Characterization of Methyltransferase from Apramycin Biosynthetic Gene Cluster and Improvement Production of Apramycin in Engineered Streptoalloteichus Tenebrarius

doi:10.21203/rs.3.rs-927201/v1

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Characterization of Methyltransferase from Apramycin Biosynthetic Gene Cluster and Improvement Production of Apramycin in Engineered Streptoalloteichus Tenebrarius

https://doi.org/10.21203/rs.3.rs-927201/v1

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Background

Apramycin is a structurally unique aminoglycoside, used in veterinary medicine or the treatment of Salmonella, Escherichia coli and Pasteurella multocida infections in farm. Although discovered and used many years ago, many biosynthetic steps of apramycin are still obscure.

Results

In this study, we identified a HemK family methyltransferase, aprI, involved in apramycin biosynthesis. The function of aprI was studied by using gene disruption and biochemical experiments, and a new aminoglycoside antibiotic demethyl-apramycin was purified from aprI disruption strain. Experiments proved that AprI converted demethyl-aprosamine to aprosamine in vitro. Based on this, the apramycin production strain was improved by overexpression the AprI to decrease the impurity production.

Conclusions

We have identified aprI is a 7’-N-methyltransferase gene in apramycin biosynthesis and confirmed the substrate of methyltransferase. Engineering of aprI resulted in a strain producing a new aminoglycoside demethyl-apramycin and apramycin mono-producing strain with less impurity production. Finally, the yield of demethyl-apramycin in apramycin mono-producing strain decreased from 196±36 mg/L to 51±9 mg/L, and the yield of apramycin increased from 2227±320 mg/L to 2331±210 mg/L.

Biomedical Engineering

Cell & Tissue Engineering

Environmental Engineering

Biotechnology and Bioengineering

Streptoalloteichus tenebrarius

apramycin biosynthesis pathway

aprI gene

methyltransferase

Apramycin, produced by Streptoalloteichus tenebrarius, is a structurally unique aminoglycoside antibiotic.¹ As frequently employed veterinary antibiotic drug, apramycin has the high antimicrobial activity on important gram-negative pathogens and has been mainly used for the treatment of Salmonella, Escherichia coli and Pasteurella multocida infections in poultry, swine or bovine.^2-3 Meanwhile, apramycin is a potent antibacterial with low ototoxicity, which properties distinct from all human used aminoglycosides. The properties attracted the attention of many research teams.^4-5

Apramycin, carbamyltobramycin and carbamylkanamycin B are isolated from S. tenebrarius.⁶ They all belong to 2-dexoystreptomycin (2-DOS) aminoglycosides. Apramycin is characterized by 2-DOS linked with an unusual bicyclic 3’-deoxyoctose moiety. Carbamyltobramycin and carbamylkanamycin B are the 4, 6-disubstituted 2-DOS aminoglycoside antibiotic like gentamicin. Moreover, both carbamyltobramycin and carbamylkanamycin B (3-deoxy-carbamyltobramycin) share the same biosynthetic gene cluster (GenBank accession number: AJ810851).⁷ Apramycin has independent biosynthetic gene cluster (GenBank accession number: AJ629123) (Figure 1A). Base on bioinformatical analysis, the genes for the common intermediates 2-deoxystreptamine and paromamine can be easily identified both in apramycin and tobramycin biosynthetic gene clusters.⁸Lividamine is the 3’ deoxygenated product of paromamine under the function of AprD3 and AprD4.^9-12 Then, lividamine was oxidized by AprQ to form 6’-oxo-lividamine which may be the intermediate of bicyclic 3’-deoxyoctose moiety.¹³ Besides, it was confirmed that AprD3 and AprD4 are responsible for carbamyltobramycin 3’-deoxygenation process (Figure 1B).¹⁴ However, the biosynthetic pathway of the unique structure of apramycin is still obscure. Here, we report the identification and characterization of a 7’-N-methyltransferase gene, aprI, and its protein substrate, demethyl-aprosamine, involved in the conversion.

As other antibiotics, the accumulation of intermediate metabolites is also observed in apramycin fermentation products. The final apramycin products contain 0.9-2.7% of demethyl-apramycin. Therefore, demethyl-apramycin is one of the main impurity, its production should be controlled. Based on the study of the biosynthetic pathways of apramycin and tobramycin, we metabolic engineering S. tenebrarius to optimize apramycin production through block tobramycin biosynthesis and decrease impurity production.

Identify function of aprI by gene deletion and complementation. Bioinformatically analyze the apramycin biosynthetic gene cluster showed that AprI possessed homology with known methyltransferase, sharing a high degree of sequence identity with the N-methyltransferase CalM of Streptomyces chartreusis (35%)¹⁸and a lower degree of sequence identity with the 3’’-N-methyltransferase GenN of Micromonospora echinospora (29%).¹⁹ All of them belong to the HemK superfamily, which catalyzes methyl group transfer of a variety of substrates. Thus, we deduce AprI is the 7’-N-methyltransferase in apramycin biosynthesis. To clarify this hypothesis, we carried out both genetic and biochemical analysis of AprI in this work.

To clarify the function of aprI, aprI gene was disrupted in the wild-type strain of S. tenebrarius. PCR amplification with wild-type strain generated a 4.8 kb fragment, while the aprI mutant strain (∆aprI) generated a 4.0 kb fragment because of a 789 bp deletion (Figure 2A and Figure 2B). The 4.0 kb fragment amplified from ∆aprI was further confirmed by DNA sequencing (data not shown). The results verified that aprI was successfully disrupted in ∆aprI. The ∆aprI was cultured to analyze its fermented products. The wild-type strain of S. tenebrarius was used as control under the same fermentation conditions. Compared to the wild-type strain, the total antibiotic production in the ∆aprI decreased from 2620 mg/L to 2183 mg/L (measured by bioassay). TLC coupled with an antimicrobial activity assay showed the ∆aprI produced a new bioactive product instead of apramycin (Figure 2C). HPLC analysis confirmed that ∆aprI accumulated a new compound 2 (Figure 2D). By HRMS analysis, the new component 2 produced by ∆aprI was considered as demethyl-apramycin (Figure S1). The demethyl product was also analyzed by MS/MS (Figure S1). The protonated fragments were similar to those of the reported mass spectra of apramycin, however, the glycoside bond of this compound cleavage formed the fragment a+b (m/z 365) instead of the ion at m/z 379,²⁰ suggesting the demethyl-apramycin might lack a methyl group at 7’-N. 2 then was purified from the fermentation broth of ∆aprI. The ¹³C-NMR analysis definitively confirmed that compound 2 lacked a 7’-N-methyl group (Figure S2 and Table S4). To preclude any possible of polar effects, we performed ∆aprI complementary. The aprI complementary plasmid pAP605, in which the entire aprI gene is under the control of the PhrdB promotor, was introduced by intergenic conjugation into ∆aprI. We screened for the erythromycin-resistance phenotype to isolate the aprI complementary strain, and we designated this strain as ∆aprI::aprI. HPLC analysis showed that the ∆aprI::aprI restored the ability to produce apramycin (Figure 2D). Thus, the possibility that aprI gene disruption had other polar effects can be ruled out. These results illustrate that AprI was the 7’-N-methyltransferase in apramycin biosynthesis.

David Crich utilized chemical synthesis methods to prepare a series of apramycin derivatives, and investigated their antibacterial activity. In these derivatives, demethyl-apramycin had good activity against E.coli and Staphylococcus aureus, and was as good as apramycin in some cases.²¹ As the main product of ∆aprI strain is demethyl-apramycin, this strain provide a convenience, efficient and continuable method for production the potentially valuable aminoglycoside antibiotic demethyl-apramycin.

Catalytic specificity of AprI. An in vivo gene disruption experiment demonstrated that the aprI gene is involved in the unique methylation process of apramycin synthesis. However, the methylation substrate and mechanism are still unknown. Thus, aprI was cloned into the expression vector pET28a(+), and recombinant AprI was expressed as an N-terminal His₆-tagged protein and expressed in E. coli BL21 as described in Methods. It is composed of 262 amino acids and is predicted to have a molecular weight of 28.8 KDa. After separation and purification on a nickel column, the molecular size was determined by SDS-PAGE to be consistent with the molecular weight of AprI (Figure 3A).

Recombinant AprI was assayed for the ability to catalyze the SAM-dependent methyl transfer reaction which convert demethyl-apramycin to apramycin. However, this recombinant protein was not able to catalyze this reaction (Figure 3C). Thus, it was speculated that the 7’-N methylation of apramycin may occur at the pseudo-trisaccharide instead of the pseudo-tetrasaccharide. We then attempted to hydrolyze demethyl-apramycin to prepare the proper substrate.²² We identified the product by HPLC and MS and named it demethyl-aprosamine (Figure S3). Using demethyl-aprosamine as substrate, the catalysis results showed that demethyl-aprosamine was converted into a new compound under the presence of both AprI and SAM (Figure 3B). It is worth noting that the reaction product showed two peaks in the HPLC analysis. As the two peaks cannot be isolated, we speculated that the two peaks might be mutually convertible isomers. To prepare the authentic aprosamine for HPLC analysis of AprI enzymatic reaction, apramycin was hydrolyzed. Furthermore, its hydrolysate also showed two peaks is identical with AprI catalytic product in the HPLC analysis (data not shown). Therefore, the two new AprI catalyzed products together were deduced as aprosamine and its isomer (Figure S4 and Figure S5). These results confirm that AprI catalyze pseudo-trisaccharide methylation rather than pseudo-tetrasaccharide (Figure 3D).

Methyl groups have stereoelectronic effects on micromolecules and biomacromolecules, thereby leading to a diversity of biological effects, containing the selectivity between biological receptors, increased potency, and protection against enzyme metabolism.²³ Methyltransferases in the biosynthesis of aminoglycoside antibiotics are mainly divided into two types. The first type is using SAM as a methyl donor to catalyze the methylation of the substrate. And the other is radical SAM enzyme catalyzing non-universal methylation. In the biosynthesis pathway of the aminoglycoside antibiotic gentamicin, there are four methyltransferases, including GenK, GenD1, GenN, and GenL.²⁴ GenK and GenD1 were identified as cobalamin-dependent radical SAM methyltransferases that transfer the methyl group to the carbon atom at the position of C6’ and C4’’ in gentamicin biosynthesis, respectively. They have a common [4Fe-4S] cluster and a highly conserved “CXXXCXXC” motif in their active sites. The two radical SAM enzymes are reported to extract a hydrogen atom from the corresponding sites of their substrates to provide a radical intermediate utilizing 5’-dAdo originating from SAM. The radical intermediate forms a C-C bond with the methyl group on methylcobalamin to obtain the C-methylated product.^25-26 GenL as the 6’-N-methyltranserase and GenN as the 3’’-N-methyltranserase in gentamicin are classified in class I SAM-dependent methyltransferase. As the terminal 6’-N-methyltrasferase, GenL has the activicity in the conversion of both C2 to C1 and of C1a to C2b. But GenL is an enzyme with low sequence similarity to GenN and other N-methyltransferases, reaction mechanism of GenL still unknown.²⁴ Similar to AprI, GenN belongs to the HemK family, which has relatively conserved “GXGXG” and “NSPT” sites to interact with SAH. The protein crystal structure of GenN has been determined.²⁷ Different from AprI, GenN can catalyze multiple pseudo-trisaccharide substrates, containing A1, A-2, Ae, and A. However, AprI act on 7’-N of the demethyl-aprosamine in this study. As a unique site methyltransferase in the biosynthesis of aminoglycoside antibiotics, substrate scopes and reaction mechanisms of AprI deserve further study and its research will provide a basis for the development of new aminoglycoside antibiotics.

Overexpression of aprI by homologous recombination in S. tenebrarius reduced demethyl-apramycin as an impurity in apramycin production.

Promoter engineering and gene knockout are the most direct and effective metabolic engineering methods for changing metabolic flow. Industrial production of apramycin is performed by microbial fermentation. Our final products contain 0.9-2.7% of demethyl-apramycin, which is the major impurity in apramycin biosynthesis. However, there is only a methyl group difference between demethyl-apramycin and apramycin, it is pretty difficult to separate demethyl-apramycin and apramycin in production process. To reduce the production of demethyl-apramycin by metabolic engineering, we intended to increase the expression of aprI. To elevate the genetic stability of engineering strains, we integrated aprI into the chromosome via homologous recombination rather than site-specific integration.

Because S. tenebrarius produce apramycin and tobramycin simultaneously, disruption tobramycin biosynthetic pathway not only can eliminate the impurity tobramycin from apramycin, but also tuned the shared biosynthetic precursors to apramycin biosynthesis. We attempted to insert the aprI gene in the tobramycin biosynthesis gene cluster to obtain strain for apramycin mono-production. In tobramycin biosynthesis gene cluster of S. tenebrarius, the tobM2 gene was considered as the second glycosyltransferase, converting the intermediate nebramine to tobramycin (Figure 1B).¹⁴To confirm tobM2 gene was unrelated to apramycin biosynthesis, the tobM2 gene was disrupted in the wild-type strain of S. tenebrarius. The tobM2 deletion plasmid pAP600 was introduced into S. tenebrarius by conjugation transfer to obtain the tobM2-disruption strain via the double crossover homologous recombination event. The PCR and DNA sequencing results confirmed that the selected strain designated ΔtobM2 does not possess the 1263 bp tobM2 sequence. The ∆tobM2 was later cultured to analyze its fermented biosynthetic products. HPLC analysis confirmed that ∆tobM2 strain still produced apramycin and did not produce tobramycin (Figure S6).

Next, aprI which are under the control of PhrdB, was inserted into the homology arm of tobM2 via homologous recombination (Figure 4A and Figure 4B). The overexpressing strain was named S. tenebrarius IB. HPLC assays demonstrated that the demethyl-apramycin yield decreased from 196±36 mg/L to 51±9 mg/L and that the percentage of demethyl-apramycin among the total apramycin products decreased from 1.17% to 0.34%. The apramycin yield of the wild-type strain was 2227±320 mg/L and that of S. tenebrarius IB was 2331±210 mg/L(Figure 4C). Through overexpression aprI, demethyl-apramycin yield was further decreased, and the purity of apramycin in the fermentation products was improved. Moreover, the tobramycin yield of the wild-type strain was 607±111 mg/L and that of S. tenebrarius IB was no more producing (Figure 4C).

In the process of constructing aprI overexpressing strain, we attempted to obtain the anticipated strain using site-specific integration plasmid pAP605. However, previous research had suggested that successive passages of strains lead to elimination of the site-specific integration plasmid.¹⁵ We compared the site-specific integration and the homologous recombination by comparing the products through five passages. The homologous recombination strain had better stability compared with the site-specific integration strain, and the production of S. tenebrarius IB is not affected by the passages (data not shown).

AprI was identified as 7’-N methyltransferase in apramycin biosynthesis by gene disruption and biochemical study. AprI had an activity to transfer a methyl group at the position of 7’-N of demethyl-aprosamine. By deletion the aprI gene, an aminoglycoside antibiotic demethyl-apramycin high producing strain was constructed. Base on the study of apramycin biosynthetic pathway and tobramycin biosynthetic pathway in S. tenebrarius, the strain overexpression of aprI was constructed to decrease the impurity production of demethyl-apramycin and tobramycin. The content of demethyl-apramycin in the aprI overexpression strain was reduced from 196±36 mg/L to 51±9 mg/L. And apramycin in the aprI overexpression strain was increased from 2227±320 mg/L to 2331±210 mg/L. The tobramycin yield of the wild-type strain was 607±111 mg/L and that of aprI overexpression strain was no more producing.

Strains and growth conditions. The plasmids used in this study are listed in Table S1. The strains used in this study are listed in Table S2. E. coli Top10 was used as cloning host, E. coli ET12567/pUZ8002 for intergeneric conjunction between E. coli and Streptomyces, and E. coli BL21 (DE3) was used for protein expression experiments. Wild-type Streptoalloteichus tenebrarius and its mutants were grown on agar medium (20 g/L soluble starch, 1 g/L beef extract, 1 g/L KNO₃, 0.5 g/L K₂HPO₄·3H₂O, 0.5 g/L NaCl, 0.5 g/L MgSO₄·7H₂O, 0.01 g/L FeSO₄·7H₂O, 15 g/L agar, pH 7.2) for sporulation and liquid CP (20 g/L glucose, 2 g/L peptone, 4 g/L yeast extract, 0.5 g/L MgSO₄·7H₂O, 0.5 g/L K₂HPO₄·3H₂O, 0.5 g/L NaCl) for mycelium. S. tenebrarius and its derivative mutants were cultured in the seed medium (10 g/L soyal bean, 10 g/L glucose, 3 g/L peptone, 1 g/L yeast powder, 5 g/L corn powder, 1 g/L CaCO₃) at 37°C for 18 hours, 3 mL(10% [v/v]) seed culture was used to inoculate the fermentation medium (40 g/L soyal bean, 20 g/L glucose, 20 g/L soluble starch, 5 g/L yeast powder, 5 g/L CaCO₃(light), 5 g/L NH₄Cl, 2 g/L MgSO₄, 0.05 g/L FeSO₄, 0.03 g/L ZnSO₄, 0.3 g/L MnCl₂) and was incubated for 5 days. E. coli strains were maintained in Luria–Bertani (LB) liquid or solid medium (10 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl) at 37°C with appropriate antibiotic selection at final concentration of 100 μg/mL ampicillin, 25 μg/mL chloramphenicol, and 50 μg/mL kanamycin.

Construction of the tobM2-disruption plasmids. Base on the sequence of the tobramycin biosynthetic gene cluster above, two DNA fragments flanking tobM2 were amplified from the genomic DNA of S. tenebrarius¹⁵ by using M2U-P1/P2 and M2D-P3/P4. The PCR products were fusion and cloned into pMD18-T (Takara), and verified by restriction endonuclease digestion and sequencing. The fragment was cloned into pSPU310to obtain the gene disruption plasmid pAP600.

Construction of the aprI-disruption plasmids. The sequence of the apramycin biosynthetic gene cluster has been deposited in the NCBI under the accession number GenBank AJ629123. For deletion of the apramycin biosynthesis gene aprI, two DNA fragments flanking aprI were amplified from the genomic DNA of S. tenebrarius by using IU-P1/P2 and ID-P3/P4. The PCR products were cloned into pIJ2925 to obtain plasmid pAP601, which was verified by restriction endonuclease digestion and sequencing. Then, the homogenous arm of aprI was cloned into pSPU310to obtain the gene disruption plasmid pAP602.

Targeted gene deletion. To create double-cross deletion mutants ΔtobM2 and ΔaprI based on wild-type strain S. tenebrarius, the corresponding plasmids pAP600 and pAP602 were introduced into S. tenebrarius by conjugation, respectively, and mutants screening were carried out using the same method described before.⁷ The desired deletion mutants were identified by PCR using the checking primers.

Complementation of ΔaprI mutant strain. For complementation of the aprI gene mutant, pAP603 was constructed as the intermediate vector by inserting aprI into pHJK241¹⁶ between the NcoI and HindIII sites. Then pAP605 was constructed by inserting aprI into the vector pEAP1¹⁷under the control of the constitutive promoter PhrdB. The plasmid pAP605 was then introduced into ΔaprI by conjugation. Complemented exconjugants were verified on agar medium containing erythromycin (100 μg/mL) and confirmed by PCR.

Construction of the aprI overexpressing strain based on homologous recombination. The fragment of PhrdB, aprI and T0 was digested from pAP603 by BglII, and then was added to plasmid pAP600 to obtain the recombinated plasmid pAP607. Plasmid pAP607 was introduced into S. tenebrarius by conjugation using the method described before. Targeted strain named S. tenebrarius IB was confirmed by PCR.

Extraction, and analysis of apramycin. Oxalic acid was added to the fermentation broths in order to precipitate calcium and magnesium ions. The pH was adjusted to 2.0 with H₂SO₄ and agitated for 1 hour. The acidified broth was centrifuged at 7000 r/min for 10 minutes, and the pH of the supernatant was readjusted to 9.0 with NH₄OH. This pretreated supernatant was then centrifuged at 7000 r/min for 10 minutes. The supernatant was readjusted to pH 5.5 with H₂SO₄, applied onto a strongly acidic resin 001×7 (Anhui Sanxing Resin Technology), and then the bound substances were eluted with 2 mol/L NH₄OH. The eluate was used for bioassays and thin-layer chromatography analysis (TLC). Propanol–methanol–25% ammonium hydroxide (20:25:23) was used for the solvent system of TLC. The bioassay was performed with Bacillus subtilis by via agar diffusion. Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed using a LC-10AT system with an evaporative light scattering detector (ELSD). Reverse C₁₈ column (4.6250 mm, 5 μm) was utilized with an evaporation temperature of 45˚C, a nitrogen pressure of 3.5 bar, and 0.2 mol/L trifluoroacetic acid as the running water.¹H and ¹³C nuclear magnetic resonance (NMR) data were recorded on a Bruker AV600 at a frequency of 600 MHz using D₂O as the solvent. High-resolution ESI-MS was performed by Department of Experiment Center at Shenyang Pharmaceutical University.

Expression and purification of AprI. The aprI gene was amplified from the genomic DNA of S. tenebrarius genomic DNA by PCR using pfu polymerase (BBI) and the oligonucleotides (Takara). The PCR product was purified by gel extraction, and inserted into vector pET-28a (+) between the NdeI and EcoRI sites to create pAP606. The resulting construct was verified by DNA sequencing, then used to transform E. coli BL21(DE3) cells using the heat-shock method. BL21(DE3)/pAP606 cells were grown in 3 mL LB medium containing kanamycin (50 mg/mL) at 37°C until an optical density (600 nm) of 0.6-0.8 was achieved, then the cultures induced by isopropylthiogalactoside (0.5 mM) at 16°C with shaking overnight. For purification, the cell pellets from 2 L culture were harvested by centrifugation at 7000 r/min for 15 minutes, and resuspended in 20 mL of binding buffer (20 mmol/L tris-HCL, 0.5 mol/L NaCl, 5 mmol/L imidazole, pH 8.0), and then opened through ultrasonication for 30 minutes on ice. Supernatant was separated from cell debris by centrifugation at 12000 r/min for 40 minutes at 4˚C and passed through a 1 mL IMAC column (GE Healthcare) charged with nickel and previously equilibrated with binding buffer. After washing the column with 30 mL of binding buffer followed by 10 mL of wash buffer (20 mmol/L tris-HCL, 0.5 mol/L NaCl, 40 mmol/L imidazole, pH 8.0), the protein was eluted with elution buffer (20 mmol/L tris-HCL, 0.5 mol/L NaCl, 200 mmol/L imidazole, pH 8.0). Fractions involving the desired product were collected and dialyzed (10 mmol/L tris-HCL, 250 mmol/L NaCl, 10% glycerol, pH 8.0). Protein was concentrated to 13 mg/mL and stored in 10% glycerol at −20°C until used.

Enzymatic assay of AprI. The in vitro enzymatic assay of AprI was performed by combining 17 mmol/L K₂HPO₄/KH₂PO₄(pH 7.4), 2 mmol/L S-adenosylmethionine (SAM), 0.4 mM substrate, and 10 μmol/L purified recombinant AprI in a 500 μL total reaction volume. The reaction was incubated at 30°C and then stopped by the addition of 1x volume of chloroform, followed by vigorous vortexing to denature the proteins. The mixture was centrifuged at 10,000 r/min for 5 minutes, and the supernatant was subjected to HPLC-ELSD and MS analysis.

2-DOS: 2-dexoystreptomycin

SAM: S-adenosylmethionine

TLC: Thin-layer chromatography analysis

HPLC: High-performance liquid chromatography

ELSD: Evaporative light scattering detector

NMR: Nuclear magnetic resonance

Availability of data and materials

The data that support the findings of this study are available from the corresponding author, upon request.

Acknowledgments

Not applicable.

Funding

This work was supported by the Scientific Research Fund of Liaoning Provincial Education Department (2017LZD05, 2019LZD03).

Author information

Affiliations

School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No.103 Wenhua Road, Shenyang, Liaoning, China

Junyang Sun, Hongjing Gao, Danyang Yan, Yu Liu, Xianpu Ni and Huanzhang Xia

Authors Contributions

J. S., H. G., X. N., and H. X. conceived the experiments; and J. S. constructed and analyzed mutants; J.S. carried out in vitro analysis; J. S., H. G., Y. L., D. Y., X. N., and H. X. analyzed the results; and H. X., X. N., and J. S. wrote the paper.

Corresponding author

Correspondence to Huanzhang Xia and Xianpu Ni.

Ethics declarations

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Not applicable.

Competing interests

The authors declare no competing interests.

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FIGS1.tif
Mass-spectra analysis of demethyl-apramycin. (A) HRMS analysis of demethyl-apramycin. (B) MS/MS analysis of demethyl-apramycin.
FIGS2.tif
13C-NMR Spectrum of demethyl-apramycin.
FIGS3.tif
Mass-spectra analysis of demethyl-aprosamine.
FIGS4.tif
Mass-spectra analysis of aprosamine.
FIGS5.tif
NMR Spectrum of aprosamine. (A) 1H-NMR Spectrum of aprosamine. (B) 13C-NMR Spectrum of aprosamine.
FIGS6.tif
Deletion of tobM2 in S. tenebrarius. (A) Schematic representation of the tobM2 deletion. (B) Verification of ΔtobM2 mutant by PCR. PCR amplification with wild-type strain chromosome generated a 5.2 kb fragment, and ΔtobM2 strain chromosome generated a 4.0 kb fragment. (C) HPLC analysis was performed by using an evaporative light-scattering detector (ELSD); (i) wild-type S. tenebrarius, and (ii) ∆tobM2.
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Characterization of Methyltransferase from Apramycin Biosynthetic Gene Cluster and Improvement Production of Apramycin in Engineered Streptoalloteichus Tenebrarius

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