Recombinant escherichia coli for synthesizing skatole from scratch, and construction method and application thereof

Abstract

This invention belongs to the field of genetic engineering and bioengineering technology, and discloses a recombinant *E. coli* strain that synthesizes skatole de novo, its construction method, and its applications. This invention involves metabolic engineering of the *E. coli* starting strain, overexpressing the skatole synthase SktA derived from *Nostoc punctata* NIES-2108, and knocking out... trpR , pheA , tyrA , tnaA Gene, overexpression of the DAHP synthase feedback inhibition mutant AroG D146N To enhance precursor supply, a two-phase fermentation method using tricresyl ester as the organic phase and glucose as the substrate was employed to produce skatole. This scheme achieved de novo microbial synthesis of skatole, with a maximum yield of 8.71 mg / L of skatole in the organic phase during two-phase fermentation. This lays the foundation for the subsequent construction of high-yield skatole engineered bacteria and can be applied to the microbial fermentation preparation of related products.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and bioengineering technology, and relates to a recombinant Escherichia coli that synthesizes skatole de novo, its construction method and application. Background Technology

[0002] Skatole, chemically known as 3-methylindole, is a substance naturally found in animal feces. It is also a valuable raw material with significant applications. In the fragrance industry, it can be used as a fixative in perfumes; in the food industry, it can be used as an additive for enhancing the aroma, preserving, and preventing color fading in products such as butter, cheese, wine, and chocolate. Currently, the industrial production of skatole mainly employs chemical synthesis. This method suffers from drawbacks such as high production costs, numerous reaction byproducts, and difficulties in separating and purifying the target product. Furthermore, the production process easily generates pollutants, which contradicts the current trend towards green chemistry.

[0003] To address the shortcomings of chemical synthesis methods, those skilled in the art have attempted to develop biosynthetic techniques. Some studies have reported that 3-indoleacetic acid can be converted into skatole under absolutely anaerobic conditions using indoleacetic acid decarboxylase. However, the conditions for this reaction are extremely demanding, requiring a strictly anaerobic environment and placing very high demands on production equipment and operations, making it difficult to achieve large-scale application. Furthermore, this method can only achieve in vitro enzymatic reactions and requires the addition of exogenous precursor substances, making it impossible to complete de novo synthesis using inexpensive carbon sources such as glucose, resulting in relatively high production costs.

[0004] Currently, no existing technology has achieved de novo microbial synthesis of skatole. There is a lack of technical solutions in this field that can open up a complete biosynthetic pathway from central carbon metabolism to skatole. At the same time, skatole itself has biotoxicity and high volatility, and the metabolic flux of precursor substances is insufficient, which also makes it difficult to advance related microbial modification work and achieve efficient and low-cost biomanufacturing of skatole. Summary of the Invention

[0005] To address the shortcomings of existing chemical synthesis methods for skatole, such as high production costs, numerous byproducts, and significant pollution, as well as the limitations of current biosynthetic technologies, such as demanding reaction conditions and the inability to utilize inexpensive carbon sources for de novo synthesis, and to solve the problems of missing biosynthetic pathways, insufficient precursor metabolic flux, and product toxicity restricting production in this field, this invention provides a recombinant Escherichia coli for de novo synthesis of skatole, its construction method, and its application. This aims to achieve efficient and green microbial de novo synthesis of skatole, reduce production costs and operational requirements, and promote the practical application of skatole biomanufacturing technology.

[0006] Therefore, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for constructing recombinant Escherichia coli that synthesizes skatole de novo, using Escherichia coli MG1655 as the starting strain, and improving it as follows: A. Overexpression of skatole synthase SktA, a diferric oxidase derived from Nostoc punctata NIES-2108, wherein the nucleotide sequence of the SktA encoding gene after codon optimization is shown in SEQ ID NO:1; codon optimization refers to replacing the codons of the foreign gene with codons frequently used by Escherichia coli according to the codon usage preferences of Escherichia coli, so as to improve gene expression efficiency; B. Knockout trpR Genes, the ones mentioned trpR The nucleotide sequence of the gene is shown in SEQ ID NO:2; C. Overexpression of the feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanolate 7-phosphate synthase D146N The AroG D146N The nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO:3; D. Knockout pheA Genes, the ones mentioned pheA The nucleotide sequence of the gene is shown in SEQ ID NO:4; E. Knockout tyrA Genes, the ones mentioned tyrA The nucleotide sequence of the gene is shown in SEQ ID NO:5; F. Knockout tnaA Genes, the ones mentioned tnaA The nucleotide sequence of the gene is shown in SEQ ID NO:6.

[0008] Furthermore, in step A of the above construction method, the skatole synthase SktA is overexpressed using pTrc99A as the expression vector.

[0009] Furthermore, in step B of the above construction method, the homologous recombination knockout is performed. trpR Gene.

[0010] Furthermore, in step C of the above construction method, the AroG is overexpressed via genome integration. D146N .

[0011] Furthermore, in steps D, E, and F of the above construction method, homologous recombination knockout is performed. pheA Gene, tyrA Genes and tnaA Gene.

[0012] Secondly, the present invention provides a recombinant Escherichia coli that synthesizes skatole de novo, wherein the recombinant Escherichia coli is constructed by the above-described construction method.

[0013] Thirdly, the present invention provides the application of the above-mentioned recombinant Escherichia coli in the fermentation preparation of skatole.

[0014] Furthermore, in the above application, the recombinant Escherichia coli is used as the fermentation strain, and glucose is used as the carbon source to carry out two-phase fermentation including an aqueous phase and an organic phase to produce skatole.

[0015] Furthermore, in the above application, the volume ratio of the aqueous phase to the organic phase is 5:1, and the organic phase is glyceryl tribaniate.

[0016] Furthermore, in the above applications, when the bacterial cells grow to OD... 600 When the concentration is 0.5~0.8, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1~1.0mM is added as an inducer to induce expression, and glyceryl tartrate is added at the same time as the inducer.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) A de novo biosynthetic pathway was established, and the de novo microbial synthesis of skatole was achieved for the first time. This invention is the first to design and successfully construct a de novo biosynthetic pathway from glucose to skatole in Escherichia coli. This was achieved through cross-species overexpression of Nostoc punctata NIES-2108 ( Nostoc punctiforme The ferric oxidase of NIES-2108, namely skatole synthase SktA (NCBI accession number: RCJ35217.1), has achieved a one-step catalytic conversion of L-tryptophan to skatole under aerobic conditions. By coupling central carbon metabolism with the metabolism of the precursor compound (L-tryptophan), a complete synthetic pathway for skatole within microorganisms has been established. This innovation solves the core problem of the lack of a microbial synthetic pathway from inexpensive carbon sources to this high-value natural product in existing technologies, fundamentally eliminating dependence on chemical synthesis methods.

[0018] (2) The supply of precursors to the pathway was enhanced, significantly increasing the yield of the target product. To overcome metabolic flux limitations, this invention further engineered the host *Escherichia coli* based on the strains that constructed the basic synthetic pathway. Specifically, this involved knocking out the protein encoding the L-tryptophan operon repressor, based on (1). trpR Genes that relieve global L-tryptophan feedback inhibition and overexpress genes derived from E. coli The MG1655 3-deoxy-D-arabino-heptanolate 7-phosphate (DAHP) synthase feedback inhibition mutant AroG D146N To enhance carbon flux in the shikimic acid pathway; key genes encoding branched-chain acid dehydrases were knocked out to synthesize phenylalanine and tyrosine. pheA Genes and cladactyl dehydrogenase tyrAThe gene disrupted the competitive pathway for carbon source diversion; finally, the gene encoding L-tryptophan hydrolase was further knocked out. tnaA The gene reduced precursor degradation and increased L-tryptophan precursor accumulation. Experimental results showed that the final strain (LSK04) after the above modifications achieved a skatole yield of 8.36 mg / L in shake-flask two-phase fermentation, which was about 8.3 times higher than that of the strain without precursor pathway enhancement (LSK02, yield of about 1.01 mg / L), laying the foundation for the subsequent construction of high-yield engineered bacteria.

[0019] (3) An efficient and controllable fermentation production method has been established, possessing potential for industrial application. This invention not only provides a recombinant Escherichia coli strain but also develops a complete method for the microbial fermentation production of skatole. This method uses the constructed recombinant Escherichia coli as the fermentation strain, glucose as the main carbon source, and is carried out under optimized fermentation medium (containing specific inorganic salts and trace metal elements, pH 6.5–7.5) and culture conditions (28–37°C, 100–500 rpm, fermentation 48–72 h). The method is achieved through mid-logarithmic growth (OD1). 600 By inducing the expression of exogenous pathways with low concentrations (0.1–1.0 mM) of IPTG (0.5–0.8 mM), a two-phase fermentation method was used to achieve efficient synthesis of skatole. This method is simple, mild, and low-cost. The fermentation products can be accurately quantified by high-performance liquid chromatography after simple processing, demonstrating its feasibility as a green and sustainable biomanufacturing platform for skatole. This provides key technical support for the future large-scale production of skatole-containing perfumes, cosmetics, and food products.

[0020] (4) The effectiveness and stability of the engineered strain and production method were verified. The verification showed that strain LSK04, under 0.5 mM IPTG induction, achieved a peak skatole yield of 8.71 mg / L during two-phase fermentation, and the yield remained relatively stable within a certain IPTG concentration range (0.10–1.0 mM), demonstrating the controllability of the production process. High-performance liquid chromatography (HPLC) analysis clearly showed a chromatographic peak in the fermentation sample with the same elution time as the skatole standard (20.889 min), which was confirmed by UV characteristic absorption wavelength, confirming the successful synthesis of the target product. These experimental data fully demonstrate the effectiveness and stability of the de novo synthetic pathway constructed in this invention. Attached Figure Description

[0021] Figure 1This diagram illustrates the constructed biosynthetic pathway and genetic engineering modification of skatole in *E. coli*. It labels the intermediate metabolites in the pathway, including: 3-phosphoglycerate (3PG), 3-phosphohydroxypyruvate (P3P), 3-phosphoserine (S3P), serine (L-SER), phosphoenolpyruvate (PEP), erythrose-4-phosphate (E4P), 3-deoxy-D-arabino-heptanone-7-phosphate (DAHP), branched acid (CHA), anthranilic acid (ANT), phenylalanine (PHE), tyrosine (TYR), indole (IND), L-tryptophan (L-TRP), and 3-methylindole (skatole). It also labels the encoding genes of related enzymes in the pathway, including phosphoglycerate dehydrogenase. SerA Phosphoserine aminotransferase SerC Phosphoserine phosphatase SerB DAHP synthase AroG L-tryptophan operon trpE , trpDCBA Branched-chain acid dehydratase PheA , cladoid dehydrogenase TyrA L-tryptophan hydrolase TnaA skatole synthase SktA The gene modification sites of this invention are also marked.

[0022] Figure 2 Agarose gel electrophoresis image of PCR identification of recombinant plasmid pSK01, where M is the DNA molecular weight marker; lane 1 is the PCR product of empty pTrc99A plasmid (318bp), and lanes 2-4 are the PCR products of pSK01 positive clones (1032bp).

[0023] Figure 3 This is a map of plasmid pSK01, a recombinant plasmid used to express skatole synthase SktA. The map shows the plasmid's origin of replication (ori), ampicillin resistance selection marker (AmpR), repressor protein gene (lacI), lactose operator gene (lacoperate), SktA skatole synthase gene and its expression cassette promoter (Trc), ribosome binding site (RBS), terminator, and other elements.

[0024] Figure 4 for trpR Agarose gel electrophoresis image of PCR identification of gene knockout strains, where M is the DNA molecular weight marker; lane 1 is the amplified genome product of wild-type Escherichia coli MG1655 (654bp), lanes 2-4 are... trpR Genomic amplification product of gene knockout positive strain (327bp).

[0025] Figure 5 for aroG D146NAgarose gel electrophoresis image of PCR identification of the genome-integrated strain, where M is the DNA molecular weight marker; lane 1 shows the wild-type MG1655 genome amplification product (703 bp), and lanes 2-3 show successful integration of the rsmG-atpI site. aroG D146N The amplification product of the gene-positive strain (1547bp).

[0026] Figure 6 for pheA , tyrA Agarose gel electrophoresis image of PCR identification of the double gene knockout strain, where M is the DNA molecular weight marker; lane 1 is the wild-type MG1655 genome amplification product (3550bp), lanes 2-4 are... pheA , tyrA Amplification product of double gene knockout positive strain (1236bp).

[0027] Figure 7 for tnaA Agarose gel electrophoresis image of PCR identification of gene knockout strains, where M is the DNA molecular weight marker; lane 1 is the wild-type MG1655 genome amplification product (1904bp), lanes 2-4 are... tnaA Amplification product of gene knockout positive strain (488bp).

[0028] Figure 8 This is a schematic diagram illustrating the skatole production process of strains LSK01~LSK04 under single-phase fermentation, used to demonstrate the skatole synthesis capacity of recombinant strains at different modification stages under single-phase fermentation conditions.

[0029] Figure 9 This is a schematic diagram illustrating the skatole production process of strains LSK02~LSK04 under two-phase fermentation conditions, used to demonstrate the skatole synthesis capacity of recombinant strains at different modification stages under two-phase fermentation conditions.

[0030] Figure 10 The figures show the liquid phase peaks of skatole standard (A), aqueous phase sample (B) from single-phase fermentation of LSK04 strain, and oil phase sample (C) from two-phase fermentation of LSK04 strain. Peak 1 is the characteristic chromatographic peak of skatole, with an elution time of 20.889 min, which is used to verify the synthesis of the target product.

[0031] Figure 11 This is a schematic diagram illustrating the two-phase fermentation production of skatole by strain LSK04 under different conditions, used to demonstrate the effect of different culture conditions on the skatole synthesis capacity of the recombinant strain. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental and detection methods in the following embodiments are conventional methods; the reagents and materials mentioned are commercially available unless otherwise specified; and the index data are measured using conventional methods unless otherwise specified.

[0034] The genotypes of the strains and plasmids involved in the examples are shown in Table 1.

[0035] Table 1. Genotypes of strains and plasmids

[0036] The primers and their specific sequences involved in the examples are shown in Table 2.

[0037] Table 2. Primer sequence listing

[0038] Culture medium components involved in the examples: 1. LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and deionized water to a final volume of 1 L.

[0039] 2. SOC resuscitation medium: 2.0 g tryptone, 0.5 g yeast extract, 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 10 mM magnesium sulfate, 20 mM glucose, diluted to 1 L with deionized water, and adjusted to pH 7.0. Preparation method: Dissolve the above ingredients in deionized water, adjust the pH to 7.0, autoclave, cool to room temperature, and finally add filtered and sterilized glucose solution.

[0040] 3. Fermentation medium: glucose 10 g / L, MgSO4 7H₂O 0.8 g / L, KH₂PO₄ 6.67 g / L, (NH₄)₂HPO₄ 4 g / L, citric acid 0.8 g / L, phenylalanine 0.04 g / L, tyrosine 0.1 g / L, sodium thiamine 0.01 g / L, trace metal salt solution 5 mL / L, pH 7.0; wherein the trace metal salt solution composition is: 10 g / L FeSO₄ 7H2O, 2.65 g / L CaCl2 2H₂O, 2.2 g / L ZnSO₄ 7H2O, 0.58 g / L MnSO4 5H2O, 1 g / L CuSO4 5H₂O, 0.1 g / L (NH₄)₆Mo₇O 24 4H₂O, 0.02 g / L Na₂B₄O₇ 10H₂O, 10 mL / L 35% HCl, deionized water as solvent; glucose and MgSO₄ 7H2O was used as the stock solution for independent sterilization, with concentrations of 500 g / L and 80 g / L, respectively. It should be noted that, due to the removal of... pheA and tyrA The strain is a phenylalanine and tyrosine auxotroph, therefore the fermentation medium is supplemented with the corresponding amino acids to meet the growth requirements of the strain.

[0041] The main reagents involved in the examples are: 1. Antibiotics: Ampicillin sodium (final concentration 100 mg / L) and chloramphenicol (final concentration 34 mg / L) are used for screening positive clones after transformation and for maintaining plasmid stability. 2. Inducers: IPTG (isopropyl-β-D-thiogalactoside, final concentration 0.1~1.0 mM) and L-arabinose (final concentration 10 mM) are used to induce the expression of exogenous proteins and recombinases, respectively. 3. Organic phase reagent: Tributyl glyceride, used to construct a two-phase fermentation system to achieve in-situ extraction of skatole and mitigate product toxicity and volatility; 4. Reagents for preparing competent cells: 10% glycerol solution, used for washing and resuspending electroporated competent cells to prepare competent cells; 5. Molecular biology reagents: Restriction endonucleases, purchased from TAKARA, used for plasmid digestion and construction to achieve plasmid linearization.

[0042] Information on the main experimental instruments involved in the examples: 1. Electroconversion instrument: used for electroconversion of Escherichia coli to introduce plasmids and homologous recombination linear fragments. In this example, the Ec1 setting is used to complete the electroconversion.

[0043] 2. PCR amplification instrument: used for amplification of target genes and homologous recombination screening fragments, as well as verification of positive clones by colony PCR.

[0044] 3. Agarose gel electrophoresis system: used for the separation and detection of nucleic acid fragments, to separate enzyme digestion products and PCR amplification products, and to provide pretreatment for gel recovery and purification.

[0045] 4. Clean bench: Provides a sterile operating environment for aseptic operations such as competent cell preparation, transformation, and strain inoculation.

[0046] 5. Constant temperature shaker: Used for seed culture and fermentation culture of strains. The culture temperature and shaking speed can be adjusted to meet the different needs of conventional strain culture at 37℃ and fermentation culture at 30℃.

[0047] 6. Constant temperature incubator: Used for culture on solid plates, screening of positive clones after transformation, and streak culture for eliminating auxiliary plasmids.

[0048] 7. Autoclave: Used for sterilization of culture media and laboratory equipment, and can be adapted to the independent sterilization requirements of fermentation culture media components.

[0049] 8. High-speed centrifuge: Used for cleaning competent cells and centrifuging fermentation samples. It can achieve high-speed centrifugation of 16,000 × g to separate the cells from the supernatant / oil phase.

[0050] 9. Ultraviolet spectrophotometer: used for bacterial culture OD 600 The value is detected to monitor the growth status of the strain and determine the timing of adding the inducer.

[0051] 10. High Performance Liquid Chromatography (Shimadzu LC-20 series): Used for quantitative detection of fermentation products, equipped with an Agilent Poroshell 120 EC-C18 (4.6×150 mm) column and a UV detector to complete the qualitative and quantitative analysis of skatole.

[0052] Example 1: Overall genetic engineering modification and synthetic pathway design of recombinant Escherichia coli.

[0053] This embodiment corresponds to the attached Figure 1 This is a schematic diagram of the biosynthesis pathway and genetic engineering modification of skatole in Escherichia coli, used to illustrate the overall genetic engineering modification process of the recombinant Escherichia coli of the present invention.

[0054] This invention uses Escherichia coli MG1655 as the starting strain and constructs a complete de novo biosynthetic pathway from glucose to skatole in Escherichia coli through metabolic engineering. The specific genetic engineering steps are as follows: A. Overexpression originates from Nostoc punctata NIES-2108 ( Nostoc punctiforme NIES-2108) ferric oxidase skatole synthase SktA This modification aims to streamline the final step of the skatole synthesis pathway from central carbon metabolism, enabling de novo microbial synthesis of skatole. By introducing an exogenous skatole synthase, SktA, the precursor L-tryptophan can be catalyzed into the target product skatole. The nucleotide sequence of the SktA encoding gene after codon optimization is shown in SEQ ID NO:1. Codon optimization refers to replacing the codons of the exogenous gene with frequently used codons by E. coli, based on their codon usage preferences, to improve gene expression efficiency.

[0055] B. Knockout trpR Gene This modification aims to remove global feedback inhibition of the L-tryptophan synthesis pathway and enhance its metabolic flux by knocking out the protein encoding the L-tryptophan operon repressor. trpR The gene relieves the transcriptional repression of the tryptophan synthesis operon by trpR, thereby increasing the expression level of the tryptophan synthesis pathway. trpR The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0056] C. Overexpression of the feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanolate 7-phosphate synthase D146N This modification aims to remove feedback inhibition from the rate-limiting enzyme in the first step of the shikimate pathway, enhancing the carbon flow supply and providing a sufficient carbon source for precursor synthesis. This is achieved by overexpressing the feedback inhibition mutant AroG. D146N This relieved the feedback inhibition of DAHP synthase and increased the metabolic flux of the shikimic acid pathway. The AroG... D146N The nucleotide sequence of the encoding gene is shown in SEQ ID NO:3.

[0057] D. Knockout pheA Gene This modification disrupts the competitive metabolic pathway that diverts branched-chain acid dehydratase to phenylalanine synthesis, reducing carbon source diversion and directing more carbon sources towards L-tryptophan synthesis. This is achieved by knocking out the enzyme encoding branched-chain acid dehydratase. pheA The gene blocks the metabolic branch from branched acid to phenylalanine, reducing the ineffective diversion of carbon sources. pheA The nucleotide sequence of the gene is shown in SEQ ID NO:4.

[0058] E. Knockout tyrA Gene This modification aims to disrupt the competitive metabolic pathway that diverts cladose acid to tyrosine synthesis, reducing carbon source diversion and directing more carbon sources towards L-tryptophan synthesis. This is achieved by knocking out the enzyme encoding cladose acid dehydrogenase. tyrAThe gene blocks the metabolic branch from branched acid to tyrosine, further reducing the ineffective diversion of carbon sources. tyrA The nucleotide sequence of the gene is shown in SEQ ID NO:5.

[0059] F. Knockout tnaA Gene This modification aims to reduce the degradation of the precursor L-tryptophan, further increasing its intracellular accumulation and providing sufficient precursors for skatole synthesis. This is achieved by knocking out the enzyme encoding L-tryptophan hydrolase. tnaA The gene reduced the degradation and consumption of L-tryptophan, increasing the intracellular concentration of the precursor. tnaA The nucleotide sequence of the gene is shown in SEQ ID NO:6.

[0060] In the synthetic pathway corresponding to the above modification strategy, starting from glucose, phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) are produced via central carbon metabolism, entering the shikimic acid pathway, and then via AroG... D146N Catalyzed by the formation of 3-deoxy-D-arabino-heptanone 7-phosphate (DAHP), it is gradually metabolized into branched acid (CHA). The branched acid enters the L-tryptophan synthesis pathway and, catalyzed by an enzyme encoded by the trpEDCBA operon, finally generates the precursor L-tryptophan (L-TRP). L-tryptophan is then converted into the target product skatole under the catalysis of skatole synthase SktA.

[0061] All of the aforementioned intermediate metabolites, related enzymes and genes, as well as the gene modification sites of this invention, are described in the appendix. Figure 1 The diagram is annotated to visually demonstrate the overall modification strategy and synthetic pathway of the present invention. Those skilled in the art can construct recombinant Escherichia coli based on this modification strategy and in conjunction with other embodiments of this application.

[0062] Example 2: This example describes the construction of the pSK01 plasmid.

[0063] The pTrc99A plasmid was digested with the restriction endonuclease NcoI to obtain a linearized plasmid vector fragment. Simultaneously, using sktA_F1 (SEQ ID NO:7) and sktA_R1 (SEQ ID NO:8) as primers, and the pUC57-sktA plasmid containing the fully synthesized sktA1 gene encoding skatole synthase as a template, the sktA gene (nucleotide sequence shown in SEQ ID NO:1) was amplified.

[0064] The linearized pTrc99A fragment and the amplified sktA1 gene fragment after enzyme digestion were subjected to agarose gel electrophoresis. The correct electrophoretic bands were excised and purified using a gel recovery kit. Then, a seamless cloning kit was used for recombination.

[0065] After transforming *E. coli* DH5α competent cells with the reaction product, the cells were plated on LB agar plates containing ampicillin sodium (final concentration 100 μg / mL). Single clones were picked from the plates and colony PCR amplification was performed using universal plasmid primers Seq-99A15K_F (SEQ ID NO:27) and Seq-99A15K_R (SEQ ID NO:28) to verify positive clones. Electrophoresis results are attached. Figure 2 As shown, lane 1 is the negative control pTrc99A empty vector plasmid band, with a fragment size of 318 bp; lanes 2-4 are the positive clone bands of the pTrc99A-sktA recombinant plasmid, with a fragment size of 1032 bp. The selected positive clones were sent to a sequencing institution for sequencing verification, confirming the successful construction of the recombinant plasmid pSK01.

[0066] A schematic diagram of plasmid pSK01 can be found here. Figure 3 The plasmid contains the origin of replication (ori), the ampicillin resistance selection marker (AmpR), the repressor protein gene (lacI), the lactose operator gene (lacoper), the SktA skatole synthase gene and its expression cassette promoter (Trc), the ribosome binding site (RBS), and the terminator.

[0067] Example 3: MG1655 trpR Gene knockout.

[0068] 1. Construction of linearized fragment KO-ΔtrpR Using pUTrc plasmid as a template and primer pairs ΔtrpR_F1 (SEQ ID NO:9) and ΔtrpR_R1 (SEQ ID NO:10) as primers, PCR amplification was performed using high-fidelity DNA polymerase to obtain a double loxP selection frame containing lox71-CmR-lox66 (chloramphenicol resistance, containing 50 bp homologous arms above and below trpR), denoted as KO-ΔtrpR1.

[0069] Using primer pairs ΔtrpR_F2 (SEQ ID NO:11) and ΔtrpR_R2 (SEQ ID NO:12) as primers and KO-ΔtrpR1 as a template, PCR amplification was performed to obtain the linearized fragment KO-ΔtrpR (containing 100 bp homologous arms upstream and downstream of trpR).

[0070] 2. Introduction of helper plasmid pKD46 E. coli Preparation of MG1655 electrocompetent states: Laboratory-preserved... E. coliMG1655 was inoculated into test tubes containing 5 mL of LB medium and cultured at 37°C with shaking at 200 rpm for 12–16 h. Then, 2% (v / v) of the culture was inoculated into 100 mL of fresh LB medium, and L-arabinose was added to a final concentration of 10 mM. The culture was then incubated at 37°C with shaking at 200 rpm for 1.5 h, until the bacterial OD reached the target concentration. 600 =0.4~0.6; centrifuge and discard the supernatant, wash the bacterial cells twice with pre-cooled 10% glycerol, and finally resuspend the bacterial cells in pre-cooled 10% glycerol to make the final volume 600~800 μL. Aliquot 100 μL into sterile 1.5 mL centrifuge tubes and freeze at -80℃ for later use.

[0071] Introduction of helper plasmid pKD46: Take one sample from a -80℃ freezer. E. coli MG1655 competent cells were thawed on ice and thoroughly mixed with approximately 50 ng of plasmid pKD46. The mixture was then transferred to pre-chilled 1 mm electroporation cuvettes and incubated on ice for 10 min. The cuvettes were dried and placed in an electroporator, where electroporation was performed using setting Ec1. Immediately after electroporation, 600 μL of pre-chilled SOC recovery medium was added and gently mixed. The entire mixture was then transferred to sterile 1.5 mL centrifuge tubes and incubated at 30°C and 200 rpm for 80 min with shaking. 80 μL of the recovered bacterial culture was then plated onto LB agar plates containing ampicillin sodium and incubated overnight at 30°C.

[0072] 3. Insertion of linearized fragment KO-ΔtrpR Pick single colonies from the plate in step 2 and inoculate them into test tubes containing 5 mL of LB broth and ampicillin sodium. Incubate at 30°C with shaking at 200 rpm for 16–20 h. Inoculate 2% of the culture volume into 15 mL of fresh LB broth and incubate at 30°C with shaking at 200 rpm for 2 h, until the bacterial culture reaches OD500. 600 =0.4~0.6; Centrifuge and discard the supernatant, wash the bacterial cells twice with pre-cooled 10% glycerol, and finally resuspend the bacterial cells in pre-cooled 10% glycerol to a final volume of 100 μL. Transfer to a sterile 1.5 mL centrifuge tube, mix thoroughly with approximately 50 ng of the KO-ΔtrpR fragment, and perform electroporation transformation in the same manner as in step 2; Spread the recovered culture mixture onto LB agar plates containing ampicillin sodium and chloramphenicol, and incubate overnight at 30°C. Perform colony PCR verification on the single clones on the plates to screen for positive clones.

[0073] 4. Elimination of helper plasmid pKD46 Streak the positive clones from step 3 onto LB agar plates containing chloramphenicol and incubate overnight at 42°C. Inoculate the single clones from the plates into 5 mL LB tubes containing or without ampicillin sodium and incubate at 37°C. Verify the elimination of plasmid pKD46 by observing the growth of the strains. Screening for ampicillin-sensitive strains completes the elimination of pKD46.

[0074] 5. Elimination of the CmR screening marker The strain with pKD46 eliminated was inoculated into test tubes containing 5 mL of LB broth and chloramphenicol, and cultured at 37°C with shaking at 200 rpm for 12–16 h. Then, it was inoculated into 15 mL of fresh LB broth at a 2% volumetric inoculation rate and cultured at 37°C with shaking at 200 rpm for 1.5 h, until the bacterial culture reached OD500. 600 =0.4~0.6; Centrifuge and discard the supernatant, wash the bacterial cells twice with pre-cooled 10% glycerol, and finally resuspend the bacterial cells in pre-cooled 10% glycerol to a final volume of 100 μL. Transfer to a sterile 1.5 mL centrifuge tube, mix thoroughly with approximately 50 ng of plasmid pjw168, and perform electroporation transformation in the same manner as in step 2; plate onto LB agar plates containing ampicillin sodium and 1 mM IPTG, and incubate overnight at 30°C. Perform colony PCR verification on single clones on the plates to screen for positive clones lacking chloramphenicol resistance.

[0075] 6. Elimination of helper plasmid pjw168 Streak the positive clones from step 5 onto LB agar plates and incubate overnight at 42°C. Inoculate the single clones from the plates into 5 mL LB tubes containing or without ampicillin sodium, and incubate at 37°C. Verify the elimination of plasmid pjw168 by observing the growth of the strains. The successfully eliminated strain is identified as strain MG1655. trpR .

[0076] 7. Strain identification PCR verification was performed using the genome identification primers Seq-KO-trpR-F (SEQ ID NO:29) and Seq-KO-trpR-R (SEQ ID NO:30). Electrophoresis results are attached. Figure 4 The amplified fragment size of wild-type MG1655 strain is 654 bp. trpR The gene knockout-positive strain amplified a fragment of 327 bp; the positive strain was sent to a sequencing institution for sequencing verification, and the gene knockout was finally confirmed. trpR Gene knockout strain MG1655ΔtrpR.

[0077] Example 4: MG1655Δ trpR middle aroG D146NGene integration.

[0078] 1. Construction of the linear editing fragment INS-aroGD146N (1) Component amplification: Using high-fidelity DNA polymerase, pUTrc plasmid was used as a template, and INS-aroG_F1 and INS-aroG_R1 (SEQ ID NO: 13~14) were used as primers for PCR amplification to obtain a double loxP selection frame containing lox71-CmR-lox66 (chloramphenicol resistance, LCL, 5' end containing an upstream rsmG-atpI 50 bp homologous arm). Simultaneously, pBBR1Tac- aroG D146N Using plasmids as templates and primer pairs aroG D146N _F1 and aroG D146N Using primers _R1 (SEQ ID NO:15~16), PCR amplification was performed to obtain... aroG D146N The target gene fragment (sequence shown in SEQ ID NO:3).

[0079] (2) Overlap Extension PCR (OEP): The LCL fragment obtained from the above amplification is combined with... aroG D146N Using the target gene fragment as a template, primers INS-aroG_F1 and aroG, designed with overlapping ends, were used. D146N _R1 was used for first-order overlap extension PCR to obtain the intermediate product INS-aroG D146N 1.

[0080] (3) Homologous arm extension: using INS-aroG D146N Using 1 as a template, nested primers INS-aroG_F2 and aroG carrying complete homologous arm sequences were used. D146N Primers _R2 (SEQ ID NO:17~18) were used for the final PCR amplification. Primer INS-aroG_F2 contains an exact homologous sequence 100 bp upstream of the rsmG-atpI site, and primer aroG... D146N _R2 contains an exact homologous sequence 100 bp downstream of this site. This step successfully obtained the final edited fragment INS-aroG. D146N Its structure is as follows: upstream 100 bp homologous arm - lox71 - CmR - lox66 - aroG D146N - Downstream 100 bp homologous arm.

[0081] 2. Introduction of helper plasmid pKD46 Following the method in step 2 of Example 3, strain MG1655ΔtrpR was prepared into an engineered strain containing pKD46. Specifically, strain MG1655ΔtrpR was prepared into electrocompetent cells, pKD46 helper plasmid was introduced, recombinase expression was induced by L-arabinose, and finally positive strains carrying pKD46 plasmid were screened.

[0082] 3. Linearized fragment INS-aroG D146N Insertion The engineered strain carrying pKD46 was taken and electrocompetent cells were prepared according to the method in step 3 of Example 3, and then reacted with approximately 50 ng of INS-aroG. D146N After mixing the fragments, electroporation transformation was performed. The transformed bacterial culture was plated onto LB agar plates containing ampicillin sodium and chloramphenicol and incubated overnight at 30°C. Single clones on the plates were verified by colony PCR, and recombinant positive clones were screened.

[0083] 4. Elimination of helper plasmid pKD46 Streak the positive clones from step 3 onto LB agar plates containing chloramphenicol and incubate overnight at 42°C. Inoculate the single clones from the plates into 5 mL LB agar tubes containing or without ampicillin sodium. Verify the elimination of plasmid pKD46 by observing the growth of the strains. Screening for ampicillin-sensitive strains completes the elimination of pKD46.

[0084] 5. Elimination of the CmR screening marker The strain with pKD46 eliminated was used to prepare electrotransformation competent cells according to step 5 of Example 3. These cells were mixed with approximately 50 ng of plasmid pjw168 and then electrotransformed. The transformed bacterial culture was plated onto LB agar plates containing ampicillin sodium and 1 mM IPTG and incubated overnight at 30°C. Colony PCR was performed on single clones on the plates to screen for positive clones lacking chloramphenicol resistance.

[0085] 6. Elimination of helper plasmid pjw168 Streak the positive clones from step 5 onto LB agar plates and incubate overnight at 42°C. Inoculate the single clones from the plates into 5 mL LB agar tubes containing or without ampicillin sodium. Verify plasmid pjw168 elimination by observing the growth of the strains. The successfully eliminated strain is identified as MG1655 ΔtrpR rsmG-atpI::aroG D146N .

[0086] 7. Strain identification Genomic PCR verification was performed on the successfully eliminated strains: amplification was performed using the upstream and downstream primers Seq-IG(rsmG-atpI)_F (SEQ ID NO:31) and Seq-IG(rsmG-atpI)_R (SEQ ID NO:32) for the rsmG-atpI site. Electrophoresis results are attached. Figure 5 As shown, lane 1 contains the rsmG-atpI site fragment of wild-type E. coli MG1655, with a size of 703 bp; lanes 2 and 3 contain rsmG-atpI site insertions. aroG D146N A positive clonal band of the gene was obtained, measuring 1547 bp. The positive clone was sent to a sequencing institution for verification, confirming the successful acquisition of the target strain MG1655 ΔtrpR rsmG-atpI::aroG. D146N .

[0087] Example 5: MG1655 ΔtrpR rsmG-atpI::aroG D146N middle pheA and tyrA Gene knockout.

[0088] 1. Construction of linear editing fragments KO-ΔpheA & tyrA Using pUTrc plasmid as a template and primer pairs ΔpheA&tyrA_F1 and ΔpheA&tyrA_R1 (SEQ ID NO:19~20) as primers, PCR amplification was performed to obtain a double loxP selection frame containing lox71-CmR-lox66 (chloramphenicol resistance, containing 50 bp homologous arms above and below pheA&tyrA), denoted as KO-ΔpheA&tyrA1.

[0089] Then, using primer pairs ΔpheA&tyrA_F2 and ΔpheA&tyrA_R2 (SEQ ID NO:21~22) as primers and KO-ΔpheA&tyrA1 as a template, PCR amplification was performed to obtain the linearized fragment KO-ΔpheA&tyrA (containing... pheA & tyrA (100 bp homologous arms upstream and downstream).

[0090] 2. Introduction of helper plasmid pKD46 Following the method in step 2 of Example 3, strain MG1655 Δ trpR rsmG-atpI::aroG D146N The engineered strain containing pKD46 was prepared by: preparing electrocompetent cells of the strain, introducing the pKD46 helper plasmid, inducing recombinase expression by L-arabinose, and finally screening to obtain positive strains carrying the pKD46 plasmid.

[0091] 3. KO-ΔpheA&tyrA Integration The engineered bacterial strain carrying pKD46 was used, and electrotransformation competent cells were prepared according to step 3 of Example 3. These cells were mixed with approximately 50 ng of the KO-ΔpheA&tyrA fragment and then electrotransformed. The transformed bacterial culture was plated onto LB agar plates containing ampicillin sodium and chloramphenicol and incubated overnight at 30°C. Single clones on the plates were verified by colony PCR, and recombinant positive clones were screened.

[0092] 4. Elimination of helper plasmid pKD46 Streak the positive clones from step 3 onto LB agar plates containing chloramphenicol and incubate overnight at 42°C. Inoculate the single clones from the plates into 5 mL LB agar tubes containing or without ampicillin sodium. Verify the elimination of plasmid pKD46 by observing the growth of the strains. Screening for ampicillin-sensitive strains completes the elimination of pKD46.

[0093] 5. Elimination of the CmR screening marker The strain with pKD46 eliminated was used to prepare electrotransformation competent cells according to step 5 of Example 3. These cells were mixed with approximately 50 ng of plasmid pjw168 and then electrotransformed. The transformed bacterial culture was plated onto LB agar plates containing ampicillin sodium and 1 mM IPTG and incubated overnight at 30°C. Colony PCR was performed on single clones on the plates to screen for positive clones lacking chloramphenicol resistance.

[0094] 6. Elimination of helper plasmid pjw168 Streak the positive clones from step 5 onto LB agar plates and incubate overnight at 42°C. Inoculate the single clones from the plates into 5 mL LB agar tubes containing or without ampicillin sodium. Verify plasmid pjw168 elimination by observing the growth of the strains. The successfully eliminated strain is identified as strain MG1655. trpR rsmG-atpI:: aroG D146N Δ pheA&tyrA .

[0095] 7. Strain identification Genomic PCR verification was performed using the identification primers Seq-Δ(pheA-tyrA)_F (SEQ ID NO:33) and Seq-Δ(pheA-tyrA)_R (SEQ ID NO:34). The electrophoresis results are attached. Figure 6 As shown: the amplified fragment length corresponding to wild-type MG1655 is 3550bp. pheA , tyrAThe amplified fragment length of the double-gene knockout positive clone was 1236 bp. The positive strain was sent to a sequencing institution for sequencing verification, confirming that the target strain was successfully constructed.

[0096] Example 6: MG1655 Δ trpR rsmG-atpI::aroG D146N Δ pheA&tyrA middle tnaA Knockout.

[0097] 1. Construction of the linear editing fragment KO-ΔtnaA Using pUTrc plasmid as a template and primer pairs ΔtnaA_F1 and ΔtnaA_R1 (SEQ ID NO:23~24) as primers, PCR amplification was performed to obtain a double loxP selection frame containing lox71-CmR-lox66 (chloramphenicol resistance, containing 50 bp homologous arms above and below tnaA), denoted as KO-ΔtnaA1.

[0098] Using primer pairs ΔtnaA_F2 and ΔtnaA_R2 (SEQ ID NO:25~26) as primers and KO-ΔtnaA1 as a template, PCR amplification was performed to obtain the linearized fragment KO-ΔtnaA (containing 100 bp homologous arms upstream and downstream of tnaA).

[0099] 2. Introduction of helper plasmid pKD46 Following the method in step 2 of Example 3, strain MG1655 Δ trpR rsmG-atpI::aroG D146N Δ pheA& tyrA The engineered strain containing pKD46 was prepared by: preparing electrocompetent cells of the strain, introducing the pKD46 helper plasmid, inducing recombinase expression by L-arabinose, and finally screening to obtain positive strains carrying the pKD46 plasmid.

[0100] 3.KO-ΔtnaA integration The engineered bacterial strain carrying pKD46 was used, and electroporation competent cells were prepared according to step 3 of Example 3. After mixing with approximately 50 ng of the KO-ΔtnaA fragment, electroporation transformation was performed. The transformed bacterial culture was plated onto LB agar plates containing ampicillin sodium and chloramphenicol and incubated overnight at 30°C. Colony PCR was performed on the single clones on the plates to verify and screen for recombinant positive clones.

[0101] 4. Elimination of helper plasmid pKD46 Streak the positive clones from step 3 onto LB agar plates containing chloramphenicol and incubate overnight at 42°C. Inoculate the single clones from the plates into 5 mL LB agar tubes containing or without ampicillin sodium. Verify the elimination of plasmid pKD46 by observing the growth of the strains. Screening for ampicillin-sensitive strains completes the elimination of pKD46.

[0102] 5. Elimination of the CmR screening marker The strain with pKD46 eliminated was used to prepare electrotransformation competent cells according to step 5 of Example 3. These cells were mixed with approximately 50 ng of plasmid pjw168 and then electrotransformed. The transformed bacterial culture was plated onto LB agar plates containing ampicillin sodium and 1 mM IPTG and incubated overnight at 30°C. Colony PCR was performed on single clones on the plates to screen for positive clones lacking chloramphenicol resistance.

[0103] 6. Elimination of helper plasmid pjw168 Streak the positive clones from step 5 onto LB agar plates and incubate overnight at 42°C. Inoculate the single clones from the plates into 5 mL LB agar tubes containing or without ampicillin sodium. Verify plasmid pjw168 elimination by observing the growth of the strains. The successfully eliminated strain is identified as MG1655 Δ. trpR rsmG-atpI::aroG D146N Δ pheA&tyrA Δ tnaA .

[0104] 7. Strain identification Genomic PCR verification was performed using identification primers seq-F-KO-tnaA (SEQ ID NO:35) and seq-R-KO-tnaA (SEQ ID NO:36). The electrophoresis results are attached. Figure 7 As shown: The amplified product of wild-type MG1655 has a fragment size of 1904 bp. tnaA The amplified fragment size of the knockout-positive strain was 488 bp. The positive strain was sent to a sequencing institution for sequencing verification to confirm its validity. tnaA The gene knockout strain was successfully constructed.

[0105] Example 7: Construction of LSK01~LSK04 strains.

[0106] E. coli strain MG1655, MG1655 Δ trpR rsmG-atpI::aroG D146N and MG1655 Δ trpR rsmG-atpI::aroG D146N Δ pheA&tyrA Δ tnaAThe strains were inoculated into test tubes containing 5 mL of LB medium and cultured with shaking at 37°C and 200 rpm for 12–16 h.

[0107] Inoculate the above-mentioned strains into 100 mL of fresh LB medium at a 2% volume inoculation rate, and incubate at 37°C and 200 rpm with shaking for 1.5 h, until the bacterial culture OD reaches 100%. 600 The concentration was increased to 0.4-0.6; the supernatant was discarded by centrifugation, and the bacterial cells were washed twice with pre-cooled 10% glycerol. Finally, the bacterial cells were resuspended in pre-cooled 10% glycerol, so that the final resuspension volumes of the three strains were 200 μL, 100 μL and 100 μL, respectively. Each 100 μL was aliquoted into a sterile 1.5 mL centrifuge tube to prepare electrocompetent cells.

[0108] Two E. coli MG1655 electrotransformation competent cells were taken and thoroughly mixed with approximately 50 ng of plasmid pTrc99A and approximately 50 ng of plasmid pSK01, respectively. Electrotransformation was performed in the same manner as in step 2 of Example 3. After transformation, the two bacterial cultures were spread onto LB agar plates containing ampicillin sodium and cultured overnight in a constant temperature incubator at 37°C to obtain strains LSK01 and LSK02, respectively.

[0109] Take 1 MG1655 Δ trpR rsmG-atpI::aroG D146N The competent cells were thoroughly mixed with approximately 50 ng of plasmid pSK01 and electroporated using the same method as in step 2 of Example 3. After transformation, the bacterial culture was spread onto LB agar plates containing ampicillin sodium and cultured overnight at 37°C to obtain strain LSK03.

[0110] Take 1 MG1655 Δ trpR rsmG-atpI::aroG D146N Δ pheA&tyrA Δ tnaA The competent cells were thoroughly mixed with approximately 50 ng of plasmid pSK01 and electroporated using the same method as in step 2 of Example 3. After transformation, the bacterial culture was spread onto LB plates containing ampicillin sodium and cultured overnight in a 37°C incubator to obtain strain LSK04.

[0111] The four strains LSK01 to LSK04 obtained above can be used for subsequent fermentation performance verification to demonstrate the skatole synthesis ability of recombinant strains at different modification stages.

[0112] Example 8: Single-phase fermentation of LSK01~LSK04 strains.

[0113] 1. Seed culture Pick a single clone from the corresponding plate or the corresponding bacterial culture frozen at -80°C and inoculate it into a test tube containing 5 mL of seed medium (LB medium), wherein ampicillin sodium is added to the seed medium to maintain plasmid stability; incubate overnight at 37°C and 200 rpm.

[0114] 2. Single-phase shake-flask fermentation The seed culture was inoculated at a volume rate of 2% into a 250 mL shake flask containing 50 mL of fermentation medium. The flask was then incubated with shaking at 30°C and 200 rpm for 60 h. Eight hours after inoculation, the OD of the bacterial culture was measured. 600 Once the concentration reaches 0.5–0.8, IPTG is added to a final concentration of 1 mM for induction. Three biological replicates are set up for each experiment.

[0115] 3. Sample processing and testing After the single-phase fermentation was completed, the aqueous phase was taken for sample processing and product detection: 100 μL of fermentation broth was taken, diluted 10 times, and the OD of the broth was measured. 600 The remaining 900 μL of fermentation broth was centrifuged at 16000×g for 10 min. The supernatant was filtered through a 0.22 μm aqueous filter and analyzed using a Shimadzu LC-20 series high-performance liquid chromatography system. The chromatographic column was an Agilent Poroshell 120 EC-C18 (4.6×150 mm). The mobile phase A was ddH2O containing 0.1% trifluoroacetic acid, and the mobile phase B was acetonitrile. The elution program was as follows: flow rate 0.65 mL / min, 0–1 min, 10% B; 1–19 min, B phase linearly increased from 10% to 60%; 19–21 min, B phase linearly increased from 60% to 70%; 21–23 min, B phase linearly decreased from 70% to 10%; 23–30 min, 10% B. The injection volume was 5 μL, the column oven temperature was 30℃, and the detection wavelength of the UV detector was 270 nm.

[0116] The test results showed that LSK02 had a skatole production of 0.13 mg / L, LSK03 had a skatole production of 0.42 mg / L, and LSK04 had a skatole production of 0.56 mg / L. These results correspond to the attached... Figure 8 .

[0117] The characteristic chromatographic peak elution time of the skatole standard was 20.889 min (see appendix). Figure 10 (A) In the single-phase fermentation aqueous sample of this embodiment, a product peak with the same elution time as the standard was detected. The detected liquid phase peak diagram corresponds to the attached... Figure 10 B in the figure verifies that the single-phase fermentation process of this embodiment successfully synthesized skatole.

[0118] Example 9: Two-phase fermentation of strains LSK02~LSK04.

[0119] 1. Seed culture The seed culture procedure in this embodiment is the same as in Example 8. Single clones from the corresponding plate or corresponding bacterial suspensions frozen at -80°C are picked and inoculated into test tubes containing 5 mL of seed culture medium (LB medium). Ampicillin sodium is added to the seed culture medium to maintain plasmid stability. The culture is carried out overnight at 37°C and 200 rpm.

[0120] 2. Two-phase shake-flask fermentation The seed culture was inoculated at a volume rate of 2% into a 250 mL shake flask containing 25 mL of fermentation medium. The flask was then incubated with shaking at 30°C and 200 rpm for 60 h. Eight hours after inoculation, the OD of the bacterial culture was measured. 600 When the water content reaches 0.5–0.8, IPTG with a final concentration of 1 mM is added for induction, and 5 mL of sterile tricresyl tartrate is added as the organic phase to construct a two-phase fermentation system with a volume ratio of aqueous phase to organic phase of 5:1. Three biological replicates are set up for each experiment.

[0121] 3. Sample processing and testing After the two-phase fermentation was completed, the lower oil phase was taken for product separation, purification, and detection: 100 μL of the upper aqueous phase was taken, diluted 10 times, and the OD of the bacterial culture was measured. 600 Values ​​were determined by centrifuging 1 mL of the lower oil phase at 16000×g for 10 min, filtering the resulting oil phase through a 0.22 μm aqueous filter membrane, and then performing high-performance liquid chromatography (HPLC) for product detection. The results showed that LSK02 produced 1.01 mg / L of skatole, LSK03 produced 5.54 mg / L, and LSK04 produced 8.36 mg / L. These results correspond to the attached... Figure 9 .

[0122] 4. High-performance liquid chromatography (HPLC) detection method The high-performance liquid chromatography (HPLC) detection method used in this embodiment is the same as in Example 8, employing a Shimadzu LC-20 series HPLC system with an Agilent Poroshell 120 EC-C18 column. Using this method, a product peak with the same elution time as the skatole standard was detected in the two-phase fermentation sample of this embodiment. The corresponding HPLC peak chromatogram is shown in the attached figure. Figure 10The value of C in the figure verifies that the two-phase fermentation process in this embodiment successfully synthesized skatole. Experimental results show that compared with single-phase fermentation, the product yield of the two-phase fermentation in this embodiment was significantly improved. Specifically, the skatole yield of the LSK04 strain during two-phase fermentation was 15 times higher than that during single-phase fermentation, proving that the two-phase fermentation system can effectively alleviate the toxicity and volatility problems of the product and significantly increase the synthesis yield of skatole.

[0123] Example 10: Fermentation of LSK04 under different conditions 1. Seed culture The seed culture procedure in this embodiment is the same as in Example 9. Single clones of strain LSK04 are picked from the corresponding plate or the corresponding bacterial solution frozen at -80°C and inoculated into a test tube containing 5 mL of seed culture medium (LB medium). Ampicillin sodium is added to the seed culture medium to maintain plasmid stability. The culture is carried out overnight at 37°C and 200 rpm.

[0124] 2. Two-phase fermentation under different induction conditions This embodiment uses the same two-phase fermentation system as in Example 9 to optimize and verify the induction conditions for strain LSK04: The seed culture was inoculated at a volume rate of 2% into a 250 mL shake flask containing 25 mL of fermentation medium. The flask was then incubated with shaking at 30°C and 200 rpm for 60 h. Eight hours after inoculation, the OD of the bacterial culture was measured. 600 To achieve a pH of 0.5–0.8, the shake flasks were divided into five groups, and IPTG was added to final concentrations of 0.10 mM, 0.25 mM, 0.50 mM, 0.75 mM, and 1.0 mM, respectively, for induction. Simultaneously, 5 mL of sterile tricresyl tartrate was added to each group as the organic phase, constructing a two-phase fermentation system with a water-to-organic phase volume ratio of 5:1. Three biological replicates were set up for each experiment.

[0125] The shake-flask fermentation medium used in this embodiment has the same composition as in Example 9, including glucose and MgSO4. The mother liquor of 7H2O was added after being independently heated and sterilized, and the composition of the trace metal salt solution was the same as in Example 9.

[0126] 3. Sample processing and testing The sample preparation and high-performance liquid chromatography detection methods in this embodiment are the same as those in Example 9, ensuring consistency in detection conditions.

[0127] The test results showed that the skatole production of strain LSK04 varied under different IPTG induction concentrations. The highest skatole production (8.71 mg / L) was observed at an IPTG concentration of 0.5 mM. This result corresponds to the attached... Figure 11 .

[0128] Experimental results show that the genetically engineered bacterium LSK04 constructed in this invention can achieve efficient accumulation of skatole under optimized induction conditions. The skatole yield in two-phase shake-flask fermentation can reach up to 8.71 mg / L, laying the foundation for the application of genetically engineered strains with high skatole production in the future.

[0129] The above embodiments can well illustrate the technical solution of the present invention, but they are only describing preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all kinds of changes and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A method for constructing recombinant Escherichia coli that synthesizes skatole de novo, characterized in that, Using Escherichia coli MG1655 as the starting strain, the following improvements were made: A. Overexpression of skatole synthase SktA, a diferric oxidase derived from Nostoc punctata NIES-2108, the nucleotide sequence of the codon-optimized gene encoding SktA is shown in SEQ ID NO:1; B. Knockout trpR Genes, the ones mentioned trpR The nucleotide sequence of the gene is shown in SEQ ID NO:2; C. Overexpression of the feedback inhibition mutant AroG of 3-deoxy-D-arabino-heptanolate 7-phosphate synthase D146N The AroG D146N The nucleotide sequence of the gene encoding the gene is shown in SEQ ID NO:3; D. Knockout pheA Genes, the ones mentioned pheA The nucleotide sequence of the gene is shown in SEQ ID NO:4; E. Knockout tyrA Genes, the ones mentioned tyrA The nucleotide sequence of the gene is shown in SEQ ID NO:5; F. Knockout tnaA Genes, the ones mentioned tnaA The nucleotide sequence of the gene is shown in SEQ ID NO:

6.

2. The construction method according to claim 1, characterized in that, In A, the skatole synthase SktA was overexpressed using pTrc99A as the expression vector.

3. The construction method according to claim 1, characterized in that, In B, the term "knockout" is achieved through homologous recombination. trpR Gene.

4. The construction method according to claim 1, characterized in that, In C, AroG is overexpressed via genome integration. D146N .

5. The construction method according to claim 1, characterized in that, In D, E, and F, knockout was achieved through homologous recombination. pheA Gene, tyrA Genes and tnaA Gene.

6. A recombinant Escherichia coli that synthesizes skatole de novo, characterized in that, The recombinant Escherichia coli was constructed using the construction method described in any one of claims 1 to 5.

7. The application of the recombinant Escherichia coli according to claim 6 in the fermentation preparation of skatole.

8. The application according to claim 7, characterized in that, Using the recombinant Escherichia coli of claim 6 as the fermentation strain and glucose as the carbon source, a two-phase fermentation including an aqueous phase and an organic phase is carried out to produce skatole.

9. The application according to claim 8, characterized in that, The volume ratio of the aqueous phase to the organic phase is 5:1, and the organic phase is glyceryl tribanilate.

10. The application according to claim 9, characterized in that, As the bacteria grow to OD 600 When the concentration is 0.5~0.8, isopropyl-β-D-thiogalactoside with a final concentration of 0.1~1.0 mM is added as an inducer to induce expression, and glyceryl tartrate is added at the same time as the inducer.

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