WO2016008332A1 - 一种提高大肠杆菌异源合成聚酮类化合物的方法和用途 - Google Patents
一种提高大肠杆菌异源合成聚酮类化合物的方法和用途 Download PDFInfo
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Definitions
- the present invention is in the field of synthetic biology and industrial biotechnology, and in particular, the present invention relates to a method and use for improving heterologous synthesis of polyketides in E. coli.
- the starting material for the synthesis of 6-dEB is propionic acid and methylmalonic acid.
- the entire process is catalyzed by polyketide synthase.
- the idea of engineering prompted researchers to construct an engineered strain for heterologous synthesis of natural products. Because E. coli metabolic network research is the most in-depth, E. coli is often used as the chassis cell of engineering bacteria.
- the pET28a was derived from the initiation site of pBR322, and pBP144 was transformed into pKOS207-129.
- the engineering strain K207-3/pKOS207-129/pBP130 was obtained in a shake flask. 22.5 mg/L of 6-dEB.
- Wang Yong et al. integrated the erythromycin polyketide synthase genes eryAI, AII, AIII into the E. coli chromosome by chromosomal recombination Red/ET method, and obtained stable strains with chromosome modification, and multiple Compared with plasmid co-expression, this strain can stably synthesize erythromycin intermediate 6-dEB.
- a method for the biosynthesis of a polyketide 6-deoxyerythrolactone of a host of a synthetic polyketide 6-deoxyerythronolide comprising:
- target gene is selected from the group consisting of:
- nucleotide synthesis and other metabolic module genes purT, lsrC, hemN, zwf, pgl, gnd, rpe, talA, talB, tktA, tktB, ulaE or yieK;
- the sugar metabolism module genes aceF, pgi, lpdA, ppk, ptsH, ptsI, glcF, glcE, fsaA or agaW;
- amino acid and protein anabolic module genes leuC, leuD, serC, serB, serA, gdhA or tnaA;
- the strain prepared in the step (1) is cultured to biosynthesize the polyketide compound 6-deoxyerythrolactone.
- Attenuating target gene expression comprises introducing an interference molecule that inhibits expression of the target gene or knocking out the target gene.
- the interfering molecule that inhibits expression of the target gene is directed against (or has its own):
- the target gene is selected from the group consisting of:
- the interfering molecule that inhibits expression of a target gene is an sRNA.
- the sRNA comprises the following structure:
- Promoter target gene inhibitory molecule (such as target gene binding sequence), terminator.
- the promoter is selected from the group consisting of: a Pr promoter (preferably having the sequence of positions 7-61 of SEQ ID NO: 52), a P BAD promoter, a T7 promoter, and a Trc promoter. child).
- the terminator is selected from the group consisting of: a TE terminator (preferably having the sequence of positions 171-110 of SEQ ID NO: 52), a T1/TE terminator, a T7 terminator, rrnB Terminator, rrnB T1 and T2 terminator).
- the interfering molecule that inhibits expression of the target gene and the terminator further comprises: a micF sequence (such as the sequence having positions 110-170 of SEQ ID NO: 52).
- the target gene inhibitory molecule is a short nucleic acid sequence, for example, 18-26 bp in length; preferably 20-24 bp; capable of complementing or binding to the mRNA of the target gene, or having the target gene mRNA A sequence that, after being transferred into a cell, expresses a sequence that is complementary or bound to the mRNA of the target gene.
- the sRNA is contained in an expression vector.
- the host bacteria of the synthetic polyketone compound 6-deoxyerythronolide is a prokaryotic bacterium capable of synthesizing the polyketide compound 6-deoxyerythronolide.
- the prokaryotic bacteria capable of synthesizing the polyketide compound 6-deoxyerythronolide is an Escherichia coli capable of synthesizing the polyketide compound 6-deoxyerythronolide;
- the propionate metabolism operon is knocked out and the phosphopantetheinyl transferase gene sfp is integrated at the knockout site; or the propionate metabolism operon is directly knocked out, Sfp integrates into any non-essential or non-functional DNA sequence region of the E. coli genome.
- the Escherichia coli is transformed with a gene encoding the erythromycin Streptomyces polyketide synthase DEBS2, a gene encoding the erythromycin Streptomyces polyketide synthase DEBS3, and a propionyl-CoA carboxylate.
- Enzyme The gene for the ⁇ -CT subunit, the gene encoding the propionyl-CoA carboxylase a-CT subunit, and the gene encoding the erythromycin Streptomyces polyketide synthase DEBS1.
- an interfering molecule which inhibits expression of a target gene which is an sRNA
- the sRNA comprising the following structure (preferably, between the interfering molecule and the terminator which inhibit expression of the target gene, Also included: micF sequence):
- Promoter target gene inhibitory molecule, terminator
- the use of the interference molecule such as sRNA, which inhibits expression of a target gene, for transforming a host strain of a synthetic polyketide 6-deoxyerythronolide, weakening a corresponding target gene, and promoting synthesis Host of the polyketide compound 6-deoxyerythronol biosynthesis polyketide 6-deoxyerythrolactone.
- a host strain for synthesizing a polyketide compound 6-deoxyerythronolide wherein the host strain is transformed with an interference molecule which inhibits expression of a target gene, or the host bacteria The target gene is knocked out; wherein the target gene is selected from the group consisting of: (a) nucleotide synthesis and other metabolic module genes purT, lsrC, hemN, zwf, pgl, gnd, rpe, talA, talB, tktA, tktB, ulaE or yieK;
- the sugar metabolism module genes aceF, pgi, lpdA, ppk, ptsH, ptsI, glcF, glcE, fsaA or agaW;
- amino acid and protein anabolic module genes leuC, leuD, serC, serB, serA, gdhA or tnaA;
- the host strain is transformed with the interference molecule that inhibits expression of the target gene, such as sRNA; and/or the host strain of the synthetic polyketide compound 6-deoxyerythronolide is a prokaryotic bacterium capable of synthesizing a polyketide compound 6-deoxyerythronol; preferably, in the Escherichia coli, a propionic acid metabolism operon is knocked out and a phosphopantethein B is integrated at the knockout site An amine transferase gene sfp; preferably, the Escherichia coli is transformed with a gene encoding the erythromycin Streptomyces polyketide synthase DEBS2, encoding a erythromycin Streptomyces polyketide synthase The gene for DEBS3, the gene encoding the propionyl-CoA carboxylase ⁇ -CT subunit, the gene encoding the propionyl-CoA carboxylase a-CT subunit, and the gene
- kits for promoting synthesis of a polyketide 6-deoxyerythritol lactone of a polyketide compound 6-deoxyerythritol lactone the reagent Included in the cassette is a sum of said host bacteria (one of which can be selected by a person skilled in the art for production); or
- the kit includes: the sum of the interfering molecules that inhibit expression of the target gene (the one skilled in the art may select one or more interfering molecules that inhibit expression of the target gene to be applied); or
- the kit includes: a sum of vectors respectively containing the interfering molecules that inhibit expression of the target gene (one of the art can select one or more of the vectors for application).
- FIG. 1 Schematic diagram of sRNA expression plasmid PJF650.
- Figure 2 Schematic diagram of the E.coli WG build process.
- FIG. 1 A 6-dEB HPLC-ELSD analysis of 40 mg/L.
- Figure 8 Effect of sRNA combination regulation on heterologous synthesis of 6-dEB in E. coli.
- the inventors have intensively studied for the first time to disclose a method for biosynthesis of a polyketide compound 6-dEB by a host bacteria which synthesizes a polyketide compound 6-dEB, by weakening or combining weakening sucC in Escherichia coli alone (expressing succinic acid)
- the expression of genes such as coenzyme A synthase can significantly increase the yield of heterologous synthetic polyketides in E. coli, and the highest yield increase rate can reach more than 60%.
- the method of the present invention enables a large accumulation of polyketone compounds during fermentation.
- interfering molecule that inhibits expression of a target gene refers to an agent that can specifically reduce the level of expression of a target gene, including various molecules known in the art to inhibit expression of a target gene, such as but not limited to antisense.
- Nucleic acids, locked nucleic acids, peptide nucleic acids, siRNA, shRNA, microRNAs, etc. (collectively referred to as target gene suppressing molecules); or constructs that carry or express antisense nucleic acids, locked nucleic acids, peptide nucleic acids, siRNA, shRNA, microRNA, and the like.
- heterologous refers to the relationship between two or more nucleic acid or protein sequences from different sources, and the relationship between proteins (or nucleic acids) from different sources and host cells is known. For example, if the combination of a nucleic acid and a host cell is generally not naturally occurring, the nucleic acid is heterologous to the host cell. A particular sequence is “heterologous” to the cell or organism into which it is inserted.
- the "host strain of synthetic polyketide 6-dEB” refers to a host or host cell known in the art to be useful for the synthesis of the polyketide 6-dEB.
- host bacteria include, but are not limited to, Escherichia coli, modified E. coli (including strains transformed into pccB and pccA, strains transformed into eryAI, AII and/or AIII, etc.).
- a preferred host strain is Escherichia coli which knocks out the propionate metabolism operon and integrates the phosphopantetheinyl transferase gene sfp at the knockout site, and wherein the expression of the polyketide encoding the erythromycin Streptomyces is transformed.
- the gene for the enzyme DEBS2 the gene encoding the erythromycin Streptomyces polyketide synthase DEBS3, the gene encoding the propionyl-CoA carboxylase ⁇ -CT subunit, the gene encoding the propionyl-CoA carboxylase a-CT subunit And a gene encoding the erythromycin Streptomyces polyketide synthase DEBS1.
- each of the target genes disclosed in the present invention is a gene known in the art, and those skilled in the art can query the sequence information of these genes through a platform such as GenBank, and therefore, those genes are readily available to those skilled in the art.
- the invention also provides a gene set comprising: (a) nucleotide synthesis and other metabolic module genes purT, lsrC, hemN, zwf, pgl, gnd, rpe, talA, talB, tktA, tktB, ulaE Or yieK; (b) pentose phosphate and glyoxylate pathway module genes yaeR, rpiA, rpiB, purH, pyrB, pyrI, cysQ, pyrC, gmk, guaA, guaB, ndk, pyrF, pyrE, pyrH or hpt; TCA cycle and oxidative phosphorylation module genes frdD, frdA, sdhA, sdhB, sdhC, sdhD, sucC, sucD, cyoA or cyoB; (d) sugar metabolism module genes ace
- One skilled in the art can select one or more genes from the set of genes according to the disclosure of the present invention, perform the operation of the method according to the present invention, and design downregulate the host cell based on the selected target gene.
- the substance expressed in it is used to promote the production of 6-dEB.
- the interference molecule which inhibits interference of a target gene is an sRNA or a construct (including an expression vector) carrying the sRNA.
- the sRNA comprises the following structures: a promoter, a target gene suppressing molecule (such as a target gene binding sequence), and a terminator; preferably, between the target gene inhibitory molecule and the terminator, Also included: micF sequence.
- the design of the promoter and terminator can be carried out according to the experience of those skilled in the art, and any suitable promoter and terminator are included in the scope of the present invention.
- the promoter is a Pr promoter and the terminator is a TE terminator.
- variations to promoters and terminators are contemplated by those skilled in the art and are still within the scope of the invention.
- the sRNA is located on an expression vector.
- the present invention also encompasses a vector comprising the sRNA.
- the expression vector usually also contains an origin of replication and/or a marker gene and the like. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. It will be appreciated that any expression vector can be used as long as the insertion of the sRNA can be achieved and the expression of the target gene can be downregulated after transformation of the cells.
- the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells. Transformation of the expression vector into a host cell can be carried out using conventional techniques well known to those skilled in the art.
- the present invention also provides a host strain for synthesizing a polyketide compound 6-dEB, which is transformed with an interference molecule which inhibits expression of a target gene.
- the host bacteria can efficiently produce the polyketide 6-dEB.
- the present invention also provides a kit for promoting the synthesis of a polyketide compound 6-dEB of a synthetic polyketide 6-dEB, the kit comprising: all of the compounds for conversion of the invention for conversion
- the sum of the host bacteria of the ketone compound 6-dEB (one of which can be selected by a person skilled in the art for production); preferably, the kit includes: a more preferred conversion with the present invention
- the sum of the host bacteria (the host strain transformed with the interfering molecule that down-regulates the gene expression of the target gene or target gene combination) of the polyketide compound 6-dEB.
- the kit includes: a sum of the sRNAs of the present invention (one of the art can select one or more sRNAs for application); preferably, the kit includes: the present invention A more preferred sum of sRNAs (downregulation of the gene expression of the target gene or target gene combination).
- the kit includes: a sum of vectors comprising the sRNA of the present invention (one of the art can select one or more of the vectors for application); preferably, the kit includes: A sum of vectors comprising a more preferred sRNA of the invention (a sRNA that down-regulates the gene expression of the target gene or target gene combination).
- strains, culture medium and related reagents used in the present invention are as follows:
- the strain E.coli DH10B was used as a cloning host, and the strain E.coli WG (pZG07/pZG08) was used as a host for the synthesis of polyketide 6-dEB (see Lu Zhiguo, Ph.D thesis, East China University of Science and Technology, 2011).
- Molecular cloning-related enzymes, DNA fragments and plasmid extraction and purification kits were supplied by NEB Corporation, TaKaRa Corporation and Axygen Corporation, respectively.
- Each medium component, antibiotics and other related reagents were purchased from Oxiod, Sinopharm Group and Shanghai Bioengineering Co., Ltd.
- the primers were synthesized by Nanjing Kingsray Biotech Co., Ltd.
- the 6-dEB fermentation medium formulation (g/L) involved in the present invention NaCl, 10; peptone, 10; yeast extract, 5; glycerin, 15; 100 mM HEPES, pH adjusted to 7.6.
- Inducing agent involved in the present invention IPTG: 24 ⁇ g/ml; precursor: sodium propionate 20 mM; induction conditions involved in the present invention: 22 ° C, 250 rpm, 100 ml shake flask solution 10 ml medium fermentation culture for 5 days.
- concentrations of the antibiotics involved in the present invention are ampicillin 100 mg/L, kanamycin 50 mg/L, and chloramphenicol 34 mg/L, respectively.
- the invention regulates the expression of a target gene by the sRNA interference technology, that is, the expression of the sRNA binds to the mRNA of the target gene, thereby suppressing the binding of the target gene mRNA to the ribosome, and then inhibiting the expression of the target gene.
- the present invention identifies target genes that are useful for increasing the synthesis of polyketides by weakening selected gene targets.
- deoB-sRNA gene sequence fragment (synthesized by Shanghai Jieduan Bioengineering Co., Ltd.), including Pr promoter and deoB target gene binding site (DNA sequence length is 24 bp, caccataataaatgcacgtttcat (SEQ ID NO: 1);
- the pentose phosphate mutase deoB gene is fully complementary to the 24 bp after the start of the ATG start sequence, the deoB gene Genbank number: NC_012971.2), the micF sequence (Genbank number: NC_000913.3) and the TE terminator, and introduced at both ends. NdeI and HindIII cleavage sites.
- the DNA sequence of this sRNA gene sequence fragment is as follows (SEQ ID NO: 52):
- sRNA expression plasmid The pACYCDuet-1 plasmid (purchased from Novagen) was used as a PCR template, and clones containing only chloramphenicol resistance were cloned by primers pACYC-F and pACYC-R (introduction of NdeI and HindIII restriction sites at both ends of the primer).
- the vector fragment of the p15A replicon was digested with NdeI and HindIII by a clean recovery kit (purchased from Axygen); the chemically synthesized deoB-sRNA sequence fragment was digested with NdeI and HindIII.
- the template plasmid pJF650 was used as a template, and the primers in Table 1 and the PCR conditions in Table 2 were used to directly obtain the TCA cycle and oxidative phosphorylation module genes frdD, frdA, sdhA by site-directed mutagenesis PCR amplification.
- sugar metabolism module genes aceF, pgi, lpdA, ppk, ptsH, ptsI, glcF, glcE, fsaA, agaW; (3) 6dEB precursor metabolism module Gene yjiM, scpA, scpB, tdcD, tdcE, pflB, pflD, PaaF, ackA, pta, ybiW; (4) pentose phosphate and glyoxylate pathway module genes yaeR, rpiA, rpiB, purH, pyrB, pyrI, cysQ, pyrC, gmk, guaA, guaB, ndk, pyrF, pyrE, pyrH, hpt;
- Target gene expression sRNA plasmid library pSJ01 target gene tdcD
- pSJ02 target gene scpB
- pSJ03 target gene scpA
- pSJ04 target gene ybiW
- pSJ05 target gene pflB
- pSJ06 target gene tdcE
- pSJ07 target gene pflD
- pSJ08 target gene paaF
- pSJ09 target gene fadJ
- pSJ10 target gene fadB
- pSJ11 target gene ackA
- pSJ12 target gene pta
- pSJ13 target gene leuD
- pSJ14 target gene leuC
- pSJ15 target gene yjiM
- pSJ16 target gene purT
- pSJ17 target gene dhaK1
- pSJ18 target gene dhaK2
- pSJ19 Target gene
- sucC-sRNA-F and sucC-sRNA-R in Table 1 are used.
- the PCR conditions in Table 2 using the template plasmid pJF650 as a template, directly mutate the 24 bp deoB target gene binding site in the pJF650 plasmid backbone to the sucC target gene binding site by site-directed mutagenesis (with the sucC gene from ATG)
- the 24 bp DNA sequence after the start of the start sequence was completely complementary), and the sRNA plasmid pSJ39 capable of weakening the expression of the sucC gene was obtained.
- the specific steps of the polyketide biosynthesis of E.coli WG strain are as follows:
- the 675 bp sfp gene (encoding phosphopantetheinylamine transferase, Genbank No.: NC_000964) was amplified by PCR using the primers sfp-F and sfp-R (Table 4) using the B. subtilis genome as a template.
- the PCR product and pET21c plasmid (purchased from Novagen) were digested with NdeI and BamHI, respectively, and ligated with T4 DNA ligase to construct plasmid pET21c-sfp;
- the plasmid pUC19-sacB/kan was used as a template, and the Kan-SacB fragment (about 2.8 Kb) of the selection marker gene carrying the homology arm was amplified with the primers SacB/Kan-F and SacB/Kan-R (Table 4). Then, using the ⁇ Red/ET homologous recombination method (Datsenki et al. PNAS, 2000, 97: 6640-6645) to integrate Kan-SacB into the propionic acid metabolism operon of the E.
- coli BL21 (DE3) chromosome, replacing propionic acid A DNA fragment of prpR, prpB, prpC and prpD in a metabolic operon; using SWG-F and WG-R as primers to amplify the SacB/Kan gene located on the chromosome of the recombinant strain for verification;
- the sfp gene carrying the T7 promoter was amplified from pET21c-sfp using sfpR-F and sfpR-R; the SacB-Kan fragment in the recombinant strain was replaced with T7-sfp-T7 again by the ⁇ Red/ET homologous recombination method.
- the fragment finally obtains the E. coli WG strain suitable for the biosynthesis of polyketides required by the present invention, and a schematic diagram of the specific modification process is shown in FIG. 2 .
- eryAII-F encoding erythromycin streptavidin polyketide synthase
- primers pccB-F, pccB-R and accA2-F, accA2-R were used to amplify the pccB gene (encoding propionyl-CoA carboxylase ⁇ -CT).
- the purified PCR products were respectively NcoI/EcoRI
- the double-digested PCR product was ligated to the pET28a plasmid digested with NcoI/EcoRI using T4 DNA ligase to construct plasmids pZG01 and pZG02; then pZG02 was digested with XbaI/EcoRI and recovered.
- the DNA fragment containing the gene accA2 was ligated with the pZG01 plasmid double-digested with SpeI/EcoRI using T4 DNA ligase to obtain plasmid pZG03.
- the eryAI gene (encoding erythromycin streptavidin polyketide synthase DEBS1, Genbank No.: NC_009142) was amplified by primer eryAI-F and eryAI-R using genomic DNA of S. erythropolis as a template, and purified by NdeI. /EcoRI double digestion, and then ligated to the plasmid pET28a double-digested with NdeI/EcoRI with T4 DNA ligase to construct plasmid pZG04;
- the linearization of pZG04 was performed by BglII, and the linearized fragment was recovered. Then, the end of the linearized fragment was digested with exonuclease I, and the viscous end after BglII digestion was converted into a blunt end, and the linearized fragment was recovered again. The fragment is digested with HindIII to recover the eryAI gene fragment;
- the pZG03 was first linearized and recovered with EcoRI, and then the viscous end of EcoRI was converted to a blunt end with exonuclease I and recovered, and finally the linearized pZG03 was digested with HindIII and recovered.
- the recovered pZG03 and eryAI gene fragments were ligated to construct plasmid pZG08, and the construction process of the plasmid pZG08 is shown in FIG.
- the plasmid pZG07, pZG08 was co-transformed into the polyketide heterologous synthetic host E.coli WG obtained above, and an engineering strain E.coli WG (pZG07/pZG08) capable of efficiently synthesizing polyketide was obtained (for the production of red mold) Precursor 6-dEB).
- a single colony was picked up in 2 ml of LB medium supplemented with 100 mg/L carbenicillin and 50 mg/L kanamycin, and cultured at 220 rpm overnight at 37 ° C to obtain a primary seed.
- the primary seed obtained above was inoculated into a 500 ml shake flask containing 50 ml of LB medium supplemented with 100 mg/L carbenicillin and 50 mg/L kanamycin at a 1% inoculum, and cultured at 37 ° C at 220 rpm.
- the OD 600 is about 1, and the secondary seed is obtained.
- the resulting secondary seeds were then inoculated at a 1% inoculum into a 5 L jar containing 4 L of fermentation medium supplemented with 100 mg/L carbenicillin and 50 mg/L kanamycin while adding a final concentration of 0.1 mM.
- IPTG, 20 mM sodium propionate was induced to culture for 5 days at 22 ° C, 250 rpm, and the fermentation was completed.
- Example 5 The fermentation broth of Example 5 was extracted three times with an equal volume of ethyl acetate, and concentrated under reduced pressure to give a crude extract.
- the crude extract was eluted in a reverse phase C-18 column using a methanol water system at a ratio of 30%, 50%, 70% and 100%, respectively.
- the separated fractions were analyzed using an Ultimate 3000 analytical HPLC. The analysis showed that the product 6-dEB was mainly present in the component of 70% methanol, and the HPLC analysis is shown in Fig. 5.
- HPLC-ELSD detection conditions were as follows: 70% methanol eluted fractions were prepared by HPLC 3000 preparative HPLC. Columns were: TSK-100V, 5 ⁇ m, 19*150mm, flow rate 15ml/min, mobile phase The 50% acetonitrile/water system was eluted isocratically.
- ELSD detector conditions Evaporative light scattering detector drift tube temperature: 95 ° C; gas flow rate: 1.6 l / min; gain: 16. Retention time t R : 10.3 min.
- the 6-dEB pure product was verified by nuclear magnetic analysis.
- the hydrogen spectrum is shown in Fig. 6.
- the chemical shift of the H spectrum of the prepared pure product is consistent with the literature report (Xin Gao, Sang Kook Woo, Michael J. Krecht, Total Synthesis of 6-Deoxyerythronolide B via CC Bond-Forming Transfer Hydrogenation, J. Am. Chem. Soc. 2013, 135, 4223-4226), this compound was determined to be 6-dEB.
- the prepared standard was formulated into a 40 mg/L methanol solution and analyzed by Ultimate 3000 analytical HPLC. Conditions: column TSK-100V, 5 ⁇ m, 4.6*150 mm; flow rate 1 ml/min; mobile phase acetonitrile/water, 50% The acetonitrile gradient eluted.
- ELSD is the detector, conditions: evaporative light scattering detector drift tube temperature: 95 ° C; gas flow rate: 1.6 l / min; gain: 16.
- the pure 6-dEB product has a very high purity and a purity of 98% or more.
- Example 1 Each of the sRNA plasmids expressed in Example 1 (using pACYCDuet-1 (purchased from Novagen) as a blank control) was transformed into the host cell E. coli WG (pZG07/pZG08) (for the production of erythromycin precursor 6 -dEB), single colonies were picked to 2ml LB medium containing carbenicillin (100mg/L), kanamycin (50mg/L) and chloramphenicol (34mg/L), 37°C, 250rpm /min was incubated overnight for 12 h, and the seeds were fermented as a shaker.
- E. coli WG pZG07/pZG08
- the above seeds were inoculated in a 100 ml shake flask containing 10 ml of 6-dEB fermentation medium (containing carboxypenicillin, kanamycin and chloramphenicol antibiotics) at a dose of 1%, and a sodium propionate at a final concentration of 20 mM was added.
- the precursor was initially induced with 100 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), and each sample was run in parallel three times. It was fermented for 5 days on a shaker at 22 ° C, 250 rpm / min. After the fermentation, the fermentation broth was poured into a 10 ml centrifuge tube and stored at -20 ° C for subsequent detection and analysis.
- IPTG isopropyl- ⁇ -D-thiogalactopyranoside
- Example 7 The fermentation broth of Example 7 was analyzed by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). Conditions: column TSK-100V, 5 ⁇ m, 4.6*150 mm; flow rate 1 ml/min; mobile phase acetonitrile/water, 50% acetonitrile isocratic elution. ELSD is the detector, conditions: evaporative light scattering detector drift tube temperature: 95 ° C; gas flow rate: 1.6 l / min; gain: 16.
- HPLC-ELSD high performance liquid chromatography-evaporative light scattering detector
- E.coli WG pZG07/pZG08/pACYCDunet-1
- weakening sucC could increase the yield of 6-dEB production by the host to 63.2%.
- the highest yield increase is E.coli WG (pZG07/pZG08/pSJ130), and its key target gene is talB (transaldolase).
- the weakening of talB can increase the yield of 6-dEB production by host to 1008.81%. .
- the sRNA method is used to regulate the metabolic network of E. coli chassis cells, and the heterologous synthesis of polyketides can be significantly improved by weakening these genes.
- Targets that increase the yield of polyketides by more than 20% by weakening are: ybiW, fadB, ackA, pta, yjiM, dhaK2, ptsH, ptsI, frdD, frdA, sdhA, sucC, sucD, glcE, lsrC, rpiA, serC, talA, talB, zwf, pyrI, cysQ, gmk, guaB, pyrH, hpt.
- the replacement resistance plasmid construction is based on the POE-PCR technique (You et al. Simple cloning via visual transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis Appl Environ Microbiol, 78(5): 1593-1595), For example, the resistance of the control plasmid pACYCDuent-1 was replaced.
- the Aparamycin-F and Aparamycin-R primers in Table 8 and PCR conditions were used, and pKC1139 (Streptomyces universal plasmid) was used as a template to directly
- the apramycin resistance gene was amplified from the pKC1139 plasmid; the pACYCDuent-1 plasmid was removed by PCR using the sRNA-Aparamycin-F and sRNA-Aparamycin-R primers in Table 8 and PCR conditions using pACYCDuent-1 as a template. Sequences other than the chloramphenicol resistance gene are amplified.
- the PCR conditions in Table 9 were used (Note: the apramycin resistance gene amplified in the first step and the pACYCDuent-1 containing no chloramphenicol resistance gene, 1 ⁇ l each) were used without primers.
- the PCR product was recovered by PCR cleaning and recovery kit, directly transformed into DH10B, and plated on an LB solid medium plate containing 50 mg/L of apramycin, and cultured overnight. A single colony was picked up in 2 ml of LB medium containing 50 mg/L of apramycin, cultured at 220 rpm, and cultured at 37 ° C overnight, and the plasmid was extracted using a plasmid extraction kit to obtain plasmid pSJ77.
- the remaining expression sRNA plasmid replacement resistance was the same as above, and the chloramphenicol resistance on the plasmid was replaced with apramycin resistance using the same primers and PCR conditions, and the sRNA plasmid information is shown in Table 10.
- Example 1 The above-described constructed sRNA plasmid (using pSJ77 as a blank control) and the expression sRNA plasmid constructed in Example 1 with a relative yield increase of 20% were combined and transferred to the host cell E.coli WG (pZG07/pZG08) (for production).
- the erythromycin precursor 6-dEB was detected by the fermentation method of Example 7 and the 6-dEB analysis in Example 8.
- the sRNA combined fermentation experiment further increased the yield of the polyketide 6-dEB by combining weakening key target genes with the 6-dEB yield of the control E. coli WG (pZG07/pZG08/pSJ39/pSJ77) being 100%.
- Example 10 weakening the two target sRNA plasmids to further increase the yield of polyketide 6-dEB
- the weakening targets for increasing the yield of polyketides by more than 550% are: nucleotide synthesis and other metabolic module genes talA, talB, zwf and pentose phosphate and glyoxylate pathway module gene pyrI, cysQ, gmk, guaB, pyrH, hpt, investigated the effect of simultaneously weakening the targets of these two modules on the yield of synthetic polyketide 6-dEB.
- sRNA scaffold targeting talB was amplified with pSJ130 as a template and sRNA-F/R as a primer (Table 12), and the PCR conditions were the same as in Table 9 in Example 9. After the PCR product was cleaned and recovered, it was digested with BamH I and Hind III; and pSJ129 was digested with Bgl II and Hind III as a carrier, and cleaned and recovered. The above PCR digested product and the pSJ129 double-cut product were ligated with T4 ligase to obtain plasmid pSJ333. The remaining plasmids were obtained in the same manner, and finally the plasmid pSJ404-pSJ421 was obtained, and the plasmid information is shown in Table 13.
- Example 8 The above constructed expression-binding sRNA plasmid (using pACYCDuent-1 as a blank control) was transferred to host cell E. coli WG (pZG07/pZG08) (for production of erythromycin precursor 6-dEB) using the fermentation method of Example 7. And 6-dEB analysis in Example 8.
- the sRNA combined fermentation experiment further increased the yield of the polyketide 6-dEB by combining weakening key target genes with the 6-dEB yield of the control E. coli WG (pZG07/pZG08/pSJ39/pSJ77) being 100%.
- Plasmid name Target of weakening sRNA plasmid 6-dEB production (mg/L) 6-dEB relative yield increase rate (%) pACYCDuent-1 Control 14.30 0.00 pSJ404 talA+pyrI 135.10 844.76
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Abstract
Description
Primer名称 | 序列(5’→3’) |
SRNA-F | CGGGATCCTAACACCGTGCGTGTTGACTATTTTA |
SRNA-R | CCCAAGCTTAGATCTACTAGTTATAAACGCAGAAAGG |
名称 | sRNA技术调控(弱化)靶点 | 抗性 |
pSJ333 | talA+talB | 氯霉素 |
pSJ334 | cysQ+guaB | 氯霉素 |
pSJ404 | talA+pyrI | 氯霉素 |
pSJ405 | talA+cysQ | 氯霉素 |
pSJ406 | talA+gmk | 氯霉素 |
pSJ407 | talA+guaB | 氯霉素 |
pSJ408 | talA+pyrH | 氯霉素 |
pSJ409 | talA+hpt | 氯霉素 |
pSJ410 | talB+pyrI | 氯霉素 |
pSJ411 | talB+cysQ | 氯霉素 |
pSJ412 | talB+gmk | 氯霉素 |
pSJ413 | talB+guaB | 氯霉素 |
pSJ414 | talB+pyrH | 氯霉素 |
pSJ415 | talB+hpt | 氯霉素 |
pSJ416 | zwf+pyrI | 氯霉素 |
pSJ417 | zwf+cysQ | 氯霉素 |
pSJ418 | zwf+gmk | 氯霉素 |
pSJ419 | zwf+guaB | 氯霉素 |
pSJ420 | zwf+pyrH | 氯霉素 |
pSJ421 | zwf+hpt | 氯霉素 |
质粒名称 | sRNA质粒弱化的靶点 | 6-dEB产量(mg/L) | 6-dEB相对产量提高率(%) |
pACYCDuent-1 | control | 14.30 | 0.00 |
pSJ404 | talA+pyrI | 135.10 | 844.76 |
pSJ405 | talA+cysQ | 128.87 | 801.19 |
pSJ406 | talA+gmk | 117.09 | 718.81 |
pSJ407 | talA+guaB | 209.38 | 1364.20 |
pSJ408 | talA+pyrH | 153.30 | 972.03 |
pSJ409 | talA+hpt | 118.84 | 731.05 |
pSJ410 | talB+pyrI | 145.38 | 916.64 |
pSJ411 | talB+cysQ | 120.11 | 739.93 |
pSJ412 | talB+gmk | 138.00 | 865.03 |
pSJ413 | talB+guaB | 60.05 | 319.93 |
pSJ414 | talB+pyrH | 149.79 | 947.48 |
pSJ415 | talB+hpt | 100.32 | 601.54 |
pSJ416 | zwf+pyrI | 152.75 | 968.18 |
pSJ417 | zwf+cysQ | 130.45 | 812.24 |
pSJ418 | zwf+gmk | 119.39 | 734.90 |
pSJ419 | zwf+guaB | 210.42 | 1371.47 |
pSJ420 | zwf+pyrH | 75.35 | 426.92 |
Claims (15)
- 一种促进合成聚酮类化合物6-脱氧红霉内酯的宿主菌生物合成聚酮类化合物6-脱氧红霉内酯的方法,其特征在于,所述方法包括:(1)在合成聚酮类化合物6-脱氧红霉内酯的宿主菌中,弱化靶基因表达;其中,所述的靶基因选自:(a)核苷酸合成和其它代谢模块基因purT、lsrC、hemN、zwf、pgl、gnd、rpe、talA、talB、tktA、tktB、ulaE或yieK;(b)磷酸戊糖和乙醛酸途径模块基因yaeR、rpiA、rpiB、purH、pyrB、pyrI、cysQ、pyrC、gmk、guaA、guaB、ndk、pyrF、pyrE、pyrH或hpt;(c)TCA循环和氧化磷酸化模块基因frdD、frdA、sdhA、sdhB、sdhC、sdhD、sucC、sucD、cyoA或cyoB;(d)糖代谢模块基因aceF、pgi、lpdA、ppk、ptsH、ptsI、glcF、glcE、fsaA或agaW;(e)6dEB前体代谢模块基因yjiM、scpA、scpB、tdcD、tdcE、pflB、pflD、PaaF、ackA、pta或ybiW;(f)脂肪酸代谢模块基因fadJ、fadB、dhaK1、dhaK2或dhaH;(g)氨基酸和蛋白质合成代谢模块基因leuC、leuD、serC、serB、serA、gdhA或tnaA;或(h)frdD+sucC组合、lsrC+frdD组合、lsrC+sucC组合、frdD+rpiA组合、talA+guaB组合或zwf+guaB组合;(2)培养步骤(1)制备的菌株,从而生物合成聚酮类化合物6-脱氧红霉内酯。
- 如权利要求1所述的方法,其特征在于,在合成聚酮类化合物6-脱氧红霉内酯的宿主菌中,弱化靶基因表达包括:引入抑制靶基因表达的干扰分子或敲除靶基因。
- 如权利要求2所述的方法,其特征在于,所述的抑制靶基因表达的干扰分子针对:sucC中SEQ ID NO:37所示的序列或其互补序列、tdcD中SEQ ID NO:2所示的序列或其互补序列、scpB中SEQ ID NO:3所示的序列或其互补序列、scpA中SEQ ID NO:4所示的序列或其互补序列、ybiW中SEQ ID NO:5所示的序列或其互补序列、pflB中SEQ ID NO:6所示的序列或其互补序列、tdcE中SEQ ID NO:7所示的序列或其互补序列、pflD中SEQ ID NO:8所示的序列或其互补序列、paaF中SEQ ID NO:9所示的序列或其互补序列、fadJ中SEQ ID NO:10所示的序列或其互补序列、fadB中SEQ ID NO:11所示的序列或其互补序列、ackA中SEQ ID NO:12所示的序列或其互补序列、pta中SEQ ID NO:13所示的序列或其互补序列、leuD中SEQ ID NO:14所示的序列或其互补序列、leuC中SEQ ID NO:15所示的序列或其互补序列、yjiM中SEQ ID NO:16所示的序列或其互补序列、purT中SEQ ID NO:17所示的序列或其互补序列、dhaK1中SEQ ID NO:18所示的序列或其互补序列、dhaK2中SEQ ID NO:19所示的序列或其互补序列、dhaH中SEQ ID NO:20所示的序列或其互补序列、ptsH中SEQ ID NO:21所示的序列或其互补序列、ptsI中SEQ ID NO:22所示的序列或其互补序列、fsaA中SEQ ID NO:23所示的序列或其互补序列、ppk中SEQ ID NO:24所示的序列或其互补序列、aceF中SEQ ID NO:25所示的序列或其互补序列、cyoA中SEQ ID NO:26所示的序列或其互补序列、frdD中SEQ ID NO:30所示的序列或其互补序列、frdA中SEQ ID NO:31所示的序列或其互补序列、pgi中SEQ ID NO:32所示的序列或其互补序列、sdhA中SEQ ID NO:33所示的序列或其互补序列、sdhB中SEQ ID NO:34所示的序列或其互补序列、sdhC中SEQ ID NO:35所示的序列或其互补序列、sdhD中SEQ ID NO:36所示的序列或其互补序列、sucD中SEQ ID NO:38所示的序列或其互补序列、tnaA中SEQ ID NO:39所示的序列或其互补序列、glcF中SEQ ID NO:40所示的序列或其互补序列、glcE中SEQ ID NO:41所示的序列或其互补序列、yaeR中SEQ ID NO:42所示的序列或其互补序列、lsrC中SEQ ID NO:43所示的序列或其互补序列、hemN中SEQ ID NO:44所示的序列或其互补序列、agaW中SEQ ID NO:45所示的序列或其互补序列、gdhA中SEQ ID NO:46所示的序列或其互补序列、cyoB中SEQ ID NO:47所示的序列或其互补序列、rpiA中SEQ ID NO:48所示的序列或其互补序列、rpiB中SEQ ID NO:49所示的序列或其互补序列、lpdA中SEQ ID NO:50所示的序列或其互补序列、serC中SEQ ID NO:51所示的序列或其互补序列、serB中SEQ ID NO:28所示的序列或其互补序列、serA中SEQ ID NO:29所示的序列或其互补序列、zwf中SEQ ID NO:174所示的序列或其互补序列、pgl中SEQ ID NO:175所示的序列或其互补序列、gnd中SEQ ID NO:176所示的序列或其互补序列、rpe中SEQ ID NO:177所示的序列或其互补序列、talA中SEQ ID NO:178所示的序列或其互补序列、talB中SEQ ID NO:179所示的序列或其互补序列、tktA中SEQ ID NO:180所示的序列或其互补序列、tktB中SEQ ID NO:181所示的序列或其互补序列、ulaE中SEQ ID NO:182所示的序列或其互补序列、yieK中SEQ ID NO:183所示的序列或其互补序列、purH中SEQ ID NO:184所示的序列或其互补序列、pyrB中SEQ ID NO:185所示的序列或其互补序列、pyrI中SEQ ID NO:186所示的序列或其互补序列、cysQ中SEQ ID NO:187所示的序列或其互补序列、pyrC中SEQ ID NO:188所示的序列或其互补序列、gmk中SEQ ID NO:189所示的序列或其互补序列、guaA中SEQ ID NO:190所示的序列或其互补序列、guaB中SEQ ID NO:191所示的序列或其互补序列、ndk中SEQ ID NO:192所示的序列或其互补序列、pyrF中SEQ ID NO:193所示的序列或其互补序列、pyre中SEQ ID NO:194所示的序列或其互补序列、pyrH中SEQ ID NO:195所示的序列或其互补序列、或hpt中SEQ ID NO:196所示的序列或其互补序列。
- 如权利要求1所述的方法,其特征在于,(1)中,所述的靶基因选自:(a)核苷酸合成和其它代谢模块基因lsrC、zwf、pgl、gnd、rpe、talA、talB、tktA、tktB、ulaE或yieK;(b)磷酸戊糖和乙醛酸途径模块基因rpiA、purH、pyrB、pyrI、cysQ、pyrC、gmk、guaA、guaB、ndk、pyrF、pyrE、pyrH或hpt;(c)TCA循环和氧化磷酸化模块基因frdD、frdA、sdhA、sucC或sucD;(d)糖代谢模块基因ptsH、ptsI或glcE;(e)6dEB前体代谢模块基因yjiM、ackA、pta或ybiW;(f)脂肪酸代谢模块基因fadB或dhaK2;(g)氨基酸和蛋白质合成代谢模块基因serC;或(h)frdD+sucC组合、lsrC+frdD组合、lsrC+sucC组合、talA+guaB组合或zwf+guaB组合。
- 如权利要求2所述的方法,其特征在于,所述的抑制靶基因表达的干扰分子是sRNA。
- 如权利要求5所述的方法,其特征在于,所述的sRNA包括以下结构:启动子、靶基因抑制分子、终止子;较佳地,所述靶基因抑制分子与终止子之间,还包括:micF序列。
- 如权利要求5所述的方法,其特征在于,所述的sRNA被包含在表达载体中。
- 如权利要求1所述的方法,其特征在于,所述的合成聚酮类化合物6-脱氧红霉内酯的宿主菌是能合成聚酮类化合物6-脱氧红霉内酯的原核细菌。
- 如权利要求8所述的方法,其特征在于,所述的能合成聚酮类化合物6-脱氧红霉内酯的原核细菌是能合成聚酮类化合物6-脱氧红霉内酯的大肠杆菌;较佳地,所述的大肠杆菌中,敲除了丙酸代谢操纵子并在该敲除位点上整合了磷酸泛酰巯基乙胺转移酶基因sfp;或直接敲除丙酸代谢操纵子,将sfp整合到大肠杆菌基因组中任意一个非必须基因或无功能DNA序列区域。
- 如权利要求9所述的方法,其特征在于,所述的大肠杆菌中转化有表达编码红霉素链霉菌聚酮合成酶DEBS2的基因、编码红霉素链霉菌聚酮合成酶DEBS3的基因、编码丙酰辅酶A羧化酶β-CT亚基的基因、编码丙酰辅酶A羧化酶a-CT亚基的基因和 编码红霉素链霉菌聚酮合成酶DEBS1的基因。
- 一种抑制靶基因表达的干扰分子,其是sRNA,其特征在于,所述的sRNA包括以下结构:启动子、靶基因抑制分子、终止子;较佳地,所述靶基因抑制分子与终止子之间,还包括:micF序列;其中,所述的靶基因抑制分子针对:sucC中SEQ ID NO:37所示的序列或其互补序列、tdcD中SEQ ID NO:2所示的序列或其互补序列、scpB中SEQ ID NO:3所示的序列或其互补序列、scpA中SEQ ID NO:4所示的序列或其互补序列、ybiW中SEQ ID NO:5所示的序列或其互补序列、pflB中SEQ ID NO:6所示的序列或其互补序列、tdcE中SEQ ID NO:7所示的序列或其互补序列、pflD中SEQ ID NO:8所示的序列或其互补序列、paaF中SEQ ID NO:9所示的序列或其互补序列、fadJ中SEQ ID NO:10所示的序列或其互补序列、fadB中SEQ ID NO:11所示的序列或其互补序列、ackA中SEQ ID NO:12所示的序列或其互补序列、pta中SEQ ID NO:13所示的序列或其互补序列、leuD中SEQ ID NO:14所示的序列或其互补序列、leuC中SEQ ID NO:15所示的序列或其互补序列、yjiM中SEQ ID NO:16所示的序列或其互补序列、purT中SEQ ID NO:17所示的序列或其互补序列、dhaK1中SEQ ID NO:18所示的序列或其互补序列、dhaK2中SEQ ID NO:19所示的序列或其互补序列、dhaH中SEQ ID NO:20所示的序列或其互补序列、ptsH中SEQ ID NO:21所示的序列或其互补序列、ptsI中SEQ ID NO:22所示的序列或其互补序列、fsaA中SEQ ID NO:23所示的序列或其互补序列、ppk中SEQ ID NO:24所示的序列或其互补序列、aceF中SEQ ID NO:25所示的序列或其互补序列、cyoA中SEQ ID NO:26所示的序列或其互补序列、frdD中SEQ ID NO:30所示的序列或其互补序列、frdA中SEQ ID NO:31所示的序列或其互补序列、pgi中SEQ ID NO:32所示的序列或其互补序列、sdhA中SEQ ID NO:33所示的序列或其互补序列、sdhB中SEQ ID NO:34所示的序列或其互补序列、sdhC中SEQ ID NO:35所示的序列或其互补序列、sdhD中SEQ ID NO:36所示的序列或其互补序列、sucD中SEQ ID NO:38所示的序列或其互补序列、tnaA中SEQ ID NO:39所示的序列或其互补序列、glcF中SEQ ID NO:40所示的序列或其互补序列、glcE中SEQ ID NO:41所示的序列或其互补序列、yaeR中SEQ ID NO:42所示的序列或其互补序列、lsrC中SEQ ID NO:43所示的序列或其互补序列、hemN中SEQ ID NO:44所示的序列或其互补序列、agaW中SEQ ID NO:45所示的序列或其互补序列、gdhA中SEQ ID NO:46所示的序列或其互补序列、cyoB中SEQ ID NO:47所示的序列或其互补序列、rpiA中SEQ ID NO:48所示的序列或其互补序列、rpiB中SEQ ID NO:49所示的序列或其互补序列、lpdA中SEQ ID NO:50所示的序列或其互补序列、serC中SEQ ID NO:51所示的序列或其互补序列、serB中SEQ ID NO:28所示的序列或其互补序列、serA中SEQ ID NO:29所示的序列或其互补序列、zwf中SEQ ID NO:174所示的序列或其互补序列、pgl中SEQ ID NO:175所示的序列或其互补序列、gnd中SEQ ID NO:176所示的序列或其互补序列、rpe中SEQ ID NO:177所示的序列或其互补序列、talA中SEQ ID NO:178所示的序列或其互补序列、talB中SEQ ID NO:179所示的序列或其互补序列、tktA中SEQ ID NO:180所示的序列或其互补序列、tktB中SEQ ID NO:181所示的序列或其互补序列、ulaE中SEQ ID NO:182所示的序列或其互补序列、yieK中SEQ ID NO:183所示的序列或其互补序列、purH中SEQ ID NO:184所示的序列或其互补序列、pyrB中SEQ ID NO:185所示的序列或其互补序列、pyrI中SEQ ID NO:186所示的序列或其互补序列、cysQ中SEQ ID NO:187所示的序列或其互补序列、pyrC中SEQ ID NO:188所示的序列或其互补序列、gmk中SEQ ID NO:189所示的序列或其互补序列、guaA中SEQ ID NO:190所示的序列或其互补序列、guaB中SEQ ID NO:191所示的序列或其互补序列、ndk中SEQ ID NO:192所示的序列或其互补序列、pyrF中SEQ ID NO:193所示的序列或其互补序列、pyre中SEQ ID NO:194所示的序列或其互补序列、pyrH中SEQ ID NO:195所示的序列或其互补序列、或hpt中SEQ ID NO:196所示的序列或其互补序列。
- 权利要求11所述的抑制靶基因表达的干扰分子的用途,其特征在于,用于转化合成聚酮类化合物6-脱氧红霉内酯的宿主菌,弱化相应靶基因,促进合成聚酮类化合物6-脱氧红霉内酯的宿主菌生物合成聚酮类化合物6-脱氧红霉内酯。
- 一种用于合成聚酮类化合物6-脱氧红霉内酯的宿主菌,其特征在于,所述的宿主菌中转化有抑制靶基因表达的干扰分子,或所述宿主菌中敲除了靶基因;其中,所述的靶基因选自:(a)核苷酸合成和其它代谢模块基因purT、lsrC、hemN、zwf、pgl、gnd、rpe、talA、talB、tktA、tktB、ulaE或yieK;(b)磷酸戊糖和乙醛酸途径模块基因yaeR、rpiA、rpiB、purH、pyrB、pyrI、cysQ、pyrC、gmk、guaA、guaB、ndk、pyrF、pyrE、pyrH或hpt;(c)TCA循环和氧化磷酸化模块基因frdD、frdA、sdhA、sdhB、sdhC、sdhD、sucC、sucD、cyoA或cyoB;(d)糖代谢模块基因aceF、pgi、lpdA、ppk、ptsH、ptsI、glcF、glcE、fsaA或agaW;(e)6dEB前体代谢模块基因yjiM、scpA、scpB、tdcD、tdcE、pflB、pflD、PaaF、ackA、pta或ybiW;(f)脂肪酸代谢模块基因靶点fadJ、fadB、dhaK1、dhaK2或dhaH;(g)氨基酸和蛋白质合成代谢模块基因leuC、leuD、serC、serB、serA、gdhA或tnaA;或(h)frdD+sucC组合、lsrC+frdD组合、lsrC+sucC组合、frdD+rpiA组合、talA+guaB 组合或zwf+guaB组合。
- 如权利要求13所述的宿主菌,其特征在于,所述的宿主菌中转化有权利要求11所述的抑制靶基因表达的干扰分子;和/或所述的合成聚酮类化合物6-脱氧红霉内酯的宿主菌是能合成聚酮类化合物6-脱氧红霉内酯的原核细菌;较佳地,所述的大肠杆菌中,敲除了丙酸代谢操纵子并在该敲除位点上整合了磷酸泛酰巯基乙胺转移酶基因sfp;较佳地,所述的大肠杆菌中转化有表达编码红霉素链霉菌聚酮合成酶DEBS2的基因、编码红霉素链霉菌聚酮合成酶DEBS3的基因、编码丙酰辅酶A羧化酶β-CT亚基的基因、编码丙酰辅酶A羧化酶a-CT亚基的基因和编码红霉素链霉菌聚酮合成酶DEBS1的基因。
- 一种用于促进合成聚酮类化合物6-脱氧红霉内酯的宿主菌生物合成聚酮类化合物6-脱氧红霉内酯的试剂盒,其特征在于,所述的试剂盒中包括:权利要求13所述的宿主菌的总和;或所述的试剂盒中包括:权利要求11所述的抑制靶基因表达的干扰分子的总和;或所述的试剂盒中包括:分别包含权利要求11所述的抑制靶基因表达的干扰分子的载体的总和。
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