Stepwise Post-glycosylation Modification of Sugar Moieties in Kanamycin Biosynthesis.

Kanamycin A is the major 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotic produced by Streptomyces kanamyceticus. The 2DOS moiety is linked with 6-amino-6-deoxy-d-glucose (6ADG) at O-4 and 3-amino-3-deoxy-d-glucose at O-6. Because the 6ADG moiety is derived from d-glucosamine (GlcN), deamination at C-2 and introduction of C-6-NH2 are required in the biosynthesis. A dehydrogenase, KanQ, and an aminotransferase, KanB, are presumed to be responsible for the introduction of C-6-NH2 , although the substrates have not been identified. Here, we examined the substrate specificity of KanQ to better understand the biosynthetic pathway. It was found that KanQ oxidized kanamycin C more efficiently than the 3''-deamino derivative. Furthermore, the substrate specificity of an oxygenase, KanJ, that is responsible for deamination at C-2 of the GlcN moiety was examined, and the crystal structure of KanJ was determined. It was found that C-6-NH2 is important for substrate recognition by KanJ. Thus, the modification of the GlcN moiety occurs after pseudo-trisaccharide formation, followed by the introduction of C-6-NH2 by KanQ/KanB and deamination at C-2 by KanJ.

Biochemical and Structural Analysis of a Dehydrogenase, KanD2, and an Aminotransferase, KanS2, That Are Responsible for the Construction of the Kanosamine Moiety in Kanamycin Biosynthesis.

Kanosamine (3-amino-3-deoxy-d-glucose) is a characteristic sugar unit found in kanamycins, a group of aminoglycoside antibiotics. The kanosamine moiety originates from d-glucose in kanamycin biosynthesis. However, the timing of the replacement of the 3-OH group of the d-glucose-derived biosynthetic intermediate with the amino group is elusive. Comparison of biosynthetic gene clusters for related aminoglycoside antibiotics suggests that the nicotinamide adenine dinucleotide (NAD+)-dependent dehydrogenase KanD2 and the pyridoxal 5'-phosphate (PLP)-dependent aminotransferase KanS2 are responsible for the introduction of the amino group at the C3 position of kanosamine. In this study, we demonstrated that KanD2 and KanS2 convert kanamycin A, B, and C to the corresponding 3″-deamino-3″-hydroxykanamycins (3″-hks) in the presence of PLP, 2-oxoglutarate, and NADH via a reverse reaction in the pathway. Furthermore, we observed that all of the 3″-hks are oxidized by KanD2 with NAD+, but d-glucose, UDP-d-glucose, d-glucose 6-phosphate, and d-glucose 1-phosphate are not. Crystal structure analysis of KanD2 complexed with 3″-hkB and NADH illustrated the selective recognition of pseudotrisaccharides, especially the d-glucose moiety with 2-deoxystreptamine, by a combination of hydrogen bonds and CH-π interactions. Overall, it was clarified that the kanosamine moiety of kanamycins is constructed after the glucosylation of the pseudodisaccharide biosynthetic intermediates in kanamycin biosynthesis.

Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation.

Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non-aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.

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.

The last step of kanamycin biosynthesis: unique deamination reaction catalyzed by the α-ketoglutarate-dependent nonheme iron dioxygenase KanJ and the NADPH-dependent reductase KanK.

Mystery solved: using heterologous expression, the activities of two enzymes exclusively belonging to the kanamycin biosynthetic pathway have been identified in vitro. A distinctive reaction mechanism to produce kanamycin is proposed and the previously unknown catalytic deamination activity of KanJ dioxygenase is uncovered.

Construction of kanamycin B overproducing strain by genetic engineering of Streptomyces tenebrarius.

Genetic engineering as an important approach to strain optimization has received wide recognition. Recent advances in the studies on the biosynthetic pathways and gene clusters of Streptomyces make stain optimization by genetic alteration possible. Kanamycin B is a key intermediate in the manufacture of the important medicines dibekacin and arbekacin, which belong to a class of antibiotics known as the aminoglycosides. Kanamycin could be prepared by carbamoylkanamycin B hydrolysis. However, carbamoylkanamycin B production in Streptomyces tenebrarius H6 is very low. Therefore, we tried to obtain high kanamycin B-producing strains that produced kanamycin B as a main component. In our work, aprD3 and aprD4 were clarified to be responsible for deoxygenation in apramycin and tobramycin biosynthesis. Based on this information, genes aprD3, aprQ (deduced apramycin biosynthetic gene), and aprD4 were disrupted to optimize the production of carbamoylkanamycin B. Compared with wild-type strain, mutant strain SPU313 (ΔaprD3, ΔaprQ, and ΔaprD4) produced carbamoylkanamycin B as a single antibiotic, whose production increased approximately fivefold. To construct a strain producing kanamycin B instead of carbamoylkanamycin B, the carbamoyl-transfer gene tacA was inactivated in strain SPU313. Mutant strain SPU314 (ΔaprD3, ΔaprQ, ΔaprD4, and ΔtacA) specifically produced kanamycin B, which was proven by LC-MS. This work demonstrated careful genetic engineering could significantly improve production and eliminate undesired products.

Establishment of a highly efficient conjugation protocol for Streptomyces kanamyceticus ATCC12853.

Kanamycin B as the secondary metabolite of wild-type Streptomyces kanamyceticus (S. kanamyceticus) ATCC12853 is often used for the synthesis of dibekacin and arbekacin. To construct the strain has the ability for kanamycin B production; the pSET152 derivatives from Escherichia coli ET12567 were introduced to S. kanamyceticus by intergeneric conjugal transfer. In this study, we established a reliable genetic manipulation system for S. kanamyceticus. The key factors of conjugal transfer were evaluated, including donor-to-recipient ratio, heat-shock, and the overlaying time of antibiotics. When spores were used as recipient, the optimal conjugation frequency was up to 6.7 × 10-6 . And mycelia were used as an alternative recipient for conjugation instead of spores; the most suitable donor-to-recipient ratio is 1:1 (107 :107 ). After incubated for only 10-12 hr and overlaid with antibiotics subsequently, the conjugation frequency can reach to 6.2 × 10-5 which is sufficient for gene knockout and other genetic operation. Based on the optimized conjugal transfer condition, kanJ was knocked out successfully. The kanamycin B yield of kanJ-disruption strain can reach to 543.18 ± 42 mg/L while the kanamycin B yield of wild-type strain was only 46.57 ± 12 mg/L. The current work helps improve the content of kanamycin B in the fermentation broth of S. kanamyceticus effectively to ensure the supply for the synthesis of several critical semisynthetic antibiotics.

Biosynthesis of 3'-deoxy-carbamoylkanamycin C in a Streptomyces tenebrarius mutant strain by tacB gene disruption.

Streptomyces tenebrarius H6 mainly produces three kinds of antibiotics: apramycin, carbamoyltobramycin and some carbamoylkanamycin B. In our present study, a dehydrogenase gene tacB in the tobramycin biosynthetic gene cluster was disrupted by in-frame deletion. The result of TLC bio-autograph analysis demonstrated the disruption mutant strain produced apramycin and a new antibiotic. The new antibiotic was identified as 3'-deoxy-carbamoylkanamycin C by MS and NMR analysis after isolation and purification. The disruption mutant was restored to produce carbamoyltobramycin in a complementation experiment by the intact tacB gene. Our studies suggested that the tacB gene encodes a 6'-dehydrogenase, which reduces the 6'-hydroxyl group of paromamine to a keto group, thus facilitating the transfer of an aminogroup to form neamine. This study is the first report on the generation of a tobramycin derivative by gene engineering, and will contribute to clarify the complete biosynthetic pathway of tobramycin.

Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering.

Both kanamycin A and kanamycin B, antibiotic components produced by Streptomyces kanamyceticus, have medical value. Two different pathways for kanamycin biosynthesis have been reported by two research groups. In this study, to obtain an optimal kanamycin A-producing strain and a kanamycin B-high-yield strain, we first examined the native kanamycin biosynthetic pathway in vivo. Based on the proposed parallel biosynthetic pathway, kanN disruption should lead to kanamycin A accumulation; however, the kanN-disruption strain produced neither kanamycin A nor kanamycin B. We then tested the function of kanJ and kanK. The main metabolite of the kanJ-disruption strain was identified as kanamycin B. These results clarified that kanamycin biosynthesis does not proceed through the parallel pathway and that synthesis of kanamycin A from kanamycin B is catalyzed by KanJ and KanK in S. kanamyceticus. As expected, the kanamycin B yield of the kanJ-disruption strain was 3268±255 µg/mL, 12-fold higher than that of the original strain. To improve the purity of kanamycin A and reduce the yield of kanamycin B in the fermentation broth, four different kanJ- and kanK-overexpressing strains were constructed through either homologous recombination or site-specific integration. The overexpressing strain containing three copies of kanJ and kanK in its genome exhibited the lowest kanamycin B yield (128±20 µg/mL), which was 54% lower than that of the original strain. Our experimental results demonstrate that kanamycin A is derived from KanJ-and-KanK-catalyzed conversion of kanamycin B in S. kanamyceticus. Moreover, based on the clarified biosynthetic pathway, we obtained a kanamycin B-high-yield strain and an optimized kanamycin A-producing strain with minimal byproduct.

Increased production of aminoglycosides associated with amplified antibiotic resistance genes.

The 6'-N-acetyltransferase derived from Streptomyces kanamyceticus strain M1164 was cloned on to the high copy plasmid vector pIJ702 and introduced into S. kanamyceticus (ATCC 12853, a kanamycin producer) and S. fradiae (ATCC 10745, a neomycin producer). In both cases transformants containing the recombinant plasmid showed increased resistance to a number of aminoglycoside antibiotics and substantially increased production of kanamycin and neomycin. This demonstrates that specific amplification of gene products associated with antibiotic biosynthesis provides a means for improving antibiotic production.

Resistance mechanisms of kanamycin-, neomycin-, and streptomycin-producing streptomycetes to aminoglycoside antibiotics.

Streptomyces kanamyceticus ISP5500, S. fradiae ISP5063 and S. griseus ISP5236, which produce kanamycin, neomycin or streptomycin respectively, were highly resistant to the antibiotics they produced. Polyphenylalanine synthesis in cell free systems was also resistant to the action of the antibiotics. Reciprocal exchange between ribosomes and S150 fractions from the three strains revealed that the S150 fraction of each strain had an enzyme activity that inactivated the appropriate antibiotic whereas the ribosomes were susceptible to the antibiotics. It was concluded that the resistance of the in vitro polyphenylalanine synthesizing systems of these antibiotics was due to the presence of inactivating enzymes. Furthermore, S. fradiae and S. kanamyceticus were highly resistant to aminocyclitol-containing aminoglycoside antibiotics other than those produced by the two strains. In these cases, the inactivating enzymes were found to have a major role in the resistance mechanism. However, the resistance of S. kanamyceticus ISP5500 to streptomycin seems to be due to resistance at the ribosomal level.

Aminoglycoside 3'-phosphotransferases I and II in Pseudomonas aeruginosa.

Aminoglycoside 3'-phosphotransferases I and II in three strains of Pseudomonas aeruginosa were studied in comparison with those in two strains of R factor-carrying Escherichia coli. The strain TI-13 of P. aeruginosa produced the former and strain H-9 the latter. Strain B-13 produced the both enzymes. The 3'-phosphotransferases of type I in P. aeruginosa TI-13, B-13 and E. coli K12 J5 R11-2 were different from each other in chromatographic behavior, molecular weight, pH optimum, and Ii. The 3'-phosphotransferase of type II in P. aeruginosa H-9 and E. coli JR66/W677 showed the same behavior.

Effect of some inhibitors on kanamycin formation by Streptomyces kanamyceticus.

The formation of kanamycin is markedly inhibited by mercuric chloride, sodium iodoacetate, 2,4-dinitrophenol, sodium arsenite and sodium azide particularly when these are added at the start of fermentation. Less inhibition of kanamycin synthesis is observed in case of sodium 5,5-diethylbarbiturate, malonic acid, sodium arsenate and sodium fluoride. Inhibition of kanamycin synthesis is associated with growth inhibition in case of 2,4-dinitrophenol, sodium arsenite and sodium azide. Bacitracin and D-cycloserine have a stimulatory effect on kanamycin synthesis with slight inhibition of cellular growth. This stimulation might be due to accumulation of cell wallintermediates--aminosugar and sugar--which are shunted to the pathway of kanamycin synthesis. Penicillin lowers kanamycin synthesis by 65 percent as compared with 19 percent reduction of cellular growth. Chloramphenicol has a stimulatory effect at lower concentration (20 mug/ml), when it is added at 24 h of fermentation. At higher concentration (50 mug/ml) chloramphenicol shows marked inhibition of both cellular growth and antibiotic biosynthesis.

Studies on aminoglycoside antibiotics: enzymic mechanism of resistance and genetics.

The kanamycin inactivating enzyme, 3'-phosphotransferase and 6'-acetyltransferase were first found in 1967 and on the basis of the enzymic mechanism of resistance a new research approach to the development of active useful derivatives was explored. The enzymic mechanism of resistance was conclusively confirmed by the synthesis of 3'-deoxykanamycin A and 3',4'-dideoxykanamycin B which did not undergo inactivation by 3'-phosphotransferase and inhibited the growth of resistant strains. Besides APH(3') and AAC(6') described above, the following enzymes were found to be involved in the mechanism of resistance to aminoglycosides: APH(3''), APH(5''), APH(6), APH(2''), AAC(3), AAC(2'), AAD(3''), AAD(2''), AAD(4'), AAD(6). Not only the removal of the group which undergoes the enzyme reaction but also the modification of the group binding to the enzyme has also given active derivatives such as amikacin etc. The substrate specificity of the enzymes, enzymes in the immobilized state, and the application of proton and 13C nmr for structure determination of reaction products are reviewed. It was noticed that all enzymes involved in resistance contain adenosine- and aminoglycoside-binding sites. These enzymes were thus suggested to be mainly different primarily in the positional relationships between these binding sites. It suggests a close evolutionary relationships of these enzymes. The role of these enzymes in the biosynthesis of aminoglycoside antibiotics is discussed and a general mode of the biosynthesis of aminoglycosides is proposed: a gene or gene set involved in biosynthesis of 2-deoxystreptamine which has no cytotoxicity is widely distributed and the deoxystreptamine produced is transformed to the final products.

[Identification of idiotrophs for 3'-deoxykanamycin B synthesis in Streptomyces tenebrarius (Higgens a. Kastner)]. / Identifikatsiia idiotrofov po sintezu 3'-dezoksikanamitsina B u Streptomyces tenebrarius (Higgens a. Kastner).

Mutants of Streptomyces tenebrarius with the blocked synthesis of 3'-deoxykanamycin B were obtained by treating the producer with NTG and chloramphenicol, or after gamma-irradiation. These mutants (idiotrophs) were distributed into three groups by means of the cosynthesis experiments on agar plates: convertors, secretors and "neutral" strains. Five idiotrophs represented five complementation groups for biosynthesis of the antibiotic. Three of these were defective in 2-deoxystreptamine synthesis, the fourth was defined as neamine-negative, and the fifth was probably blocked in regulation of enzymes responsible for conversion of neamine or paromamine into kanamycins. Localization of mutations has been shown on the scheme of kanamycins' biosynthesis.

[Isolation and study of the properties of Streptomyces kanamyceticus mutants with disorders of kanamycin biosynthesis]. / Poluchenie i izuchenie svoistv mutantov Streptomyces kanamyceticus s narusheniiami biosinteza kanamitsinov.

The strains of Streptomyces kanamyceticus 1375 and 1 were exposed to N-nitroso-N-methyl urea, ethidium bromide and acriflavine and mutants with impaired biosynthesis of kanamycin (Kan-) were isolated. The majority of the mutants were genetically unstable and segregated with the formation of both the Kan- and Kan+ clones. The stable Kan- mutants were tested in pairs for the cosynthesis. The capacity of the mutants for the recovery of the kanamycin synthesis in media supplemented with kanamycin precursors such as 2-desoxystreptamine and D-glucoseamine was studied. Four classes of the Kan- mutants involving various genes controlling the kanamycin biosynthesis in S. kanamyceticus were identified by the results of the tests.

[Metabolites produced by Streptomyces kanamyceticus mutants with impaired biosynthesis of kanamycin]. / Metabolity, obrazuemye mutantami Streptomyces kanamyceticus s narushennym biosintezom kanamitsina.

Metabolites produced by Streptomyces kanamyceticus mutants with impaired kanamycin biosynthesis (kan mutants) were investigated by thin layer chromatography. With spectrophotometric scanning of the chromatograms the quantitative content of kanamycin A and 2-desoxystreptamine (2-DOS) in the culture fluid was determined. Five groups of the S.kanamyceticus mutants with impaired kanamycin biosynthesis at various stages were identified: kanA produced no D-glucosamine, kanB and kanC produced no 2-DOS, kanD was not able to transfer 2-DOS to the metabolites with the antibiotic activity, kanG synthesized no kanosamine.

[Electrophoretic separation of spores of producers of kanamycin and tylosin]. / Elektroforeticheskoe razdelenie spor produtsentov kanamitsina i tilozina.

The stages of spore development, the most favourable for electrophoretic separation of the spores, were studied in the cultures producing kanamycin and tylosin. The stage of sporulating was shown to be the optimal. The step-by-step procedure for preparing the spores for electrophoresis is described. There were observed distinctions in the fatty acid spectrum of the cells in the cultures grown from electrophoretic fractions of the spores with different antibiotic production capacity.

[Analysis of genome rearrangements in Streptomyces kanamyceticus mutants]. / Analiz perestroek genoma u mutantov Streptomyces kanamyceticus.

Studies with the use of pulsed electrophoresis showed that Asel and Dral restriction endonucleases segregated chromosomal DNA of the initial strain Streptomyces kanamyceticus 1 into 16 and 12 fragments, respectively. The total size of the strain chromosomal DNA was from 7715 (the total of the Dral fragment sizes) to 7788 kb (the total of the Asel fragment sizes). Chromosomal DNA rearrangements were detected in the unstable Kan- mutant kan12 as well as in mutant genR10 which was characterized by higher resistance to kanamycin and gentamicin. As compared to DNA of the initial strain S.kanamyceticus 1 and the stable mutant kanC782, mutant kan12 lacked 98-kb and 220-kb Asel fragments and contained additional 297-kb and 450-kb fragments. DNA of genR10 lacked 420-kb Asel fragment but contained an additional 450-kb fragment. Kan12 and genR10 as well as two more mutants (genR8 and genR8.1) resistant to the above antibiotics contained amplifications of gene kmr determining resistance of S.kanamyceticus 1 to kanamycin. The most intensive amplification was detected in the most resistant mutant genR8.1.

[Comparative study of Streptomyces--producer of aminoglycoside antibiotics, monomycin and kanamycin, and the strain 344 obtained by fusion of their protoplasts by the method of restrictive total DNA fingerprinting]. / Sravnitel'noe izuchenie streptomitsetov--produtsentov monomitsina a kanamitsina i shtamma 344, poluchennogo pri zliianii ikh proto- plastov metodom restriktsionnykh "fingerprintov" total'noi DNK.

The method of total DNA restriction finger prints was applied to the study of Streptomyces monomycini INA 1465 producing monomycin, Streptomyces kanamyceticus INA K-13 producing kanamycin and strain 344 isolated after fusion of the protoplasts of strain 1465 and K-13, which produced albofungin and chloralbofungin, aminoglycoside antibiotics. For preparing the finger prints of the strains splitting by endonucleases BamHI, PstI, PvuII, and BgIII was used. The finger prints showed that strain 344 was related to the strain of S. monomycini and markedly differed from the strain of S. kanamyceticus. Strain 344 was likely to result from reconstruction (probably 20-kb deletion) of the genome of S. monomycini INA 1465 induced by the preparation and regeneration of its protoplasts. The reconstruction could affect the genome area with localization of the genes involved in monomycin biosynthesis and monomycin resistance genes.