WO2012166408A2 - Souches de e. coli génétiquement modifiées pour la production d'analogues d'érythromycine - Google Patents

Souches de e. coli génétiquement modifiées pour la production d'analogues d'érythromycine Download PDF

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WO2012166408A2
WO2012166408A2 PCT/US2012/038780 US2012038780W WO2012166408A2 WO 2012166408 A2 WO2012166408 A2 WO 2012166408A2 US 2012038780 W US2012038780 W US 2012038780W WO 2012166408 A2 WO2012166408 A2 WO 2012166408A2
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gene
coli strain
module
substitute
binding domain
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WO2012166408A3 (fr
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Blaine Pfeifer
Haoran Zhang
Sung-Hee Park
Ming Jiang
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Tufts University
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01094—6-Deoxyerythronolide-B synthase (2.3.1.94)

Definitions

  • Erythromycin produced by Saccharopolyspora erythraea, is a family of macrolide antibiotics effective against a broad spectrum of bacterial pathogens. Erythromycin biosynthesis involves two complicated pathways, i.e., (1) the polyketide synthesis pathway for producing 6-deoxyerythronolide B (6dEB), the macrocyclic core of erythromycin, and (2) the sugar synthesis and attachment/ tailoring pathway for synthesizing the erythromycin sugar moieties and attaching them to the macrocyclic core.
  • 6dEB 6-deoxyerythronolide B
  • erythromycin Since its discovery in 1949, erythromycin has been used as an important antibiotic to treat various bacterial infections. However, it causes side effects, such as diarrhea, loss of appetite, nausea, stomach pain, vomiting, and allergic responses. In addition, an increasing number of patients harbor erythromycin-resistant bacterial strains, resulting in low efficacy of erythromycin treatment in these patients.
  • One aspect of the present invention features a genetically modified E. coli strain for producing an analog of 6-deoxyerythronolide B (6dEB), the macrocyclic core of erythromycin.
  • This E. coli strain carries genetically engineered S. erythraea 6-deoxyerythronolide B synthase (DEBS) genes coding for a modified DEBS that includes one or more of (i) a substitute loading region containing the loading region of a non-erythromycin polyketide synthase, such as a rifamycin polyketide synthase from, e.g., Amycolatopsis mediterranei; (ii) a substitute NADPH binding domain in at least one module, the substitute NADPH binding domain being a NADPH binding domain from a different module; and (iii) a substitute module (e.g., Module 2) that contains a functional domain (e.g., a dehydratase domain) of a rapamycin polyketide
  • a substituted domain is a domain in the modified DEBS of this invention that replaces the wild-type counterpart.
  • the modified DEBS contains a substitute NADHP binding domain in one or both of Module 2 and Module 6, the substitute NADPH binding domain being a NADPH binding domain from a wild-type Module 3.
  • the genetically modified E. coli strain can further contain one or more of a sfp gene, an accAllaccA2 gene, a pccB gene, a birA gene, and a prpE gene.
  • the genetically modified E. coli strain of this invention can contain genes for producing erythromycin analogs, including an eryBII gene, an eryBIII gene, an eryBIV gQUQ, an eryBV gQUQ, an eryBVI gene, an eryBVII gene, an eryCI gene, an eryCII gene, an eryCIII gene, an eryCIV gene, an eryCV gene, an eryCVI gene, and one or more of (i) an oleU gene, (ii) an o/eFgene, (iii) a tylB gene and a tylMI gene, and (iv) a lom33 gene, a lom30 gene, a lom36 gene, and a lom32 gene.
  • genes for producing erythromycin analogs including an eryBII gene, an eryBIII gene, an eryBIV gQUQ, an eryBV gQUQ, an eryBVI gene, an eryB
  • this E. coli strain further contains an eryF gene, an eryG gene, an eryK gene, a glycosyltransferase gene (e.g., DesVII from S. Venezuelae) and an accompanying chaperon gene (e.g., DesVIII from S. Venezuelae), an ermE gene, or a combination thereof.
  • it is eryF, eryG, or eryK negative.
  • E. coli strains described above can also contain one or more exogenous genes each coding for a chaperone protein, such as dnaK, dnaJ, grpE, groES, groEL, and tig.
  • exogenous genes listed above is operably linked with an E. coli promoter so that it is expressed in the E. coli strain of this invention.
  • Another aspect of this invention features a method of producing an erythromycin analog or a 6dEB analog by cultivating one of the genetically modified E. coli strains described above in a suitable medium and collecting the medium for isolation of the erythromycin/6dEB analog.
  • the medium can contain a 6dEB substrate as an intermediate for synthesis of an erythromycin analog.
  • the 6dEB substrate can be 6dEB produced in an E. coli strain carrying wild type S. erythraea DEBS genes.
  • it is a 6dEB analog produced in an E. coli strain expressing the modified S. erythraea 6dEB synthase described above.
  • Fig. 1 is a schematic illustration of the erythromycin A biosynthesis pathway.
  • Panel A depicts the erythrymycin A gene cluster as organized in the S. erythraea chromosome.
  • Panel B shows the structure of S. erythraea 6dEB synthase and the erythromycin synthesis biopathway.
  • Fig. 2 is a diagram illustrating a biosynthetic scheme for producing erythromycin analogs.
  • Panel A shows modifications to DEBS. The filled ovals refer to functional domains from a non-erythromycin polyketide synthase.
  • Panel B shows a modified erythromycin sugar synthesis pathway, in which one or more of L-olivose, L-oleandrose consider D-mycaminose, and pyrrolosamine sugar residues, instead of the erythromycin D-desosamine sugar residue, are synthesized.
  • Panel C lists a number of erythromycin analogs to be produced in the genetically engineered E. coli strains of this invention.
  • Panels D and E are diagrams demonstrating synthesis of two erythromycin analogs in two exemplary genetically modified E. coli strains described herein via mass spectrum.
  • Erythromycin biosynthesis includes two complicated pathways: (1) the polyketide synthesis pathway for producing the 6dEB macrocyclic core of erythromycin, and (2) the sugar synthesis and attachment pathway for synthesizing the erythromycin sugar moieties and attaching them to the 6dEB core.
  • the polyketide synthesis pathway is catalyzed by DEBS, a megadalton ⁇ 2 ⁇ 2 ⁇ 2 complex containing ⁇ , ⁇ , and ⁇ subunits, which are encoded by DEBS1, DEBS2, and DEBS3 genes, respectively. See Pfeifer et al., Science 291 : 1790- 1792, 2001 ; and Fig. 1.
  • the sugar synthesis/attachment pathway involves a number of enzymes, including the gene products of eryBII, eryBIII, eryBIV, eryBV, eryBVI, eryBVII gene, eryCI gene, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryF, and optionally, one or both of eryG and eryK. See Fig. 1 and US Patent Application No. 12/764,230. The organization of these genes in the S. erythraea chromosome is shown in Fig. 1, Panel A.
  • Described herein is a genetically engineered E. coli strain carrying one or both of (a) a modified 6dEB synthase that is designed for producing 6dEB analogs, and (b) a modified sugar synthesis/attachment/tailoring pathway that involves additional genes for synthesis one or more non-erythromycin sugar moieties.
  • DEBS consists of three large subunits, DEBS1, DEBS2 and DEBS3, which are encoded, respectively, by EryAI, EryAII, EryAIII (also known as DEBS1, DEBS2, and DEBS3, respectively). See Table 1 below and also McDaniel et al, Proc, Natl. Acad. Sci. USA
  • Each of the DEBS1, DEBS2 and DEBS3 subunits contains two modules, Modules 1 and 2, Modules 3 and 4, and Modules 5 and 6, respectively. See Fig.1.
  • Each module contains at least three essential domains: a ketosynthase domain (KS), an acyl transferase (AT) domain, and an acyl carrier protein (ACP) domain.
  • the AT domain selects the appropriate carbon extender unit and transfers the units from acyl-CoA onto the
  • the KS domain accepts the polyketide chain from the previous module and catalyzes chain elongation reaction by adding an ACP-bound extender unit through decarboxylative condensation.
  • Each of the modules also includes a ketoreductase domain (K ).
  • DEBS also includes a two-domain loading region located in DEBS 1 , N-terminal to Module 1. This loading region is responsible for priming the synthase with a proprionate starter unit.
  • DEBS includes a single-domain releasing region located C-terminal to Module 6, i.e., the thioesterase (TE) domain.
  • TE thioesterase
  • GenBank accession numbers of the genes coding for the three subunits of the DEBS from S. erythraea NR L 2338 is shown in Table 1 below:
  • DEBS genes from other S. erythraea strains can be retrieved from a public database, i.e., the GenBank.
  • a wild-type DEBS, as described above, can be modified in one or more of the following manners to produce 6dEB analogs.
  • the two-domain loading region in a wild-type DEBS can be replaced with a loading region from a non-erythromycin polyketide synthase, an enzyme complex involved in biosynthesis of a non-erythromycin polyketide.
  • the DEBS thus modified is capable of synthesizing a 6dEB analog using a starter unit specific to the replacement loading fragment, in stead of the proprionate starter unit recognizable by the loading region of wild-type DEBS.
  • replacement loading regions examples include, but are limited to, the loading region from a rifamycin polyketide synthase or an avermectin polyketide synthase.
  • a replacement loading region uses an aryl-containing molecule as the starter unit. It is known that certain aryl derivatives of erythromycin possess antibiotic activities, presumably by binding to a separate domain of the 23S RNA within the 50S ribosomal component and compensating for lost macrolide binding affinity as a result of ribosomal methylation (the primary form of macrolide resistance).
  • the replacement loading region is from S. roseosporus NRRL 15998 rifamycin polyketide synthase (GenBank accession number ZP 06582601) or from A. mediterranei rifamycin polyketide synthase (GenBank accession number AF040570). See Pfeifer et al, Science
  • an NADPH-binding domain in at least one of the modules is replaced with the concomitant sequence from another module.
  • the NADPH-binding domain in Mocule 3 (responsible for the C9 ketone group shnthesis) is used to replace the corresponding NADPH-binding domain in Module 2 and/or Module 6 for synthesizing a 6dEB analog with a C3 ketone group, which subsitutes for the hydroxyl group at C3 in 6dEB.
  • Such an analog mimics the successful 3 rd -generation ketolide compounds (see Douthwaite et al, J Antimicrob Chemother 48 Suppl Tl : 1-8, 2001).
  • replacing the hydroxyl group with a ketone group at the C3 position in the macrocylic core of erythromycin has proven useful in overcoming macrolide resistance (particularly, efflux resistance). See Ma et al, J Med Chem
  • a functional domain from a rapamycin polyketide synthase (e.g., GenBank accession number CAA60460.1 or CAA60459.1), such as a dehydratase domain (DH) or an AT domain, is inserted into one of the six modules (e.g., Module 2) in the DEBS to generate a substitute module.
  • This substitute module contains the rapamycin polyketide synthase functional domain, as well as the functional domains of the wild-type counterpart.
  • Suitable rapamycin polyketide synthase functional domains to be used in this invention include, but are not limited to, those described in McDaniel et al
  • the DEBS thus modified can synthesize a 6dEB analog having a double bond between CI 1 and C12.
  • An E. coli strain carrying genes coding for any of the mofieid DEBS enzymes described above can be prepared via conventional recombinant technology. Genes of interst can be isolated from their natural sources and cloned into suitable vectors. Any of the modifications described above can be introduced into one or more of DEBSI, DEBS2, and DEBS3 by methods known in the art. Genetically engineered DEBS genes for expressing a modified DEBS can be cloned into one or more expression cassettes, in which each of the genes is in operative linkage to an E. coli promoter. When necessary, codon optimization can be applied to the modified DEBS genes so as to enhance their expression levels in E. coli.
  • a promoter sequence is a nucleotide sequence containing elements that initiates the transcription of an operably linked nucleic acid sequence.
  • a promoter contains an RNA polymerase binding site. It can further contain one or more enhancer elements which, by definition, enhances transcription, or one or more regulatory elements that control the on/off status of the promoter.
  • An E. coli promoter is a promoter that functions within E. coli.
  • E. coli promoters include the ⁇ -lactamase and lactose promoter systems (see Chang et al., Nature 275:615-624, 1978), the SP6, T3, T5, and T7 RNA polymerase promoters (Studier et al., Meth. Enzymol. 185:60-89, 1990), the lambda promoter (Elvin et al., Gene 87: 123-126, 1990), the trp promoter (Nichols and Yanofsky, Meth. in Enzymology 101 : 155-164, 1983), the tac and trc promoters (Russell et al., Gene 20:231-243, 1982), and pCold (see US Patent No. 6,479,260).
  • the expression cassette(s) mentioned above, included in one or more plasmids, can be introduced into an E. coli host stain, such as JM109, BL21(DE3), DH5a, and MCI 061, via conventional recombinant technology.
  • the resultant positive transformants can be examined to determine their expression of the genetically engineered DEBS genes by, e.g., Western blot, SDS-PAGE, or enzymatic activity analysis.
  • the expression cassette(s) is incorporated into the chromosome of the host strain via homologous recombination. In another example, it is extra-chromosomal.
  • the E. coli strain carrying coding sequences for 5 the modified DEBS can further contain one or more of the genes coding for phosphopantetheinyl transferase, propionyl-CoA synthetase, acyl-CoA carboxylase complex A subunit,
  • propionyl-CoA carboxylase complex B subunit propionyl-CoA carboxylase complex B subunit
  • carboxylase holoenzyme synthetase See Pfeifer et al., Science 291(5509): 1790-2, 2001.
  • Such genes can be obtained from a suitable microorganism, e.g., E. coli, S. erythraea, S. coelicolor, and B. subtilis.
  • both native genes and their functional variants can be used to construct the E. coli strain of this invention.
  • Functional variants include degenerative variants of their native counterparts (e.g., those produced by codon optimization) and nucleotide sequences coding for functional equivalents of the proteins encoded by the native genes.
  • a functional equivalent of a protein refers to a polypeptide that shares at least 85% (e.g., 90%, 95%, or 99%) sequence identity to the protein and has the same bioactivity as the protein.
  • Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25:3389-3402, 1997.
  • the default parameters of the respective programs e.g., BLASTX and BLASTN
  • BLASTX and BLASTN the default parameters of the respective programs
  • any of the E. coli strains described above can be cultured in a medium in the presence of a suitable starter substrate under suitable conditions (e.g., at 22 °C) to produce a 6dEB analog.
  • a suitable starter substrate e.g., at 22 °C
  • the starter substrate depends upon the type of the loading region in the modified DEBS. For example, when the loading region is from a rifamycin polyketide synthase, the starter substrate is a benzyl compound. After a suitable culturing period, the medium is harvested for isolation of the 6dEB analog, which can be used as a substrate for preparing a corresponding erythromycin analog.
  • the erythromycin sugar synthesis/attachment pathway can be modified in one or more of the following manners for erythromycin analog production.
  • the eryF gene needed for hydroxylation of the C6 position of 6dEB, can be inactivated or deleted via a routine method for producing erythromycin analogs with improved in vivo stability and reduced side effects. It has been found that such erythromycin analogs have reduced potential to form hemiketal-type byproducts, which limit potency and contribute to side -reactions like stomach cramps. See Itoh et al, Am J Physiol 247(6 Pt l):G688-94, 1984; Weber et al, Science 252(5002):114-7, 1991; and Katz et al, Chem Rev 105(2):499-528, 2005.
  • non-erythromycin deoxysugars i.e., sugar moieties not present in a native erythromycin
  • D-desosamine distained in a native erythromycin
  • the S. erythraea genes involved in erythromycin sugar synthesis i.e., eryBII, eryBIII, eryBIV, eryBV, eryBVI, eryBVII, eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, and optionally, one or more of eryF, eryG, eryK, and ermE
  • genes involved in synthesis of non-erythromycin sugar moieties e.g., those listed in Table 3 above
  • Both native genes and their functional variants e.g., degenerative variants such as codon optimized variants
  • the expression cassettes, included in one or more plasmids, can be introduced into an E. coli host stain via conventional recombinant technology.
  • the E. coli host strain also carries the modified DEBS genes described above.
  • the resultant positive transformants can be examined to determine their expression of the genes of interest by, e.g., Western blot, SDS-PAGE, or enzymatic activity analysis.
  • the expression cassettes are incorporated into the chromosome of the host strain via homologous recombination. In another example, they remain extra-chromosomal.
  • any of the E. coli strains carrying the genes responsible for both erythromycin sugar synthesis/attachment and non-erythromycin sugar synthesis can further contain one or more genes each coding for a glycosyltransferase.
  • the glycosyltransferase is DesVII (and an accompanying chaperone DesVIII) from S. venezuelae (GenBank accession numbers
  • AAC68677,1 and AAC68676.1 which has been shown to have relaxed specitifity for both polyketide aglycone and deoxysugar substrates. See Borisova et al., Angew Chem Int Ed Engl 45(17):2748-53, 2006; and Xue et al, Proc Natl Acad Sci USA 95(21): 12111-6, 1998.
  • Other suitable glycosyltransferases include, but are not limited to those from the original S.
  • one or more of them can be fused with a nucleotide sequence coding for a protein tag, preferably a hexa-His tag.
  • E. coli strains described above can be further transformed by one or more plasmids for expression of one or more chaperone proteins.
  • E. coli chaperone proteins are well known in the art. Examples include, but are not limited to, dnaK, dnaJ, grpE, groES, groEL, and tig.
  • An E. coli strain described above, designed for producing an erythromycin analog can be cultured in a medium in the presence of a 6dEB substrate under suitable conditions (e.g., at 22 °C) to produce erythromycin.
  • 6-dEB substrate refers to 6dEB and its analogs, i.e., compounds having the same macrocylic core as 6dEB with one or more of the side groups in 6dEB (except the two hydroxyl groups for sugar attachment) replaced with a suitable group(s), e.g., alkyl group, an alkenyl group, a ketone group, a hydroxyl group, akyl , benzyl, or other starter groups, and any combination of chirality associated with the previously mentioned groups (where applicable).
  • a 6dEB substrate can be prepared either by chemical synthesis or by a genetically modified microorganism (e.g., E. coli as described above) designed for producing such (see Pfeifer et al., Science 291 : 1790- 1792, 2001. After a suitable period of time, the culturing medium is collected and the erythromycin analog contained in it is purified via a conventional method, e.g., HPLC.
  • a genetically modified microorganism e.g., E. coli as described above
  • EXAMPLE Producing Benzyl-erythromycin Using Genetically Modified E. coli Strains
  • E. coli cell cultures for molecular biology and SDS-PAGE analysis were conducted in Luria-Bertani (LB) medium at 37°C and 250 rpm.
  • Heterologous 6dEB and erythromycin biosynthesis were conducted in a production medium at 22°C and 250 rpm.
  • One liter of the production medium contained 5 g yeast extract, 10 g tryptone, 15 g glycerol, 10 g sodium chloride, 3 mL 50% v/v Antifoam B, 100 mM 4-(2-hydroxyethyl)-l-piperazineethanesuffonic acid (HEPES) buffer, and was adjusted to pH 7.6 by 5 M NaOH before use.
  • Erythromycin tailoring and resistance genes shown in Table 5 below, were PCR-amplified from S. erythraea genomic DNA using the primers also listed in Table 5.
  • the Ndel/EcoRl digested fragments of the PCR products (except for genes eryBIIl, BV, and CIII) were individually inserted into plasmid pET21c digested with the same restriction enzymes.
  • the eryBIIl PCR product was digested by Nhel/EcoRl and inserted into pET21c using the corresponding restriction sites.
  • the eryBV and eryCIII PCR products were digested and inserted by Ndel/Sacl and Ndel/HindlU, respectively.
  • kanamycin-resistant pET28a plasmid Genes eryCI, CII, CIII, CIV, CV, CVI, eryF, eryG, and eryK were combined (in order) into one operon by sequential Xbal/Spel and HmdIII digestion and ligation.
  • the resulting plasmid pHZT2 was a derivative of the ampicillin-resistant pET21c plasmid. Table 5.
  • plasmid carrying an additional eryK gene In order to construct a plasmid carrying an additional eryK gene, PCR and NdellXhol digestion preceded ligation into the streptomycin-resistant pCDFDuet-1 vector.
  • the resulting plasmid was named pHZT4.
  • the chaperone expression plasmids pG-KJE8, pGro7, pKJE7, pG-Tf2, and pTfl6 were individually co-transformed with plasmids pHZTl, pHZT2, and (when indicated) pHZT4 into E. coli BL21(DE3) to test the effects each chaperone or chaperone combination had on erythromycin biosynthesis.
  • Plasmids pHZTl, pHZT2, and pHZT4, in combination with one or more of the chaperone plasmids and eryK-pCOF, were transformed into BL21(DE3) and STAR(DE3) to generate genetically modified E. coli strains capable of producing erythromycin compounds.
  • BAPl(pBP173/pBP144), capable of producing 6dEB analogs were constructed as follows.
  • the loading domain of a wild-type EryAI was replaced with the di-loading domain from A. mediterranei rifamycin polyketide synthase, following the method described in Pfeifer et al., Science 291 :1790- 1792, 2001.
  • the mutated EryA I gene, cloned into an expression plasmid was introduced into an E. coli host cell, together with the wild-type EryAII and EryAIII genes, resulting in E. coli strain BAPl(pBP130/pBP165), which is capable of producing a benzyl analog of 6dEB.
  • the acyltransferase domain of eryAIII Module 6 was replaced with the acyltransferase domain of rapamycin polyketide synthase Module 2, following the method described in Liu et al. J. Am. Chem. Soc. 119: 10553-10554, 1997.
  • the mutated eryAIII gene thus generated was cloned together with the wild-type eryAII gene into an expression plasmid to produce plasmid pBP173.
  • This plasmid was then introduced into an E. coli host cell, together with plasmid pBP144, which carries a wild-type eryAI gene, resulting in E. coli strain
  • E. coli strain BAPl(pBP130/pBP165) described above was cultured in 100 mL production medium containing 100 mg/L carbenicillin, 50 mg/L kanamycin, 100 ⁇ isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG), 20 mM propionate, and 20 mM of benzoate (stock solution adjusted to pH 7) at 22°C for 7 days to produce a benzyl-6dEB analog.
  • IPTG isopropyl ⁇ -D-l-thiogalactopyranoside
  • benzoate stock solution adjusted to pH 7 days to produce a benzyl-6dEB analog.
  • E. coli strain BAPl(pBP173/pBP144) also described above was cultured in 100 mL a production medium containing 100 mg/L carbenicillin, 50 mg/L kanamycin, 100 ⁇ IPTG, and 20 mM propionate at 22°C for 7 days to produce a 2-desmethyl-6dEB analog.
  • the 6dEB analogs produced as described above were extracted with ethyl acetate, dried, and resuspended in methanol. These compounds were quantified using an HPLC -Evaporative Light Scattering Detector and the 6dEB analogs were quantified by MS as previously described (with purified 6dEB as a standard). See Zhang et al. Biotechnol Bioeng 105(3):567-573, 2010.
  • E. coli strain BL21(DE3)(pHZTl/pHZT2/pHZT4/pGro7) designed for producing erythromycin compounds, was inoculated (5% v/v) into a production medium containing 100 mg/L carbenicillin, 50 mg/L kanamycin, 20 mg/L chloramphenicol, 50 mg/L streptomycin, 100 ⁇ IPTG, and 2 mg/mL arabinose (pGro7 required arabinose induction) and cultured at 22°C for 24 hours.
  • the E. coli cells were cultured for an additional 3 days at 22°C to produce an erythromycin analog. See Fig. 2, Panels D and E.
  • an LC-MS calibration curve was prepared using commercially available erythromycin A as an external standard and roxithromycin as an internal standard.
  • erythromycin A was added to E. coli BL21(DE3) or BAP1 cultures grown under the same conditions described above.
  • the samples were dissolved in 100 of methanol containing 2.5 mg/L roxithromycin and subjected to LC-MS analysis to prepare the calibration curve.
  • a portion of the extracts resulting from the erythromycin analog producing E. coli cultures was similarly mixed with roxithromycin and analyzed against the calibration curve.
  • the ratio of the erythromycin analog and roxithromycin standard peak areas was correlated to quantify experimental erythromycin production with a suitable calibration curve made before every experimental analysis. All reported titers represent at least three independent experiments. Negative controls included 1) experiments reliant on strain BAP1 without the required production plasmids, 2) uninduced cultures, and 3) the replacement of pHZT2 with an empty pET21c expression vector.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des souches de E. coli génétiquement modifiées pour la production d'analogues d'érythromycine ou de 6-déoxyérythronolide B. Chacune de ces souches de E. coli exprime (a) une synthase de 6-déoxyérythronolide B (6dEB) de S. erythraea modifiée apte à synthétiser un analogue de 6dEB et/ou (b) des gènes mis en jeu dans la voie de synthèse/attachement/confection de sucre érythromycine et ceux mis en jeu dans la synthèse d'une ou plusieurs fractions sucrées non érythromycine, telles que L-olivose, L-oléandrose, D-mycaminose et pyrrolosamine.
PCT/US2012/038780 2011-05-27 2012-05-21 Souches de e. coli génétiquement modifiées pour la production d'analogues d'érythromycine WO2012166408A2 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5998194A (en) * 1995-12-21 1999-12-07 Abbott Laboratories Polyketide-associated sugar biosynthesis genes
US20040087003A1 (en) * 2002-06-13 2004-05-06 Zhihao Hu Methods and cells for improved production of polyketides
US20040214276A1 (en) * 2002-07-31 2004-10-28 Chaitan Khosla Production of glycosylated macrolides in E. coli
US20110262971A1 (en) * 2010-04-21 2011-10-27 Tufts University Genetically Modified E. coli Strains for Producing Erythromycin

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5998194A (en) * 1995-12-21 1999-12-07 Abbott Laboratories Polyketide-associated sugar biosynthesis genes
US20040087003A1 (en) * 2002-06-13 2004-05-06 Zhihao Hu Methods and cells for improved production of polyketides
US20040214276A1 (en) * 2002-07-31 2004-10-28 Chaitan Khosla Production of glycosylated macrolides in E. coli
US20050287587A1 (en) * 2002-07-31 2005-12-29 The Board Of Trustees Of The Leland Stanford Junior University Production of glycosylated macrolides in E. coli
US20110262971A1 (en) * 2010-04-21 2011-10-27 Tufts University Genetically Modified E. coli Strains for Producing Erythromycin

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
EDUARDO RODRIGUEZ ET AL.: 'Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains' JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY. vol. 30, no. 8, August 2003, ISSN 1367-5435 pages 480 - 488 *
HAORAN ZHANG ET AL.: 'Complete biosynthesis of erythromycin A and designed analogs using E. coli as a heterologous host' CHEMISTRY AND BIOLOGY vol. 17, no. 11, 24 November 2010, ISSN 1074-5521 pages 1232 - 1240 *
JEFFREY D. KITTENDORF ET AL.: 'Developing tools for engineering hybrid polyketide synthetic pathways' CURRENT OPINION IN BIOTECHNOLOGY. vol. 17, no. 6, 2006, ISSN 0958-1669 pages 597 - 605 *
SUMATI MURLI ET AL.: 'Metabolic engineering of Escherichia coli for improved 6-deoxyerythronolide B production' JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY. vol. 30, no. 8, August 2003, ISSN 1367-5435 pages 500 - 509 *
XUE GAO ET AL.: 'Engineering polyketide biosynthesis and biocatalysis in Escherichia coli' APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. vol. 88, no. 6, December 2010, ISSN 0175-7598 pages 1233 - 1242 *

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