Method for improving heterologous synthesis of Escherichia coli into polyketides and use of same

The present invention falls within the fields of synthetic biology and industrial biotechnology, in particular, the present invention relates to a method for improving the heterologous synthesis of a polyketide by E. coli and use thereof.

Natural products play an important role in the process of drug development and discovery. Over the past decade, researchers have used natural products heterologously synthesized and the products have shown great potential. The heterologous biosynthesis of polyketids is one of the branches researched deeply and progressed rapidly in the current field of synthetic biology. Donadio proposed a synthetic model of 6-deoxyerythromycin lactone B in 1991, and studies on erythromycin polyketide synthases (PKSs) have thus become a paradigm for Type I PKSs. The biosynthesis of erythromycin is divided into two parts: the first is the formation of 6-deoxyerythronolide-B (6-dEB), the parent nucleus of erythromycin; and the second is the sidechain glycosylation of 6-dEB to synthesize erythromycin. 6-dEB is the first intermediate that can be isolated during the synthesis of erythromycin, and the starting materials for the synthesis of 6-dEB are propionic acid and methylmalonic acid. The entire process is catalyzed by polyketide synthases. Engineering ideas promote researchers to construct an engineered strain for the heterologous synthesis of natural products. As the metabolic network of E. coli has been researched most deeply, E. coli is often used as the chassis cell for engineered strains.

In 2001, Pfeifer et al. heterologously synthesized 6-dEB, the first precursor of erythromycin, using E. coli , opening the first line of the heterologous biosynthesis of erythromycin. Later researchers have also made many attempts to obtain erythromycin efficiently through E. coli , for example, in order to improve plasmid stability, Murli et al., in 2003, obtained an engineered strain K207-3/pKOS207-129/pBP130, with 22.5 mg/L of 6-dEB being obtained by culturing the same in a shake flask, by modifying pBP144 to pKOS207-129 through integrating genes pccB and pccA into the YgfG site encoding methylmalonyl-CoA decarboxylase in E. coli and replacing the starting site of pET28a derived from pBR322 with the replication origin of RSF1010. In order to improve the stability of the host itself, WANG Yong et al. obtained a chromosome-modified stable strain by integrating erythromycin polyketide synthase genes eryAI, AII and AIII into the chromosome of E. coli through a chromosome recombination Red/ET method, wherein the strain may stably synthesize 6-dEB, an intermediate of erythromycin, compared with the co-expression of multiple plasmids.

Over the recent decade, the heterologous synthesis of polyketids with erythromycin as the representative in E. coli has made a series of significant progresses, and erythromycin A was successfully synthesized in E. coli . However, there are still many problems, for example, the yield of erythromycin in E. coli is too low, compared with the original strain, Saccharopolyspora erythraea . MENG Hailin et al., in 2011, found that the actual yield of polyketids synthesized by E. coli was only about 1/10 of the theoretical yield currently after analyzing the metabolic network of E. coli using an in silico analysis and research platform, indicating that the heterogeneous synthetic pathway of polyketids is regulated by the global network of E. coli . Therefore, by modifying the global network of E. coli , it is possible to further improve its ability to heterologously synthesize polyketids.

An object of the present invention is to provide a method for improving the heterologous synthesis of a polyketide by E. coli and use thereof.

In a first aspect of the present invention, provided is a method for promoting a host strain for synthesizing the polyketide 6-deoxyerythronolide to synthesize the polyketide 6-deoxyerythronolide biologically, the method comprising:

(1) attenuating the expression of a target gene in the host strain for synthesizing the polyketide 6-deoxyerythronolide;

wherein, the target gene is selected from:

(a) a gene for nucleotide synthesis and other metabolism modules, purT, lsrC, hemN, zwf, pgl, gnd, rpe, talA, talB, tktA, tktB, ulaE or yieK;

(b) a gene for pentose phosphate and glyoxylate pathway modules, yaeR, rpiA, rpiB, purH, pyrB, pyrI, cysQ, pyrC, gmk, guaA, guaB, ndk, pyrF, pyrE, pyrH or hpt;

(c) a gene for TCA cycle and oxidative phosphorylation modules, frdD, frdA, sdhA, sdhB, sdhC, sdhD, sucC, sucD, cyoA or cyoB;

(d) a gene for carbohydrate metabolism module, aceF, pgi, lpdA, ppk, ptsH, ptsI, glcF, glcE, fsaA or agaW;

(e) a gene for 6-dEB precursor metabolism module, yjiM, scpA, scpB, tdcD, tdcE, pflB, pflD, PaaF, ackA, pta or ybiW;

(f) a gene target for fatty acid metabolism module, fadJ, fadB, dhaK1, dhaK2 or dhaH;

(g) a gene for amino acid and protein synthetic metabolism modules, leuC, leuD, serC, serB, serA, gdhA or tnaA; or

(h) the combination of frdD+sucC, the combination of lsrC+frdD, the combination of lsrC+sucC, the combination of frdD+rpiA, the combination of talA+guaB or the combination of zwf+guaB; and

(2) culturing the strain prepared in step (1), thereby synthesizing the polyketide 6-deoxyerythronolide biologically.

In a preferred embodiment, attenuating the expression of a target gene in the host strain for synthesizing the polyketide 6-deoxyerythronolide includes: introducing an interfering molecule that inhibits the expression of the target gene or knocking out the target gene.

In another preferred embodiment, the interfering molecule that inhibits the expression of the target gene is directed to the following (or has the same itself):

a sequence shown in SEQ ID NO: 37 in sucC or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 2 in tdcD or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 3 in scpB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 4 in scpA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 5 in ybiW or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 6 in pflB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 7 in tdcE or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 8 in pflD or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 9 in paaF or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 10 in fadJ or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 11 in fadB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 12 in ackA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 13 in pta or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 14 in leuD or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 15 in leuC or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 16 in yjiM or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 17 in purT or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 18 in dhaK1 or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 19 in dhaK2 or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 20 in dhaH or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 21 in ptsH or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 22 in ptsI or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 23 in fsaA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 24 in ppk or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 25 in aceF or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 26 in cyoA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 30 in frdD or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 31 in frdA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 32 in pgi or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 33 in sdhA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 34 in sdhB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 35 in sdhC or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 36 in sdhD or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 38 in sucD or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 39 in tnaA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 40 in glcF or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 41 in glcE or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 42 in yaeR or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 43 in lsrC or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 44 in hemN or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 45 in agaW or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 46 in gdhA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 47 in cyoB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 48 in rpiA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 49 in rpiB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 50 in lpdA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 51 in serC or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 28 in serB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 29 in serA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 174 in zwf or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 175 in pgl or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 176 in gnd or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 177 in rpe or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 178 in talA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 179 in talB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 180 in tktA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 181 in tktB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 182 in ulaE or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 183 in yieK or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 184 in purH or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 185 in pyrB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 186 in pyrI or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 187 in cysQ or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 188 in pyrC or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 189 in gmk or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 190 in guaA or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 191 in guaB or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 192 in ndk or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 193 in pyrF or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 194 in pyre or a complementary sequence thereof,

a sequence shown in SEQ ID NO: 195 in pyrH or a complementary sequence thereof, or

a sequence shown in SEQ ID NO: 196 in hpt or a complementary sequence thereof.

In another preferred embodiment, in (1) of the method, the target gene is selected from:

(a) a gene for nucleotide synthesis and other metabolism modules, lsrC, zwf, pgl, gnd, rpe, talA, talB, tktA, tktB, ulaE or yieK;

(b) a gene for pentose phosphate and glyoxylate pathway modules, rpiA, purH, pyrB, pyrI, cysQ, pyrC, gmk, guaA, guaB, ndk, pyrF, pyrE, pyrH or hpt;

(c) a gene for TCA cycle and oxidative phosphorylation modules, frdD, frdA, sdhA, sucC or sucD;

(d) a gene for carbohydrate metabolism module, ptsH, ptsI or glcE;

(e) a gene for 6-dEB precursor metabolism module, yjiM, ackA, pta or ybiW;

(f) a gene target for fatty acid metabolism module, fadB or dhaK2;

(g) a gene for amino acid and protein synthetic metabolism modules, serC; or

(h) the combination of frdD+sucC, the combination of lsrC+frdD, the combination of lsrC+sucC, the combination of talA+guaB or the combination of zwf+guaB.

In another preferred embodiment, the interfering molecule that inhibits the expression of the target gene is sRNA.

In another preferred embodiment, the sRNA comprises the following structure:

a promoter, a target gene-inhibiting molecule (e.g., a target gene-binding sequence) and a terminator.

In another preferred embodiment, the promoter is selected from: Pr promoter (preferably, having the sequence of position 7 to position 61 in SEQ ID NO: 52), P BAD promoter, T7 promoter and Trc promoter.

In another preferred embodiment, the terminator is selected from: TE terminator (preferably, having the sequence of position 171 to position 310 in SEQ ID NO: 52), T1/TE terminator, T7 terminator, rrnB terminator, rrnB T1 and T2 terminators.

In another preferred embodiment, the following is also included between the interfering molecule that inhibits the expression of the target gene and the terminator: micF sequence (e.g., the sequence of position 110 to position 170 in SEQ ID NO: 52).

In another preferred embodiment, the target gene-inhibiting molecule is a short nucleic acid sequence, such as 18-26 bp in length; and preferably 20-24 bp; which can be complementary or bind to the mRNA of the target gene, or which has a stretch of the sequence of the mRNA of the target gene, wherein the stretch of the sequence can express a sequence that is complementary or binds to the mRNA of the target gene after being transferred into a cell.

In another preferred embodiment, the sRNA is included in an expression vector.

In another preferred embodiment, the host strain for synthesizing the polyketide 6-deoxyerythronolide is a prokaryotic bacterium capable of synthesizing the polyketide 6-deoxyerythronolide.

In another preferred embodiment, the prokaryotic bacterium capable of synthesizing the polyketide 6-deoxyerythronolide is E. coli capable of synthesizing the polyketide 6-deoxyerythronolide;

preferably, in the E. coli , the operon for propionic acid metabolism is knocked out and phosphopantetheinyl transferase gene sfp is integrated into the knockout site; or the operon for propionic acid metabolism is directly knocked out and sfp is integrated into any non-essential gene or a non-functional DNA sequence region in the genome of E. coli.

In another preferred embodiment, the E. coli is transformed with a gene encoding polyketide synthetase DEBS2 of Streptomyces erythreus , a gene encoding polyketide synthetase DEBS3 of Streptomyces erythreus , a gene encoding propionyl-CoA carboxylase β-CT subunit, a gene encoding propionyl-CoA carboxylase a-CT subunit and a gene encoding polyketide synthetase DEBS1 of Streptomyces erythreus.

In another aspect of the present invention, provided is an interfering molecule that inhibits the expression of a target gene, which is an sRNA comprising the following structure (preferably, the following is also included between the interfering molecule that inhibits the expression of the target gene and the terminator: micF sequence):

a promoter, a target gene-inhibiting molecule and a terminator.

In another aspect of the present invention, provided is use of the interfering molecule (e.g., sRNA) that inhibits the expression of a target gene, for transforming a host strain for synthesizing the polyketide 6-deoxyerythronolide, attenuating the corresponding target gene or promoting a host strain for synthesizing the polyketide 6-deoxyerythronolide to synthesize the polyketide 6-deoxyerythronolide biologically

In another aspect of the present invention, provided is a host strain for synthesizing the polyketide 6-deoxyerythronolide, wherein the host strain is transformed with an interfering molecule that inhibits the expression of a target gene, or the host strain has the target gene knocked out; wherein, the target gene is selected from: (a) a gene for nucleotide synthesis and other metabolism modules, purT, lsrC, hemN, zwf, pgl, gnd, rpe, talA, talB, tktA, tktB, ulaE or yieK;

(b) a gene for pentose phosphate and glyoxylate pathway modules, yaeR, rpiA, rpiB, purH, pyrB, pyrI, cysQ, pyrC, gmk, guaA, guaB, ndk, pyrF, pyrE, pyrH or hpt;

(c) a gene for TCA cycle and oxidative phosphorylation modules, frdD, frdA, sdhA, sdhB, sdhC, sdhD, sucC, sucD, cyoA or cyoB;

(d) a gene for carbohydrate metabolism module, aceF, pgi, lpdA, ppk, ptsH, ptsI, glcF, glcE, fsaA or agaW;

(e) a gene for 6-dEB precursor metabolism module, yjiM, scpA, scpB, tdcD, tdcE, pflB, pflD, PaaF, ackA, pta or ybiW;

(f) a gene target for fatty acid metabolism module, fadJ, fadB, dhaK1, dhaK2 or dhaH;

(g) a gene for amino acid and protein synthetic metabolism modules, leuC, leuD, serC, serB, serA, gdhA or tnaA; or

(h) the combination of frdD+sucC, the combination of lsrC+frdD, the combination of lsrC+sucC, the combination of frdD+rpiA, the combination of talA+guaB or the combination of zwf+guaB.

In another preferred embodiment, the host strain is transformed with the interfering molecule (e.g., sRNA) that inhibits the expression of the target gene; and/or the host strain for synthesizing the polyketide 6-deoxyerythronolide is a prokaryotic bacterium capable of synthesizing the polyketide 6-deoxyerythronolide; preferably, in the E. coli , the operon for propionic acid metabolism is knocked out and phosphopantetheinyl transferase gene sfp is integrated into the knockout site; and preferably, the E. coli is transformed with a gene encoding polyketide synthetase DEBS2 of Streptomyces erythreus , a gene encoding polyketide synthetase DEBS3 of Streptomyces erythreus , a gene encoding propionyl-CoA carboxylase β-CT subunit, a gene encoding propionyl-CoA carboxylase a-CT subunit and a gene encoding polyketide synthetase DEBS1 of Streptomyces erythreus.

In another aspect of the present invention, provided is a kit for promoting a host strain for synthesizing the polyketide 6-deoxyerythronolide to synthesize the polyketide 6-deoxyerythronolide biologically, the kit comprising: the sum of the host strains (from which one or more strains those skilled in the art can select for production); or

the kit comprising: the sum of the interfering molecules that inhibit the expression of a target gene (from which one or more interfering molecules that inhibit the expression of the target gene those skilled in the art can select for application); or

the kit comprising: the sum of vectors that respectively comprise the interfering molecule that inhibits the expression of a target gene (from which one or more vectors those skilled in the art can select for application).

Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein.

FIG. 1 , a schematic diagram of sRNA-expressing plasmid PJF650.

FIG. 2 , a schematic diagram of the construction process of E. coli WG.

FIG. 3 , the construction process of plasmid pZG07.

FIG. 4 , the construction process of plasmid pZG08.

FIG. 5 , HPLC analysis of isolated fractions of 6-dEB.

FIG. 6 , hydrogen NMR spectrum of pure 6-dEB prepared.

FIG. 7 , HPLC-ELSD analysis diagram of 6-dEB at 40 mg/L.

FIG. 8 , effect of sRNA combinatorial regulation on the heterologous synthesis of 6-dEB by E. coli.

After an in-depth research, the present inventors have first disclosed a method for promoting a host strain for synthesizing the polyketide 6-dEB to synthesize the polyketide 6-dEB biologically, by attenuating individually or in combination the expression of sucC (which expresses succinyl-CoA synthetase) and other genes in E. coli , to significantly increase the yield of the polyketide heterologously synthesized in E. coli , wherein the highest yield increase rate can reach 60% or more. The present method enables massive accumulation of the polyketide in the fermentation process.

As used herein, the “interfering molecule that inhibits the expression of a target gene” refers to an agent that can specifically reduce the expression level of the target gene, including a variety of molecules known in the art that can inhibit the expression of the target gene, such as, but not limited to, antisense nucleic acids, locked nucleic acids, peptide nucleic acids, siRNAs, shRNAs and microRNAs (collectively referred to as target gene-inhibiting molecules); or constructs that carry or express antisense nucleic acids, locked nucleic acids, peptide nucleic acids, siRNAs, shRNAs, microRNAs, etc.

As used herein, the “heterologous” refers to the relationship between two or more nucleic acids or protein sequences from different sources, or the relationship between a protein (or nucleic acid) and a host cell from different sources. For example, if the combination of a nucleic acid and a host cell is usually 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.

As used herein, the “host strain for synthesizing the polyketide 6-dEB” refers to a host strain or host cell known in the art that can be applied to synthesize the polyketide 6-dEB. The host strain includes, but is not limited to, E. coli and improved E. coli (including strains transformed with pccB and pccA, strains transformed with eryAI, AII and/or AIII, etc.). Preferred host strain is E. coli which has its operon for propionic acid metabolism knocked out and phosphopantetheinyl transferase gene sfp integrated into the knockout site, and which is transformed with a gene encoding polyketide synthetase DEBS2 of Streptomyces erythreus , a gene encoding polyketide synthetase DEBS3 of Streptomyces erythreus , a gene encoding propionyl-CoA carboxylase β-CT subunit, a gene encoding propionyl-CoA carboxylase a-CT subunit and a gene encoding polyketide synthetase DEBS1 of Streptomyces erythreus.

In order to optimize the formation of the polyketide 6-dEB, the present inventors have made extensive studies to find a suitable target gene for improvement, down-regulation of the expression of which helps to promote the formation of 6-dEB. The various target genes disclosed in the present invention are genes known in the art, those skilled in the art can query the sequence information of these genes through GenBank and other platforms, and it is thus easy for those skilled in the art to obtain these genes.

The present invention also provides a set of genes, wherein the set of genes comprises: (a) a gene for nucleotide synthesis and other metabolism modules, purT, lsrC, hemN, zwf, pgl, gnd, rpe, talA, talB, tktA, tktB, ulaE or yieK; (b) a gene for pentose phosphate and glyoxylate pathway modules, yaeR, rpiA, rpiB, purH, pyrB, pyrI, cysQ, pyrC, gmk, guaA, guaB, ndk, pyrF, pyrE, pyrH or hpt; (c) a gene for TCA cycle and oxidative phosphorylation modules, frdD, frdA, sdhA, sdhB, sdhC, sdhD, sucC, sucD, cyoA or cyoB; (d) a gene for carbohydrate metabolism module, aceF, pgi, lpdA, ppk, ptsH, ptsI, glcF, glcE, fsaA or agaW; (e) a gene for 6-dEB precursor metabolism module, yjiM, scpA, scpB, tdcD, tdcE, pflB, pflD, PaaF, ackA, pta or ybiW; (f) a gene target for fatty acid metabolism module, fadJ, fadB, dhaK1, dhaK2 or dhaH; and (g) a gene for amino acid and protein synthetic metabolism modules, leuC, leuD, serC, serB, serA, gdhA or tnaA. Those skilled in the art, according to the present disclosure, may select one or more genes from the set of genes to carry out the operation of the method as described in the present invention and design a material for down-regulating its expression in a host cell on the basis of the selected target gene, thereby promoting the formation of 6-dEB.

As a preferred embodiment of the present invention, the interfering molecule that inhibits the interference of a target gene is sRNA or a construct (including expression vector) carrying the sRNA. Preferably, the sRNA comprises the following structure: a promoter, a target gene-inhibiting molecule (e.g., a target gene-binding sequence) and a terminator; and preferably, the following is also included between the target gene-inhibiting molecule and the terminator: micF sequence.

The design of the promoter and terminator may be carried out according to the experience of those skilled in the art, and any suitable promoter and terminator are encompassed within the scope of the present invention. As a preferred embodiment of the present invention, the promoter is Pr promoter, and the terminator is TE terminator. However, those skilled in the art may consider changing the promoter and terminator, which are still encompassed within the scope of the present invention.

Typically, the sRNA is located in an expression vector. Accordingly, the present invention also encompasses a vector comprising the sRNA. Generally, the expression vector also contains a replication origin and/or a marker gene, etc. Methods well known to those skilled in the art can be used to construct an expression vector required for the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. It will be appreciated that any expression vector may be selected as long as the sRNA can be inserted and the expression of the target gene can be down-regulated after transforming a cell. In addition, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selecting a transformed host cell. Transformation of a host cell with an expression vector 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 the polyketide 6-dEB, wherein the host strain is transformed with an interfering molecule that inhibits the expression of a target gene. The host strain can efficiently produce the polyketide 6-dEB.

The present invention also provides a kit for promoting a host strain for synthesizing the polyketide 6-dEB to synthesize the polyketide 6-dEB biologically, the kit comprising: transformed with the sum of all the host strains for synthesizing the polyketide 6-dEB of the present invention (from which one or more strains those skilled in the art can select for production); and preferably, the kit comprising: transformed with the sum of the more preferred host strains for synthesizing the polyketide 6-dEB (host strains transformed with an interfering molecule that down-regulates the gene expression of the target gene or the target gene combination) of the present invention.

Or, the kit comprises: the sum of the sRNAs of the present invention (from which one or more sRNAs those skilled in the art can select for application); and preferably, the kit comprises: the sum of the more preferred sRNAs (sRNAs that down-regulate the gene expression of the target gene or the target gene combination) of the present invention.

The kit comprises: the sum of vectors that comprise the sRNA of the present invention (from which one or more vectors those skilled in the art can select for application); and preferably, the kit comprises: the sum of vectors that comprise the more preferred sRNAs (sRNAs that down-regulate the gene expression of the target gene or the target gene combination) of the present invention.

The present invention will be further illustrated with reference to specific examples below. It is to be understood that these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods not specified for the specific conditions in the following examples are generally carried out in accordance with conventional conditions, such as the conditions described in J. Sambrook et al. (eds), Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or in accordance with the conditions recommended by the manufacturer.

The strains, culture media and related reagents used in the present invention were as follows.

The strain E. coli DH10B was used as the clone host, and the strain E. coli WG (pZG07/pZG08) was used as the host for synthesizing the polyketide 6-dEB (see, L U Zhiguo, Doctoral Dissertation of East China University of Science and Technology, 2011). The molecular cloning-related enzymes and kits for the extraction and purification of DNA fragments and plasmids were provided by NEB, TaKaRa and Axygen, respectively. The various medium components, antibiotics and other related reagents were purchased from Oxiod, Sinopharm Group and Shanghai Bioengineering Co., Ltd. The primers were synthesized by GenScript (Nanjing) Co., Ltd.

The formulation of the 6-dEB fermentation medium involved in the present invention was as follows (g/L): NaCl, 10; peptone, 10; yeast extract, 5; glycerin, 15; and 100 mM HEPES, with pH adjusted to 7.6. The inducers involved in the present invention were as follows: IPTG: 24 μg/ml; and precursor: sodium propionate 20 mM. The induction conditions involved in the present invention were as follows: 10 ml of culture medium charged in a 100 ml shake flask was fermented and cultured for 5 days at 22° C., 250 rpm.

The antibiotic concentrations involved in the present invention were ampicillin 100 mg/L, kanamycin 50 mg/L and chloramphenicol 34 mg/L, respectively.