Engineered cell for heterologous synthesis of erythromycin, synthesis method and use

Abstract

Provided are an engineered cell for heterologous synthesis of erythromycin, a synthesis method and the use. An essential gene for heterologous synthesis of erythromycin is integrated into the chromosome of a prokaryotic cell; and on the basis of the gene, the yield of erythromycin A is greatly improved by means of targeted knockout of a set of genes. A new way is provided for the breeding of high-yield Escherichia coli strains for erythromycin production and industrial application thereof.

Description

Engineering cells, synthesis methods and uses for heterologous synthesis of erythromycin

[0001] This application claims priority to patent application No. CN 202410199752.9, filed on February 22, 2024; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the field of synthetic biology and industrial biotechnology, and in particular relates to an engineering cell for heterologous synthesis of erythromycin, a synthesis method and application. Background Art

[0003] Erythromycin was isolated from Saccharopolyspora erythraea by McGuire et al. in 1952 (McGuire JM, Bunch RL, Anderson RC, et al. Ilotycin, a new antibiotic [J]. Schweizerische medizinische Wochenschrift, 1952, 82(41): 1064-1065). It consists of six components: erythromycin A, B, C, D, E, and F. Erythromycin A has the best antibacterial activity and is widely used clinically as an important drug for the treatment of Gram-positive bacterial infections. Erythromycin is not only an important first-generation macrolide antibiotic, but also a key raw material for the synthesis of second to fourth-generation macrolide antibiotics.

[0004] Currently, Saccharopolyspora erythrae remains the only bacterial species used in industrial production of erythromycin. However, despite years of breeding high-yield strains, significant breakthroughs in erythromycin fermentation titers have been difficult to achieve. Given the unique advantages of prokaryotic hosts like Escherichia coli in recombinant expression technology, some researchers hope to leverage the rapid growth of E. coli to achieve breakthroughs in erythromycin synthesis efficiency. In 2010, the Pfeifer group cloned genes related to the erythromycin A biosynthesis pathway into expression plasmids and introduced them into Escherichia coli, achieving de novo synthesis of erythromycin A in E. coli. This demonstrated the theoretical feasibility of heterologous synthesis of erythromycin A or other macrolide compounds in E. coli (Zhang H, Wang Y, Wu J, Skalina K, Pfeifer BA. Complete biosynthesis of erythromycin A and designed analogs using E. coli as a heterologous host. Chem Biol. 2010; 17(11): 1232-1240. doi: 10.1016 / j.chembiol.2010.09.013). However, the yield of de novo synthesis of erythromycin A in a single strain in this work was only 0.6 mg / L, which was too low.

[0005] In the previous work of the present inventors, the production of intermediates in the erythromycin synthesis pathway was studied, and some limiting factors were found and improved; however, it is still difficult to screen suitable high-yielding strains and to achieve an increase in the yield of erythromycin de novo synthesis.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide an engineering cell for heterologous synthesis of erythromycin, a synthesis method and use.

[0008] In a first aspect of the present invention, a method for producing erythromycin using prokaryotic cells is provided, comprising: (1) integrating an erythromycin synthesis gene into the genome of the prokaryotic cells and down-regulating a group of target genes; wherein the erythromycin synthesis gene includes: a gene for synthesizing 6dEB, a gene for synthesizing erythromycin glycosyl side chains and genes for hydroxylation and methylation modification; wherein the down-regulated target genes include: zwf, rfbC, vioA, and vioB; and (2) culturing the prokaryotic cells to produce erythromycin.

[0009] In one or more embodiments, the genes for synthesizing 6dEB include: DEBS1, DEBS2, DEBS3; preferably also include: accA1, pccB, sfp, prpE.

[0010] In one or more embodiments, the genes for synthesizing erythromycin sugar side chains and hydroxylation and methylation modifications include: eryF, eryBVI, eryBII, eryBVII, eryBIII, eryBIV, eryBV, eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryG, and eryK.

[0011] In one or more embodiments, the downregulated target gene further comprises a gene selected from: pgi, yihX, gcd, galU, wecD or wecE.

[0012] In one or more embodiments, the method further comprises integrating the molecular chaperone gene into the genome.

[0013] In one or more embodiments, the molecular chaperone gene includes: groES or groEL.

[0014] In one or more embodiments, the method further comprises: integrating the erythromycin resistance gene ermE into the genome.

[0015] In one or more embodiments, the prokaryotic cell is an Escherichia coli cell

[0016] In one or more embodiments, the erythromycin synthesis gene is integrated at multiple locations in the genome.

[0017] In one or more embodiments, the erythromycin synthesis gene is integrated into the cell genome at the location of the araA, thiQ, lacY, and malT genes.

[0018] In one or more embodiments, the expression cassettes of DEBS2 and DEBS3 are integrated into the position of the araA gene; preferably, "promoter-rbs-DEBS2-rbs-DEBS3-terminator" is introduced at this position.

[0019] In one or more embodiments, the expression cassettes of pccB, accA1, and DEBS1 are integrated into the thiQ gene position; preferably, "promoter-pccB-accA1-promoter-rbs-DEBS1-terminator" is introduced into this position.

[0020] In one or more embodiments, the expression cassettes of eryF, eryBVI, eryBII, AeeryBVII, AeeryBIII, eryBIV, eryBV, and optionally groES, groEL, ermE are integrated into the lacY gene position; preferably, "promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-terminator" is introduced at this position.

[0021] In one or more embodiments, the expression cassettes of eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryG, and eryK are integrated into the malT gene position; preferably, "promoter-rbs-eryCI-rbs-eryCII-rbs-eryCIII-rbs-eryCIV-rbs-eryCV-rbs-eryCVI-rbs-eryG-rbs-eryK-terminator" is introduced at this position.

[0022] In one or more embodiments, the promoter is a T7 promoter, and the terminator is a T7 terminator.

[0023] In one or more embodiments, the integration is performed using CRISPR-Cas (such as Cas9)-mediated gene editing methods, homologous recombination, transposon or phage-mediated methods.

[0024] In one or more embodiments, the downregulation is performed using CRISPR-Cas (such as Cas9)-mediated gene editing methods, homologous recombination, transposon or phage-mediated gene blocking methods.

[0025] In one or more embodiments, the gene integration and gene downregulation method is a CRISPR-Cas9-mediated gene editing method.

[0026] In one or more embodiments, in step (2), an erythromycin synthesis substrate is added to the culture system (culture medium); preferably, the erythromycin synthesis substrate is a substrate for producing propionyl-CoA.

[0027] In one or more embodiments, the erythromycin synthesis substrate includes: propionic acid, propionate; more preferably, the erythromycin synthesis substrate is sodium propionate.

[0028] In another aspect of the present invention, a prokaryotic cell for producing erythromycin is provided, wherein the erythromycin synthesis gene is integrated into the cell's genome, and a set of target genes are downregulated. The erythromycin synthesis gene includes genes for synthesizing 6dEB and genes for synthesizing erythromycin glycosyl side chains and hydroxylation and methylation modifications. The downregulated target genes include zwf, rfbC, vioA, and vioB. Preferably, the 6dEB synthesis gene includes DEBS1, DEBS2, and DEBS3; preferably, the gene also includes accA1, pccB, sfp, and prpE. The genes for synthesizing erythromycin glycosyl side chains and hydroxylation and methylation modifications include eryF, eryBVI, eryBII, eryBVII, eryBIII, eryBIV, eryBV, eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryG, and eryK. Preferably, the downregulated target genes also include genes selected from the group consisting of pgi, yihX, gcd, galU, wecD, and wecE. Optionally, a molecular chaperone gene is integrated into the genome; preferably, the molecular chaperone gene includes groES or groEL. Optionally, an erythromycin resistance gene ermE is integrated into the genome.

[0029] In one or more embodiments, the prokaryotic cell is an Escherichia coli cell; preferably, the erythromycin synthesis gene is integrated into multiple locations in the genome; more preferably, it is integrated into the araA, thiQ, lacY, and malT gene locations in the cell genome.

[0030] In another aspect of the present invention, a method for producing the prokaryotic cell is provided, comprising: integrating an erythromycin synthesis gene and down-regulating a group of target genes into the genome of the prokaryotic cell; optionally, further integrating a molecular chaperone gene and / or an erythromycin resistance gene ermE into the genome of the prokaryotic cell; wherein the erythromycin synthesis gene, the down-regulated target gene, and the molecular chaperone gene are as defined above.

[0031] In another aspect of the present invention, the use of the prokaryotic cell is provided for producing erythromycin.

[0032] In another aspect of the present invention, a kit for producing erythromycin is provided, comprising the prokaryotic cell.

[0033] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1. Erythromycin synthesis pathway in engineered E. coli sZG75. In the figure, PCC is encoded by genes accA1 and pccB; the genes encoding PrpE and Sfp are carried by E. coli WG.

[0035] Figure 2. Mass spectrometry detection results of the fermentation products of strain E. coli sZG9.

[0036] Fig. 3. Fermentation test results of strains with different genotypes. DETAILED DESCRIPTION

[0037] This invention discloses a method and use for heterologous synthesis of erythromycin in prokaryotic cells. This invention, for the first time, integrates essential genes for heterologous erythromycin synthesis into prokaryotic chromosomes. Furthermore, by targetedly knocking out a group of these genes, erythromycin A production is significantly increased. This invention provides a new approach for the breeding of high-erythromycin-producing Escherichia coli strains and their industrial application.

[0038] The inventors discovered that the synthesis of erythromycin in Escherichia coli using plasmids is a bottleneck leading to low erythromycin production. This process also presents several disadvantages, including significant inconvenience in the subsequent breeding of high-yield strains, the additional cost of antibiotics to maintain the plasmid, and plasmid stability issues. The present invention, however, integrates the genes required for erythromycin synthesis into the host cell genome, further combined with targeted gene knockout, to achieve stable and efficient production of erythromycin A.

[0039] The inventors also discovered that if all synthetic genes were integrated at single sites or at inappropriate sites, the integration effect would be less than ideal. However, optimizing the placement / distribution of integration sites can effectively improve the integration effect.

[0040] the term

[0041] As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources. For example, a promoter is exogenous to a gene of interest if the combination of the promoter and the gene sequence does not normally occur in nature. A particular sequence is "exogenous" or "heterologous" to the cell or organism into which it is inserted.

[0042] As used herein, the terms "expression cassette," "transgene cassette," and "transgene expression cassette" are used interchangeably to refer to a gene expression system comprising all necessary elements for expressing a polypeptide / protein of interest, typically including the following exogenous elements: a promoter, a gene sequence encoding a polypeptide, a terminator, and optionally a signal peptide coding sequence. These elements are operably linked.

[0043] As used herein, "operably linked" or "operably associated" refers to the functional spatial arrangement of two or more nucleic acid / protein regions or nucleic acid / protein sequences. For example, a promoter region is "operably linked" to a target nucleic acid sequence when it is positioned at a specific position relative to the target nucleic acid sequence such that transcription of the nucleic acid sequence is directed by the promoter region.

[0044] As used herein, the terms “contain,” “have,” or “include” include “comprise,” “mainly consist of,” “substantially consist of,” and “consist of”; “mainly consist of,” “substantially consist of,” and “consist of” are subordinate concepts of “contain,” “have,” or “include.”

[0045] As used herein, the "kit" refers to a package / box that is convenient for commercial-scale production and use, which usually contains a container / small package, and the culture medium, solution, single component or component of the present invention can be placed in the container / small package.

[0046] As used herein, the term "conservative variant polypeptide" refers to a polypeptide that substantially retains the same biological function or activity as the polypeptide. The "conservative variant polypeptide" may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0047] As used herein, the term "variant" or "mutant" refers to a peptide or polypeptide that has an amino acid sequence that has been altered by insertion, deletion, or substitution of one or more amino acids compared to a reference sequence, but retains at least one biological activity. The mutants described in any embodiment herein include amino acid sequences that have at least 50%, 60%, or 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with a reference sequence (such as the natural sequence of any enzyme described herein) and retain the biological activity of the reference sequence (such as activity as a CoA ligase, etc.). The sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. Mutants also include amino acid sequences that have one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of the reference sequence while still retaining the biological activity of the reference sequence. The multiple mutations generally refer to within 1-20, such as 1-15, 1-10, 1-8, 1-5, or 1-3. The substitutions are preferably conservative substitutions. For example, in the art, conservative substitutions with amino acids with similar or similar properties generally do not alter the function of proteins or polypeptides. "Amino acids with similar or similar properties" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in a polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

[0048] Genes and expression systems

[0049] In the present invention, the erythromycin synthesis gene is integrated into the genome of the prokaryotic cell, and the gene is constructed into a suitable expression cassette. Information on some erythromycin synthesis genes is shown in Table 1.

[0050] The erythromycin synthesis genes include the genes DEBS1, DEBS2, and DEBS3 for synthesizing 6dEB, and optionally, also include genes sfp, accA1, pccB, and prpE.

[0051] Among them, the erythromycin synthesis gene also includes the genes eryF, eryBVI, eryBII, eryBVII, eryBIII, eryBIV, eryBV, eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryG, and eryK for synthesizing erythromycin glycosyl side chains and hydroxylation and methylation modifications.

[0052] As a preferred embodiment of the present invention, the genes required for erythromycin synthesis also include molecular chaperone genes, preferably groES and / or groEL.

[0053] As a preferred embodiment of the present invention, the genes required for erythromycin synthesis also include the erythromycin resistance gene ermE.

[0054] Table 1

[0055] The erythromycin synthesis gene or polypeptide (protein) of the present invention may be naturally occurring, for example, it may be isolated or purified from an animal, plant, or microorganism. Furthermore, the gene or polypeptide may also be artificially prepared, for example, by obtaining the gene using conventional genetic engineering recombinant techniques, or by artificial synthesis, and encoding a polypeptide.

[0056] The amino acid sequences of the various proteins (enzymes) described in the present invention are preferably the sequences listed in Table 1, and also include "conservative variant polypeptides" having the same functions as the enzymes listed in Table 1. The present invention also includes fragments, derivatives and analogs of the polypeptides. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the polypeptides. The gene required for the synthesis of erythromycin may also be a gene having the same function as the above-mentioned gene, such as a mutant of the same gene or a gene with the same function from a different species.

[0057] The "conservative variant polypeptides (proteins)" described herein may include (but are not limited to): deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as additions or deletions of one or more (e.g., up to 50, more preferably up to 20 or 10, and more preferably up to 5) amino acids at the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, additions of one or more amino acids to the C-terminus and / or N-terminus generally do not alter protein function. The present invention also provides analogs of the polypeptides described herein. These analogs may differ from the native polypeptide in amino acid sequence, in modifications that do not affect the sequence, or in a combination of these. These polypeptides include natural or induced genetic variants. Induced variants can be obtained through various techniques, such as random mutagenesis via radiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biology techniques. Analogs also include analogs with residues other than natural L-amino acids (e.g., D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0058] The polynucleotide encoding the polypeptide of the present invention may be in the form of DNA or RNA. DNA forms include eDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide or a polynucleotide further comprising additional coding and / or non-coding sequences.

[0059] The present invention also relates to vectors comprising the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors or polypeptide coding sequences of the present invention, and methods for producing the polypeptides of the present invention by recombinant technology.

[0060] The present invention relates to nucleic acid constructs, or expression cassettes, comprising the erythromycin synthesis genes of the present invention and one or more regulatory elements or elements required for genomic homologous recombination operatively linked to these genes. The polynucleotides of the present invention can be manipulated in a variety of ways to ensure expression of the polypeptide or protein. Manipulations of the nucleic acid constructs can be performed prior to insertion into a vector, depending on the specific expression vector or requirements.

[0061] The method of integrating the expression cassette into the host cell chromosome is a commonly used method in the art, including but not limited to CRISPR-Cas9-mediated gene editing methods, homologous recombination, transposon- or phage-mediated gene integration methods, preferably, CRISPR-Cas9-mediated gene editing methods are used.

[0062] A gene knock-in vector can be used to integrate the polynucleotide sequence described herein into the region of interest of the genome. Typically, in addition to containing the polynucleotide sequence, the gene knock-in vector may also contain a genomic homology arm sequence. When using a gene knock-in vector, CRISPR / Cas technology can be used simultaneously to homologously recombine the polynucleotide sequence to a specific position. CRISPR / Cas technology guides the Cas nuclease to modify the genome at the insertion position by designing a guide RNA for the target gene, thereby increasing the efficiency of homologous recombination in the gene-modified region and homologously recombine the target fragment contained in the gene knock-in vector to the target site. In the expression regulatory sequence or expression cassette, an inducible or constitutive promoter can be used according to different needs. The inducible promoter can achieve more controllable protein expression and compound production, which is conducive to industrial application.

[0063] The technical solution of the present invention further includes targeted downregulation of a group of genes selected from the group consisting of zwf, rfbC, vioA, vioB, pgi, yihX, gcd, galU, wecD, and wecE in a host cell harboring an erythromycin biosynthesis gene. Information on these genes is shown in Table 2. In a more preferred embodiment, the targeted downregulation includes the following genes: zwf, rfbC, vioA, and vioB.

[0064] Table 2

[0065] Methods for downregulating the gene include, but are not limited to, homologous recombination, CRISPR-Cas9-mediated gene editing, and transposon- or phage-mediated gene blocking methods.

[0066] As a preferred embodiment of the present invention, CRISPR / Cas (such as Cas9) system is used for targeted gene editing, thereby knocking out the gene. After determining the target gene and target site, known methods can be used to introduce sgRNA and Cas into the cell. The nucleic acid capable of forming the sgRNA is a nucleic acid construct or an expression vector, or the nucleic acid capable of forming the Cas mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into the cell to form active sgRNA and Cas mRNA in the cell.

[0067] In the present invention, the host cell used to construct the expression system is a prokaryotic cell, such as a bacterial cell. Preferably, the host cell is Escherichia coli. The Escherichia coli can be, for example, BL21 (DE3), JM109 (DE3), MG1655, or the like.

[0068] In a preferred embodiment, when an expression cassette is introduced into Escherichia coli, the expression cassette includes a combination of a promoter, an RBS, a CDS sequence of a gene, and a terminator suitable for working in Escherichia coli. Preferably, the promoter is a T7 promoter or other promoter that can work in Escherichia coli, and the terminator is a T7 terminator or other terminator that can work in Escherichia coli.

[0069] The integration of single sites or integration of inappropriate sites makes the integration effect less than ideal, while the optimized setting / allocation of integration sites effectively improves the integration effect. As a preferred embodiment of the present invention, the expression cassettes of DEBS2 and DEBS3 are integrated into the araA gene position; the expression cassettes of pccB, accA1, and DEBS1 are integrated into the thiQ gene position; the expression cassettes of eryF, eryBVI, eryBII, AeeryBVII, AeeryBIII, eryBIV, eryBV, and optional groES, groEL, and ermE are integrated into the lacY gene position; the expression cassettes of eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryG, and eryK are integrated into the malT gene position.

[0070] Production of erythromycin

[0071] The expression system of the present invention can be used to produce erythromycin, comprising: culturing the prokaryotic cells constructed according to the present invention in a suitable culture system (culture medium) supplemented with an erythromycin synthesis substrate; and isolating and purifying erythromycin from the culture medium or cells. The erythromycin synthesis substrate is a substrate for producing propionyl-CoA; more preferably, the erythromycin synthesis substrate comprises propionic acid or propionate; and even more preferably, the erythromycin synthesis substrate is sodium propionate.

[0072] After obtaining the fermentation product, erythromycin or its intermediates can be extracted from the fermentation product using known techniques. For example, high performance liquid chromatography can be used to analyze and identify the product to confirm that the desired compound has been obtained.

[0073] The present invention also provides a kit containing the recombinant expression system constructed according to the present invention. Other reagents commonly used for transgenic manipulation may also be included in the kit to facilitate use by those skilled in the art. In addition, the kit may also include instructions for use to guide those skilled in the art.

[0074] The present invention provides a method and application for improving heterologous erythromycin synthesis in Escherichia coli. By integrating genes required for heterologous erythromycin synthesis into the chromosome and targetedly deleting some genes, the yield of erythromycin A is significantly increased. Furthermore, for the first time, all genes required for heterologous erythromycin A synthesis are integrated into the host cell chromosome, resulting in minimal byproducts. This avoids the numerous disadvantages of synthesizing erythromycin A in cells using plasmids, including instability when multiple plasmids coexist, the additional cost of adding antibiotics to maintain the plasmids, and the inconveniences in breeding high-yield strains caused by the presence of multiple plasmids.

[0075] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0076] Materials and methods

[0077] 1. Sequence information of polynucleotides used for construction

[0078] >G0219526-1A (SEQ ID NO: 1)

[0079] >G0219526-1B (SEQ ID NO: 2)

[0080] >G0219526-1C (SEQ ID NO: 3)

[0081] 2. Use of DNA fragments

[0082] The homology arms used in the construction process are listed in Table 3.

[0083] Table 3

[0084] The schematic diagram of the synthesis pathway of erythromycin in the engineered bacterium E. coli sZG75 is shown in Figure 1.

[0085] 3. Specific materials and instruments

[0086] Oligonucleotide primers, antibiotics, the nucleic acid dye ethidium bromide (EB), and the standard compound erythromycin were purchased from Sangon Biotech Co., Ltd. The polymerase chain reaction (PCR) gel recovery kit and plasmid extraction kit were both Axygen products. The high-fidelity PCR enzyme PrimeSTAR Max DNA Polymerase was from Takara Bio, Japan. PCR was performed using an Arktik Thermal Cycler (Thermo Fisher Scientific).

[0087] E. coli DH10B strain was used for plasmid construction; E. coli WG strain (BL21 (DE3) ΔprpRBCD:: T7prom-sfp, T7prom-prpE) and plasmids pZG07 and pZG08 refer to Wang Yong, Xiong Zhiqiang, Song Shujie, et al. A method and use for improving heterologous synthesis of polyketides in Escherichia coli [P]. Shanghai: CN106191155A, 2016-12-07; plasmid pZF230 refer to Liu Z, Xu J, Feng Z, Wang Y. Multi-strategy engineering unusual sugar TDP-1-mycarose biosynthesis to improve the production of 3-O-α-mycarosylerythronolide B in Escherichia coli. Synth Syst Biotechnol. 2022; 7(2): 756-764. Published 2022 Mar 20.doi:10.1016 / j.synbio.2022.03.002; DNA fragments G0219526-1A, G0219526-1B, and G0219526-1C were synthesized by General Biotechnology (Anhui) Co., Ltd.; plasmid pET21c was purchased from Novagen.

[0088] Example 1. Acquisition of Escherichia coli strain E. coli sZG2

[0089] (1) Integrate the DNA fragment E8arm into the chromosome of the strain E. coli WG

[0090] The Escherichia coli strain E. coli WG was gene-edited using a CRISPR-Cas9-mediated gene editing method. See Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang SA modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021; 53(5): 620-627. doi: 10.1093 / abbs / gmab036.

[0091] Plasmid pZG33, expressing a sgRNA targeting the target gene araA (Genbank accession numbers CP053602, 69640-71142), was constructed by PCR using primers E8F and E8R, using plasmid pTargetF as a template. The PCR reaction system and conditions are shown in Table 4, and the primers used are shown in Table 5.

[0092] After gel recovery of the PCR product, the plasmid template was removed using the endonuclease DpnI. The digested product was electrophoresed and gel recovered again. A 5-μl aliquot was transformed into DH10B cells and plated onto a solid plate containing LB medium at a final concentration of 50 mg / L. The cells were incubated overnight at 37°C. A single clone was randomly selected for plasmid extraction and, after verification by sequencing, the resulting plasmid, pZG33, was obtained (which expresses the sgRNA targeting araA).

[0093] Using the E. coli WG genome as a template, PCR amplification with primers E8arm_U1F and E8arm_U1R yielded E8arm_U1. Using pZG07 as a template, PCR amplification with primers E8arm_U2F and E8arm_U2R yielded E8arm_U2. Using pZG07 as a template, PCR amplification with primers E8arm_D2F and E8arm_D2R yielded E8arm_D2. Using E. coli WG as a template, PCR amplification with primers E8arm_D1F and E8arm_D1R yielded E8arm_D1. Fusion PCR of E8arm_U1 and E8arm_U2 yielded E8arm_U. Fusion PCR of E8arm_D2 and E8arm_D1 yielded E8arm_D. Finally, fusion PCR of E8arm_U and E8arm_D yielded E8arm. PCR reaction systems and conditions are shown in Table 4. All PCR products were gel-purified. Plasmid pZG33 and DNA fragment E8arm were electroporated into E. coli strain E. coli WG containing plasmid pEcCas.

[0094] For electroporation, E. coli WG transformed with the pEcCas plasmid was cultured at 37°C in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose (to induce expression of the λ-Red system carried by the pEcCas plasmid) until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0095] The electroporated bacterial suspension was plated onto LB plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was selected as a template for PCR with primers E8-ckF and E8-ckR. Integration of the DNA fragment E8arm was confirmed by the presence of a 3282 bp DNA band on an agarose gel. The confirmed strain was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG33.

[0096] The strain from which plasmid pZG33 had been removed was then cultured in liquid LB medium containing 5 g / L glucose at 37°C overnight, and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured at 37°C overnight to remove plasmid pEcCas.

[0097] The E. coli strain constructed in this way is E. coli WG araA::E8arm, denoted as E. coli sZG1.

[0098] Table 4. PCR reaction system and conditions

[0099] Table 5. Primers used for integration of E8arm

[0100] (2) Integrate the DNA fragment T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator into the chromosome of E. coli sZG1

[0101] Plasmid pZG34, which expresses an sgRNA targeting the target DNA fragment E8arm, was constructed by PCR using primers E9F and E9R (Table 6) and plasmid pTargetF as a template. The specific construction steps of plasmid pZG34 are similar to those of plasmid pZG33.

[0102] Plasmid pZG07 was double-digested with restriction endonucleases HpaI and PacI, and the digestion products were recovered on gel to obtain a DNA fragment of 21890 bp containing T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator.

[0103] Plasmid pZG34 and a 21890 bp DNA fragment containing T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator were electroporated into E. coli sZG1 containing plasmid pEcCas.

[0104] For electroporation, E. coli sZG1 transformed with the pEcCas plasmid was cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0105] The electroporated bacterial suspension was spread onto LB plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was selected as a template for PCR using primers E8-ckF and E9-ckR. Integration of the DNA fragment T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator was confirmed by the presence of a 2479 bp DNA band on an agarose gel. The confirmed strain was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove plasmid pZG34.

[0106] The strain from which plasmid pZG34 had been removed was then cultured in liquid LB medium containing 5 g / L glucose at 37°C overnight, and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured at 37°C overnight to remove plasmid pEcCas.

[0107] The E. coli strain constructed in this way is E. coli WG araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, denoted as E. coli sZG2.

[0108] Table 6. Primers used for integration of T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator

[0109] Example 2: Obtaining E. coli strain E. coli sZG4

[0110] (1) Integrate the DNA fragment Earm1 into the chromosome of E. coli sZG2

[0111] Plasmid pZG55, which expresses an sgRNA targeting the target gene thiQ (Genbank accession numbers CP053602, 75034-75732), was constructed by PCR using primers E26F and E26R (Table 7) with plasmid pTargetF as a template. The specific construction steps for plasmid pZG55 were similar to those for plasmid pZG33 in Example 1.

[0112] Using E.coli WG as template, Earm1_U1F and Earm1_U1R as primers were used to amplify by PCR to obtain Earm1_U1; using pZG08 as template, Earm1_U2F and Earm1_U2R as primers were used to amplify by PCR to obtain Earm1_U2; using pZG08 as template, Earm1_D2F and Earm1_D2R as primers were used to amplify by PCR to obtain Earm1_D2; using E.coli WG as template, Earm1_D1F and Earm1_D1R as primers were used to amplify by PCR to obtain Earm1_D1; Earm1_U1 and Earm1_U2 were fused by PCR to obtain Earm1_U1_U2; Earm1_D2 and Earm1_D1 were fused by PCR to obtain Earm1_D2_D1; and Earm1_U1_U2 and Earm1_D2_D1 were fused by PCR to obtain Earm1. The PCR reaction system and conditions are shown in Table 4. PCR products were gel-purified. Plasmid pZG55 and DNA fragment Earm1 were electroporated into E. coli WG containing plasmid pEcCas.

[0113] For electroporation, E. coli sZG2 transformed with the pEcCas plasmid was cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0114] The electroporated bacterial suspension was plated onto LB plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was selected as a template for PCR with primers Earm1_U1F and Earm1_D1R. Integration of the Earm1 DNA fragment was confirmed by the presence of a 3367 bp DNA band on an agarose gel. The strain that passed this confirmation was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG55. The pZG55-free strain was then cultured overnight at 37°C in liquid LB medium containing 5 g / L glucose and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured overnight at 37°C to remove the plasmid pEcCas.

[0115] The E. coli strain constructed in this way is E. coli WG thiQ::Earm1,araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, denoted as E. coli sZG3.

[0116] Table 7. Primers used for integration of Earm1

[0117] (2) Integrate the DNA fragment T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator into the chromosome of E. coli sZG3

[0118] By PCR using primers E28F and E28R (Table 8), plasmid pTargetF was used as a template to construct plasmid pZG57 capable of expressing sgRNA targeting the target DNA fragment Earm1. The specific construction steps of plasmid pZG57 are similar to those of plasmid pZG33 in Example 1.

[0119] Plasmid pZG08 was double-digested with the restriction endonucleases HpaI and PacI, and the digestion product was recovered from gel filtration, yielding a 15,660-bp DNA fragment containing the T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator. Plasmid pZG57 and the 15,660-bp DNA fragment containing the T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator were co-electroporated into E. coli strain sZG3 harboring the plasmid pEcCas.

[0120] For electroporation, E. coli sZG3 transformed with the pEcCas plasmid was cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0121] The electroporated bacterial suspension was plated onto LB plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was selected as a template for PCR using primers Earm1_U1F and E10-ckR3. Integration of the DNA fragment T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator was confirmed by the presence of a 2605 bp DNA band on an agarose gel. The confirmed strain was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG57. The strain, from which plasmid pZG57 had been removed, was then cultured overnight at 37°C in liquid LB medium containing 5 g / L glucose and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured overnight at 37°C to remove plasmid pEcCas. The resulting E. coli strain, E. coli WG thiQ::T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator, araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, was designated E. coli sZG4.

[0122] Table 8. Primers used for integration of T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator

[0123] Example 3 Obtaining E. coli strain E. coli sZG6

[0124] (1) Integrate the DNA fragment Earm3c into the chromosome of E. coli sZG4

[0125] Plasmid pZG60, which expresses an sgRNA targeting the target gene lacY (Genbank accession numbers CP053602, 332759-334012), was constructed by PCR using primers E31F and E31R (Table 9) with plasmid pTargetF as a template. The specific construction steps for plasmid pZG60 were similar to those for plasmid pZG33 in Example 1.

[0126] Using E.coli WG as template, Earm3c_U1F and Earm3c_U1R as primers, PCR amplification was performed to obtain Earm3c_U1; using pZF230 as template, Earm3c-U2F and Earm3c_U2R as primers, PCR amplification was performed to obtain Earm3c_U2; using pZF230 as template, Earm3c_D2_UF and 230arm_D2_UR as primers, PCR amplification was performed to obtain Earm3c_D2_U; using pZF230 as template, 230arm_D2_DF and Earm3c_D2_DR as primers, PCR amplification was performed to obtain Earm3c_D2_D; using E.coli WG was used as a template, and PCR amplification with primers Earm3c_D1F and Earm3c_D1R yielded Earm3c_D1. Fusion PCR of Earm3c_D2_D and Earm3c_D1 yielded Earm3c_D2_D_D1. Fusion PCR of Earm3c_U1 and Earm3c_U2 yielded Earm3c_U1_U2. Fusion PCR of Earm3c_D2_U and Earm3c_D2_D_D1 yielded Earm3c_D2_U_D2_D_D1. Fusion PCR of Earm3c_U1_U2 and Earm3c_D2_U_D2_D_D1 yielded Earm3c. PCR reaction systems and conditions are shown in Table 4. PCR products were gel-purified. Plasmid pZG60 and the Earm3c DNA fragment were electroporated into E. coli strain sZG4 containing the plasmid pEcCas.

[0127] For electroporation, E. coli sZG4 transformed with the pEcCas plasmid was cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0128] The electroporated bacterial suspension was plated onto LB plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was selected as a template for PCR with primers Earm3c_ckF and Earm3c_ckR. Integration of the Earm3c DNA fragment was confirmed by the presence of a 3770 bp DNA band on an agarose gel. The strain that passed this confirmation was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG60. The pZG60-free strain was then cultured overnight at 37°C in liquid LB medium containing 5 g / L glucose and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured overnight at 37°C to remove the plasmid pEcCas. The E. coli strain constructed in this way is E. coli WG thiQ::T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator, araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, ΔlacY::Earm3c, and is denoted as E. coli strain E. coli sZG5.

[0129] Table 9. Primers used for integration of Earm3c

[0130] (2) Integrate the DNA fragment T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator into the chromosome of E. coli strain sZG5

[0131] The specific construction of plasmid pZG55 is shown in Example 2.

[0132] The plasmid pZF230 was double-digested with restriction endonucleases XbaI and SpeI, and the digestion products were recovered on gel to obtain a DNA fragment with a size of 12746 bp containing T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs--eryBII--rbs-AeeryBVII--rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator. Plasmid pZG55 and a 12746 bp DNA fragment containing T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7 terminator were electroporated into E. coli strain sZG5 containing plasmid pEcCas.

[0133] For electroporation, E. coli sZG5 transformed with the pEcCas plasmid was cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0134] The electroporated bacterial solution was spread on an LB plate containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride resistance and cultured overnight at 37°C. A single colony was picked as a template, and PCR was performed with primers Earm3c_U1F and 230arm-ckR2. The integration of the DNA fragment T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbseryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator was confirmed by observing the presence of a 1751 bp DNA band in the agarose gel. The strain confirmed by this was cultured overnight at 37°C in a liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG55.

[0135] The strain from which plasmid pZG55 had been removed was then cultured in liquid LB medium containing 5 g / L glucose at 37°C overnight, and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured at 37°C overnight to remove plasmid pEcCas.

[0136] The E. coli strain constructed in this way is E. coli WG thiQ::T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator, araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, ΔlacY::T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator, and is denoted as E. coli strain E. coli sZG6.

[0137] The primers used for integrating T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator are shown in Table 10.

[0138] Table 10

[0139] Example 4. Obtaining E. coli strain E. coli sZG9

[0140] (1) Integrate the DNA fragment 247Aarm into the chromosome of E. coli sZG6

[0141] Plasmid pZG64, which expresses an sgRNA targeting the target gene malT (Genbank accession numbers CP053602, 3414679-3417384), was constructed by PCR using primers E35F and E35R (Table 11) and plasmid pTargetF as a template. The specific construction steps for plasmid pZG64 were similar to those for plasmid pZG33 in Example 1.

[0142] Using E. coli WG as template and primers 247A_U1F and 247A_U1R as PCR amplification, 247A_U1 was obtained; using pET21c as template and primers 247A_U2F and 247A_U2R as PCR amplification, 247A_U2 was obtained; using G0219526-1-A as template and primers 247A_D2F and 247A_D2R as PCR amplification, 247A_D2 was obtained; using E. coli WG as template and primers 247A_U1F and 247A_U1R as PCR amplification, 247A_U1 was obtained; using pET21c as template and primers 247A_U2F and 247A_U2R as PCR amplification, 247A_D2 was obtained; WG was used as a template, and PCR amplification with primers 247A_D1F and 247A_D1R yielded 247A_D1. 247A_U1 and 247A_U2 were fused to yield 247A_U1_U2; 247A_D2 and 247A_D1 were fused to yield 247A_D2_D1; and 247A_U1_U2 and 247A_D2_D1 were fused to yield 247Aarm. PCR reaction systems and conditions are shown in Table 4. PCR products were gel-purified. Plasmid pZG64 and DNA fragment 247Aarm were electroporated into E. coli strain sZG6, harboring the pEcCas plasmid.

[0143] For electroporation, E. coli sZG6 transformed with the pEcCas plasmid was cultured at 37°C in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose until the OD600 reached 0.5–0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at an electric field strength of 18 kV cm⁻¹.

[0144] The electroporated bacterial suspension was plated onto LB plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was selected as a template for PCR with primers 247A_D2F and 247A_D2R, and integration of the DNA fragment 247Aarm was confirmed by the presence of a 3452 bp DNA band on an agarose gel. The strain that passed this confirmation was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG64. The pZG64-removed strain was then cultured overnight at 37°C in liquid LB medium containing 5 g / L glucose and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured overnight at 37°C to remove the plasmid pEcCas.

[0145] The E. coli strain constructed in this way is E. coli WG thiQ::T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator, araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, ΔlacY::T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator, ΔmalT::247A, and is denoted as E. coli strain E. coli sZG7.

[0146] Table 11. Primers used for integration of 247Aarm

[0147] (2) Integrate the DNA fragment 247Barm2 ​​into the chromosome of E. coli sZG7

[0148] The specific construction of plasmid pZG55 is shown in Example 2.

[0149] PCR amplification was performed using G0219526-1A as a template and primers 247A-ckF5 and 247B_UR (Table 12) to obtain 247Barm2_U; PCR amplification was performed using G0219526-1B as a template and primers 247B_MF and 247B_MR to obtain 247Barm2_M; PCR amplification was performed using E. coli WG as a template and primers 247B_DF and 247B_D1R to obtain 247Barm2_D; fusion PCR of 247Barm2_U and 247Barm2_M was performed to obtain 247Barm2_U_M; and fusion PCR of 247Barm2_U_M and 247Barm2_D was performed to obtain 247Barm2. PCR reaction systems and conditions are shown in Table 4. All PCR products were gel-purified. Plasmid pZG55 and DNA fragment 247Barm2 ​​were electroporated into E. coli strain sZG7 containing plasmid pEcCas.

[0150] For electroporation, E. coli sZG7 transformed with the pEcCas plasmid was cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0151] The electroporated bacterial suspension was plated onto LB plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was selected as a template for PCR with primers 247A-ckF5 and 247B_D1R. Integration of the DNA fragment 247Barm2 ​​was confirmed by the presence of a 4144 bp DNA band on an agarose gel. The strain that passed this confirmation was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG55. The pZG55-free strain was then cultured overnight at 37°C in liquid LB medium containing 5 g / L glucose and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured overnight at 37°C to remove the plasmid pEcCas.

[0152] The E. coli strain constructed in this way is E. coli WG thiQ::T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator, araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, ΔlacY::T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator, ΔmalT::247AB, and is denoted as E. coli strain E. coli sZG8.

[0153] Table 12. Primers used for integration of 247Barm2

[0154] (3) Integrate the DNA fragment 247Carm5 into the chromosome of E. coli sZG8

[0155] The specific construction of plasmid pZG57 is shown in Example 2.

[0156] Using G0219526-1B as template and primers 247C_U1F2 and 247C_U1R (Table 13) as primers, PCR amplification was performed to obtain 247C5_U1; using G0219526-1C as template and primers 247C_U2F and 247C_U2R4 as primers, PCR amplification was performed to obtain 247C5_U2; using pET21c as template and primers 247C_D2F4 and 247C_D2R as primers, PCR amplification was performed to obtain 247C5_D2; using E. coli as template, PCR amplification was performed to obtain 247C5_U1; using G0219526-1C as template and primers 247C_U2F and 247C_U2R4 as primers, PCR amplification was performed to obtain 247C5_U2; using E. coli as template, PCR amplification was performed to obtain 247C5_D2; WG was used as a template, and PCR amplification with primers 247C_D1F and 247B_D1R yielded 247C5_D1. 247C5_U1 and 247C5_U2 were fused to yield 247C5_U1_U2; 247C5_D2 and 247C5_D1 were fused to yield 247C5_D2_D1; and 247C5_U1_U2 and 247C5_D2_D1 were fused to yield 247Carm5. PCR reaction systems and conditions are shown in Table 4. PCR products were gel-purified. Plasmid pZG57 and DNA fragment 247Carm5 were electroporated into E. coli strain sZG8 harboring the pEcCas plasmid.

[0157] For electroporation, E. coli sZG8 transformed with the pEcCas plasmid was cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0158] The electroporated bacterial suspension was plated onto LB plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was selected as a template for PCR with primers 247C_U1F2 and 247B_D1R. Integration of the DNA fragment 247Carm5 was confirmed by the presence of a 4139 bp DNA band on an agarose gel. The strain that passed this confirmation was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG57. The pZG57-free strain was then cultured overnight at 37°C in liquid LB medium containing 5 g / L glucose and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured overnight at 37°C to remove the plasmid pEcCas.

[0159] The E. coli strain constructed in this way is E. coli WG thiQ::T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator, araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, ΔlacY::T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator, ΔmalT::T7 promoter-rbs-eryCI--rbs--eryCII-rbs--eryCIII-rbs--eryCIV-rbs--eryCV--rbs--eryCVI-rbs-eryG-rbs--eryK-T7 terminator, which is denoted as E. coli strain. sZG9.

[0160] Table 13. Primers used for integration of 247Carm5

[0161] Example 5. Obtaining E. coli strain E. coli sZG75

[0162] (1) Knockout of genes rfbC, wzx, vioA, and vioB (the four genes are located adjacently and can be knocked out at once) in E. coli sZG9

[0163] Plasmid pZG101, expressing an sgRNA targeting the target gene wzx (Genbank accession numbers CP053602, 2008492-2009916), was constructed by PCR using primers E66F and E66R (Table 14) with plasmid pTargetF as the template. The construction steps for plasmid pZG101 were similar to those for plasmid pZG33 in Example 1.

[0164] wzx2_U was obtained by PCR amplification using E. coli WG as template and vioAB_UF and wzx2_UR as primers;

[0165] Using E. coli WG as a template and primers wzx2_DF and rfbC_DR, PCR amplification was performed to generate wzx2_D. wzx2_U and wzx2_D were then fused to generate wzx2arm. The PCR reaction system and conditions are shown in Table 4. All PCR products were gel-purified. Plasmid pZG101 and the wzx2arm DNA fragment were then electroporated into E. coli strain sZG9 harboring the pEcCas plasmid.

[0166] For electroporation, E. coli sZG9 transformed with the pEcCas plasmid was cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0167] The electroporated bacterial solution was spread onto an LB plate containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride resistance and cultured overnight at 37°C. A single colony was picked as a template and PCR was performed with primers vioAB-ck and rfbC-ckR. The knockout of the genes rfbC (Genbank accession number CP053602, 2009920-2010465), wzx, and vioAB (Genbank accession number CP053602, 2006790-2008482) was confirmed by observing a 1240 bp DNA band in an agarose gel. The strain that passed this confirmation was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG101. The strain from which plasmid pZG101 had been removed was then cultured in liquid LB medium containing 5 g / L glucose at 37°C overnight, and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured at 37°C overnight to remove plasmid pEcCas.

[0168] The E. coli strain thus constructed is E. coli WG thiQ::T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator, araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, ΔlacY::T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator,ΔmalT::T7 promoter-rbs-eryCI--rbs--eryCII-rbs-eryCIII-rbs-eryCIV-rbs--eryCV--rbs--eryCVI--rbs--eryG--rbs-eryK-T7 terminator, ΔrfbCΔwzxΔvioAB, denoted as E. coli strain sZG66.

[0169] Table 14. Primers used to knock out rfbC, wzx, and vioAB

[0170] (2) Knockout of gene zwf in E. coli strain sZG66

[0171] Plasmid pZG28, which can express sgRNA targeting the target gene zwf, was constructed by PCR using primers E4F and E4R (Table 15) and plasmid pTargetF as a template. The specific construction steps of plasmid pZG28 are similar to those of plasmid pZG33 in Example 1.

[0172] Using E. coli WG as a template, PCR amplification with primers zwf_UF and zwf_UR yielded zwf_U. Using E. coli WG as a template, PCR amplification with primers zwf_DF and zwf_DR yielded zwf_D. Fusion PCR of zwf_U and zwf_D yielded zwf_arm. PCR reaction systems and conditions are shown in Table 4. PCR products were gel-purified. Plasmid pZG28 and the zwf_arm DNA fragment were electroporated into E. coli strain sZG66 harboring the pEcCas plasmid.

[0173] For electroporation, E. coli sZG66 transformed with the pEcCas plasmid was cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.5-0.7. The culture was transferred to a centrifuge tube, incubated on ice for 30 minutes, washed three times with ice-cold sterile distilled water, and finally resuspended in ice-cold 10% glycerol for use. Electroporation was performed at 18 kV / cm -1 The test was carried out under the electric field strength of .

[0174] The electroporated bacterial solution was spread onto an LB plate containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride, and cultured overnight at 37°C. A single colony was selected as a template and the primers zwf_UF

[0175] PCR was performed with zwf_DR, and knockout of the zwf gene was confirmed by the presence of a 1206 bp DNA band on an agarose gel. The strain that passed this confirmation was cultured overnight at 37°C in liquid LB medium containing 50 mg / L kanamycin and 10 mM rhamnose to remove the plasmid pZG28. The strain from which the plasmid pZG28 had been removed was then cultured overnight at 37°C in liquid LB medium containing 5 g / L glucose and then transferred to solid LB medium containing 5 g / L glucose and 10 g / L sucrose and cultured overnight at 37°C to remove the plasmid pEcCas.

[0176] The E. coli strain thus constructed is E. coli WG thiQ::T7 promoter-pccB-accA1-T7 promoter-rbs-DEBS1-T7 terminator, araA::T7 promoter-rbs-DEBS2-rbs-DEBS3-T7 terminator, ΔlacY::T7 promoter-rbs-SaeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-T7terminator,ΔmalT::T7 promoter-rbs--eryCI--rbs--eryCII--rbs-eryCIII-rbs--eryCIV-rbs-eryCV--rbs--eryCVI--rbs--eryG--rbs--eryK-T7 terminator, ΔrfbCΔwzxΔvioABΔzwf, denoted as E. coli strain E. coli sZG75.

[0177] Table 15. Primers used to knock out zwf

[0178] Example 6: Fermentation test of strains with different genotypes

[0179] The strain E. coli sZG9 in Example 4 and the strain E. coli sZG75 in Example 5 were inoculated into 4 mL of LB medium, respectively, and cultured at 37° C. and 250 rpm to serve as precultures.

[0180] After 12 h, 100 μL of the preculture was inoculated into 10 mL of HEPES fermentation medium, IPTG was added to a final concentration of 0.5 mM, and sodium propionate was added to a final concentration of 5 mM. The culture was then shaken at 22° C. and 250 rpm for 7 days.

[0181] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is distilled water, pH natural.

[0182] HEPES fermentation medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 5 g / L glycerol, 23.8 g / L HEPES, solvent is distilled water, pH 7.6.

[0183] After the fermentation is completed, 1 mL of fermentation broth was taken to measure OD600; 1 mL of fermentation broth was extracted three times with 1 mL of ethyl acetate, the supernatants were combined and evaporated to dryness, and then re-dissolved with 100 μL of methanol for subsequent detection.

[0184] Mass spectrometry detection was performed using a Q Exactive quadruple triplet orbitrap mass spectrometer (Thermo Scientific, MA, USA) in the positive ESI mode with a detection mass-to-charge ratio (m / z) ranging from 100 to 1000.

[0185] The mass spectrometry results are shown in FIG2 , indicating that erythromycin A is produced in the fermentation broth of the strain E. coli sZG9.

[0186] HPLC-ELSD detection was performed. The HPLC-ELSD detection conditions were as follows: the HPLC detection system consisted of a Dionex UltiMate 3000 analytical HPLC (Thermo Scientific, MA, USA) and an ELSD detector (U3000). The liquid phase column was a SilGreen ODS column, φ4.6×250 mm, S-5 μM (Greenherbs Co., Ltd., Beijing, China). Mobile phase A was acetonitrile (containing 0.1% formic acid) and mobile phase B was 50 mM formic acid ammonia water. The specific detection method was as follows: the initial mobile phase 0% A was gradually increased to 95% A and 5% B within 30 minutes, and then returned to the initial mobile phase within 1 minute and continued to equilibrate for 4 minutes. The flow rate was 1 mL / min. The setting parameters of ELSD were: temperature 60°C, gas flow 1.6 L / min, and gain 1.

[0187] The HPLC-ELSD test results are shown in FIG3 . The erythromycin A production in E. coli sZG9 was 2.06 mg / L, and the erythromycin A production in E. coli sZG75 was 4.67 mg / L, indicating that the erythromycin A production capacity of strain E. coli sZG75 was 126.70% higher than that of E. coli sZG9.

[0188] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. A method for producing erythromycin using prokaryotic cells, comprising: (1) Integrate the erythromycin synthesis gene into the prokaryotic cell genome and downregulate a group of target genes; The erythromycin synthesis gene includes: a gene for synthesizing 6dEB, a gene for synthesizing erythromycin glycosyl side chain and a gene for hydroxylation and methylation modification; Among them, the downregulated target genes included: zwf, rfbC, vioA, and vioB; (2) Cultivating the prokaryotic cell according to claim 1 to produce erythromycin.

2. The method according to claim 1, wherein The genes for synthesizing 6dEB include: DEBS1, DEBS2, DEBS3; preferably also include: accA1, pccB, sfp, prpE; The genes for synthesizing erythromycin saccharide side chains and hydroxylation and methylation modifications include: eryF, eryBVI, eryBII, eryBVII, eryBIII, eryBIV, eryBV, eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryG, and eryK.

3. The method according to claim 1, wherein The downregulated target gene further comprises a gene selected from: pgi, yihX, gcd, galU, wecD or wecE.

4. The method according to claim 1, wherein The method further comprises: Integrate the molecular chaperone gene into the genome; preferably, the molecular chaperone gene includes: groES or groEL; and / or The erythromycin resistance gene ermE was integrated into the genome.

5. The method according to claim 1, wherein The prokaryotic cell is an Escherichia coli cell; preferably, the erythromycin synthesis gene is integrated into multiple positions in the genome; more preferably, it is integrated into the positions of araA, thiQ, lacY, and malT genes in the cell genome.

6. The method according to claim 5, wherein Integrate the expression cassettes of DEBS2 and DEBS3 into the position of the araA gene; preferably, introduce "promoter-rbs-DEBS2-rbs-DEBS3-terminator" at this position; Integrate the expression cassettes of pccB, accA1, and DEBS1 into the thiQ gene position; preferably, introduce "promoter-pccB-accA1-promoter-rbs-DEBS1-terminator" into this position; The expression cassettes of eryF, eryBVI, eryBII, AeeryBVII, AeeryBIII, eryBIV, eryBV, and optionally groES, groEL, and ermE are integrated into the position of the lacY gene; preferably, "promoter-rbs-AeeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-terminator" is introduced at this position; Integrate the expression cassettes of eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryG, and eryK into the position of the malT gene; preferably, introduce "promoter-rbs-eryCI-rbs-eryCII-rbs-eryCIII-rbs-eryCIV-rbs-eryCV-rbs-eryCVI-rbs-eryG-rbs-eryK-terminator" at this position; More preferably, the promoter is a T7 promoter, and the terminator is a T7 terminator.

7. The method according to claim 1, wherein The integration is performed using CRISPR-Cas mediated gene editing, homologous recombination, transposon or phage mediated methods; and / or The downregulation is performed using CRISPR-Cas mediated gene editing, homologous recombination, transposon or phage mediated gene disruption methods; Preferably, the gene integration and gene downregulation method is a CRISPR-Cas9-mediated gene editing method.

8. The method according to claim 1, wherein In step (2), an erythromycin synthesis substrate is added to the culture system; preferably, the erythromycin synthesis substrate is a substrate for producing propionyl-CoA; more preferably, the erythromycin synthesis substrate includes: propionic acid, propionate; more preferably, the erythromycin synthesis substrate is sodium propionate.

9. A prokaryotic cell for producing erythromycin, wherein the erythromycin synthesis gene is integrated into the genome and a set of target genes are downregulated; in, The erythromycin synthesis genes include: genes for synthesizing 6dEB, genes for synthesizing erythromycin glycosyl side chains and genes for hydroxylation and methylation modification; Among them, the downregulated target genes include: zwf, rfbC, vioA, and vioB.

10. The prokaryotic cell according to claim 9, wherein The genes for synthesizing 6dEB include: DEBS1, DEBS2, DEBS3; preferably also include: accA1, pccB, sfp, prpE; the genes for synthesizing erythromycin glycosyl side chains and hydroxylation and methylation modifications include: eryF, eryBVI, eryBII, eryBVII, eryBIII, eryBIV, eryBV, eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryG, eryK; Optionally, the downregulated target gene further comprises a gene selected from: pgi, yihX, gcd, galU, wecD or wecE; Optionally, a molecular chaperone gene is also integrated into its genome; preferably, the molecular chaperone gene includes: groES or groEL; Optionally, the erythromycin resistance gene ermE is also integrated into its genome.

11. The prokaryotic cell according to claim 9, wherein The prokaryotic cell is an Escherichia coli cell; preferably, the erythromycin synthesis gene is integrated into multiple positions in the genome; more preferably, it is integrated into the positions of araA, thiQ, lacY, and malT genes in the cell genome.

12. The prokaryotic cell according to claim 11, wherein The expression cassettes of DEBS2 and DEBS3 are integrated into the position of araA gene; preferably, "promoter-rbs-DEBS2-rbs-DEBS3-terminator" is introduced into this position; The expression cassettes of pccB, accA1, and DEBS1 are integrated into the thiQ gene position; preferably, "promoter-pccB-accA1-promoter-rbs-DEBS1-terminator" is introduced into this position; The expression cassettes of eryF, eryBVI, eryBII, AeeryBVII, AeeryBIII, eryBIV, eryBV, and optionally groES, groEL, and ermE are integrated into the lacY gene position; preferably, "promoter-rbs-AeeryF-rbs-groES-groEL-rbs-eryBVI-rbs-eryBII-rbs-AeeryBVII-rbs-AeeryBIII-rbs-eryBIV-rbs-eryBV-rbs-ermE-terminator" is introduced at this position; The expression cassettes of eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryG, and eryK are integrated into the malT gene position; preferably, "promoter-rbs-eryCI-rbs-eryCII-rbs-eryCIII-rbs-eryCIV-rbs-eryCV-rbs-eryCVI-rbs-eryG-rbs-eryK-terminator" is introduced at this position; More preferably, the promoter is a T7 promoter, and the terminator is a T7 terminator.

13. A method for producing the prokaryotic cell according to any one of claims 9 to 12, comprising: Integrate erythromycin biosynthesis genes into the prokaryotic genome and downregulate a set of target genes; Optionally, a molecular chaperone gene and / or an erythromycin resistance gene ermE are integrated into the genome of the prokaryotic cell; wherein the erythromycin synthesis gene, the down-regulated target gene, and the molecular chaperone gene are as defined in claims 9 to 12.

14. Use of the prokaryotic cell according to any one of claims 9 to 12 for producing erythromycin.

15. A kit for producing erythromycin, comprising: The prokaryotic cell according to any one of claims 9 to 12.

Images