WO2009008664A2 - A producing method for gentamicin a2 or the precursors and recombinant microorganisms producing the same - Google Patents

A producing method for gentamicin a2 or the precursors and recombinant microorganisms producing the same Download PDF

Info

Publication number
WO2009008664A2
WO2009008664A2 PCT/KR2008/004033 KR2008004033W WO2009008664A2 WO 2009008664 A2 WO2009008664 A2 WO 2009008664A2 KR 2008004033 W KR2008004033 W KR 2008004033W WO 2009008664 A2 WO2009008664 A2 WO 2009008664A2
Authority
WO
WIPO (PCT)
Prior art keywords
gentamicin
paromamine
genes
producing
gnta
Prior art date
Application number
PCT/KR2008/004033
Other languages
French (fr)
Other versions
WO2009008664A3 (en
WO2009008664A4 (en
Inventor
Jea Jeong Kim
Si Kyu Lim
Yeo Joon Yoon
Je Won Park
Mi Ok Lee
Bo-Mi Lee
Dong Hwan Kim
Jeoung Hyun Ryu
Keum Soon Lee
Original Assignee
Genotech Co., Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genotech Co., Ltd filed Critical Genotech Co., Ltd
Publication of WO2009008664A2 publication Critical patent/WO2009008664A2/en
Publication of WO2009008664A3 publication Critical patent/WO2009008664A3/en
Publication of WO2009008664A4 publication Critical patent/WO2009008664A4/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes

Definitions

  • the present invention relates to aminoglycoside-based antibiotics, specifically gentamicin, useful as a medication for treatment of infectious diseases. More particularly, the present invention relates to a method of producing gentamicin A2 via systematic recombination and expression of its biosynthetic genes obtained from Micromonospora echinospora or Micromonospora purpurea that is a native production organism or strain of gentamicin A2 in a heterologous host, specifically in Streptomyces venezuelae. The present invention also relates to precursors thereof.
  • Gentamicin is an anminoglycoside antibiotic complex produced by a Micromonospora species. Aminoglycosides act on the 3OS subunit of the bacterial ribosome, hindering the translation stage of protein synthesis to inhibit the growth of bacteria. Structurally, gentamicin is a 4, 6- disubstituted , aminoglycoside composed of 2-deoxystreptamine (2-DOS) to which purpurosamine and garosamine aminosugars are bonded at C-4 and C-6 positions, respectively.
  • 2-DOS 2-deoxystreptamine
  • Micromonospora echinospora is a strain which produces gentamicin A, Al, A2, X2, G418, J1-20A, J1-20B, B, Bl, Cl, CIa, C2, C2b, and C2a.
  • Commercially available gentamicin sulfate is a complex mixture of C-form gentamicin, i.e., Cl, CIa, C2, C2b, and C2a, containing a small amount of A-form gentamicin, i.e., A2 and Al. This is because complete separation and purification of various forms of gentamicins by a common purifying process is difficult since the respective gentamicin components have similar physical and chemical properties.
  • An object of the present invention is to provide a method of producing gentamicin and precursors thereof in a heterologous host by recombining gentamicin biosynthetic genes suitable for expression in the heterologous host, and a recombinant microorganism of the same.
  • the present invention provides a biosynthetic method for gentamicin and precursors thereof through systematic recombination and introduction of gentamicin biosynthetic genes to Streptomyces venezuelae, a non- aminoglycoside producing actinobacteria.
  • the present invention elucidates genes necessary for biosynthesis of gentamicin A2, 2-deoxystreptamine (2- DOS), 2'-/V-acetyl-paromamine and paromamine, which are crucial antibiotics and precursors for the biosynthesis of gentamicin, and the biosynthetic mechanism thereof, and therefore systematically dissolve and introduce the genes into a heterologous host, thereby produce gentamicin A2, 2-DOS, and paromamine. Further, the present invention provides a method of producing aminoglycoside antibiotics, and more particularly, a method of producing an antibiotic compound in a heterologous host of gentamicin.
  • the present invention provides direct evidence for the biosynthesis of 4,6-disubstituted aminoglycoside antibiotics and allows for the assignment of functions of each gene through an in-vivo study of the expression of gentamicin biosynthetic genes in a heterologous host. Actually, gentamicin and precursors thereof were produced in the heterologous host. In addition, the results of glycosyltransferases GntZ and GntD to substrates in the present invention enable development of novel aminoglycosides using the same. [Description of Drawings]
  • Fig. 1 shows LC/ESI-MS/MS chromatograms of culture fluids of Streptomyces venezuelae mutant strains transformed with plasmids (a): pYJ495 expressing gntF, gntA, gntB, and gntP, (b): pYJ497 expressing gntF, gntB, and gntF, (c): pYJ496 expressing gntA, gntB, and gntP ⁇ (d): pYJ498 expressing gntA, gntB, gntP, and gntZ', (e): pYJ501 expressing gntA, gntB, gntP, gntZ, kacA', and (f): pYJ503 expressing gntA, gntB, gn
  • Fig. 2 shows HPLC-ESI-MS chromatograms of gentamicin A2 (4 in Fig. 5) and 2'-7V-acetyl-paromamine (2 in Fig. 5) by the glycosyltransferase.
  • Fig. 3 shows HPLC-ESI-MS chromatograms, in which ⁇ 2i> (a): 2-deoxystreptamine (1 in Fig. 5)
  • Fig. 4 is H NMR spectra of
  • Fig. 5 is a biosynthetic pathway of gentamicin. [Best Mode]
  • the above and other object of the present invention can be accomplished by providing an effective producing method of gentamicin and precursors thereof by estimating a biosynthetic process of gentamicin.
  • a recombinant microorganism producing at least one selected from the group consisting of gentamicin, paromamine, 2'-vV-acetyl-paromamine and 2- deoxystreptamine is formed by recombining a heterologous host that do not biosynthesize gentamicin with genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neol ⁇ .
  • a vector producing at least one selected from the group consisting of gentamicin, paromamine, 2'-/V-acetyl-paromamine, and 2-deoxystreptamine which includes gentamicin biosynthetic genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neol ⁇ .
  • the recombinant microorganism or the vector may further include a gentamicin resistance gene.
  • the heterologous host may comprise a Streptomyces species.
  • a producing method for paromamine from 2'-./V-acetyl-paromamine comprises expressing genes btrD, kacA, and neol ⁇ in a heterologous host that do not biosynthesize gentamicin.
  • a producing method for at least one selected from the group consisting of gentamicin, paromamine, 2'-yV-acetyl-paromamine and 2-deoxystreptamine comprises recombining a heterologous host that do not biosynthesize gentamicin with genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neol ⁇ .
  • the method may further comprise adding at least one of a gene gntD and an enzyme GntD and UDP-D- ⁇ ylose to produce a large amount of gentamicin A2.
  • a 2- deoxystreptamine producing recombinant microorganism is formed by recombining a heterologous host that do not biosynthesize gentamicin with genes gntB, gntA, and gntP.
  • a 2- deoxystreptamine producing vector comprises gentamicin biosynthetic genes gntB, gntA, and gntP.
  • a 2'-jV-acetyl-paromamine producing recombinant microorganism forms by recombining a heterologous host which do not biosynthesize gentamicin with genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO.
  • a 2'-vV-acetyl-paromamine producing vector comprises gentamicin biosynthetic genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO.
  • a paromamine producing recombinant microorganism forms by recombining a non- gentamicin biosynthesizing heterologous host with genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO and at least one selected from the group O
  • a paromamine producing vector comprises gentamicin biosynthetic genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO and at least one selected from the group consisting of btrD, kacA, and neol ⁇ .
  • gentamicin precursor refers to 2- deoxystreptamine (hereinafter, also referred to as "2-DOS”), 2'-yV-acetyl- D-paromamine, and/or paromamine.
  • gentamicin biosynthetic genes refers to biosynthetic genes separated from a gentamicin biosynthesizing strain of Micromonospora echinospora, which is already known in the art and is obtained from pGENOl (GenBank Accession No. AJ575934 (M.K. Kharel, D.B. Basnet, HX. Lee, K. Liou, Y.H. Moon, J.J.
  • heterologous host refers to strains of microorganisms other than Micromonospora echinospora that is a native gentamicin producing strain, specifically to Streptomyces venezuelae and the like that are non-aminoglycoside producing strains.
  • ⁇ 48> Genes were amplified from pGENOl using a DNA polymerase chain reaction (PCR) enzyme. A primer including an appropriate restriction enzyme for each gene was used. The amplified genes were properly introduced to pSE34 plasmid (N. Smirnova, K. A. Reynolds, J. Bacteriol. 2001, 183, 2335-2342.) to be controlled by an ermE promoter which is a strong actinomycetal promoter. Expression vectors were prepared as in Table 2. The production method will be described in detail in the examples.
  • PCR DNA polymerase chain reaction
  • the prepared genes were introduced to Streptomyces venezuelae YJ003 (J. S. J. Hong, S. H. Park, C. Y. Choi, J. K. Sohng, Y. J. Yoon, FEMS Microbiol. Lett. 2004, 238, 391-399).
  • Analytical HPLC-ESI-MS was carried out for metabolite analysis. For more accurate analysis, preparative HPLC was carried out to fractionize the product. The fractions were analyzed by H, C, 2D H-H COSY NMR spectrum to identify products.
  • gentamicin biosynthesis begins from D-glucose-6-phosphate (Glc-6-P) modified to 2- deoxy-scy/Zo-inosose (2-DOI) by GntB enzyme (Korean Patent Registration No. 10-0470743) through carbocyclization. Two aminotransferase reactions and a single dehydrogenase reaction were then required to synthesize 2- deoxy-scy/Arinosamine (2-DOIA) and l-keto-2-deoxy-scj7/o-inosamine (keto- 2-DOIA) (M.K. Kharel, D. B. Basnet, HX. Lee, K.
  • a prerequisite for biosynthesis of gentamicin in a heterologous host is to impart gentamicin resistance to the heterologous host.
  • pYJ489 ⁇ grmA, 0 and gntO was introduced into the heterologous host.
  • the genes grwA and grmO encode bacterial ribosome methylases, and the gntO gene is assumed to be a transporter of aminoglycoside to impart resistance to the heterologous host without structurally modifying antibiotic expression.
  • transformants harbouring pYJ489 were resistant up to 100 ⁇ g/ml of added gentamicin whereas the growth of non-transformants was completely inhibited.
  • the added gentamicin was recollected for analysis, so that gentamicin was changed in neither structure nor activity due to the introduction of the genes.
  • GntD and GntZ were combined with 2-DOS biosynthetic genes, gntA, B, and P, and resistance genes, grmA, 0 and gntO, to prepare pYJ498.
  • the transformants of pYJ498 did not produce paromamine but synthesized 2'-yV-acetyl-D-paromamine as was detected by HPLC-ESI-MS at 59.8 mimutes with an m/z 366 (Fig. Id).
  • NDP-GIcNAc NDP-N-acetyl-D-glucosamine
  • NDP-GIcN NDP-D- glucosamine
  • UDP-GIcNAc incorporation of UDP- GIcNAc into 2-DOS was verified as the amount of 2'-yV-acetyl-D-paromamine increased (Fig. 2a, b, c).
  • a putative glycosyltransferase using UDP-GIcNAc as a substrate is BtrM from the cluster of butirosin biosynthetic genes, and its homologues of Neo8, KanF, and GntZ from neomycin, kanamycin, and gentamicin biosynthetic gene clusters, respectively, are suggested to use UDP-GIcNAc as a substrate (N. M. Llewellyn, J. B. Spencer, Nat. Prod. Rep. 2006, 23, 864-874).
  • GntZ is a UDP-GIcNAc glycosyltransferase.
  • BtrD a paromamine deacetylase, eliminates an acetyl group from 2'-./ ⁇ -acetyl-D- paromamine to produce D-paromamine (A. W. Truman, F. Huang, N. M. Llewellyn, J. B. Spencer, Angew. Chem. Int. Ed. 2007, 46, 1462-1464).
  • the present invention imported btrD, kacA, and neol ⁇ from Bacillus circulans, Streptomyces kanamyceticus, and Streptomyces fradiae, respectively. Then, btrD, kacA and neol ⁇ was each combined with separate constructs containing 2-DOS biosynthetic genes, glycosyltransferase, and resistance genes to produce plasmids pYJ500, pYJ ⁇ Ol, and pYJ502, respectively.
  • paromamine and 2'-yV-acetyl-D-paromamine were identified in-vivo, thereby verifying that 2'-/ ⁇ -acetyl-D-paromamine is a direct precursor of aminoglycoside antibiotics such as gentamicin.
  • the second glycosylation by NDP-D- ⁇ ylose is required for the formation of gentamicin A2 from paromamine.
  • the combination of gntD and a paromamine biosynthetic gene prepared pYJ503.
  • the transformant of pYJ503 produced gentamicin A2 at a retention time of 73.3 minutes with an m/z 456, together with paromamine and 2'-jV-acetyl-D-paromamine (Fig. If). Therefore, GntD is a glycosyltransferase to transfer NDP-D- ⁇ ylose to paromamine. A large amount of paromamine remained and the yield of gentamicin A2 was only 4.5 ⁇ g/L, for which the heterlogous host had a limiting amount of NDP-D- ⁇ ylose.
  • NDP-D- ⁇ ylose was a substrate actually incorporated into paromamine to produce gentamicin A2
  • a cell-free extract of the heterlogous host harbouring pYJ503 was prepared and incubated with different concentrations of exogeneous UDP-D- xylose. It was observed that the amount of gentamicin A2 gradually increases as the exogenous UDP-D- ⁇ ylose increases in concentration (Fig. 2d, 2e, 2f). In this regard, it was confirmed that UDP-D- ⁇ ylose incorporated into gentamicin A2.
  • the present invention completed gentamicin A2 and the minimal gene set involved in the formation of precursors thereof.
  • 2-DOS is synthesized by enzymes encoded by gntA, gntB and gntP.
  • the gntZ gene that encodes a glycosyltransferase, actually functions in transferring N-acetyl-D-glucosamine to produce 2'-/V-acetyl-D-paromamine.
  • a gentamicin biosynthetic gene which encodes a deacetylase, modify 2'-N- acetyl-D-paromamine into D-paromamine still remains to be found, while BtrD of a deacetylase and its homologues KacA and Neol ⁇ were able to carry out its function instead.
  • GntD having an activity of NDP-xylose transferase is necessary to produce gentamicin A2.
  • the clarified functions of the genes are listed in Table 3.
  • E. coli Escherichia coli
  • DHlOB Escherichia coli
  • plasmid Litmus28 New England Biolabs
  • Gene btrD was amplified from Bacillus circulans genomic DNA using btrDHF and btrD-R.
  • Gene kacA was amplified from Streptomyces kanamyceticus genomic DNA using kacA-F and kacA-R.
  • Gene neol ⁇ was amplified from Streptomyces fradiae genomic DNA using neol6-F and neol6-R.
  • the gene amplification was carried out according to general gene amplification enzyme methods. The amplified genes were introduced into appropriate restriction sites of plasmid Litmus 28 and analyzed to confirm that it has the same nucleotide sequence as the native genes.
  • Streptomyces and pSE34 of an E Streptomyces and pSE34 of an E.
  • Resistance gene expressing plasmid vector ⁇ 109>
  • the resistance genes grmA, gntO and grmO were employed to confer gentamicin resistance to Streptomyces venezuelae.
  • the PCR-amplified gntO and grmO genes were digested with Sbfl/Hindlll and Sb ⁇ /BstBl, respectively, and introduced to Litmus 28 at the same time.
  • the PCR- amplified grmA was introduced to Ncol/Xbal restriction site.
  • the gntO-grmO gene was digested with SnaBl/fl/ndlll, and the grmO gene was digested with Xbal/SnaBl.
  • the two digested fragments were simultaneously cloned to pSE34 digested with Xbal/HindiU, thereby preparing pYJ489.
  • pYJ489 ⁇ grmA-gntO- grmO was inoculated to obtain resistance to an antibiotic used for production of gentamicin in a heterologous host. It is believed that the grmA and grmO genes encoded methylases of ribosomes of bacteria and the gntO gene product was a putative aminoglycoside transporter imparting resistance to the antibiotic without structurally modifying the antibiotic to be expressed.
  • transformants harbouring ⁇ YJ489 were resistant up to 100 ⁇ g/ml of added gentamicin whereas growth of non- transformants was completely inhibited. Furthermore, it was not observed that the introduction of the genes (antibiotic resistance genes) changed gentamicin in either structure or activity.
  • Example 3 Preparation of expression vector for production of 2-DOS
  • the gntF, gntA, gntB, gntC, and gntP genes which had been PCR-amp1 ified and introduced to Litmus 28, were digested with appropriate restriction enzymes to construct a set of genes including gntFABC, gntFABP, gntABP, and gntFBP in Litmus 28.
  • Plasmids harbouring the set were digested with BglW/Xbal and introduced into pSE34 digested with the same enzyme, thereby preparing pYJ494, ⁇ YJ495, pYJ496, and pYJ497.
  • transformants of pYJ495 and pYJ496 produced 2-DOS.
  • the genes grmA and gntZ were digested with appropriate restriction enzymes and simultaneously cloned into Litmus 28. Then, the grmA-gntZ and gntO-grmO genes were each digested with Xbal/SnaBl and SnaBl/ff ⁇ ndlll and o
  • the genes grmA and gntD were digested with appropriate restriction enzymes and cloned into Litmus 28. Then, the grmA-gntD and gntO-grmO genes were each digested with Xbal/SnaBl and SnaBl/Hindlll and were ligated into the Xbal/Hindlll digested pYJ496, produced in Example 3, to produce pYJ499.
  • Example 5 Preparation of plasmid vector for simultaneous expression of deacetylase gene-incorporated 2-DOS biosynthetic genes, resistance genes, and glycosyltransferase genes
  • a deacetylase gene brtD was amplified from a Bacillus circulans genome.
  • a SnaBl/BstBl digested brtD gene, the BstBl/Hindlll digested gntO- grmO fragment, and the Xbal/SnaBl digested grmA-gntD fragment were incorporated into the Xbal/SnaBl digested pYJ496, produced in Example 3, thereby producing pYJ500.
  • a deacetylase gene kacA was amplified from a Streptomyces kanamyceticus genome.
  • a SnaBl/BstBl digested kacA gene, the BstBl/Hindi11 digested gntO-grmO fragment, and the Xbal/SnaBl digested grmA-gntD fragment were incorporated into the Xbal/SnaBl digested pYJ496, produced in Example 3, thereby producing ⁇ YJ501.
  • a deacetylase gene neol ⁇ was amplified from a Streptomyces fradiae genome.
  • a SnaBl/BstBl digested neol ⁇ , the BstBl/Hindlll digested gntO-grmO fragment, and the Xbal/SnaBl digested grmA-gntD fragment were incorporated into the Xbal/SnaBl digested pYJ496, produced in Example 3, thereby producing pYJ502.
  • the gntD gene encoding a glycosyltransferase was introduced into pYJ501, produced in Example 5, to produce pYJ503.
  • the gntD gene was digested with Sb ⁇ /Spel and incorporated into the Litmus 28 containing the grmA-grntZ.
  • the grmA-gntZ-gntD gene was digested with Xbal/SnaBl and incorporated into the Xbal/Hindlll digested pYJ496, produced from the SnaBl/Hindlll digested pYJ501 in Example 4, thereby producing pYJ503.
  • Streptomyces venezuelae YJ003 is a mutant strain which lacks TDP-D- desosamine biosynthetic ability (J.S.J. Hong, S.H. Park, CY. Choi, J.K. Sohng, Y.J. Yoon, FEMS Microbiol. Lett. 2004, 238, 391-399).
  • Preparations of spores and protoplasts, introduction of plasmids into protoplasts, and selection of plasmids were conducted by methods proposed in practical streptomyces genetics (T. Kaieser, M. J. Bibb, M. J. Buttner, K. F. Chater, D. A. Hopwood, John Innes Centre, Norwich, UK, 2000).
  • Streptomyces venezuelae was cultured in liquid R2YE to prepare protoplasts. Then, pSE34, pYJ489 of Example 2, pYJ494, pYJ495, pYJ496, and pYJ497 of Example 3, pYJ498 and pYJ499 of Example 4, pYJ500, pYJ501, and pYJ502 of Example 5, and pYJ503 of Example 6 were introduced into the protoplasts, respectively. Regeneration of the protoplasts and selection of the plasmids were carried out in agar containing 30 ⁇ g/mL thiostrepton.
  • the plasmids of Streptomyces venezuelae were cultivated at 30°C for 3 to 4 days in a 1 L flask containing 300 ml of an R2YE medium supplemented with 30 ⁇ g/mL thiostrepton.
  • Example 8 Separation of products from transformants and analysis Analytical HPLC-ESI-MS was performed on a Waters/Micromass Quattro micro/W) interface using two XTerra MS Ci 8 (3.5 ⁇ s ⁇ , Waters) columns, 150mm
  • Preparative HPLC was performed with a Spherisorb S5 0DS2 (250mmx20 mm, Waters) semi-prep column using the same solvent as employed in the elution of Example 8 at a flow rate of 12 mL/min for 100 minutes. This eluent was fractionized into 5 mL fractions, which in turn were monitored by analytical HPLC-ESI-MS, as described in Example 8, to confirm the presence of gentamicin precursors. The fractions containing the desired product were pooled and extracted by Solid-Phase Extraction (SPE) with SPE.
  • SPE Solid-Phase Extraction
  • Paromamine was obtained by acid hydrolysis of reference paromomycin (Sigma), titration of the hydrolysate to pH 6.0, purification with an SPE cartridge, and freeze-drying according to a method proposed by a previous study (T. H. Haskell, J. C. French, Q. R. Bartz, /. Am. Chem. Soc. 1959, 81, 3480-3481).
  • the retention time of paromamine from the HPLC analysis was 74.8 minutes (Fig. 3c) and the MS/MS fragmentation pattern is shown in Fig. 3g.
  • a compound produced by plasmid pYJ501 harbouring plasmid with gntA, B, P, Zand kacA genes was paromamine; H NMR (500 MHz, D 2 O): ⁇ 1.74
  • Gentamicin A2 was isolated and purified from the extract of wild- type Micromonospora echinospora ATCC 15835 by preparative HPLC. The retention time of gentamicin A2 by the HPLC analysis was 73.3 minutes (Fig. 3d), and the purified gentamicin A2 was a pale yellow solid. The MS/MS fragmentation pattern shown in Fig. 3h is confirmed by the NMR data acquired in earlier studies. A compound produced from the mutant strain of Streptomyces venezuelae harbouring pYJ503 was identified to be gentamicin
  • Gentamicin biosynthetic precursors produced by Streptotnyces venezuelae mutant strains were extracted using an OASIS MCX cartridge, as previously described, and were analyzed by HPLC-ESI-MS described in Example 9.
  • the quantification of the analytes was conducted using a multiple reactions monitoring (MRM) mode. This was done by detecting the selected analytes with respect to a set of two mass ions, which are a parent ion and a specific product ion: 2-DOS, 163 > 102; 2'-N- acetylparomamine, 366 > 163; paromamine, 324 > 163; and gentamicin A2, 456 > 163.
  • glycosyltransferases GntZ and GntD each function to diphosphate sugar substrates such as UDP-yV-acetyl-D- glucosamine (UDP-GlcNAc, GeneChem) and UDP4)- ⁇ ylose (CarboSource Services)
  • cell-free extracts of two mutant strains of Streptomyces venezuelae harboring pYJ498 and pYJ503 were prepared by glass-bead homogenization.
  • Mycelium (6 g wet weight) from each of the Streptomyces venezuelae mutant strains, cultivated in an R2YE medium and harvested by centrifugation followed by washing with 20ml of 0.1 M Tris-HCl (pH 7.6) twice, was resuspended in 20 mL of an extraction buffer (0.1 M Tris-HCl, 10 mM MgCl 2 , 6 mM 2-mercaptoethanol , 1 mM phenylmethylsulphonyl fluoride, pH 7.6). After mixing with 10 g of pre-cooled glass beads (150 to 212 ⁇ m in diameter, Sigma), the mycelium was disrupted by vigorous agitation using a Vortex mixer.
  • Disruptions for 30 sec were repeated 10 times in ice.
  • the glass beads were eliminated by low-speed centrifugation, and cellular debris was removed by centrifugation at 18,000Xg for 20 min.
  • the whole process of cell-free extract preparation carried out at 4°C to prevent protein denaturation.
  • About 15 mL of the resulting supernatant was used for in-vitro reaction experiments.
  • the glycosyltransfer reactions were initiated by adding activated sugar substrates to the cell-free extracts.
  • 0, 1, and 5mg of UDP-GIcNAc were added to the reactions to give final concentrations between 0 and 0.55 mM, respectively.
  • the present invention produces gentamicin A2 and precursors thereof, separation and purification of which are difficult using a conventional method, by combining various gentamicin biosynthetic genes into recombinant microorganisms and vectors as necessary via recognition of functions of gentamicin biosynthetic enzymes, and thus can be applied to development of novel aminoglycoside antibiotics.
  • sequence listing of the present invention relates to primers required for amplification of protein genes involved in biosynthesis of gentamicin A2 and precursors thereof.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Disclosed herein is a method of producing aminoglycoside antibiotics containing 2-deoxystrept amine, paromamine, 2'-N-acetyl-paromamine, or gentamicin A2 by the recombination of biosynthetic genes in a heterologous host.

Description

[DESCRIPTION]
[Invention Title]
A PRODUCING METHOD FOR GENTAMICIN A2 OR THE PRECURSORS AND RECOMBINANT MICROORGANISMS PRODUCING THE SAME
[Technical Field]
<i> The present invention relates to aminoglycoside-based antibiotics, specifically gentamicin, useful as a medication for treatment of infectious diseases. More particularly, the present invention relates to a method of producing gentamicin A2 via systematic recombination and expression of its biosynthetic genes obtained from Micromonospora echinospora or Micromonospora purpurea that is a native production organism or strain of gentamicin A2 in a heterologous host, specifically in Streptomyces venezuelae. The present invention also relates to precursors thereof.
[Background Art]
<2> Gentamicin is an anminoglycoside antibiotic complex produced by a Micromonospora species. Aminoglycosides act on the 3OS subunit of the bacterial ribosome, hindering the translation stage of protein synthesis to inhibit the growth of bacteria. Structurally, gentamicin is a 4, 6- disubstituted , aminoglycoside composed of 2-deoxystreptamine (2-DOS) to which purpurosamine and garosamine aminosugars are bonded at C-4 and C-6 positions, respectively.
[Chemistry Figure 1]
Figure imgf000004_0001
2-Deoxystreptamine
<3>
<4> [Table 1]
<5>
Figure imgf000004_0002
Micromonospora echinospora is a strain which produces gentamicin A, Al, A2, X2, G418, J1-20A, J1-20B, B, Bl, Cl, CIa, C2, C2b, and C2a. Commercially available gentamicin sulfate is a complex mixture of C-form gentamicin, i.e., Cl, CIa, C2, C2b, and C2a, containing a small amount of A-form gentamicin, i.e., A2 and Al. This is because complete separation and purification of various forms of gentamicins by a common purifying process is difficult since the respective gentamicin components have similar physical and chemical properties. There is no antibiotic formed of a single form of gentamicin except for isepamicin, a semi-synthetic antibiotic extracted from gentamicin. It is not easy to develop a Micromonospora echinospora strain that produces a single form of gentamicin. This is because gentamicin biosynthesis pathway is a considerably complicated process that related with a large number of genes involving main biosynthesis and various modifications in a producing strain. Gentamicin biosynthetic genes have been registered with US GenBank as Accession No. AY524043 (J. Unwin, S. Standage, D. Alexander, T. Hosted Jr, AX. Horan, E.M. Wellington, /. Antibiot. 2004, 57, 436-445.) and No. AJ575934 (M.K. Kharel, D.B. Basnet, HX. Lee, K. Liou, Y.H. Moon, J.J. Kim, J.S. Woo, J.K. Sohng, MoI. Cells 2004, 18, 71-78). However, detailed functions of gentamicin biosynthetic genes have yet to be elucidated via genetic modification due to complication in genetic modification of Micromonospora echinospora. The biosynthetic process of gentamicin has only been estimated by adding and fermenting a precursor to a mutant obtained from random mutation (R.T. Testa, B.C. Til ley, J. Antibiot. 1976, 29, 140-146; R.T. Testa, BX. Tilley, Jpn. J. Antibiot. 1979, 32 Suppl, S47-S59; H. Kase, Y. Odakura, K. Nakayama, J. Antibiot. 1982, 35, 1-9.). Or, it has been inferred from the functions of proteins elucidated in the biosynthesis of kanamycin, butyrosin, etc., which are similar aminoglycosides to gentamicin(M. K. Kharel, B. Subba, D. B. Basnet, J.S. Woo, HX. Lee, K. Liou, J.K. Sohng, Arch. Biochem. Biophys. 2004, 429, 204-214. N. M. Llewellyn, J. B. Spencer, Nat. Prod. Rep. 2006, 23, 864- 874).
[Disclosure]
[Technical Problem]
<6> An object of the present invention is to provide a method of producing gentamicin and precursors thereof in a heterologous host by recombining gentamicin biosynthetic genes suitable for expression in the heterologous host, and a recombinant microorganism of the same.
[Technical Solution] <7> The present invention provides a biosynthetic method for gentamicin and precursors thereof through systematic recombination and introduction of gentamicin biosynthetic genes to Streptomyces venezuelae, a non- aminoglycoside producing actinobacteria. The present invention elucidates genes necessary for biosynthesis of gentamicin A2, 2-deoxystreptamine (2- DOS), 2'-/V-acetyl-paromamine and paromamine, which are crucial antibiotics and precursors for the biosynthesis of gentamicin, and the biosynthetic mechanism thereof, and therefore systematically dissolve and introduce the genes into a heterologous host, thereby produce gentamicin A2, 2-DOS, and paromamine. Further, the present invention provides a method of producing aminoglycoside antibiotics, and more particularly, a method of producing an antibiotic compound in a heterologous host of gentamicin.
[Advantageous Effects]
<9> The present invention provides direct evidence for the biosynthesis of 4,6-disubstituted aminoglycoside antibiotics and allows for the assignment of functions of each gene through an in-vivo study of the expression of gentamicin biosynthetic genes in a heterologous host. Actually, gentamicin and precursors thereof were produced in the heterologous host. In addition, the results of glycosyltransferases GntZ and GntD to substrates in the present invention enable development of novel aminoglycosides using the same. [Description of Drawings]
<io> Fig. 1 shows LC/ESI-MS/MS chromatograms of culture fluids of Streptomyces venezuelae mutant strains transformed with plasmids (a): pYJ495 expressing gntF, gntA, gntB, and gntP, (b): pYJ497 expressing gntF, gntB, and gntF, (c): pYJ496 expressing gntA, gntB, and gntP\ (d): pYJ498 expressing gntA, gntB, gntP, and gntZ', (e): pYJ501 expressing gntA, gntB, gntP, gntZ, kacA', and (f): pYJ503 expressing gntA, gntB, gntP, gntZ, kacA, and gntD, in which pYJ501 and 503 co-express the resistance genes grmA, grmO, and gntO.
<π> Fig. 2 shows HPLC-ESI-MS chromatograms of gentamicin A2 (4 in Fig. 5) and 2'-7V-acetyl-paromamine (2 in Fig. 5) by the glycosyltransferase.
<i2> (a), (b), (c): GntZ substrate reaction product using cell-free extracts from 5. venezuelae transformed with pYJ498
<i3> (a): without UDP-N-acetyl-D-glucosamine (UDP-GIcNAc)
<i4> (b): with addition of 0.11 mM UDP-GIcNAc
<i5> (c): with addition of 0.55 mM UDP-GIcNAc
<16> (d), (e), (f): GntD substrate reaction product using cell-free extracts from 5. venezuelae transformed with pYJ503
<i7> (d): without UDP-D-χylose (UDP-XyD
<i8> (e): with addition of 0.12 mM UDP-XyI
<19> (f): with addition of 0.60 mM UDP-XyI.
<20> Fig. 3 shows HPLC-ESI-MS chromatograms, in which <2i> (a): 2-deoxystreptamine (1 in Fig. 5)
<22> (b) : 2'-jV-acetylparomamine (2 in Fig. 5)
<23> (c): paromamine (3 in Fig.5)
<24> (d): gentamicin A2 (4 in Fig. 5)
<25> (e-h): mass spectra of (a), (b), (c), and (d)
<26> Fig. 4 is H NMR spectra of
<27> (a): standard 2-deoxystreptamine!
<28> (b): 2'-vV-acetylparomamine produced from S. venezuelae YJ003 transformed with pYJ498;
<29> (c): paromamine produced from paromomycin; and
<30> (d): gentamicin A2 produced from wild-type Micromonospora echinospora.
<3i> Fig. 5 is a biosynthetic pathway of gentamicin. [Best Mode]
<32> In accordance with an aspect of the present invention, the above and other object of the present invention can be accomplished by providing an effective producing method of gentamicin and precursors thereof by estimating a biosynthetic process of gentamicin.
<33> In accordance with another aspect of the present invention, a recombinant microorganism producing at least one selected from the group consisting of gentamicin, paromamine, 2'-vV-acetyl-paromamine and 2- deoxystreptamine is formed by recombining a heterologous host that do not biosynthesize gentamicin with genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neolβ.
<34> In accordance with a further aspect of the present invention, a vector producing at least one selected from the group consisting of gentamicin, paromamine, 2'-/V-acetyl-paromamine, and 2-deoxystreptamine, which includes gentamicin biosynthetic genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neolβ.
<35> The recombinant microorganism or the vector may further include a gentamicin resistance gene. <36> The heterologous host may comprise a Streptomyces species.
<37> In accordance with another aspect of the present invention, a producing method for paromamine from 2'-./V-acetyl-paromamine comprises expressing genes btrD, kacA, and neolβ in a heterologous host that do not biosynthesize gentamicin.
<38> In accordance with yet another aspect of the present invention, a producing method for at least one selected from the group consisting of gentamicin, paromamine, 2'-yV-acetyl-paromamine and 2-deoxystreptamine comprises recombining a heterologous host that do not biosynthesize gentamicin with genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neolβ.
<39> The method may further comprise adding at least one of a gene gntD and an enzyme GntD and UDP-D-χylose to produce a large amount of gentamicin A2.
<40> In accordance with yet another aspect of the present invention, a 2- deoxystreptamine producing recombinant microorganism is formed by recombining a heterologous host that do not biosynthesize gentamicin with genes gntB, gntA, and gntP.
<4i> In accordance with yet another aspect of the present invention, a 2- deoxystreptamine producing vector comprises gentamicin biosynthetic genes gntB, gntA, and gntP.
<42> In accordance with yet another aspect of the present invention, a 2'-jV-acetyl-paromamine producing recombinant microorganism forms by recombining a heterologous host which do not biosynthesize gentamicin with genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO.
<43> In accordance with yet another aspect of the present invention, a 2'-vV-acetyl-paromamine producing vector comprises gentamicin biosynthetic genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO.
<44> In accordance with yet another aspect of the present invention, a paromamine producing recombinant microorganism forms by recombining a non- gentamicin biosynthesizing heterologous host with genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO and at least one selected from the group O
consisting of btrD, kacA, and neolβ.
<45> In accordance with yet another aspect of the present invention, a paromamine producing vector comprises gentamicin biosynthetic genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO and at least one selected from the group consisting of btrD, kacA, and neolβ.
<46> Next, exemplary embodiments of the present invention will be described in detail.
<47> Herein, the term "gentamicin precursor" refers to 2- deoxystreptamine (hereinafter, also referred to as "2-DOS"), 2'-yV-acetyl- D-paromamine, and/or paromamine. The term "gentamicin biosynthetic genes" refers to biosynthetic genes separated from a gentamicin biosynthesizing strain of Micromonospora echinospora, which is already known in the art and is obtained from pGENOl (GenBank Accession No. AJ575934 (M.K. Kharel, D.B. Basnet, HX. Lee, K. Liou, Y.H. Moon, J.J. Kim, J.S. Woo, J.K. Sohng, MoI. Cells 2004, 18, 71-78)). The term "heterlogous host" refers to strains of microorganisms other than Micromonospora echinospora that is a native gentamicin producing strain, specifically to Streptomyces venezuelae and the like that are non-aminoglycoside producing strains.
<48> Genes were amplified from pGENOl using a DNA polymerase chain reaction (PCR) enzyme. A primer including an appropriate restriction enzyme for each gene was used. The amplified genes were properly introduced to pSE34 plasmid (N. Smirnova, K. A. Reynolds, J. Bacteriol. 2001, 183, 2335-2342.) to be controlled by an ermE promoter which is a strong actinomycetal promoter. Expression vectors were prepared as in Table 2. The production method will be described in detail in the examples.
<49> [Table 2] <50>
Figure imgf000011_0001
The prepared genes were introduced to Streptomyces venezuelae YJ003 (J. S. J. Hong, S. H. Park, C. Y. Choi, J. K. Sohng, Y. J. Yoon, FEMS Microbiol. Lett. 2004, 238, 391-399). The Streptomyces venezuelae YJ003, into which the constructed vectors has been inserted, was cultured in an R2YE medium at 30°C for 3 ~ 4 days to analyze metabolites. Analytical HPLC-ESI-MS was carried out for metabolite analysis. For more accurate analysis, preparative HPLC was carried out to fractionize the product. The fractions were analyzed by H, C, 2D H-H COSY NMR spectrum to identify products.
<51> The inventors suggested that an early stage of gentamicin biosynthesis begins from D-glucose-6-phosphate (Glc-6-P) modified to 2- deoxy-scy/Zo-inosose (2-DOI) by GntB enzyme (Korean Patent Registration No. 10-0470743) through carbocyclization. Two aminotransferase reactions and a single dehydrogenase reaction were then required to synthesize 2- deoxy-scy/Arinosamine (2-DOIA) and l-keto-2-deoxy-scj7/o-inosamine (keto- 2-DOIA) (M.K. Kharel, D. B. Basnet, HX. Lee, K. Liou, Y.H. Moon, J.J. Kim, J. S. Woo, J.K. Sohng, MoI. Cells 2004, 18, 71-78; J. Unwin, S. Standage, D. Alexander, T. Hosted Jr, AX. Horan, E. M. Wellington, J. Antibiot.2004, 57, 436-445.) (Fig.5).
<52> <53> The inventors tried to express the genes gntF, A, B, and C in a heterologous host to produce 2-DOS, in which gntF, A, B, and C are presumed to encode an inosose synthase (GntB), two putative aminotransferases (GntA and GntF), and a dehydrogenase (GntC) as included in pYJ494. However, 2-DOS was not detected in the transformants where ρYJ494 was expressed. Llewellyn and Spencer predicted that a 2-DOIA dehydrogenase involved in the 2-DOS biosynthesis would be GntP having an amino acid sequence homology with Neo5 required for neomycin biosynthesis (N.M. Llewellyn, J.B. Spencer, Nat. Prod. Rep. 2006, 23, 864-874). pYJ495 with gntP having replaced gntC was transformed to express gntF, A, B, and P in a heterologous host. Extracts from the pYJ495 transformants were analyzed by HPLC-ESI-MS, thereby successfully detecting 2-DOS with an m/z of 163 at a retention time of 61.4 minutes (Fig. Ia).
<54> A study on the biosynthesis of butirosin produced from Bacillus circulans reported that an enzyme, BtrS, was involved in two transamination reactions for biosyntheses of 2-DOS (K. Yokoyama, F. Kudo, M. Kuwahara, K. Inomata, H. Tamegai , T. Eguchi , K. Kakinuma, J. Am. Chem. Soc. 2005, 127, 5869-5874). Considering that 2-DOS involved in the gentamicin biosynthesis would be a similar case, the inventors of the present invention prepared pYJ496 containing gntA, B, and P and pYJ497 containing gntF, B, and P. As an examination result of the transformants where the foregoing genes had been expressed, the expression of GntA, GntB, and GntP produced 2-DOS (Fig. Ic), whereas the expression of GntF, GntB, and GntP did not produce 2-DOS (Fig. Ib). Accordingly, it is presumed that a single transaminase of GntA acted in both steps of transamination for the biosynthesis of 2-DOS (Fig. 5) and GntF was involved in transamination at a subsequent stage to the 2-DOS biosynthesis in the biosynthesis of gentamicin.
<55> A prerequisite for biosynthesis of gentamicin in a heterologous host is to impart gentamicin resistance to the heterologous host. In order to ensure the resistance of the host to gentamicin, pYJ489 {grmA, 0 and gntO) was introduced into the heterologous host. The genes grwA and grmO encode bacterial ribosome methylases, and the gntO gene is assumed to be a transporter of aminoglycoside to impart resistance to the heterologous host without structurally modifying antibiotic expression. As a result of analysis, transformants harbouring pYJ489 were resistant up to 100 μg/ml of added gentamicin whereas the growth of non-transformants was completely inhibited. The added gentamicin was recollected for analysis, so that gentamicin was changed in neither structure nor activity due to the introduction of the genes.
<56> Two glycosylation reactions are required for gentamicin biosynthesis. It is presumed that nucleotidyl diphosphate (NDP) glucosamine is added to 2-DOS to form paromamine as a pseudodisaccharide intermediate, and NDP-D-χylose is subsequently added thereto to form gentamicin A2 (Fig. 5). In the cluster of gentamicin biosynthetic genes, gntD and gntZ have been identified to encode putative glycosyltransferases. In order to identify the roles of two glycosyltransferases, GntD and GntZ, gntZ was combined with 2-DOS biosynthetic genes, gntA, B, and P, and resistance genes, grmA, 0 and gntO, to prepare pYJ498. The transformants of pYJ498 did not produce paromamine but synthesized 2'-yV-acetyl-D-paromamine as was detected by HPLC-ESI-MS at 59.8 mimutes with an m/z 366 (Fig. Id). The transformants of pYJ499 in which gntZ was replaced by gntD produce neither 2'-jV-acetyl- D-paromamine nor paromamine. Therefore, GntZ seems to be responsible for accepting NDP-N-acetyl-D-glucosamine (NDP-GIcNAc) rather than NDP-D- glucosamine (NDP-GIcN) as a substrate for attachment to 2-DOS. To confirm that NDP-GIcNAc is the substrate, a cell-free extract of the transformants harbouring pYJ498 was prepared and incubated with different concentrations of exogeneous uridine diphospho (UDP)-GIcNAc. Then, incorporation of UDP- GIcNAc into 2-DOS was verified as the amount of 2'-yV-acetyl-D-paromamine increased (Fig. 2a, b, c). A putative glycosyltransferase using UDP-GIcNAc as a substrate is BtrM from the cluster of butirosin biosynthetic genes, and its homologues of Neo8, KanF, and GntZ from neomycin, kanamycin, and gentamicin biosynthetic gene clusters, respectively, are suggested to use UDP-GIcNAc as a substrate (N. M. Llewellyn, J. B. Spencer, Nat. Prod. Rep. 2006, 23, 864-874). Although these enzymes have yet to be functionally characterized using purified enzymes, the present invention provides significant evidence that GntZ is a UDP-GIcNAc glycosyltransferase. In the biosynthesis of butirosin, Truman et al . have recently shown that BtrD, a paromamine deacetylase, eliminates an acetyl group from 2'-./^-acetyl-D- paromamine to produce D-paromamine (A. W. Truman, F. Huang, N. M. Llewellyn, J. B. Spencer, Angew. Chem. Int. Ed. 2007, 46, 1462-1464). Since no BtrD homologue was found in the gentamicin biosynthetic cluster, the present invention imported btrD, kacA, and neolβ from Bacillus circulans, Streptomyces kanamyceticus, and Streptomyces fradiae, respectively. Then, btrD, kacA and neolβ was each combined with separate constructs containing 2-DOS biosynthetic genes, glycosyltransferase, and resistance genes to produce plasmids pYJ500, pYJδOl, and pYJ502, respectively. Expression of all three plasmids in a heterologous host led to the production of paromamine with an m/z 324 at a retention time of 74.8 minutes, along with 2'-jV-acetyl-D-paromamine. Both of transformants of kacA and neolβ genes produced about 150 μg/L of paromamine, whereas a strain expressing btrD yielded a slightly lower amount of paromamine, possibly due to the fact that BtrD is derived from Bascillus while KacA and Neol6 are derived from Streptomyces. The in-vivo results of KacA and Neolδ verify the report of Truman et al. (A. W. Truman, F. Huang, N. M. Llewellyn, J. B. Spencer, Angew. Chem. Int. Ed. 2007, 46, 1462-1464). In a previous report, 2'-;V-acetyl-D-paromamine was synthesized in-vitro using a 2'-yV-acetyltransferase from Providencia stuartii and used to verify the deacetylation activity of six histidine-tagged BtrD. In the present invention, meanwhile, paromamine and 2'-yV-acetyl-D-paromamine were identified in-vivo, thereby verifying that 2'-/¥-acetyl-D-paromamine is a direct precursor of aminoglycoside antibiotics such as gentamicin. <57> The second glycosylation by NDP-D-χylose is required for the formation of gentamicin A2 from paromamine. The combination of gntD and a paromamine biosynthetic gene prepared pYJ503. The transformant of pYJ503 produced gentamicin A2 at a retention time of 73.3 minutes with an m/z 456, together with paromamine and 2'-jV-acetyl-D-paromamine (Fig. If). Therefore, GntD is a glycosyltransferase to transfer NDP-D-χylose to paromamine. A large amount of paromamine remained and the yield of gentamicin A2 was only 4.5 βg/L, for which the heterlogous host had a limiting amount of NDP-D-χylose. To further confirm that NDP-D-χylose was a substrate actually incorporated into paromamine to produce gentamicin A2, a cell-free extract of the heterlogous host harbouring pYJ503 was prepared and incubated with different concentrations of exogeneous UDP-D- xylose. It was observed that the amount of gentamicin A2 gradually increases as the exogenous UDP-D-χylose increases in concentration (Fig. 2d, 2e, 2f). In this regard, it was confirmed that UDP-D-χylose incorporated into gentamicin A2.
<58> In conclusion, the present invention completed gentamicin A2 and the minimal gene set involved in the formation of precursors thereof.
<59> 2-DOS is synthesized by enzymes encoded by gntA, gntB and gntP. The gntZ gene that encodes a glycosyltransferase, actually functions in transferring N-acetyl-D-glucosamine to produce 2'-/V-acetyl-D-paromamine. A gentamicin biosynthetic gene which encodes a deacetylase, modify 2'-N- acetyl-D-paromamine into D-paromamine still remains to be found, while BtrD of a deacetylase and its homologues KacA and Neolβ were able to carry out its function instead. GntD having an activity of NDP-xylose transferase is necessary to produce gentamicin A2. The clarified functions of the genes are listed in Table 3.
<60> [Table 3] <61>
Figure imgf000016_0001
[Mode for Invention]
<62> Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to the examples disclosed below but can be implemented in various types.
<63> <64> <Example 1> Cloning of gentamicin biosynthetic gene from Micromonospora echinospora
<65> Escherichia coli (E. coli) DHlOB and plasmid Litmus28 (New England Biolabs) were used for subcloning. The E. coli was cultured in an LB medium to which 50 μg/ml of ampicillin was added for selection of plasmids. Primers for gene amplification containing proper restriction sites shown in Table 4 were employed for corresponding genes. Genes were amplified from cosmid pGENOl cloned from Micromonospora echinospora using grmA-F and grmA-R for amplification of grwA, grm0-F and grm0-R for grmO, gntO-F and gntO-R for gntO, gntA-F and gntA-R for gntA, gntB-F and gntB-R for gntB, gntC-F and gntC-R for gntC, gntF-F and gntF-R for gntF, gntD-F and gntD-R for gntB, and gntZ-F and gntZ-R for gntZ. Gene btrD was amplified from Bacillus circulans genomic DNA using btrDHF and btrD-R. Gene kacA was amplified from Streptomyces kanamyceticus genomic DNA using kacA-F and kacA-R. Gene neolβ was amplified from Streptomyces fradiae genomic DNA using neol6-F and neol6-R. The gene amplification was carried out according to general gene amplification enzyme methods. The amplified genes were introduced into appropriate restriction sites of plasmid Litmus 28 and analyzed to confirm that it has the same nucleotide sequence as the native genes. In the present invention, Streptomyces and pSE34 of an E. coli shuttle vector were basically used for expressing the genes. It should be noted that the configuration of the genes is crucial for the present invention, and that vectors, E. coli, primers, amplification enzymes and/or amplification conditions in examples can be easily modified or changed by a person having ordinary knowledge in the art to which the invention pertains, and do not limit the present invention. <66> [Table 4]
<67>
<68>
<89>
<73>
<7B>
<SΘ>
<82>
<8β>
<88>
<91B>
<9I>
<9β>
<*99>
<103>
<108>
Figure imgf000018_0001
<108> <Example 2> Resistance gene expressing plasmid vector <109> The resistance genes grmA, gntO and grmO were employed to confer gentamicin resistance to Streptomyces venezuelae. The PCR-amplified gntO and grmO genes were digested with Sbfl/Hindlll and SbΔ/BstBl, respectively, and introduced to Litmus 28 at the same time. The PCR- amplified grmA was introduced to Ncol/Xbal restriction site. The gntO-grmO gene was digested with SnaBl/fl/ndlll, and the grmO gene was digested with Xbal/SnaBl. The two digested fragments were simultaneously cloned to pSE34 digested with Xbal/HindiU, thereby preparing pYJ489. pYJ489 {grmA-gntO- grmO) was inoculated to obtain resistance to an antibiotic used for production of gentamicin in a heterologous host. It is believed that the grmA and grmO genes encoded methylases of ribosomes of bacteria and the gntO gene product was a putative aminoglycoside transporter imparting resistance to the antibiotic without structurally modifying the antibiotic to be expressed. As a result of analysis, transformants harbouring ρYJ489 were resistant up to 100 μg/ml of added gentamicin whereas growth of non- transformants was completely inhibited. Furthermore, it was not observed that the introduction of the genes (antibiotic resistance genes) changed gentamicin in either structure or activity.
<Example 3> Preparation of expression vector for production of 2-DOS To express 2-DOS biosynthetic genes, the gntF, gntA, gntB, gntC, and gntP genes, which had been PCR-amp1 ified and introduced to Litmus 28, were digested with appropriate restriction enzymes to construct a set of genes including gntFABC, gntFABP, gntABP, and gntFBP in Litmus 28. Plasmids harbouring the set were digested with BglW/Xbal and introduced into pSE34 digested with the same enzyme, thereby preparing pYJ494, ρYJ495, pYJ496, and pYJ497. As a result of expression of these plasmids, transformants of pYJ495 and pYJ496 produced 2-DOS.
<Example 4> Preparation of plasmid vector for simultaneous expression of 2-DOS biosynthetic genes, resistance genes, and glycosyltransferase genes
The genes grmA and gntZ were digested with appropriate restriction enzymes and simultaneously cloned into Litmus 28. Then, the grmA-gntZ and gntO-grmO genes were each digested with Xbal/SnaBl and SnaBl/ffϊndlll and o
were ligated into Xbal/Hindlll digested pYJ496, produced in Example 3, to produce pYJ498.
The genes grmA and gntD were digested with appropriate restriction enzymes and cloned into Litmus 28. Then, the grmA-gntD and gntO-grmO genes were each digested with Xbal/SnaBl and SnaBl/Hindlll and were ligated into the Xbal/Hindlll digested pYJ496, produced in Example 3, to produce pYJ499.
<Example 5> Preparation of plasmid vector for simultaneous expression of deacetylase gene-incorporated 2-DOS biosynthetic genes, resistance genes, and glycosyltransferase genes
A deacetylase gene brtD was amplified from a Bacillus circulans genome. A SnaBl/BstBl digested brtD gene, the BstBl/Hindlll digested gntO- grmO fragment, and the Xbal/SnaBl digested grmA-gntD fragment were incorporated into the Xbal/SnaBl digested pYJ496, produced in Example 3, thereby producing pYJ500.
A deacetylase gene kacA was amplified from a Streptomyces kanamyceticus genome. A SnaBl/BstBl digested kacA gene, the BstBl/Hindi11 digested gntO-grmO fragment, and the Xbal/SnaBl digested grmA-gntD fragment were incorporated into the Xbal/SnaBl digested pYJ496, produced in Example 3, thereby producing ρYJ501.
A deacetylase gene neolβ was amplified from a Streptomyces fradiae genome. A SnaBl/BstBl digested neolβ, the BstBl/Hindlll digested gntO-grmO fragment, and the Xbal/SnaBl digested grmA-gntD fragment were incorporated into the Xbal/SnaBl digested pYJ496, produced in Example 3, thereby producing pYJ502.
<Example 6> Preparation of plasmid vector for production of gentamicin A2
For the production of gentamicin A2, the gntD gene encoding a glycosyltransferase was introduced into pYJ501, produced in Example 5, to produce pYJ503. The gntD gene was digested with SbΔ/Spel and incorporated into the Litmus 28 containing the grmA-grntZ. Then, the grmA-gntZ-gntD gene was digested with Xbal/SnaBl and incorporated into the Xbal/Hindlll digested pYJ496, produced from the SnaBl/Hindlll digested pYJ501 in Example 4, thereby producing pYJ503.
<Example 7> Introduction of expression vector into streptomyces venezuelae and cultivation
Streptomyces venezuelae YJ003 is a mutant strain which lacks TDP-D- desosamine biosynthetic ability (J.S.J. Hong, S.H. Park, CY. Choi, J.K. Sohng, Y.J. Yoon, FEMS Microbiol. Lett. 2004, 238, 391-399). Preparations of spores and protoplasts, introduction of plasmids into protoplasts, and selection of plasmids were conducted by methods proposed in practical streptomyces genetics (T. Kaieser, M. J. Bibb, M. J. Buttner, K. F. Chater, D. A. Hopwood, John Innes Centre, Norwich, UK, 2000). That is, Streptomyces venezuelae was cultured in liquid R2YE to prepare protoplasts. Then, pSE34, pYJ489 of Example 2, pYJ494, pYJ495, pYJ496, and pYJ497 of Example 3, pYJ498 and pYJ499 of Example 4, pYJ500, pYJ501, and pYJ502 of Example 5, and pYJ503 of Example 6 were introduced into the protoplasts, respectively. Regeneration of the protoplasts and selection of the plasmids were carried out in agar containing 30 μg/mL thiostrepton. To identify the production of gentamicin and gentamicin derivatives, the plasmids of Streptomyces venezuelae were cultivated at 30°C for 3 to 4 days in a 1 L flask containing 300 ml of an R2YE medium supplemented with 30 μg/mL thiostrepton.
<Example 8> Separation of products from transformants and analysis Analytical HPLC-ESI-MS was performed on a Waters/Micromass Quattro micro/W) interface using two XTerra MS Ci8 (3.5 μsα, Waters) columns, 150mm
X2.1mm and 250mmx2.1 mm, connected in tandem. The analytes were eluted at a flow rate of 60 μl/min with a 30% acetonitrile aqueous solution with cAJ
10 mM heptafluorobutyric acid (HFBA, Fluka), and monitored by ESI in the positive mode.
<Example 9> Fractionization and analysis of gentaniicin precursors
General experimental procedures
Preparative HPLC was performed with a Spherisorb S5 0DS2 (250mmx20 mm, Waters) semi-prep column using the same solvent as employed in the elution of Example 8 at a flow rate of 12 mL/min for 100 minutes. This eluent was fractionized into 5 mL fractions, which in turn were monitored by analytical HPLC-ESI-MS, as described in Example 8, to confirm the presence of gentamicin precursors. The fractions containing the desired product were pooled and extracted by Solid-Phase Extraction (SPE) with
OASIS MCX (Waters) to eliminate impurities, followed by freeze-drying. H,
13C, and 2D 1H-1H COSY NMR spectra were acquired with a Varian INOVA 500 spectrometer at 298 K. Chemical shifts were reported in ppm by comparison with 3-(trimethylsilyl)-l-propane sulfonic acid (TSP) as an internal reference. The assignment of each compound was carried out by comparison with the previously assigned H NMR spectra (T. L. Nagabhushan, W. N. Turner, P. J. L. Daniels, J. B. Morton, J. Org. Chem.1975, 40, 2830-2834; T. L. Nagabhushan, P. J. L. Daniels, R. S. Jaret, J. B. Morton, J. Org. Chem. 1975, 40, 2835-2836; A. W. Truman, F. Huang, N. M. Llewellyn, J. B. Spencer, Angew. Chem. Int. Ed. 2007, 46, 1462-1464.) and by a combination of ID and 2D NMR experiments. All NMR data processing was done with MESTREC software.
2-deoxystreptamine
The retention time of 2-deoxystreptamine (2-DOS) was 61.2 minutes (Fig. 3a) and the MS/MS fragmentation pattern is shown in Fig. 3e. Based on comparisons of the retention time and the MS/MS spectra with those of the 2-DOS standards, a compound produced by plasmids pYJ495 and pYJ496 expressing gntA-gntB-gntP genes was found to be 2-DOS; H NMR (500 MHz, D2O): δ 1.72 (2H, dt, /= 9.6, 6.0 Hz, 2-H2), 2.59 (2H, q, J= 10.2 Hz, 1- H, 3-H), 3.31 (IH, t, J= 10.0 Hz, 5-H), 3.39 (2H, t, J = 9.6 Hz, 4-H, 6- H); 13C NMR (100 MHz, D2O): δ 37.4, 53.0, 53.3, 74.6, 79.6, 79.9; HRMS
(ESI) m/z 163.1882 [M+H]+ (calculated for C6Hi4N2O3, 163.1870) (Fig.4a ).
2'-jV-acetylparomamine
2'-yV-acetylparomamine was isolated from the Streptomyces venezuelae mutant strain harbouring pYJ498. The product was a pale yellow solid and had a retention time of 59.8 minutes in the HPLC analysis (Fig. 3b). The MS/MS fragmentation pattern is shown in Fig. 3f. By comparing the assigned NMR data (A. W. Truman, F. Huang, N. M. Llewellyn, J. B. Spencer, Angew. Chem. Int. Ed. 2007, 46, 1462-1464) and the results of MS/MS spectra of various gentamicin precursors from previous studies, a compound produced by plasmid pYJ498 harboring gntA, B, P and Z genes was identified as 2'-/V- acetyl-paromamine; 1H NMR (500 MHz, D2O): δ 1.75 (2H, dt , J= 12.0, 5.1 z, 2-H2), 2.03 (3H, s, 7' -H3), 2.65 (IH, q, J = 13.1 Hz, 1-H), 2.88 (IH, q, J
= 12.1 Hz, 3-H), 3.11 (IH, t, J= 8.6 Hz, 4-H), 3.40 (IH, t, J= 9.3 Hz, 4 ' -H), 3.48 (IH, t, J= 9.2 Hz, 6-H), 3.55 (IH, t, J= 9.3 Hz, 5-H), 3.73 (IH, m, 5' -H), 3.79 (2H, m, 6' -H2), 4.03 (IH, t, J= 9.7 Hz, 3' -H), 4.12
(IH, t, J = 9.6 Hz, 2' -H), 5.56 (IH, d, J= 3.3 Hz, 1' -H); 13C NMR (100 MHz, D2O): δ 23.3, 35.4, 49.3, 51.4, 57.1, 62.2, 71.3, 72.5, 72.7, 77.4,
78. 1 , 86.0 , 99. 1 , 170.4; HRMS (ESI ) m/z 366. 1807 [M+H]+(calculated for Ci4H27N3O8 , 366. 1795) (Fig. 4b) .
Paromamine
Paromamine was obtained by acid hydrolysis of reference paromomycin (Sigma), titration of the hydrolysate to pH 6.0, purification with an SPE cartridge, and freeze-drying according to a method proposed by a previous study (T. H. Haskell, J. C. French, Q. R. Bartz, /. Am. Chem. Soc. 1959, 81, 3480-3481). The retention time of paromamine from the HPLC analysis was 74.8 minutes (Fig. 3c) and the MS/MS fragmentation pattern is shown in Fig. 3g. A compound produced by plasmid pYJ501 harbouring plasmid with gntA, B, P, Zand kacA genes was paromamine; H NMR (500 MHz, D2O): δ 1.74
(2H, dt, J = 12.5, 5.2 Hz, 2-H2), 2.60 (IH, q, J = 13.1 Hz, 1-H), 2.85 (IH, q, J = 12.1 Hz, 3-H), 3.10 ? 3.13 (2H, m, 2' -H, 4-H), 3.42 (IH, t, J = 9.6 Hz, 4' -H), 3.45 (IH, t, J= 9.3 Hz, 6-H), 3.57 (IH, t, J= 9.4 Hz, 5- H), 3.63 (IH, t, J= 9.6 Hz, 3' -H), 3.74 (IH, m, 5' -H), 3.78 (2H, m, 6'
-H2), 5.14 (IH, d, J= 3.6 Hz, 1' -H); 13C NMR (100 MHz, D2O): δ 35.5, 49.1,
51.2 , 55.3 , 61.2 , 72.4, 72.7, 74.2 , 77.4, 78.2 , 86.3 , 102.2 ; HRMS (ESI ) m/z 324.3440 [M+H]+ (calculated for Ci2H25N3O7, 324.3428) (Fig. 4c) .
Gentamicin A2
Gentamicin A2 was isolated and purified from the extract of wild- type Micromonospora echinospora ATCC 15835 by preparative HPLC. The retention time of gentamicin A2 by the HPLC analysis was 73.3 minutes (Fig. 3d), and the purified gentamicin A2 was a pale yellow solid. The MS/MS fragmentation pattern shown in Fig. 3h is confirmed by the NMR data acquired in earlier studies. A compound produced from the mutant strain of Streptomyces venezuelae harbouring pYJ503 was identified to be gentamicin
A2; 1H NMR (500 MHz, D2O): δ 1.74 (2H, dt, J= 12.3, 4.2 Hz, 2-H2), 2.83
(2H, q, J = 12.8 Hz, 1-H, 3-H), 3.10 - 3.14 (3H, dt , J= 10.3, 5.7 Hz, 4-H, 6-H, 2' -H) 3.38 - 3.42 (2H, m, 4' -H, 4"-H), 3.49 (IH, t, J= 8.2 Hz, 3"- H), 3.64 (IH, t, J= 9.1 Hz, 3' -H), 3.73 (IH, t, J= 9.1 Hz, 2"-H), 3.76 (IH, m, 5' -H), 3.79 (2H, m, 6' -H2), 3.81 (IH, t, J= 6.2 Hz, 5-H), 3.88 (2H , d, J = 3.6 Hz , 5"-H2) , 5.03 (IH, d, J = 3.7 Hz , 1"-H) , 5.18 ( IH, d, J=
3.8 Hz , 1' -H) ; 13C NMR (100 MHz , D2O) : 5 35.5, 49.2 , 49.5, 54.8 , 61.1 , 66.2 , 70.3 , 72.3 , 73.0 , 73.6 , 74.0 , 75.8 , 77.4, 86.2 , 86.4 , 102.4, 104.1 , ; HRMS (ESI ) m/z 456.1123 [M+H]+ (calculated for Ci7H33N3On, 456. 1115) (Fig.
4d) .
<Example 10> Extraction and analysis of gentamicin biosynthetic precursors
Gentamicin biosynthetic precursors produced by Streptotnyces venezuelae mutant strains were extracted using an OASIS MCX cartridge, as previously described, and were analyzed by HPLC-ESI-MS described in Example 9. The quantification of the analytes was conducted using a multiple reactions monitoring (MRM) mode. This was done by detecting the selected analytes with respect to a set of two mass ions, which are a parent ion and a specific product ion: 2-DOS, 163 > 102; 2'-N- acetylparomamine, 366 > 163; paromamine, 324 > 163; and gentamicin A2, 456 > 163.
<Example 11> In vitro glycosyltransfer reactions using cell-free extracts
In order to determine whether glycosyltransferases GntZ and GntD each function to diphosphate sugar substrates such as UDP-yV-acetyl-D- glucosamine (UDP-GlcNAc, GeneChem) and UDP4)-χylose (CarboSource Services), cell-free extracts of two mutant strains of Streptomyces venezuelae harboring pYJ498 and pYJ503 were prepared by glass-bead homogenization. Mycelium (6 g wet weight) from each of the Streptomyces venezuelae mutant strains, cultivated in an R2YE medium and harvested by centrifugation followed by washing with 20ml of 0.1 M Tris-HCl (pH 7.6) twice, was resuspended in 20 mL of an extraction buffer (0.1 M Tris-HCl, 10 mM MgCl2, 6 mM 2-mercaptoethanol , 1 mM phenylmethylsulphonyl fluoride, pH 7.6). After mixing with 10 g of pre-cooled glass beads (150 to 212 μm in diameter, Sigma), the mycelium was disrupted by vigorous agitation using a Vortex mixer. Disruptions for 30 sec were repeated 10 times in ice. The glass beads were eliminated by low-speed centrifugation, and cellular debris was removed by centrifugation at 18,000Xg for 20 min. The whole process of cell-free extract preparation carried out at 4°C to prevent protein denaturation. About 15 mL of the resulting supernatant was used for in-vitro reaction experiments. The glycosyltransfer reactions were initiated by adding activated sugar substrates to the cell-free extracts. In order to investigate the substrate specificty of GntZ, 0, 1, and 5mg of UDP-GIcNAc were added to the reactions to give final concentrations between 0 and 0.55 mM, respectively. For GntD, 0, 1, and 5 mg of UDP-D-χylose were added to the reactions to give final concentrations between 0 and 0.60 mM, respectively. The reaction mixtures were incubated at 30°C for 10 hours, and the reaction was quenched with 15 mL of ice-cold phenol/chloroform/isoamyl alcohol (25:24:1) to denature protein. The reaction solution was centrifuged to separate a water layer, and the remaining product was purified with OASIS MCX SPE. Finally, the gentamicin precursors were dissolved in 200 μi of water and subjected to HPLC-ESI-MS analysis. The foregoing experimental process was repeated twice.
[Industrial Applicability]
As described above, the present invention produces gentamicin A2 and precursors thereof, separation and purification of which are difficult using a conventional method, by combining various gentamicin biosynthetic genes into recombinant microorganisms and vectors as necessary via recognition of functions of gentamicin biosynthetic enzymes, and thus can be applied to development of novel aminoglycoside antibiotics.
[Sequence List Text] The sequence listing of the present invention relates to primers required for amplification of protein genes involved in biosynthesis of gentamicin A2 and precursors thereof.

Claims

[CLAIMS]
[Claim 1]
A recombinant microorganism for producing at least one material selected from the group consisting of gentamicin, paromamine, 2'-jV-acetyl- paromamine, and 2-deoxystreptamine, the recombinant microorganism being formed by recombining a non-gentamicin biosynthesizing heterologous host with genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neolβ.
[Claim 2]
A vector for producing at least one material selected from the group consisting of gentamicin, paromamine, 2'-/V-acetyl-paromamine, and 2- deoxystreptamine, the vector comprising gentamicin biosynthetic genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neolβ.
[Claim 3]
The recombinant microorganism according to claim 1, further comprising a gentamicin resistance gene.
[Claim 4]
The vector according to claim 2, further comprising a gentamicin resistance gene.
[Claim 5]
The recombinant microorganism according to claim 1, wherein the heterologous host comprises a Streptomyces species.
[Claim 6]
A method of producing paromamine from 2'-;V-acetyl-paromamine, comprising: expressing genes btrD, kacA, and neolβ in a non-gentamicin biosynthesizing heterologous host.
[Claim 7]
A method of producing at least one selected from the group consisting of gentamicin, paromamine, 2'-jV-acetyl-paromamine, and 2-deoxystreρtamine, the method comprising: recombining a non-gentamicin biosynthesizing heterologous host with genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neolβ.
[Claim 8]
<i64> The method according to claim 7, further comprising: adding at least one of a gene gntD or an enzyme GntD and UDP-D-xylose to produce a large amount of gentamicin A2.
[Claim 9]
<i65> A 2-deoxystreptamine producing recombinant microorganism formed by recombining a non-gentamicin biosynthesizing heterologous host with genes gntB, gntA, and gntP.
[Claim 10]
<i66> A 2-deoxystreptamine producing vector comprising: gentamicin biosynthetic genes gntB, gntA, and gntP.
[Claim 11]
<i67> A 2 ' -Λf-acetyl-paromamine producing recombinant microorgani sm formed by recombining a non-gent ami c in biosynthesizing heterologous host with genes gntB, gntA, gntP, gntZ, grwA, grmO, and gntO.
[Claim 12]
<168> A 2'-/V-acetyl-paromamine producing vector comprising gentamicin biosynthetic genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO.
<169>
[Claim 13]
<17O> A paromamine producing recombinant microorganism formed by recombining a non-gentamicin biosynthesizing heterologous host with genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO and at least one selected from the group consisting of btrD, kacA, and neolβ.
[Claim 14]
<i7i> A paromamine producing vector comprising gentamicin biosynthetic genes gntB, gntA, gntP, gntZ, grmA, grmO, and gntO and at least one selected from the group consisting of btrD, kacA, and neolβ.
PCT/KR2008/004033 2007-07-09 2008-07-09 A producing method for gentamicin a2 or the precursors and recombinant microorganisms producing the same WO2009008664A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2007-0068464 2007-07-09
KR1020070068464A KR20090005436A (en) 2007-07-09 2007-07-09 A producing method for gentamicin a2 and the precursors and a recombinant microorganism producing the substances

Publications (3)

Publication Number Publication Date
WO2009008664A2 true WO2009008664A2 (en) 2009-01-15
WO2009008664A3 WO2009008664A3 (en) 2009-03-19
WO2009008664A4 WO2009008664A4 (en) 2009-05-07

Family

ID=40229272

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2008/004033 WO2009008664A2 (en) 2007-07-09 2008-07-09 A producing method for gentamicin a2 or the precursors and recombinant microorganisms producing the same

Country Status (2)

Country Link
KR (1) KR20090005436A (en)
WO (1) WO2009008664A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110358718A (en) * 2019-07-19 2019-10-22 福州市鼓楼区荣德生物科技有限公司 The building and its application of main product Gentamicin C1a engineering bacteria
CN113403237A (en) * 2021-07-27 2021-09-17 青岛安惠仕生物制药有限公司 Gentamicin sulfate prepared by enhanced microbial fermentation and application method thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040026067A (en) * 2002-09-17 2004-03-27 주식회사 대웅 Nucleotide sequences and amino acid sequences of deoxy-scyllo-inosose synthase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040026067A (en) * 2002-09-17 2004-03-27 주식회사 대웅 Nucleotide sequences and amino acid sequences of deoxy-scyllo-inosose synthase

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JAMIE UNWIN ET AL.: 'Gene cluster in micromonospora echinospora ATCC15835 of the biosynthesis of the gentamicin C complex' THE JOURNAL OF ANTIBIOTICS vol. 57, no. 7, August 2004, pages 436 - 445 *
NICHOLAS ET AL.: 'Biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics' NATURAL PRODUCT REPORTS vol. 23, September 2006, pages 864 - 874 *
PNAISER ET AL.: 'EM17 the distribution of the gantamicin gene cluster in nature' SOCIETY FOR GENERAL MICROBIOLOGY-153RD MEETING-UMIST 2003, page 31 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110358718A (en) * 2019-07-19 2019-10-22 福州市鼓楼区荣德生物科技有限公司 The building and its application of main product Gentamicin C1a engineering bacteria
CN113403237A (en) * 2021-07-27 2021-09-17 青岛安惠仕生物制药有限公司 Gentamicin sulfate prepared by enhanced microbial fermentation and application method thereof
CN113403237B (en) * 2021-07-27 2021-12-28 青岛安惠仕生物制药有限公司 Gentamicin sulfate prepared by enhanced microbial fermentation and application method thereof

Also Published As

Publication number Publication date
KR20090005436A (en) 2009-01-14
WO2009008664A3 (en) 2009-03-19
WO2009008664A4 (en) 2009-05-07

Similar Documents

Publication Publication Date Title
Gross et al. Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads
US10337044B2 (en) Kanamycin compound, kanamycin-producing Streptomyces species bacterium, and method of producing kanamycin
Lombo et al. Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin
Koběrská et al. Sequence analysis and heterologous expression of the lincomycin biosynthetic cluster of the type strain Streptomyces lincolnensis ATCC 25466
EP2766389B1 (en) Gene cluster for biosynthesis of griselimycin and methylgriselimycin
Li et al. Mining of a streptothricin gene cluster from Streptomyces sp. TP-A0356 genome via heterologous expression
Kharel et al. Molecular cloning and characterization of a 2-deoxystreptamine biosynthetic gene cluster in gentamicin-producing Micromonospora echinospora ATCC15835
CN112111439A (en) Saccharopolyspora spinosa for high yield of spinosad and method for improving yield of spinosad
JP5422387B2 (en) Method for producing non-natural antibiotics
WO2009008664A2 (en) A producing method for gentamicin a2 or the precursors and recombinant microorganisms producing the same
WO2011145211A1 (en) Ketoreductase mutant
Han et al. Biosynthesis of glycosylated derivatives of tylosin in Streptomyces venezuelae
Lamichhane et al. Heterologous production of spectinomycin in Streptomyces venezuelae by exploiting the dTDP-D-desosamine pathway
Nepal et al. Heterologous production of paromamine in Streptomyces lividans TK24 using kanamycin biosynthetic genes from Streptomyces kanamyceticus ATCC12853
EP1443113B1 (en) Method of obtaining indolocarbazoles using biosynthetic rebeccamycin genes
Tornus et al. Identification of four genes from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22 involved in the biosynthesis of L-rhodinose
JP5601788B2 (en) Ketreductase mutant
JOONG et al. The Role of a Second Protein (Des VIII) in Glycosylation for the Biosynthesis of Hybrid Macrolide Antibiotics in Streptomyces venezuelae
WO2000037608A2 (en) Micromonospora echinospora genes encoding for biosynthesis of calicheamicin and self-resistance thereto
KR20140142648A (en) Construct for biosynthesis of resveratrol glucoside derivatives and method for producing it using thereof
WO2011009416A1 (en) Biosynthetic gene cluster of nocathiacins and use thereof
Lei et al. In vivo investigation of the substrate recognition capability and activity affecting amino acid residues of glycosyltransferase FscMI in the biosynthesis of candicidin
KR20150065439A (en) Flavonoid-O-methyltransferase and Method for methylation of flavones
Du et al. Selective biosynthesis of a rhamnosyl nosiheptide by a novel bacterial rhamnosyltransferase
CN109266662B (en) Gene cluster for biosynthesis of innovative mycin or ring-opening innovative mycin

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08778694

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08778694

Country of ref document: EP

Kind code of ref document: A2