Recombinant escherichia coli mixed culture system for synthesizing indigo from scratch and construction method thereof

Abstract

This application relates to the field of microbial co-culture fermentation technology, specifically disclosing a recombinant Escherichia coli mixed system for de novo indigo synthesis and a method for co-culturing de novo indigo synthesis. This application reduces the metabolic burden of producing indigo from a single recombinant Escherichia coli by constructing two recombinant Escherichia coli strains and then constructing a recombinant Escherichia coli mixed system for de novo indigo synthesis. The clear division of labor among the bacterial groups in this application allows the product-producing bacteria to focus on the precursor produced by the precursor-synthesizing bacteria, resulting in a significant increase in indigo yield. Using this method, the indigo yield reaches over 8.3 g / L. The two recombinant Escherichia coli strains in this application synthesize indigo from a single strain using glycerol as a substrate, effectively solving the problems of high metabolic load, low conversion efficiency, and low yield associated with single strains in existing technologies. It also has the advantages of being green, environmentally friendly, safe, stable, and free from chemical pollution.

Description

Technical Field

[0001] This application relates to the field of microbial co-culture fermentation technology, and in particular to a recombinant Escherichia coli mixed system for de novo synthesis of indigo and its construction method. Background Technology

[0002] Indigo, a natural dye derived from plants, was originally extracted and processed from the indigo plant. However, cultivating this plant requires a large amount of arable land, has a long cultivation cycle, and is greatly affected by weather conditions. With industrial development, indigo is now often prepared chemically using aniline as a raw material. However, this raw material is highly toxic, the reaction requires strong acids and bases, and the reaction temperature is high, posing significant harm to the environment and human health. As people's living standards improve and their health awareness increases, the search for healthier, more environmentally friendly, and natural indigo pigments, returning to a natural and healthy ecological state, has become a popular aspiration.

[0003] Currently, strains used for microbial synthesis of indigo are usually constructed by knocking out genes on the host that compete with tryptophan synthesis and metabolism, and expressing genes encoding enzymes in the indigo biosynthesis pathway. Although they can achieve microbial synthesis of indigo, the yield has not been satisfactory. Therefore, constructing a genetically engineered strain and fermentation method for high-yield indigo is crucial for increasing indigo yield and realizing environmentally friendly and pollution-free industrial production of indigo.

[0004] The production of indigo using a single strain of *E. coli* has limitations and can easily lead to potential metabolic burden. Co-culture engineering shows promising applications in the biosynthesis of complex chemicals, but the stability of co-culture systems depends on the correlation between different bacterial communities. Furthermore, there are currently no reports of producing indigo using co-culture of different recombinant *E. coli* strains. Summary of the Invention

[0005] The purpose of this application is to overcome the shortcomings of the prior art and provide a recombinant Escherichia coli mixed bacterial system for de novo synthesis of indigo and its construction method; this application achieves efficient conversion from carbon source to indigo without adding any intermediates.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a recombinant Escherichia coli mixed bacterial system for de novo synthesis of indigo, the recombinant Escherichia coli mixed bacterial system comprising precursor synthesizing bacteria and product synthesizing bacteria; The precursor synthesizing bacteria include: knocking out the repressor protein encoding gene, phosphotransacetase encoding gene, tryptophanase encoding gene, and lactate dehydrogenase encoding gene of Escherichia coli; overexpressing the glycerol permease encoding gene, glycerol kinase encoding gene, glycerol 3-phosphate dehydrogenase encoding gene, triose phosphate isomerase encoding gene, 3-deoxy-D-arabinohepenolate-7-phosphate synthase encoding gene, and anthranilate synthase encoding gene to obtain the precursor synthesizing bacteria; The product-synthesizing bacteria include: Escherichia coli overexpressing the tryptophanase encoding gene, the styrene monooxygenase encoding gene, and the single-domain oxygen-binding hemoglobin encoding gene, to obtain the product-synthesizing bacteria.

[0007] The recombinant Escherichia coli mixed bacterial system provided in this application includes precursor synthesizers and product synthesizers. The precursor synthesizers can relieve feedback inhibition of intermediates and products, block the conversion of acetyl-CoA to acetic acid in E. coli, block the conversion of pyruvate to lactic acid, block further conversion of precursors, enhance the utilization of carbon source glycerol, strengthen the supply of key intermediate E4P, maintain the metabolic flux balance of the pentose phosphate pathway, and promote the synthesis of precursor tryptophan. The product synthesizers can enhance the conversion efficiency of precursor tryptophan, improve oxygen uptake and utilization, strengthen the supply of cofactor NADPH, and provide sufficient ATP to maintain the efficient synthesis of indigo. Co-culturing with the above-mentioned recombinant E. coli mixed bacterial system can synthesize indigo de novo.

[0008] As a preferred embodiment of the recombinant Escherichia coli mixed bacterial system for de novo synthesis of indigo described in this application, the precursor synthesizing bacteria also overexpress the transketolase encoding gene, the transaldolase encoding gene, and the D-ribulose-5-phosphate-3-epimerase encoding gene. And / or, the product-producing bacteria also overexpress the gene encoding membrane-bound pyridine nucleotide transhydrogenase and the gene encoding polyphosphate kinase.

[0009] Overexpression of the aforementioned coding gene in the precursor and product synthesizing bacteria of this application can better form a recombinant Escherichia coli mixed system for de novo indigo synthesis, thereby significantly increasing indigo yield to over 8.3 g / L.

[0010] As a preferred embodiment of the recombinant Escherichia coli mixed system for de novo synthesis of indigo described in this application, the repressor protein encoding gene includes the repressor protein encoding gene trpR; the phosphoacetyltransferase encoding gene includes the phosphoacetyltransferase encoding gene pta; the tryptophanase encoding gene includes the tryptophanase encoding gene tnaA; the lactate dehydrogenase encoding gene includes the lactate dehydrogenase encoding gene ldhA; the glycerol permease encoding gene includes the glycerol permease encoding gene glpF; the glycerol kinase encoding gene includes the glycerol kinase encoding gene glpK; the 3-phosphoglycerol dehydrogenase encoding gene includes the 3-phosphoglycerol dehydrogenase encoding gene glpD; the triose phosphate isomerase encoding gene includes the triose phosphate isomerase encoding gene tpiA; and the 3-deoxy-D-arabinohepylulonate-7-phosphate synthase encoding gene includes the 3-deoxy-D-arabinohepylulonate-7-phosphate synthase encoding gene aroG. S180F The anthranilic acid synthase encoding gene includes the anthranilic acid synthase encoding gene trpE. S40F ; And / or, the styrene monooxygenase encoding gene includes the styrene monooxygenase encoding gene SMO; the single-domain oxygen-binding hemoglobin encoding gene includes the single-domain oxygen-binding hemoglobin encoding gene vgb.

[0011] In a preferred embodiment of the recombinant Escherichia coli mixed bacterial system for de novo synthesis of indigo described in this application, the transketolase encoding gene includes the transketolase encoding gene tktA; the transaldolase encoding gene includes the transaldolase encoding gene talB; and the D-ribulose-5-phosphate-3-epimerase encoding gene includes the D-ribulose-5-phosphate-3-epimerase encoding gene rpe. And / or, The membrane-bound pyridine nucleotide transhydrogenase encoding gene includes the membrane-bound pyridine nucleotide transhydrogenase encoding gene pntAB; the polyphosphate kinase encoding gene includes the polyphosphate kinase encoding gene ppk.

[0012] In the technical solution of this application, the precursor synthetic bacteria (recombinant Escherichia coli TRP-07) integrates glpF, glpK, glpD, tpiA, and aroG by knocking out trpR, pta, tnaA, and ldhA genes. S180F trpE S40FThe tktA, talB, and rpe genes can relieve feedback inhibition of intermediates and products, block the conversion of acetyl-CoA to acetic acid in E. coli, block the conversion of pyruvate to lactate, block further conversion of precursors, enhance the utilization of the carbon source glycerol, strengthen the supply of the key intermediate E4P, maintain the metabolic flux balance of the pentose phosphate pathway, and promote the synthesis of the precursor tryptophan. The product-synthesizing strain (recombinant E. coli DL-02), by integrating the tnaA, SMO, vgb, pntAB, and ppk genes, can enhance the conversion efficiency of precursor tryptophan, improve oxygen uptake and utilization, strengthen the supply of the cofactor NADPH, and provide sufficient ATP to maintain the efficient synthesis of indigo.

[0013] In some specific embodiments, the D-ribulose-5-phosphate-3-epimerase encoding gene, the styrene monooxygenase encoding gene, and the single-domain oxygen-binding hemoglobin encoding gene can be derived from any species.

[0014] As a preferred embodiment of the recombinant Escherichia coli mixed bacterial system for de novo synthesis of indigo described in this application, the nucleotide sequence of the repressor protein encoding gene trpR is shown in SEQ ID NO: 1; the nucleotide sequence of the phosphotransacetase encoding gene pta is shown in SEQ ID NO: 2; the nucleotide sequence of the tryptophanase encoding gene tnaA is shown in SEQ ID NO: 3; the nucleotide sequence of the lactate dehydrogenase encoding gene ldhA is shown in SEQ ID NO: 4; the nucleotide sequence of the glycerol permease encoding gene glpF is shown in SEQ ID NO: 5; the nucleotide sequence of the glycerol kinase encoding gene glpK is shown in SEQ ID NO: 6; the nucleotide sequence of the 3-phosphoglycerate dehydrogenase encoding gene glpD is shown in SEQ ID NO: 7; the nucleotide sequence of the triose phosphate isomerase encoding gene tpiA is shown in SEQ ID NO: 8; and the 3-deoxy-D-arabinohepenolate-7-phosphate synthase encoding gene aroG is shown in SEQ ID NO: 8. S180F The nucleotide sequence is shown in SEQ ID NO: 9; the anthranilate synthase encoding gene trpE S40F The nucleotide sequence of the styrene monooxygenase encoding gene SMO is shown in SEQ ID NO: 10; the nucleotide sequence of the single-domain oxygen-binding hemoglobin encoding gene vgb is shown in SEQ ID NO: 14; and the nucleotide sequence of the single-domain oxygen-binding hemoglobin encoding gene vgb is shown in SEQ ID NO: 15.

[0015] As a preferred embodiment of the recombinant Escherichia coli mixed bacterial system for de novo synthesis of indigo described in this application, the nucleotide sequence of the transketolase encoding gene tktA is shown in SEQ ID NO: 11; the nucleotide sequence of the transaldolase encoding gene talB is shown in SEQ ID NO: 12; the nucleotide sequence of the D-ribulose-5-phosphate-3-epimerase encoding gene rpe is shown in SEQ ID NO: 13; the nucleotide sequence of the membrane-bound pyridine nucleotide transhydrogenase encoding gene pntAB is shown in SEQ ID NO: 16; and the nucleotide sequence of the polyphosphate kinase encoding gene ppk is shown in SEQ ID NO: 17.

[0016] In some specific embodiments, the D-ribulose-5-phosphate-3-epimerase encoding gene rpe is derived from Bacillus subtilis, and its nucleotide sequence is shown in SEQ ID NO: 13; the styrene monooxygenase encoding gene SMO is derived from Pseudomonas putida, and its nucleotide sequence is shown in SEQ ID NO: 14; the single-domain oxygen-binding hemoglobin encoding gene vgb is derived from Vibrio hygroscopicus, and its nucleotide sequence is shown in SEQ ID NO: 15.

[0017] As a preferred embodiment of the method for de novo synthesis of indigo from a recombinant Escherichia coli mixed system described in this application, the Escherichia coli includes Escherichia coli BL21(DE3).

[0018] As a preferred embodiment of the method for constructing the recombinant Escherichia coli mixed bacterial system for de novo synthesis of indigo as described in this application, the method for constructing the precursor synthesizing bacteria includes the following steps: 1) The repressor protein encoding gene trpR in Escherichia coli BL21(DE3) was knocked out using gene editing to obtain engineered strain A; 2) The pta gene, which encodes phosphotransacetase, was knocked out on the engineered bacterium A obtained in step 1) using gene editing to obtain engineered bacterium B; 3) The tryptophanase encoding gene tnaA on the engineered bacterium B obtained in step 2) was knocked out using gene editing to obtain engineered bacterium C; 4) The lactate dehydrogenase encoding gene ldhA on the engineered bacteria C obtained in step 3) was knocked out using gene editing to obtain engineered bacteria D; 5) Using gene editing, the glycerol permease encoding gene glpF, the glycerol kinase encoding gene glpK, and the glycerol 3-phosphate dehydrogenase encoding gene glpD were integrated into the engineered bacteria D obtained in step 4) to obtain engineered bacteria E; 6) Using gene editing, the gene encoding triose phosphate isomerase tpiA and the gene encoding DAHP synthase mutant aroG were modified. S180F and the trpE gene encoding anthranilic acid synthaseS40F Integrate into the engineered bacteria E obtained in step 5) to obtain engineered bacteria F; 7) Using gene editing, the transketolase encoding gene tktA, the transaldolase encoding gene talB, and the D-ribulose-5-phosphate-3-epimerase encoding gene rpe were integrated into the engineered bacteria F obtained in step 6) to obtain engineered bacteria G, which is the precursor synthesizer.

[0019] In some specific embodiments, the D-ribulose-5-phosphate-3-epimerase encoding gene rpe described in step 7) is integrated into engineered bacteria F after codon optimization.

[0020] As a preferred embodiment of the method for constructing the recombinant Escherichia coli mixed bacterial system for de novo synthesis of indigo as described in this application, the method for constructing the product-synthesizing bacteria includes the following steps: 1) Using gene editing, the tryptophanase encoding gene tnaA, the styrene monooxygenase encoding gene SMO, and the single-domain oxygen-binding hemoglobin encoding gene vgb were integrated into Escherichia coli BL21(DE3) to obtain engineered strain I; 2) Using gene editing, the membrane-bound pyridine nucleotide transhydrogenase encoding gene pntAB and the polyphosphate kinase encoding gene pppk were integrated into the engineered bacteria I obtained in step 1) to obtain the product-synthesizing bacteria.

[0021] In some specific embodiments, in step 1), the styrene monooxygenase encoding gene SMO and the single-domain oxygen-binding hemoglobin encoding gene vgb are integrated into Escherichia coli BL21(DE3) after codon optimization.

[0022] This application also provides a method for co-culturing a recombinant Escherichia coli mixed system to synthesize indigo de novo, wherein the secondary seed culture of the recombinant Escherichia coli mixed system for de novo indigo synthesis is inoculated into a fermentation medium for fermentation to obtain indigo.

[0023] Preferably, the secondary seed culture of the precursor synthesizing bacteria and the secondary seed culture of the product synthesizing bacteria are simultaneously inoculated into the fermentation medium for fermentation to obtain indigo.

[0024] In this application, precursor-synthesizing bacteria and product-synthesizing bacteria are simultaneously inoculated into the fermentation medium to obtain a recombinant Escherichia coli mixed system for de novo indigo synthesis. This method reduces the metabolic burden of a single recombinant Escherichia coli producing indigo from glucose, providing the necessary amino acid precursors for the recombinant Escherichia coli to synthesize indigo. The various bacterial groups used in this application have clear divisions of labor and cooperate with each other, thereby increasing indigo yield.

[0025] In some specific embodiments, the OD of the secondary seed culture of the precursor synthesizing bacteria 600=2.4; OD of the secondary seed culture of the product-synthesizing bacteria 600 =0.8.

[0026] As a preferred embodiment of the method for constructing the recombinant Escherichia coli mixed system for de novo synthesis of indigo described in this application, the fermentation conditions include: fermentation at a fermentation temperature of 30°C and a rpm of 300-900 rpm for 72 h.

[0027] As a preferred embodiment of the method for de novo synthesis of indigo from a recombinant Escherichia coli mixed bacterial system described in this application, the inoculation ratio of the secondary seed liquid of the precursor synthesizing bacteria and the secondary seed liquid of the product synthesizing bacteria is (1~4):1, or 1:(1~4), preferably 3:1.

[0028] This application provides a novel method for synthesizing indigo. By constructing two recombinant *E. coli* strains separately, and then co-culturing a mixed system of recombinant *E. coli* to synthesize indigo de novo, the metabolic burden of producing indigo with a single recombinant *E. coli* strain is reduced. In this application, the different bacterial groups have clearly defined roles, with the product-producing bacteria focusing on the precursors generated by the precursor-producing bacteria, resulting in a significant increase in indigo yield. Using this method, the indigo yield reaches over 8.3 g / L.

[0029] Compared with the prior art, this application has the following beneficial effects: This application provides a method for de novo synthesis of indigo using a mixed system of recombinant Escherichia coli. This method constructs two recombinant Escherichia coli strains and then a mixed system of them to synthesize indigo de novo, thus reducing the metabolic burden of producing indigo with a single recombinant Escherichia coli strain. The different bacterial groups in this application have clearly defined roles; the product-producing strain (recombinant Escherichia coli DL-02) focuses on the precursor produced by the precursor-synthesizing strain (recombinant Escherichia coli TRP-07), significantly increasing indigo yield. Using this method, the indigo yield reaches over 8.3 g / L. The two recombinant Escherichia coli strains in this application synthesize indigo de novo using glycerol as a substrate, effectively solving the problems of high metabolic load, low conversion efficiency, and low yield associated with single strains in existing technologies. It also has the advantages of being green, environmentally friendly, safe, stable, and free from chemical pollution. Attached Figure Description

[0030] Figure 1 A flowchart illustrating the de novo synthesis of indigo in a mixed bacterial co-culture system; Figure 2 Figure 1 shows the fermentation results of indigo with different combinations of recombinant Escherichia coli. Figure 3 Figure 1 shows the fermentation results of indigo at different inoculum ratios; Figure 4 The image shows the results of co-culturing and fermenting two recombinant Escherichia coli strains in a 5L reactor to synthesize indigo. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0032] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0033] Unless otherwise specified, all materials and reagents used in this application are commercially available.

[0034] Escherichia coli DH5α (hereinafter referred to as DH5α) used for vector construction can be obtained commercially.

[0035] Escherichia coli BL21 (DE3) was purchased from Shanghai Beinuo Biotechnology Co., Ltd.

[0036] Plasmids pTargetF, pCas9, pACYCDuet, pETDuet, and pET-28a were all purchased from BioWind.

[0037] High-fidelity DNA polymerase, restriction endonuclease, and In-Snap Assembly Master Mix were purchased from TAKARA.

[0038] Plasmid extraction kit, DNA purification kit, gel extraction kit, and bacterial genomic DNA extraction kit were purchased from OMEGA.

[0039] The culture medium formulation involved in this application is as follows: LB (Luria-Bertani) liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, sterilized at 121℃ for 20 min.

[0040] LB (Luria-Bertani) solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder. Sterilize at 121℃ for 20 min. Cool the solid medium to about 50℃, add the required antibiotics, pour the plate, and let it solidify. Then, place it at 4℃ for later use.

[0041] TB medium: 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 2.313 g / L potassium dihydrogen phosphate, 12.54 g / L dipotassium hydrogen phosphate.

[0042] Fermentation medium: 12 g / L peptone, 24 g / L yeast extract, 8.95 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 15 g / L glycerol, 1 g / L glucose, 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 1.9 g / L magnesium sulfate, 0.005 g / L vitamin B1, 50 mM sodium hexametaphosphate.

[0043] Feeding medium: 750 g / L glycerol.

[0044] Unless otherwise specified, the plasmid transformation into E. coli DH5α in the following examples and effect examples are all chemical transformations. The chemical transformation includes the following steps: the transformation system is added to DH5α competent cells that have been thawed on ice, incubated on ice for 30 min, then heat-shocked at 42°C for 1 min, incubated on ice again for 2 min, then 900 μL of pre-cooled LB medium is added, and incubated at 37°C and 220 rpm for 60 min; 100 μL of the incubated bacterial solution is added to LB medium containing the corresponding resistance and cultured overnight at 37°C.

[0045] Electroporation transformation includes the following steps: 250 μg of the fragment to be transformed is added to the prepared electroporation competent cells, gently mixed evenly, and then added to an electroporation cuvette and incubated on ice for 5-10 min; after 2-3 electroporations at 1.8 KV, LB medium preheated to 42℃ is quickly added, and the cells are heat-shocked in a 42℃ water bath for 1 min; then the heat-shocked transformation solution is incubated in a shaker at 37℃ for 2 hours; after incubation, 100 μL of the transformation solution is spread on an LB solid plate and incubated in a 37℃ incubator for 48 h.

[0046] The Gibson assembly technique (hereinafter referred to as Gibson) is operated with reference to the following literature: Gibson, DG, et al. (2009). Enzymatic assembly of DNA molecules upto several hundred kilobases. Nature Methods, 6(5), 343-345. Gibson, DG, et al. (2010). Creation of a bacteria1 cell controlled by a chemically synthesized genome. Science, 329(5987), 52-56. In the following embodiments, the nucleotide sequence of the repressor protein encoding gene trpR is shown in SEQ ID NO: 1; the nucleotide sequence of the phosphoryltransferase encoding gene pta is shown in SEQ ID NO: 2; the nucleotide sequence of the tryptophanase encoding gene tnaA is shown in SEQ ID NO: 3; the nucleotide sequence of the lactate dehydrogenase encoding gene ldhA is shown in SEQ ID NO: 4; the nucleotide sequence of the glycerol permease encoding gene glpF is shown in SEQ ID NO: 5; the nucleotide sequence of the glycerol kinase encoding gene glpK is shown in SEQ ID NO: 6; the nucleotide sequence of the 3-phosphoglycerate dehydrogenase encoding gene glpD is shown in SEQ ID NO: 7; the nucleotide sequence of the triose phosphate isomerase encoding gene tpiA is shown in SEQ ID NO: 8; and the 3-deoxy-D-arabinohepenolate-7-phosphate synthase encoding gene aroG is shown in SEQ ID NO: 8. S180F The nucleotide sequence is shown in SEQ ID NO: 9; the anthranilate synthase encoding gene trpE S40F The nucleotide sequences of the gene are shown in SEQ ID NO: 10; the nucleotide sequence of the transketolase encoding gene tktA is shown in SEQ ID NO: 11; the nucleotide sequence of the transaldolase encoding gene talB is shown in SEQ ID NO: 12; the nucleotide sequence of the D-ribulose-5-phosphate-3-epimerase encoding gene rpe is shown in SEQ ID NO: 13; the nucleotide sequence of the styrene monooxygenase encoding gene SMO is shown in SEQ ID NO: 14; the nucleotide sequence of the single-domain oxygen-binding hemoglobin encoding gene vgb is shown in SEQ ID NO: 15; the nucleotide sequence of the membrane-bound pyridine nucleotide transhydrogenase encoding gene pntAB is shown in SEQ ID NO: 16; and the nucleotide sequence of the polyphosphate kinase encoding gene ppk is shown in SEQ ID NO: 17.

[0047] Example 1: Construction of engineered bacteria TRP-01 In this embodiment, an engineered bacterium TRP-01 was constructed, and the repressor protein coding gene trpR was knocked out.

[0048] Specifically, the following steps are included: 1) Based on the trpR sequence of the E. coli repressor protein encoding gene in the NCBI database, the N20 sequence was designed, and primers N20-trpR-F and N20-trpR-R were designed accordingly. Then, using N20-trpR-F and N20-trpR-R as primers and pTargetF as a template, PCR amplification was performed to obtain the linear vector pTargetF-trpR plasmid. This linear vector was phosphorylated and self-ligated. The ligation product was transformed into DH5α competent cells, plated on LB plates containing streptomycin resistance, transformants were picked, plasmids were extracted, and sequenced for verification, yielding the plasmid pTargetF-trpR.

[0049] 2) Primers U-trpR-F, U-trpR-R, D-trpR-F, and D-trpR-R were designed based on the phosphate repressor protein encoding gene trpR. Using the BL21(DE3) genome as a template, the upstream fragment U-trpR and the downstream fragment D-trpR were amplified, respectively. The upstream and downstream fragments were fused using primers U-trpR-F and D-trpR-R to form the trpR knockout fragment ΔtrpR.

[0050] 3) ΔtrpR and plasmid pTargetF-trpR were electroporated into BL21(DE3) competent cells containing pCas9 plasmid. After rapid recovery in 1 mL LB medium at 37°C and 150 rpm for 1 h, the cells were plated onto LB agar plates containing kanamycin and streptomycin antibiotics. After incubation upside down for 24 h, positive transformants were identified by colony PCR using primers U-trpR-F and D-trpR-R.

[0051] 4) Pick a single colony, add IPTG and incubate for 12 hours to eliminate the pTargetF-trpR plasmid. Spread the colony on an LB solid medium plate containing kanamycin, select a single colony, and obtain the strain that has eliminated the pTargetF-trpR plasmid.

[0052] 5) Select single colonies and inoculate them into LB liquid medium. After overnight culture at 42°C, screen for single colonies that can grow on non-resistance plates but not on kanamycin-containing plates. Use primers U-trpR-F and D-trpR-R to verify the colony PCR. The verified strain is named TRP-01.

[0053] Example 2: Construction of engineered bacteria TRP-02 In this embodiment, an engineered bacterium TRP-02 was constructed, and the pta gene, which encodes phosphate transacetylase, was knocked out.

[0054] Specifically, the following steps are included: 1) Based on the pta sequence encoding the E. coli phosphotransacetase gene in the NCBI database, the N20 sequence was designed, and primers N20-pta-F and N20-pta-R were designed accordingly. Then, using N20-pta-F and N20-pta-R as primers and pTargetF as a template, PCR amplification was performed to obtain the linear vector pTargetF-pta plasmid. This linear vector was phosphorylated and self-ligated. The ligation product was transformed into DH5α competent cells, plated on LB plates containing streptomycin resistance, transformants were picked, plasmids were extracted, and sequenced for verification, yielding the plasmid pTargetF-pta.

[0055] 2) Primers U-pta-F, U-pta-R, D-pta-F, and D-pta-R were designed based on the phosphotransacetase encoding gene trpR. Using the BL21(DE3) genome as a template, the upstream fragment U-pta and the downstream fragment D-pta were amplified, respectively. The upstream and downstream fragments were fused together using primers U-pta-F and D-pta-R to form the pta knockout fragment Δpta.

[0056] 3) Electroporate Δpta and plasmid pTargetF-pta into TRP-01 competent cells containing pCas9 plasmid. After rapid recovery in 1 mL LB medium at 37°C and 150 rpm for 1 h, the cells were plated onto LB agar plates containing kanamycin and streptomycin antibiotics. After incubation upside down for 24 h, positive transformants were identified by colony PCR using primers U-pta-F and D-pta-R.

[0057] 4) Pick a single colony, add IPTG and incubate for 12 hours to eliminate the pTargetF-pta plasmid. Spread the colony on an LB solid medium plate containing kanamycin, select a single colony, and obtain the strain that has eliminated the pTargetF-pta plasmid.

[0058] 5) Select single colonies and inoculate them into LB liquid medium. After overnight culture at 42°C, screen for single colonies that can grow on non-resistance plates but not on kanamycin-containing plates. Use primers U-pta-F and D-pta-R to verify the colonies by PCR. The verified strains are named TRP-02.

[0059] Example 3: Construction of engineered bacteria TRP-03 In this embodiment, an engineered bacterium TRP-03 was constructed, and the tryptophanase encoding gene tnaA was knocked out.

[0060] Specifically, the following steps are included: 1) Based on the tnaA sequence of the *E. coli* tryptophanase encoding gene in the NCBI database, the N20 sequence was designed, and primers N20-tnaA-F and N20-tnaA-R were designed accordingly. Then, using N20-tnaA-F and N20-tnaA-R as primers and pTargetF as a template, PCR amplification was performed to obtain the linear vector pTargetF-tnaA plasmid. This linear vector was phosphorylated and self-ligated. The ligation product was transformed into DH5α competent cells, plated on LB plates containing streptomycin resistance, transformants were picked, plasmids were extracted, and sequenced for verification, yielding the plasmid pTargetF-tnaA.

[0061] 2) Primers U-tnaA-F, U-tnaA-R, D-tnaA-F, and D-tnaA-R were designed based on the tryptophanase-encoding gene tnaA. Using the BL21(DE3) genome as a template, the upstream fragment U-tnaA and the downstream fragment D-tnaA were amplified, respectively. The upstream and downstream fragments were fused using primers U-tnaA-F and D-tnaA-R to form the tnaA knockout fragment ΔtnaA.

[0062] 3) Electroporate ΔtnaA and plasmid pTargetF-tnaA into TRP-02 competent cells containing pCas9 plasmid. After rapid recovery in 1 mL LB medium at 37°C and 150 rpm for 1 h, plate the cells onto LB agar plates containing kanamycin and streptomycin antibiotics. Incubate upside down for 24 h, then identify positive transformants using colony PCR with primers U-tnaA-F and D-tnaA-R.

[0063] 4) Pick a single colony, add IPTG and incubate for 12 hours to eliminate the pTargetF-tnaA plasmid. Spread the colony on an LB solid medium plate containing kanamycin, select a single colony, and obtain the strain that has eliminated the pTargetF-tnaA plasmid.

[0064] 5) Select single colonies and inoculate them into LB liquid medium. After overnight culture at 42°C, screen for single colonies that can grow on non-resistance plates but not on kanamycin-containing plates. Use primers U-tnaA-F and D-tnaA-R to verify the colony PCR. The verified strain is named TRP-03.

[0065] Example 4: Construction of engineered bacteria TRP-04 In this embodiment, an engineered bacterium TRP-04 was constructed, and the lactate dehydrogenase encoding gene ldhA was knocked out.

[0066] Specifically, the following steps are included: 1) Based on the ldhA sequence of the *E. coli* tryptophanase encoding gene in the NCBI database, the N20 sequence was designed, and primers N20-ldhA-F and N20-ldhA-R were designed accordingly. Then, using N20-ldhA-F and N20-ldhA-R as primers and pTargetF as a template, PCR amplification was performed to obtain the linear vector pTargetF-ldhA plasmid. This linear vector was phosphorylated and self-ligated. The ligation product was transformed into DH5α competent cells, plated on LB plates containing streptomycin resistance, transformants were picked, plasmids were extracted, and sequenced for verification, yielding the plasmid pTargetF-ldhA.

[0067] 2) Primers U-ldhA-F, U-ldhA-R, D-ldhA-F, and D-ldhA-R were designed based on the lactate dehydrogenase encoding gene ldhA. Using the BL21(DE3) genome as a template, the upstream fragment U-ldhA and the downstream fragment D-ldhA were amplified, respectively. The upstream and downstream fragments were fused using primers U-ldhA-F and D-ldhA-R to form the ldhA knockout fragment ΔldhA.

[0068] 3) Electroporate ΔldhA and plasmid pTargetF-ldhA into TRP-03 competent cells containing pCas9 plasmid. After rapid recovery in 1 mL LB medium at 37°C and 150 rpm for 1 h, plate the cells onto LB agar plates containing kanamycin and streptomycin antibiotics. Incubate upside down for 24 h, then identify positive transformants using colony PCR with primers U-ldhA-F and D-ldhA-R.

[0069] 4) Pick a single colony, add IPTG and incubate for 12 hours to eliminate the pTargetF-ldhA plasmid. Spread the colony on an LB solid medium plate containing kanamycin, select a single colony, and obtain the strain that has eliminated the pTargetF-ldhA plasmid.

[0070] 5) Select single colonies and inoculate them into LB liquid medium. After overnight culture at 42°C, screen for single colonies that can grow on non-antibiotic plates but not on kanamycin-containing plates. Use primers U-ldhA-F and D-ldhA-R to verify the colony PCR. The verified strain is named TRP-04.

[0071] Example 5: Construction of engineered bacteria TRP-05 In this embodiment, an engineered bacterium TRP-05 was constructed, which overexpressed the glycerol permease encoding gene glpF, the glycerol kinase encoding gene glpK, and the glycerol-3-phosphate dehydrogenase encoding gene glpD.

[0072] Specifically, the following steps are included: 1) Using the Escherichia coli BL21(DE3) genome as a template, PCR amplification was performed using pET-28a-glpF-F and pET-28a-glpF-R primers to obtain the glpF fragment encoding the glycerol permease gene (nucleotide sequence as shown in SEQ ID NO: 5); PCR amplification was performed using NcoI-glpK-F and glpK-glpD-R primers to obtain the glpK fragment encoding the glycerol kinase gene (nucleotide sequence as shown in SEQ ID NO: 6); PCR amplification was performed using glpK-glpD-F and glpD-NcoI-R primers to obtain the glpD fragment encoding the glycerol 3-phosphate dehydrogenase gene (nucleotide sequence as shown in SEQ ID NO: 7).

[0073] 2) Using the commercially available plasmid pET-28a as a substrate, the pET-28a linearized vector was obtained by digestion with the restriction endonuclease Xba I. The pET-28a linearized vector and the glpF gene fragment with homologous arms were ligated using a seamless cloning ligation kit from Takara Bio Inc. The ligation product was transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the ligation product was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for approximately 16 hours. Colony PCR was performed using primers glpF-YZ-F and glpF-YZ-R for verification. Strains with correct PCR verification were cultured, and the recombinant plasmid was extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The plasmid with correct sequencing was identified as pET-28a-glpF.

[0074] 3) Using the obtained gene fragments glpK and glpD as templates, fusion PCR was performed using primers NcoI-glpK-F and glpD-NcoI-R to obtain the gene fragment glpK-glpD.

[0075] 4) Using pET-28a-glpF plasmid as a substrate, the pET-28a-glpF linearized vector was obtained by restriction endonuclease Nco I digestion and recovery. The pET-28a-glpF linearized vector and the glpK-glpD gene fragment with homologous arms were ligated using Takara Bio's seamless cloning ligation kit. The ligation product was transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the product was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for approximately 16 h. Colony PCR was performed using primers glpK-glpD-YZ-F and glpK-glpD-YZ-R. Strains with correct PCR results were cultured, and recombinant plasmids were extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The plasmid with correct sequencing was identified as pET-28a-glpF-glpK-glpD.

[0076] The pET-28a-glpF-glpK-glpD plasmid constructed in the above steps was transformed into bacteria TRP-04 using a chemical transformation method. After resuscitation and culture, the plasmid was plated onto LB agar plates containing 50 μg / mL kanamycin resistance and incubated at 37°C for approximately 16 h. The single colony growing on the LB agar plate was recombinant Escherichia coli BL21(DE3) / pET-28a-glpF-glpK-glpD, named TRP-05.

[0077] Example 6: Construction of engineered bacteria TRP-06 In this embodiment, an engineered bacterium TRP-06 was constructed, which overexpressed the triose phosphate isomerase encoding gene tpiA and the DAHP synthase encoding gene aroG. S180F With the anthranilate synthase encoding gene trpE S40F .

[0078] Specifically, the following steps are included: 1) Using the *E. coli* BL21(DE3) genome as a template, PCR amplification was performed using pETDuet-tpiA-F and pETDuet-tpiA-R primers to obtain the tpiA fragment (nucleotide sequence shown in SEQ ID NO: 8) encoding the triose phosphate isomerase gene; pETDuet-aroG was used as a primer. S180F -F and pETDuet-aroG S180F PCR amplification was performed using primer -R to obtain the gene encoding 3-deoxy-D-arabinohepenoic acid-7-phosphate synthase, aroG. S180F Fragment (nucleotide sequence as shown in SEQ ID NO: 9); using pETDuet-trpE S40F -F and pETDuet-trpE S40F PCR amplification was performed using primers -R to obtain the anthranilic acid synthase encoding gene trpE. S40F Fragment (nucleotide sequence as shown in SEQ ID NO: 10).

[0079] 2) Using the commercially available plasmid pETDuet as a substrate, the pETDuet linearized vector was obtained by digestion with the restriction endonuclease Xba I. The pETDuet linearized vector and the tpiA gene fragment with homologous arms were ligated using a seamless cloning ligation kit from Takara Bio Inc. The ligation product was then transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the ligation product was plated onto LB agar plates containing 50 μg / mL ampicillin-resistant medium and incubated at 37°C for approximately 16 h. Colony PCR was performed using primers tpiA-YZ-F and tpiA-YZ-R for verification. Strains with correct PCR verification were cultured, and the recombinant plasmid was extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The plasmid with correct sequencing was identified as pETDuet-tpiA.

[0080] 3) Using pETDuet-tpiA plasmid as a substrate, the pETDuet-tpiA linearized vector was obtained by digestion with the restriction endonuclease EcoR I. The pETDuet-tpiA linearized vector and aroG containing homologous arms were then combined. S180F Gene fragments were ligated using a seamless cloning and ligation kit from Takara Bio Inc. The ligation products were then transformed into *E. coli* DH5α via chemical transformation. After resuscitation and culture, the ligation products were plated onto LB agar plates containing 50 μg / mL ampicillin and incubated at 37°C for approximately 16 h. Primers aroG were used. S180F -YZ-F and aroG S180F -YZ-R was used for colony PCR verification. Strains that passed PCR verification were cultured, recombinant plasmids were extracted, and sent to Qingke Biotechnology Co., Ltd. for sequencing. Strains with correct sequencing were identified as pETDuet-tpiA-aroG. S180F Plasmid.

[0081] 4) Using pETDuet-tpiA-aroG S180F Using plasmid as substrate, pETDuet-tpiA-aroG was obtained by restriction endonuclease Kpn I digestion and recovery. S180F Linearization of the vector. pETDuet-tpiA-aroG S180F Linearized vectors and trpE with homologous arms S40F Gene fragments were ligated using a seamless cloning and ligation kit from Takara Bio Inc. The ligation products were then transformed into *E. coli* DH5α via chemical transformation. After resuscitation and culture, the ligation products were plated onto LB agar plates containing 50 μg / mL ampicillin and incubated at 37°C for approximately 16 h. Primers trpE were used. S40F -YZ-F and trpE S40F-YZ-R was used for colony PCR verification. Strains that passed PCR verification were cultured, recombinant plasmids were extracted, and sent to Qingke Biotechnology Co., Ltd. for sequencing. Strains with correct sequencing were identified as pETDuet-tpiA-aroG. S180F -trpE S40F Plasmid.

[0082] 5) The pETDuet-tpiA-aroG constructed in the above steps was converted using a chemical transformation method. S180F -trpE S40F The plasmid was transformed into bacterial strain TRP-05. After resuscitation and culture, it was plated onto LB agar plates containing 50 μg / mL ampicillin resistance and incubated at 37°C for approximately 16 h. Single colonies growing on the LB agar plates were recombinant *Escherichia coli* BL21(DE3) / pET-28a-glpF-glpK-glpD / pETDuet-tpiA-aroG. S180F -trpE S40F It was named TRP-06.

[0083] Example 7: Construction of engineered bacteria TRP-07 In this embodiment, an engineered bacterium TRP-07 was constructed, which overexpressed the transketolase encoding gene tktA, the transaldolase encoding gene talB, and the D-ribulose-5-phosphate-3-epimerase encoding gene rpe.

[0084] Specifically, the following steps are included: 1) Using the Escherichia coli BL21(DE3) genome as a template, PCR amplification was performed using pACYCDuet-tktA-F and tktA-talB-R primers to obtain the tktA fragment (nucleotide sequence shown in SEQ ID NO: 11) of the transketolase encoding gene; PCR amplification was performed using tktA-talB-F and pACYCDuet-talB-R primers to obtain the talB fragment (nucleotide sequence shown in SEQ ID NO: 12) of the transaldolase encoding gene; the D-ribulose-5-phosphate-3-epimerase encoding gene rpe (nucleotide sequence shown in SEQ ID NO: 13) was synthesized by Qingke Biotechnology Co., Ltd. after codon optimization, and PCR amplification was performed using pACYCDuet-rpe-F and pACYCDuet-rpe-R primers to obtain the rpe fragment of the D-ribulose-5-phosphate-3-epimerase encoding gene containing homologous arms.

[0085] 2) Using the obtained gene fragments tktA and talB as templates, fusion PCR was performed using primers pETDuet-tktA-F and pETDuet-talB-R to obtain the gene fragment tktA-talB.

[0086] 3) Using the commercially available plasmid pACYCDuet as a substrate, the pACYCDuet linearized vector was obtained by digestion with the restriction endonuclease EcoRI. The pACYCDuet linearized vector and the tktA-talB gene fragment with homologous arms were ligated using a seamless cloning ligation kit from Takara Bio Inc. The ligation product was transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the product was plated onto LB agar plates containing 50 μg / mL chloramphenicol and incubated at 37°C for approximately 16 h. Colony PCR was performed using primers tktA-talB-YZ-F and tktA-talB-YZ-R. Strains with correct PCR results were cultured, and recombinant plasmids were extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The plasmid with correct sequencing was identified as pACYCDuet-tktA-talB.

[0087] 4) Using pACYCDuet-tktA-talB plasmid as a substrate, the pACYCDuet-tktA-talB linearized vector was obtained by restriction endonuclease Xho I digestion and recovery. The pACYCDuet-tktA-talB linearized vector and the rpe gene fragment with homologous arms were ligated using Takara Bio's seamless cloning ligation kit. The ligation product was transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the product was plated onto LB agar plates containing 50 μg / mL chloramphenicol-resistant medium and incubated at 37°C for approximately 16 h. Colony PCR was performed using primers rpe-YZ-F and rpe-YZ-R. Strains with correct PCR results were cultured, and the recombinant plasmid was extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmid was identified as pACYCDuet-tktA-talB-rpe.

[0088] 5) The pACYCDuet-tktA-talB-rpe plasmid constructed in the above steps was transformed into bacteria TRP-06 using a chemical transformation method. After resuscitation and culture, it was plated onto LB agar plates containing 50 μg / mL chloramphenicol-resistant medium and incubated at 37°C for approximately 16 h. The single colonies growing on the LB agar plates are recombinant Escherichia coli BL21(DE3) / pET-28a-glpF-glpK-glpD / pETDuet-tpiA-aroG S180F -trpE S40F / pACYCDuet-tktA-talB-rpe, named TRP-07.

[0089] Example 8: Construction of engineered bacteria DL-01 In this embodiment, an engineered bacterium DL-01 was constructed, which overexpressed the tryptophanase encoding gene tnaA, the styrene monooxygenase encoding gene SMO, and the single-domain oxygen-binding hemoglobin encoding gene vgb.

[0090] Specifically, the following steps are included: 1) Using the Escherichia coli BL21(DE3) genome as a template, PCR amplification was performed using pETDuet-tnaA-F and pETDuet-tnaA-R primers to obtain the tnaA fragment of the tryptophanase encoding gene (nucleotide sequence as shown in SEQ ID NO: 3); the styrene monooxygenase encoding gene SMO (nucleotide sequence as shown in SEQ ID NO: 14) was synthesized by Qingke Biotechnology Co., Ltd. after codon optimization, and PCR amplification was performed using pETDuet-SMO-F and pETDuet-SMO-R primers to obtain the SMO fragment of the styrene monooxygenase encoding gene containing homologous arms; the single-domain oxygen-binding hemoglobin encoding gene vgb (nucleotide sequence as shown in SEQ ID NO: 15) was synthesized by Qingke Biotechnology Co., Ltd. after codon optimization, and PCR amplification was performed using pETDuet-vgb-F and pETDuet-vgb-R primers to obtain the vgb fragment of the single-domain oxygen-binding hemoglobin encoding gene containing homologous arms.

[0091] 2) Using the commercially available plasmid pETDuet as a substrate, the pETDuet linearized vector was obtained by digestion with the restriction endonuclease Xba I. The pETDuet linearized vector and the tnaA gene fragment with homologous arms were ligated using a seamless cloning ligation kit from Takara Bio Inc. The ligation product was then transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the culture was plated onto LB agar plates containing 50 μg / mL ampicillin-resistant medium and incubated at 37°C for approximately 16 h. Colony PCR was performed using primers tnaA-YZ-F and tnaA-YZ-R for verification. Strains with correct PCR verification were cultured, and the recombinant plasmid was extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmid was identified as the pETDuet-tnaA plasmid.

[0092] 3) Using pETDuet-tnaA plasmid as a substrate, the pETDuet-tnaA linearized vector was obtained by restriction endonuclease EcoRI digestion and recovery. The pETDuet-tnaA linearized vector and the SMO gene fragment with homologous arms were ligated using a seamless cloning ligation kit from Takara Bio Inc. The ligation product was transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the product was plated onto LB agar plates containing 50 μg / mL ampicillin-resistant medium and incubated at 37°C for approximately 16 h. Colony PCR was performed using primers SMO-YZ-F and SMO-YZ-R for verification. Strains with correct PCR verification were cultured, and the recombinant plasmid was extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmid was identified as pETDuet-tnaA-SMO plasmid.

[0093] 4) Using pETDuet-tnaA-SMO plasmid as a substrate, the pETDuet-tnaA-SMO linearized vector was obtained by restriction endonuclease Sla I digestion and recovery. The pETDuet-tnaA-SMO linearized vector and the vgb gene fragment with homologous arms were ligated using a seamless cloning ligation kit from Takara Bio Inc. The ligation product was transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the product was plated onto LB agar plates containing 50 μg / mL ampicillin-resistant medium and incubated at 37°C for approximately 16 h. Colony PCR was performed using primers vgb-YZ-F and vgb-YZ-R for verification. Strains with correct PCR verification were cultured, and the recombinant plasmid was extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmid was identified as pETDuet-tnaA-SMO-vgb.

[0094] 5) The pETDuet-tnaA-SMO-vgb plasmid constructed in the above steps was transformed into bacteria BL21(DE3) using a chemical transformation method. After resuscitation and culture, it was plated onto LB agar plates containing 50 μg / mL ampicillin-resistant medium and incubated at 37°C for approximately 16 h. The single colony growing on the LB agar plate is the recombinant Escherichia coli BL21(DE3) / pETDuet-tnaA-SMO-vgb, named DL-01.

[0095] Example 9: Construction of engineered bacteria DL-02 In this embodiment, an engineered bacterium DL-02 was constructed, which overexpressed the membrane-bound pyridine nucleotide transhydrogenase gene pntAB and the polyphosphate kinase gene ppk.

[0096] Specifically, the following steps are included: 1) Using the Escherichia coli BL21(DE3) genome as a template, PCR amplification was performed using pET-28a-pntAB-F and pET-28a-pntAB-R primers to obtain the pntAB fragment encoding the membrane-bound pyridine nucleotide transhydrogenase gene (nucleotide sequence as shown in SEQ ID NO: 16); PCR amplification was performed using pET-28a-ppk-F and pET-28a-ppk-R primers to obtain the ppk fragment encoding the polyphosphate kinase gene (nucleotide sequence as shown in SEQ ID NO: 17).

[0097] 2) Using the commercially available plasmid pET-28a as a substrate, the pET-28a linearized vector was obtained by digestion with the restriction endonuclease Xba I. The pET-28a linearized vector and the pntAB gene fragment with homologous arms were ligated using a seamless cloning ligation kit from Takara Bio Inc. The ligation product was then transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the ligation product was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for approximately 16 h. Colony PCR was performed using primers pntAB-YZ-F and pntAB-YZ-R. Strains with correct PCR results were cultured, and the recombinant plasmid was extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmid was identified as pET-28a-pntAB.

[0098] 3) Using pET-28a-pntAB plasmid as a substrate, the pET-28a-ppk linearized vector was obtained by restriction endonuclease Nco I digestion and recovery. The pET-28a-ppk linearized vector and the ppk gene fragment with homologous arms were ligated using Takara Bio's seamless cloning ligation kit. The ligation product was transformed into *E. coli* DH5α using chemical transformation. After resuscitation and culture, the ligation product was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for approximately 16 hours. Colony PCR was performed using primers ppk-YZ-F and ppk-YZ-R for verification. Strains with correct PCR verification were cultured, and the recombinant plasmid was extracted and sent to Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmid was identified as pET-28a-pntAB-ppk.

[0099] 4) The pET-28a-pntAB-ppk plasmid constructed in the above steps was transformed into bacteria DL-01 using a chemical transformation method. After resuscitation and culture, it was plated onto LB agar plates containing 50 μg / mL kanamycin-resistant medium and incubated at 37°C for approximately 16 h. The single colony growing on the LB agar plate is the recombinant Escherichia coli BL21(DE3) / pET-28a-pntAB-ppk, named DL-02.

[0100] The primers used in Examples 1-9 are shown in Table 1.

[0101] Table 1 Primers used in the construction of engineered bacteria Example 10: Validation of a shake-flask fermentation system for producing indigo from recombinant Escherichia coli TRP-07 and DL-02. (1) Seed culture: Recombinant Escherichia coli TRP-07 was streaked in three zones on LB solid plates to activate the seed culture, and cultured at 37℃ for 24h; single colonies were picked and inoculated into LB liquid medium, and cultured at 37℃ for 12h as the primary seed culture of recombinant Escherichia coli TRP-07; the primary seed culture was then divided according to OD 600 =0.6 was inoculated into 25 mL of TB medium and cultured at 30℃ and 200 rpm for 8 h to obtain a secondary seed culture of recombinant Escherichia coli TRP-07; Recombinant Escherichia coli DL-02 was streaked into three zones on LB agar plates for seed culture and incubated at 37°C for 24 h. Single colonies were picked and inoculated into LB liquid tubes and incubated at 37°C for 12 h to obtain the primary seed culture of recombinant Escherichia coli DL-02. The primary seed culture was then divided according to OD... 600 =0.6 was inoculated into 25 mL of TB medium and cultured at 30℃ and 200 rpm for 8 h to obtain the secondary seed culture of recombinant Escherichia coli DL-02 strain; (2) Co-culture: The secondary seed culture of recombinant Escherichia coli TRP-07 was cultured according to OD... 600 =2.4 and the secondary seed culture of recombinant Escherichia coli DL-02 according to OD 600 =0.8 1 mL each was simultaneously inoculated into 50 mL of fermentation medium to obtain a mixed system of recombinant Escherichia coli TRP-07 and recombinant Escherichia coli DL-02. Fermentation was carried out at 30℃ and 200 rpm for 72 h, with 1.34 mL of feed medium added at 24 h and 48 h respectively.

[0102] Example 11: 5L bioreactor fermentation system for producing indigo from recombinant Escherichia coli TRP-07 and DL-02. verify (1) Seed culture: Recombinant Escherichia coli TRP-07 was streaked in three zones on LB solid plates to activate the seed culture, and cultured at 37℃ for 24h; single colonies were picked and inoculated into LB liquid medium, and cultured at 37℃ for 12h as the primary seed culture of recombinant Escherichia coli TRP-07; the primary seed culture was then divided according to OD 600 =0.6 was inoculated into 100 mL TB medium and cultured at 30℃ and 200 rpm for 8 h to obtain secondary seed culture of recombinant Escherichia coli TRP-07; Recombinant Escherichia coli DL-02 was streaked into three zones on LB agar plates for seed culture and incubated at 37°C for 24 h. Single colonies were picked and inoculated into LB liquid tubes and incubated at 37°C for 12 h to obtain the primary seed culture of recombinant Escherichia coli DL-02. The primary seed culture was then divided according to OD... 600 =0.6 was inoculated into 100 mL of TB medium and cultured at 30 °C and 200 rpm for 8 h to obtain the secondary seed culture of recombinant Escherichia coli DL-02 strain; (2) Co-culture: The secondary seed culture of recombinant Escherichia coli TRP-07 was cultured according to OD... 600 =2.4 and the secondary seed culture of recombinant Escherichia coli DL-02 according to OD 600 =0.8 100 mL each were simultaneously inoculated into 2 L of fermentation medium to obtain a mixed system of recombinant Escherichia coli TRP-07 and recombinant Escherichia coli DL-02. Fermentation was carried out at 30℃ and 200 rpm gradually increased to 900 rpm for 72 h. At 12 h of fermentation, fed medium was started at a feeding rate of 2 g / L / h until fermentation was completed.

[0103] Example 1: The recombinant bacterial combination was co-cultured and fermented with indigo according to the method in Example 10. This example compares the effects of co-culturing fermentation with different combinations of precursor-synthesizing and product-synthesizing bacteria. The experimental steps are the same as in Example 10. The different co-culturing combinations of recombinant bacteria are as follows: Combination 1: TRP-04 and DL-01; Combination 2: TRP-04 and DL-02; Combination 3: TRP-05 and DL-01; Combination 4: TRP-05 and DL-02; Combination 5: TRP-06 and DL-01; Combination 6: TRP-06 and DL-02; Combination 7: TRP-07 and DL-01; Combination 8: TRP-07 and DL-02.

[0104] OD values ​​were measured at 606 nm using indigo standard solutions at concentrations of 1, 2, 4, 8, 12, 16, and 18 μg / mL. The indigo concentration was plotted on the ordinate. 606 A standard curve was plotted on the x-axis, resulting in the curve Y = 14.68X - 0.1968, R0. 2 =0.9996.

[0105] The OD values ​​of different samples at 606 nm were measured using a UV spectrophotometer. The yield of indigo was calculated based on the standard curve. The results are shown in [Figure number missing]. Figure 2 The synthetic route for recombinant E. coli co-culture is shown in [link to relevant documentation]. Figure 1 .

[0106] Figure 2 The results showed that the recombinant bacterial co-culture system of this application can effectively utilize glycerol to synthesize indigo. Among them, the combination of recombinant strains TRP-07 and DL-02 showed the best effect, with a yield of 1.41 g / L after 72 h of fermentation.

[0107] The comparative results showed that the tryptophan synthesis capacity of the precursor bacteria was the key factor determining the yield of this co-culture system. Overexpression of the DAHP synthase encoding gene aroG, which resists feedback inhibition, was crucial. S180F With the anthranilate synthase encoding gene trpE S40F Subsequently, indigo yield showed a significant increase, with combination 5 increasing by 5.82 times compared to combination 4. Further enhancement of intermediate supply resulted in a 49.3% increase in yield (combination 7). The highest yield in shake flasks was achieved by enhancing the supply of cofactors (NADPH and ATP) in the product-synthesizing bacteria, demonstrating that sufficient cofactors are required for indigo synthesis.

[0108] Example 2: The different inoculation ratios of the two recombinant bacteria in Comparative Example 2 were carried out according to the method in Example 10. Indigo co-culture yeast This example compares the effects of co-culturing and fermenting two recombinant strains at different biomass ratios.

[0109] The experimental procedure was the same as in Example 10, with TRP-07 and DL-02 inoculated at the following biomass ratios: Method 1: TRP-07:DL-02 = 1:4; Method 2: TRP-07:DL-02 = 1:3; Method 3: TRP-07:DL-02 = 1:2; Method 4: TRP-07:DL-02 = 1:1; Method 5: TRP-07:DL-02 = 2:1; Method 6: TRP-07:DL-02 = 3:1; Method 7: TRP-07:DL-02 = 4:1.

[0110] The detection method is the same as in Example 1, and the results are as follows: Figure 3 As shown.

[0111] Figure 3The results showed that the inoculum size of the two strains in the co-culture system significantly affected the yield of the final target product. The best fermentation effect was observed when the inoculum ratio of precursor-synthesizing bacteria to product-synthesizing bacteria was 3:1, with a yield of 1.48 g / L in shake flasks after 72 hours. Comparative results indicated that a low inoculum size of the precursor-synthesizing bacteria had the most significant impact on yield; when the inoculum ratio was 1:4 (Method 1), the indigo yield was only 0.52 g / L, approximately one-third of the highest yield. Therefore, ensuring the synthesis and supply of the precursor tryptophan is crucial for improving the efficiency of the co-culture system.

[0112] Example 3: The engineered bacteria from Example 7 and the engineered bacteria from Example 9 were subjected to indigo treatment according to the method in Example 11. Blue Synthesis Fermentation The detection method is the same as in Example 1, and the results are as follows: Figure 4 As shown.

[0113] Figure 4 The results showed that the co-culture system could still efficiently synthesize indigo from glycerol in a 5L reactor. Furthermore, due to the more favorable fermentation conditions provided by the reactor, the indigo yield reached 8.3 g / L after 72 hours of fermentation. The process trend in the figure shows that the indigo synthesis efficiency was lower in the early stages, but the synthesis rate was faster in the middle and later stages. This may be related to the tryptophan production by the precursor synthesizer in the co-culture system. When the precursor supply increased, the product began to accumulate more rapidly, indicating that the rapid growth of the precursor synthesizer TRP-07 and the precursor synthesis in the co-culture system determined the product synthesis rate, consistent with the conclusions of Example 2.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A recombinant Escherichia coli mixed culture system for synthesizing indigo de novo, characterized by, The recombinant Escherichia coli mixed bacterial system includes precursor synthesizing bacteria and product synthesizing bacteria; The precursor synthesizing bacteria include: knocking out the repressor protein encoding gene, phosphotransacetase encoding gene, tryptophanase encoding gene, and lactate dehydrogenase encoding gene of Escherichia coli; overexpressing the glycerol permease encoding gene, glycerol kinase encoding gene, glycerol 3-phosphate dehydrogenase encoding gene, triose phosphate isomerase encoding gene, 3-deoxy-D-arabinohepenolate-7-phosphate synthase encoding gene, and anthranilate synthase encoding gene to obtain the precursor synthesizing bacteria; The product-synthesizing bacteria include: Escherichia coli overexpressing the tryptophanase encoding gene, the styrene monooxygenase encoding gene, and the single-domain oxygen-binding hemoglobin encoding gene, to obtain the product-synthesizing bacteria.

2. The recombinant Escherichia coli mixed culture system synthesizing indigo de novo according to claim 1, characterized in that, The precursor synthesizing bacteria also overexpressed the transketolase encoding gene, the transaldolase encoding gene, and the D-ribulose-5-phosphate-3-epimerase encoding gene; And / or, the product-producing bacteria also overexpress the gene encoding membrane-bound pyridine nucleotide transhydrogenase and the gene encoding polyphosphate kinase.

3. The recombinant Escherichia coli mixed culture system synthesizing indigo de novo according to claim 1, characterized in that, The repressor protein encoding gene includes the repressor protein encoding gene trpR; the phosphoacetyltransferase encoding gene includes the phosphoacetyltransferase encoding gene pta; the tryptophanase encoding gene includes the tryptophanase encoding gene tnaA; the lactate dehydrogenase encoding gene includes the lactate dehydrogenase encoding gene ldhA; the glycerol permease encoding gene includes the glycerol permease encoding gene glpF; the glycerol kinase encoding gene includes the glycerol kinase encoding gene glpK; the 3-phosphoglycerol dehydrogenase encoding gene includes the 3-phosphoglycerol dehydrogenase encoding gene glpD; the triose phosphate isomerase encoding gene includes the triose phosphate isomerase encoding gene tpiA; and the 3-deoxy-D-arabinoheptanulonate-7-phosphate synthase encoding gene includes the 3-deoxy-D-arabinoheptanulonate-7-phosphate synthase encoding gene aroG. S180F The anthranilic acid synthase encoding gene includes the anthranilic acid synthase encoding gene trpE. S40F ; And / or, the styrene monooxygenase encoding gene includes the styrene monooxygenase encoding gene SMO; the single-domain oxygen-binding hemoglobin encoding gene includes the single-domain oxygen-binding hemoglobin encoding gene vgb.

4. The recombinant Escherichia coli mixed culture system synthesizing indigo de novo according to claim 2, wherein, The transketolase encoding gene includes the transketolase encoding gene tktA; the transaldolase encoding gene includes the transaldolase encoding gene talB; the D-ribulose-5-phosphate-3-epimerase encoding gene includes the D-ribulose-5-phosphate-3-epimerase encoding gene rpe; And / or, The membrane-bound pyridine nucleotide transhydrogenase encoding gene includes the membrane-bound pyridine nucleotide transhydrogenase encoding gene pntAB; the polyphosphate kinase encoding gene includes the polyphosphate kinase encoding gene ppk.

5. The recombinant Escherichia coli mixed culture system synthesizing indigo de novo according to claim 3, wherein, The nucleotide sequence of the repressor protein encoding gene trpR is shown in SEQ ID NO: 1; the nucleotide sequence of the phosphoryltransferase encoding gene pta is shown in SEQ ID NO: 2; the nucleotide sequence of the tryptophanase encoding gene tnaA is shown in SEQ ID NO: 3; the nucleotide sequence of the lactate dehydrogenase encoding gene ldhA is shown in SEQ ID NO: 4; the nucleotide sequence of the glycerol permease encoding gene glpF is shown in SEQ ID NO: 5; the nucleotide sequence of the glycerol kinase encoding gene glpK is shown in SEQ ID NO: 6; the nucleotide sequence of the 3-phosphoglycerate dehydrogenase encoding gene glpD is shown in SEQ ID NO: 7; the nucleotide sequence of the triose phosphate isomerase encoding gene tpiA is shown in SEQ ID NO: 8; and the 3-deoxy-D-arabinohepenolate-7-phosphate synthase encoding gene aroG is shown in SEQ ID NO:

8. S180F The nucleotide sequence is shown in SEQ ID NO: 9; the anthranilate synthase encoding gene trpE S40F The nucleotide sequence of the styrene monooxygenase encoding gene SMO is shown in SEQ ID NO: 10; the nucleotide sequence of the single-domain oxygen-binding hemoglobin encoding gene vgb is shown in SEQ ID NO: 14; and the nucleotide sequence of the single-domain oxygen-binding hemoglobin encoding gene vgb is shown in SEQ ID NO:

15.

6. The recombinant Escherichia coli mixed culture system synthesizing indigo de novo according to claim 4, wherein, The nucleotide sequence of the transketolase encoding gene tktA is shown in SEQ ID NO: 11; the nucleotide sequence of the transaldolase encoding gene talB is shown in SEQ ID NO: 12; the nucleotide sequence of the D-ribulose-5-phosphate-3-epimerase encoding gene rpe is shown in SEQ ID NO: 13; the nucleotide sequence of the membrane-bound pyridine nucleotide transhydrogenase encoding gene pntAB is shown in SEQ ID NO: 16; and the nucleotide sequence of the polyphosphate kinase encoding gene ppk is shown in SEQ ID NO:

17.

7. The recombinant Escherichia coli mixed culture system synthesizing indigo de novo according to claim 1, wherein, The Escherichia coli includes Escherichia coli BL21(DE3).

8. The method for constructing a recombinant Escherichia coli mixed culture system for de novo synthesis of indigo according to any one of claims 1 to 7, characterized in that, The method for constructing the precursor synthesizing bacteria includes the following steps: 1) The repressor protein encoding gene trpR in Escherichia coli BL21(DE3) was knocked out using gene editing to obtain engineered strain A; 2) The pta gene, which encodes phosphotransacetase, was knocked out on the engineered bacterium A obtained in step 1) using gene editing to obtain engineered bacterium B; 3) The tryptophanase encoding gene tnaA on the engineered bacterium B obtained in step 2) was knocked out using gene editing to obtain engineered bacterium C; 4) The lactate dehydrogenase encoding gene ldhA on the engineered bacteria C obtained in step 3) was knocked out using gene editing to obtain engineered bacteria D; 5) Using gene editing, the glycerol permease encoding gene glpF, the glycerol kinase encoding gene glpK, and the glycerol 3-phosphate dehydrogenase encoding gene glpD were integrated into the engineered bacteria D obtained in step 4) to obtain engineered bacteria E; 6) using gene editing to integrate phosphotriose isomerase encoding gene tpiA, DAHP synthase mutant encoding gene aroG, and anthranilate synthase encoding gene trpE S180F and anthranilate synthase encoding gene trpE S40F into the resulting engineered bacteria E of step 5), to obtain engineered bacteria F; 7) Using gene editing, the transketolase encoding gene tktA, the transaldolase encoding gene talB, and the D-ribulose-5-phosphate-3-epimerase encoding gene rpe were integrated into the engineered bacteria F obtained in step 6) to obtain engineered bacteria G, which is the precursor synthesizer.

9. The method for constructing a recombinant Escherichia coli mixed culture system for de novo synthesis of indigo according to claim 8, wherein The method for constructing the product-synthesizing bacteria includes the following steps: 1) Using gene editing, the tryptophanase encoding gene tnaA, the styrene monooxygenase encoding gene SMO, and the single-domain oxygen-binding hemoglobin encoding gene vgb were integrated into Escherichia coli BL21(DE3) to obtain engineered strain I; 2) Using gene editing, the membrane-bound pyridine nucleotide transhydrogenase encoding gene pntAB and the polyphosphate kinase encoding gene pppk were integrated into the engineered bacteria I obtained in step 1) to obtain the product-synthesizing bacteria.

10. A method for de novo synthesis of indigo by co-culturing a recombinant Escherichia coli mixed culture system, characterized by, The secondary seed culture of the recombinant Escherichia coli mixed system for de novo synthesis of indigo as described in any one of claims 1 to 7 is inoculated into a fermentation medium for fermentation to obtain indigo.

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