flavin-linked dehydrogenase GenQ. Surprisingly, GenQ acts in both branches of the pathway, likewise oxidizing G418 to an analogous ketone. Amination of these intermediates, catalyzed mainly by aminotransferase GenB1, produces the known intermediates JI-20A and JI-20B, respectively. Other pyridoxal phosphate-dependent enzymes (GenB3 and GenB4) act in enigmatic dehydroxylation steps that convert JI-20A and JI-20B into the gentamicin C complex or (GenB2) catalyze the epimerization of gentamicin C2a into gentamicin C2.

GenK-catalyzed C-6' methylation in the biosynthesis of gentamicin: isolation and characterization of a cobalamin-dependent radical SAM enzyme.

The existence of cobalamin (Cbl)-dependent enzymes that are members of the radical S-adenosyl-l-methionine (SAM) superfamily was previously predicted on the basis of bioinformatic analysis. A number of these are Cbl-dependent methyltransferases, but the details surrounding their reaction mechanisms have remained unclear. In this report we demonstrate the in vitro activity of GenK, a Cbl-dependent radical SAM enzyme that methylates an unactivated sp(3) carbon during the biosynthesis of gentamicin, an aminoglycoside antibiotic. Experiments to investigate the stoichiometry of the GenK reaction revealed that 1 equiv each of 5'-deoxyadenosine and S-adenosyl-homocysteine are produced for each methylation reaction catalyzed by GenK. Furthermore, isotope-labeling experiments demonstrate that the S-methyl group from SAM is transferred to Cbl and the aminoglycoside product during the course of the reaction. On the basis of these results, one mechanistic possibility for the GenK reaction can be ruled out, and further questions regarding the mechanisms of Cbl-dependent radical SAM methyltransferases, in general, are discussed.

Construction of a gentamicin C1a-overproducing strain of Micromonospora purpurea by inactivation of the gacD gene.

Gentamicin C1a is the precursor of the semi-synthetic antibiotic etimicin and has the highest antibacterial activity in the clinically important gentamicin C mixture. To obtain a gentamicin C1a-overproducing strain, we inactivated gacD gene in Micromonospora purpurea. The gacD was presumed to encode a C6' methyltransferase by sequence analysis, and plays a role in the conversion of the gentamicin intermediate X2 to G418. So the inactivation of gacD blocks the metabolic pathways from X2 to G418 and leads to the accumulation of gentamicin C1a.The resulting recombination strain produced gentamicin C1a more than 10-fold compared to the wild type strain. Moreover, the wild-type strain produced 4 main production components, C1a, C2, C2a and C1, while the recombination strain produced only 2 components, C1a and C2b, making the purification of gentamicin C1a easier. The recombination strain was genetically stable and should be useful for the industrial production of gentamicin C1a.

Characterization of a key aminoglycoside phosphotransferase in gentamicin biosynthesis.

Gentamicin is an aminoglycoside antibiotic obtained from cultures of Micromonospora as the important anti-infective agents. Gentamicin which lacks 3'-hydroxyl group can avoid the attack from the modification enzymes of antibiotic-resistant bacteria in clinic. Consequently, C-3' dehydroxylation is the key step in gentamicins biosynthesis. We suppose that there are some enzymes responsible for converting intermediate JI-20A to 3',4'-bisdehydroxylated final product gentamicin C(1a), while phosphorylation of 3'-OH is possibly the first step for C-3' dehydroxylation. The gentamicin biosynthetic gene gntI, encoding an aminoglycoside phosphotransferase, was cloned from Micromonospora echinospora ATCC15835 and overexpressed in Escherichia coli. The resulting phosphotransferase was purified, and the kinetic parameters for Kanamycin A, Kanamycin B, Neomycin B and Amikacin were determined. Elucidation of NMR data of phosphorylated kanamycin B has unambiguously demonstrated a regiospecific phosphorylation of 3'-hydroxyl of the 6-aminohexose ring. The results described here partly confirm that the 3'-dehydroxylation step is preceded by a 3' phosphorylation step. It is predicted that GntI belongs to a new aminoglycoside phosphotransferase group involved with aminoglycoside antibiotics biosynthesis pathway.

Identification of gntK, a gene required for the methylation of purpurosamine C-6' in gentamicin biosynthesis.

Gentamicin and sisomicin are two different aminoglycoside antibiotics. The comparison of their chemical structure and biosynthetic gene clusters, coupled with bioinformatic analysis, suggested that the gntK gene would be associated with methylation. The gntK gene fragment in M. purpurea G1008 was inactivated by genetic engineering and its mutant strain M. purpurea GK1101 (ΔgntK) was screened. The metabolites of G1008 and GK1101 was analyzed by HPLC-MS, which revealed that GK1101 no longer produced gentamicin C(1) or C(2), while mainly synthesizing gentamicin C(1a), and the production of C(1a) increased significantly. This indicated that the metabolic flow for the gentamicin C(1) and C(2) biosynthesis was blocked by disrupting the gntK gene, which substantiated that the gntK gene encoded the enzyme that catalyzes the methylation of purpurosamine C-6'. The mutant GK1101 has good prospects for industrial application. In addition, our study provides information that can be used to clarify the function of a single gene and simplify the targeted genetic breeding of important pharmaceutical microorganisms.

Microbial biosynthesis and applications of gentamicin: a critical appraisal.

Gentamicin is an aminoglycoside antibiotic produced by various species of the genus Micromonospora and has received much attention in the recent years as a broad-spectrum antibiotic for treatment of various infections. It exists as a complex of closely related aminoglycoside structures and the clinically significant one is the gentamicin C complex. This review article focuses attention on the present status of knowledge and the main advancements achieved in the last few decades on the subject of gentamicin with regard to its production, biosynthetic pathway, mode of action, and uses. The various nutritional and environmental parameters affecting gentamicin production and the factors affecting the release of bound gentamicin are discussed. Further, strain improvement using UV and/or chemical mutagenesis can be applied to augment the efficiency of the producer strain and a number of case studies are presented. Different detection and quantitative methods for gentamicin estimation and the mode of action of gentamicin are discussed in detail. This antibiotic finds extensive use in combination chemotherapy and as a drug for different delivery agents for treatment of osteomyelitis and other recent applications in gene therapy.

Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set.

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.