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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.

A structurally unique aminoglycoside produced in Streptoalloteichus tenebrarius, Apramycin is used in veterinary medicine or the treatment of Salmonella, Escherichia coli, and Pasteurella multocida infections. Although apramycin was discovered nearly 50 years ago, many biosynthetic steps of apramycin remain unknown. In this study, we identified a HemK family methyltransferase, AprI, to be the 7’-N-methyltransferase in apramycin biosynthetic pathway. Biochemical experiments showed that AprI converted demethyl-aprosamine to aprosamine. Through gene disruption of aprI, we identified a new aminoglycoside antibiotic demethyl-apramycin as the main product in aprI disruption strain. The demethyl-apramycin is an impurity in apramycin product. In addition to demethyl-apramycin, carbamyltobramycin is another major impurity. However, unlike demethyl-apramycin, tobramycin is biosynthesized by an independent biosynthetic pathway in S. tenebrarius. The titer and rate of apramycin were improved by overexpression of the aprI and disruption of the tobM2, which is a crucial gene for tobramycin biosynthesis. The titer of apramycin increased from 2227 ± 320 mg/L to 2331 ± 210 mg/L, while the titer of product impurity demethyl-apramycin decreased from 196 ± 36 mg/L to 51 ± 9 mg/L. Moreover, the carbamyltobramycin titer of the wild-type strain was 607 ± 111 mg/L and that of the engineering strain was null. The rate of apramycin increased from 68% to 87% and that of demethyl-apramycin decreased from 1.17% to 0.34%.

Background Tetramycin is a 26-member tetraene antibiotic used in agriculture. It has two components, tetramycin A and tetramycin B. Tetramycin B is obtained by the hydroxylation of tetramycin A on C4. This reaction is catalyzed by the cytochrome P450 monooxygenase TtmD. The two components of tetramycin have different antifungal activities against different pathogenic fungi. Therefore, the respective construction of high-yield strains of tetramycin A and tetramycin B is conducive to more targeted action on pathomycete and has a certain practical value. Results Streptomyces ahygroscopicus S91 was used as the original strain to construct tetramycin A high-yield strains by blocking the precursor competitive biosynthetic gene cluster, disrupting tetramycin B biosynthesis, and overexpressing the tetramycin pathway regulator. Eventually, the yield of tetramycin A in the final strain was up to 1090.49 ± 136.65 mg·L− 1. Subsequently, TtmD, which catalyzes the conversion from tetramycin A to tetramycin B, was overexpressed. Strains with 2, 3, and 4 copies of ttmD were constructed. The three strains had different drops in tetramycin A yield, with increases in tetramycin B. The strain with three copies of ttmD showed the most significant change in the ratio of the two components. Conclusions A tetramycin A single-component producing strain was obtained, and the production of tetramycin A increased 236.84% ± 38.96% compared with the original strain. In addition, the content of tetramycin B in a high-yield strain with three copies of ttmD increased from 26.64% ± 1.97 to 51.63% ± 2.06%.

Recombinant human thrombopoietin (rhTPO) was approved by the National Medical Products Administration in 2010 for the treatment of thrombocytopenia in patients with immune thrombocytopenic purpura and chemotherapy-induced thrombocytopenia. Nevertheless, no method for determining rhTPO bioactivity has been recorded in different national/regional pharmacopoeia. Novel methods for lot release and stability testing are needed that are simpler, quicker, and more accurate. Here, we developed a novel reporter gene assay (RGA) for rhTPO bioassay with Ba/F3 cell lines that stably expressed human TPO receptor and luciferase reporter driven by sis-inducible element, gamma response region, and gamma-interferon activated sequence. During careful optimization, the RGA method demonstrated high performance characteristics. According to the International Council for Harmonization Q2 (R1) guidelines and the Chinese Pharmacopoeia 2020 edition, the validation results demonstrated that this method is highly time-saving, sensitive, and robust for research, development, manufacture, and quality control of rhTPO.

Additional file 3: Figure S3. Inactivation of nysB in S.ahygroscopicus S91. a. Construction of the recombinant plamid pDNB; b. Double crossover validation of the recombinant strain S91-ΔNB; c.Verification of sequencing in the recombinant strain S91-ΔNB.

Additional file 4: Figure S4. Inactivation of ttmD in S.ahygroscopicus S91-ΔNB. a. Construction of the recombinant plasmid pDTD; b. Double crossover validation of the recombinant strain S91-ΔNBΔTD; c. Verification of sequencing in the recombinant strain S91-ΔNBΔTD.

Additional file 8: Table S2. Strains, plasmids and primers used in this study.

Additional file 5: Figure S5. Cloning and overexpression of ttmRIV. a. Construction of the recombinant plamid pETRIV; b. PCR analysis of the recombinant strain S91-ΔNBΔTD::TRIV, M. DL2000 (2.0k, 1.0k, 0.75k, 0.5k, 0.25k, 0.1k), 1. S91-ΔNBΔTD::pETRIV/PB-1& TRIV-R (1.1k), 2. S91-ΔNBΔTD/PB-1&TRIV-R, 3. S91-ΔNBΔTD::pSET152/PB-1&TRIV-R.

Additional file 7: Table S1. Production analysis in S.ahygroscopicus S91 and its mutants.

Additional file 6: Figure S6. Cloning and overexpression of ttmD. a. Construction of the recombinant plamid p2ETD; b. PCR analysis of the multicope ttmD recombinant strains, M. DL2000, 1. S91-ΔNB::pETD/PB-1&TD-R (1.7k), 2. S91-ΔNB::p2ETD/PB-1&TD-R (1.7k), 3. S91-ΔNB::p3ETD/PB-1&TD-R (1.7k), 4. S91-ΔNB/PB-1&TD-R, 5. S91-ΔNB::pSET152/PB-1&TD-R.

Additional file 1: Figure S1. Biosynthesis of tetramycin.

Additional file 2: Figure S2. LC-MS analysis of tetramycin and nystatin in Streptomyces ahygroscopicus S91. a. The HPLC profile of Streptomyces ahygroscopicus S91 fermentation products; b. The UV absorption spectra and MS of tetramycin B; c. The UV absorption spectra and MS of nystatin; d. The UV absorption spectra and MS of tetramycin A.

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 ΔgenK, 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.

Additional file 9: Figure S9.1H NMR and 13C NMR spectroscopic analyses of compound (8) reduced with NaBH4 or NaBD4. (a) 1H NMR spectroscopic analysis of compound (8) reduced with NaBD4. 1H NMR (600 MHz, D2O) δ 5.40 (d, J = 3.5 Hz, 1H), 5.01 (d, J = 3.7 Hz, 1H), 4.14 (dd, J = 10.9, 3.7 Hz, 1H), 3.95–3.92 (m, 1H), 3.91 (d, J = 12.9 Hz, 1H), 3.81–3.76 (m, 1H), 3.72 – 3.66 (m, 3H), 3.56 (d, J = 1.3 Hz, 1H), 3.56 – 3.53 (m, 1H), 3.48 (ddd, J = 11.9, 6.2, 3.3 Hz, 4H), 3.44–3.37 (m, 2H), 2.83 (s, 3H), 2.45 (dt, J = 12.6, 4.2 Hz, 1H), 1.96–1.92 (m, 1H), 1.88 (dd, J = 12.6, 4.1 Hz, 1H), 1.84 (t, J = 8.7 Hz, 1H), 1.72 (dd, J = 14.0, 2.8 Hz, 1H), 1.48–1.41 (m, 1H), 1.26 (s, 3H). (b) 13C NMR spectroscopy analysis of the compound (8) reduced with NaBD4. 13C NMR (151 MHz, D2O) δ 101.14, 96.49, 83.43, 80.38, 73.63, 72.02, 71.10, 69.83, 64.76 (d, J = 432.03 Hz), 63.32, 62.44, 49.50, 49.06, 48.81, 34.42, 27.80, 23.78, 21.23, 20.82.

Additional file 1: Figure S1. In-frame deletion of genB4 in M. echinospora. (a) Schematic representation of the in-frame deletions in wild-type M. echinospora; (b) Confirmation of M. echinospora △genB4 by PCR with the primers, B4Y1 and B4Y4. The arrows indicate the expected size of the PCR fragments in the wild type and mutants. (c) Confirmation of M. echinospora △genK△genB4 by PCR with the primers, B4Y1 and B4Y4. The arrows indicate the expected size of the PCR fragments in the wild type and mutants.

Additional file 2: Figure S2. (1) and (6) reduced with NaBH4. (1) and (6) had the same retention times in HPLC-ELSD when separated by cation-exchange chromatography. (1) was reduced by NaBH4, whereas (6) was not.

Additional file 8: Figure S8. Characterization of purified recombinant GenB4. (a) SDS-PAGE analysis of purified GenB4 (51.9 kDa). The production of N-His6-tagged GenB4 was carried out in E. coli BL21(DE3). The acrylamide percentage of the SDS-PAGE gels was 12%. (b) UV–vis absorption spectrum of the purified recombinant proteins, His6-GenB4.

Additional file 5: Figure S5.1H NMR and 13C NMR spectroscopic analyses of intermediate (1). (a) 1H NMR spectroscopic analysis of intermediate (1). 1H NMR (600 MHz, D2O) δ 6.30 (t, J = 4.1 Hz, 1H), 5.49 (s, 1H), 4.96 (d, J = 3.7 Hz, 1H), 4.10 (dd, J = 10.9, 3.6 Hz, 1H), 3.94–3.89 (m, 1H), 3.86 (d, J = 12.7 Hz, 2H), 3.69–3.61 (m, 2H), 3.46–3.39 (m, 2H), 3.39–3.32 (m, 2H), 2.79 (d, J = 5.3 Hz, 3H), 2.59 (s, 2H), 2.49 (dt, J = 20.1, 4.2 Hz, 1H), 2.43 (dt, J = 12.6, 4.2 Hz, 1H), 2.26 (s, 3H), 1.82 (q, J = 12.7 Hz, 1H), 1.22 (s, 3H). (b) 13C NMR (151 MHz, D2O) δ 195.94 (s), 146.75 (s), 113.70 (s), 101.19 (s), 97.10 (s), 83.14 (s), 80.08 (s), 73.35 (s), 69.83 (s), 67.62 (s), 66.24 (s), 63.36 (s), 48.15 (s), 45.69 (s), 38.65 (s), 34.38 (s), 27.52 (s), 24.45 (s), 23.99 (s), 20.80 (s).

Additional file 4: Figure S4. Mass-spectra analysis of the intermediates in the mutants.

Additional file 3: Figure S3. Mass-spectra analysis of the intermediates in the mutants.

Additional file 7: Figure S7. HPLC-ELSD analysis of fermentation production by complementation strains and the original strains.

Additional file 6: Figure S6. Complementation of the genB4 disrupting strain. (a) Map of the genetic complementation vector, pEAP1B4. The phrdb1 and B4dn are primers used to check the complementation strains. (b) Confirmation of the complementation strain by PCR. The arrows indicate the expected size of the PCR fragments in the original strain and the mutants.

Tumor necrosis factor alpha (TNFα) is now regarding as a key role in the pathogenesis of immune-mediated disease such as Rheumatoid arthritis (RA), Crohn's Disease, Psoriatic arthritis and Plaque Psoriasis. HERE we have successfully developed an anti-hTNFα monoclonal rabbit antibody(HZ3M) with high binding and neutralizing activity based on RabMAbs platform. Rabbit hybridomas, immunized subcutanrously with 0.4 mg human TNFα, were generated from the rabbit splenocytes and a total of 142 hybridoma clones with specific binding to human TNFa were obtained. The anti-TNFa RabMAbs showed better neutralizing activity and higher antigen binding affinity compared to Humira and Remicade, the elimination phase half-life 58.2h respectively. In vivo efficay studies, normal mice or human TNF-alpha transgenic mice were injected with 1.0 mg/kg Humira (positive control), HZ3M at 0.33?1.0 or 3.0 mg/kg, or solvent (negative control), showed that HZ3M is able to bind and neutralize hTNFα in transgenic and normal mice as well as normal rabbits.Clearly dose-dependent response can be determined. Compared to marketed anti-TNFa drug Humira, the efficacy of HZ3M is seems to show significant longer holding time.Our observations indicate that the HZ3M derived from RabMAb preclinical safety study, and might have a therapeutic role in RA treatment.

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.

A putative regulatory gene ttmRIV located in the tetramycin biosynthetic gene cluster was found in Streptomyces ahygroscopicus. In-frame deletion of ttmRIV led to abolishment of tetramycin and significant enhancement of nystatin A1, whose production reached 2.1-fold of the H42 parental strain. Gene complementation by an integrative plasmid carrying ttmRIV restored tetramycin biosynthesis revealed that ttmRIV was indispensable to tetramycin biosynthesis. Gene expression analysis of the H42 strain and its mutant strain ΔttmRIV via reverse transcriptase-PCR of the tetramycin gene cluster demonstrated that the expression levels of most biosynthetic genes were reduced in ΔttmRIV. Results of electrophoretic mobility shift assays showed that TtmRIV bound the putative promoters of several genes in the tetramycin pathway. Thus, TtmRIV is a pathway-specific positive regulator activating the transcription of the tetramycin gene cluster in S. ahygroscopicus. Providing an additional copy of ttmRIV under the control of the ermEp* promoter in the H42 strain boosted tetramycin A production to 3.3-fold.

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used to treat life-threatening bacterial infections. The gentamicin C complex consists of gentamicin C1, gentamicin C1a, and epimers gentamicin C2 and gentamicin C2a. At present there is a generally accepted pathway of gentamicin biosynthesis, except for detailed understanding of the epimerization process involving gentamicins C2 and C2a. Here we have investigated the biosynthesis of these epimers. JI-20B-an intermediate in the gentamicin biosynthetic pathway-and its epimer JI-20Ba were generated by in-frame deletion within genP, which encodes a phosphotransferase that catalyzes the first step of 3',4'-bisdehydroxylation in gentamicin biosynthesis. GenB1 and GenB2 are aminotransferases with different substrate specificities and enantioselectivities. JI-20Ba, containing a 6'S chiral amine, a precursor of gentamicin C2a, was synthesized from G418 by GenQ/GenB1 through sequential oxidation/transamination at C-6'. GenQ/GenB2 catalyzed the synthesis of JI-20B, containing a 6'R chiral amine, a precursor of gentamicin C2, from G418. GenB2 catalyzed the epimerization of JI-20Ba/JI-20B and of gentamicins C2a/C2.

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.

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.

Complete and independent tetramycin and nystatin gene clusters containing varying lengths of type I polyketide synthase (PKS) genes were isolated from Streptomyces ahygroscopicus, a producer of tetramycin (a tetraene) in large amounts and nystatin A1 (a heptaene) in small amounts. Tetramycin was similar to pimaricin, and nystatin A1 was similar to amphotericin. All these polyene macrolide antibiotics possessed the same macrolactone ring biosynthesized from coenzyme A precursors by PKSs but had different number of atoms in the macrolactone ring and side groups. Because tetramycin and nystatin shared limited coenzyme A precursors in the same producer organism, blocking the consumption of precursors in tetramycin pathway may increase the coenzyme A pool. Thus, we genetically manipulated the tetramycin PKS to enhance nystatin production. The type I PKS ttmS1 gene mutant abolished production of tetramycin and had a beneficial effect on the production of nystatin A1. For the mutant, the yield of nystatin A1 was increased by 10-fold compared to that of the wild-type. Thus, deletion of the tetramycin pathway redirected precursor metabolic fluxes and provided an easy genetic approach to manipulate organisms and to increase production levels of a precise target.

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.

Three putative regulatory genes, namely, ttmRI , ttmRII , and ttmRIII , which are present in the tetramycin ( ttm ) biosynthetic gene cluster, were found in Streptomyces ahygroscopicus . Disruption of ttmRI , ttmRII , or ttmRIII reduced tetramycin production, and their complementation restored their production at varying degrees. Gene expression analysis of the wild-type (WT) and mutant strains through reverse transcriptase-polymerase chain reaction (RT-PCR) of the ttm gene cluster showed that the expression levels of most of the biosynthetic genes were reduced in Δ ttmRI , Δ ttmRII , and Δ ttmRIII . Electrophoretic mobility shift assays demonstrated that TtmRI, TtmRII, and TtmRIII bound the promoters of several genes in the ttm gene cluster. This study found that these three proteins are a group of positive regulators that activate the transcription of the ttm gene cluster in S. ahygroscopicus . In addition, the Δ ttmRII reduced the degree of grey pigmentation. Thus, TtmRII demonstrated a pleiotropic regulatory function on tetramycin biosynthetic pathway and on development.

Background 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. 2ʹ-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2ʹ-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 C2ʹ 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. Results 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 μg/ml, which was 14-fold higher than that of M. echinospora ∆P. The enzymes KanJ and KanK necessary to convert 2ʹ-NH2 into 2ʹ-OH from the kanamycin biosynthetic pathway were heterologously expressed in M. echinospora ΔKΔP to transform JI-20A into gentamicin B. The strain with kanJK under PermE* could produce 80 μg/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 μg/ml. Conclusion 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. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0402-6) contains supplementary material, which is available to authorized users.

Additional file 2: Figure S2. 1H NMR spectrum of the new compound from kanJK expression strains.

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.

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.

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.