Recombinant escherichia coli with high yield of l-tryptophan and application thereof

Abstract

The application discloses a recombinant escherichia coli with high yield of L-tryptophan and application thereof, and belongs to the technical field of bioengineering.The application provides the recombinant escherichia coli with high yield of L-tryptophan to solve the problem that traditional microbial fermentation methods in the prior art depend on known target directional modification, result in a bottleneck in yield improvement of the strain, and cannot meet the industrial high-yield requirement.The preservation number of the recombinant escherichia coli is CGMCC No.38624, glucose is used as a carbon source, the L-tryptophan yield reaches 11.0 g / L after 24 h of fermentation in a 5L bioreactor, the L-tryptophan yield of a starting strain cultured under the same condition is only 3.4 g / L, the strain is stable in genetic traits, high in fermentation efficiency, and can be used for high-efficiency production of L-tryptophan.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a recombinant Escherichia coli strain that produces high levels of L-tryptophan and its applications. Background Technology

[0002] L-Tryptophan is an essential aromatic amino acid for humans and animals, widely used in food, feed, and pharmaceuticals. In feed, L-Tryptophan serves as an important nutritional fortifier, enhancing livestock growth performance and immunity. In the pharmaceutical field, L-Tryptophan is a precursor to neurotransmitters such as serotonin and melatonin, and is also an important raw material for amino acid infusions and antidepressants. In the food industry, L-Tryptophan serves as a flavor and nutritional regulator, possessing significant economic value.

[0003] The industrial production of L-tryptophan mainly includes chemical synthesis, enzymatic catalysis, and microbial fermentation. Among these, chemical synthesis is cumbersome, difficult to achieve chiral resolution, and causes significant pollution, and has been gradually replaced. Enzymatic catalysis requires high substrate purity and is costly, making large-scale application difficult. Microbial fermentation, using renewable raw materials such as glucose as carbon sources and utilizing hosts such as E. coli for fermentation, offers advantages such as low cost, mild conditions, and environmental friendliness, and has become the mainstream technology for L-tryptophan production.

[0004] Currently, the construction of high-yield Escherichia coli strains mostly adopts a rational optimization strategy of metabolic pathways: through knockout... trpR, tnaAB The gene unblocks the repression and degradation pathways, enhances the shikimic acid pathway and central carbon metabolism, blocks the byproduct branch, and overexpresses [the gene]. trpEDCBA Operators can improve synthetic throughput. However, traditional rational modification often relies on targeted modification of known targets, which makes it difficult to achieve global synergistic optimization of complex metabolic networks. This results in a significant bottleneck in increasing strain yield, making it difficult to meet the high-yield requirements of industrialization.

[0005] Combining genome-wide evolution with high-throughput screening can yield multi-gene synergistic mutations at a global level, effectively overcoming the limitations of rational modification. Biosensors can specifically identify intracellular metabolites, enabling rapid screening of high-yield strains, significantly improving evolutionary efficiency, and providing new ideas for efficient strain modification. Summary of the Invention

[0006] This invention addresses the problem that traditional microbial fermentation methods rely on targeted modification of known targets, which leads to a bottleneck in increasing strain yield and fails to meet the demands of high-yield industrial production. It provides a recombinant Escherichia coli strain that produces high levels of L-tryptophan and its applications.

[0007] One objective of this invention is to provide a recombinant Escherichia coli strain that produces high levels of L-tryptophan. This recombinant Escherichia coli is named TrpPC01, with accession number CGMCC No. 38624, and is classified as Escherichia coli. Escherichia Coli It is deposited at the China General Microbiological Culture Collection Center, with a deposit date of May 11, 2026.

[0008] In a preferred embodiment of the present invention, the screening method for the recombinant Escherichia coli is as follows: using knockout... trpR, tnaAB The gene was introduced from the *E. coli* strain. trpEDCBA Simultaneously constructing a basic plasmid and an MP6 mutant plasmid based on... tnaC A tryptophan biosensor was coupled with chloramphenicol resistance, and recombinant Escherichia coli with high L-tryptophan production was obtained under continuous induced mutation and resistance screening conditions. The trpR The nucleotide sequence of the gene is shown in SEQ ID NO.1. tnaA The nucleotide sequence of the gene is shown in SEQ ID NO.2. tnaB The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0009] The second objective of this invention is to provide the application of the above-mentioned recombinant Escherichia coli in the fermentation production of L-tryptophan.

[0010] The third objective of this invention is to provide a method for fermenting and producing L-tryptophan, wherein the method comprises: inoculating the above-mentioned recombinant Escherichia coli into a seed culture medium and incubating it with shaking at 37°C and 200 rpm for 4-6 h until OD reaches 100%. 600 Once the concentration reaches 5-6, a seed culture is obtained. The seed culture is then inoculated into a fermentation medium for fermentation. After fermentation, L-tryptophan is obtained by detection and separation.

[0011] In a preferred embodiment of the present invention, the inoculation amount of the seed liquid is 10%.

[0012] In a preferred embodiment of the present invention, the seed culture medium comprises: 21.6 g / L dipotassium hydrogen phosphate, 8.64 g / L potassium dihydrogen phosphate, 13.68 g / L yeast extract, 4.5 g / L ammonium sulfate, with the remainder being water, and a pH of 7.15.

[0013] In a preferred embodiment of the present invention, a sugar solution is added to the seed culture medium at a volume ratio of 10%, wherein the sugar solution is 30 g / L glucose monohydrate and 1 g / L magnesium sulfate heptahydrate.

[0014] In a preferred embodiment of the present invention, the fermentation medium comprises: 3.2 g / L potassium dihydrogen phosphate, 4.8 g / L ammonium sulfate, 0.0605 g / L ferrous sulfate, 1.6 g / L citric acid, 0.8 mL trace elements, 32 g / L MOPS, 3.2 mL phenol red solution, with the remainder being water, and a pH of 7.15.

[0015] In a preferred embodiment of the present invention, a sugar solution is added to the fermentation medium at a volume ratio of 10%, wherein the sugar solution is 25 g / L glucose monohydrate and 3 g / L magnesium sulfate heptahydrate.

[0016] In a preferred embodiment of the present invention, the method for producing L-tryptophan by fermentation is shake flask fermentation or fermenter fermentation.

[0017] Compared with the prior art, the beneficial effects of the present invention are: the present invention uses knockout trpR, tnaAB The gene was derived from the *E. coli* strain BW25113, and the gene was synthesized through a genome-wide mutation of MP6. tnaC A biosensor-coupled directed evolutionary screening system yielded a recombinant *E. coli* strain with high L-tryptophan production. The recombinant *E. coli* strain provided by this invention exhibits a 3.4-fold increase in production compared to the original starting strain, demonstrating that this screening system can effectively tap into the high-yield potential of strains; further optimization is possible. trpEDCBA After expressing the plasmid, the strain yield was significantly increased, breaking through the yield bottleneck of traditional rational metabolic engineering.

[0018] The recombinant *E. coli* strain provided by this invention, using glucose as a carbon source, achieved an L-tryptophan yield of 11.0 g / L after 24 h of fermentation in a 5 L bioreactor system, compared to only 3.4 g / L for the starting strain cultured under the same conditions. This strain exhibits stable genetic traits and high fermentation efficiency, making it suitable for the efficient production of L-tryptophan.

[0019] The screening method constructed in this invention can efficiently mine co-mutations at the whole genome level, and at the same time reveal the adaptability differences between the genome of the evolved strain and the exogenous plasmid, providing a new strategy for constructing high-tryptophan-producing strains. The strains have good genetic stability and have important prospects for industrial application.

[0020] [Bio-preservation information]: The recombinant Escherichia coli is named TrpPC01, with accession number CGMCC No. 38624, and is classified as Escherichia coli. Escherichia Coli It is deposited at the China General Microbiological Culture Collection Center, with a deposit date of May 11, 2026. Attached Figure Description

[0021] Figure 1 Based on different tryptophan concentrationstnaC Functional verification diagram of the tryptophan biosensor; Figure 2 A graph comparing the shake flask yields of recombinant Escherichia coli and the starting chassis strain; Figure 3 A comparison of shake-flask fermentation production performance of the starting chassis strain and recombinant Escherichia coli under optimized plasmid background. Figure 4 A comparison of fermenter production performance between the starting chassis strain and recombinant Escherichia coli under plasmid background; A represents the starting chassis strain, and B represents the recombinant Escherichia coli. Detailed Implementation

[0022] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0024] The recombinant Escherichia coli described in the following examples is named TrpPC01, with accession number CGMCC No. 38624, and classified as Escherichia coli. Escherichia Coli It is deposited at the China General Microbiological Culture Collection Center, with a deposit date of May 11, 2026.

[0025] The LB liquid culture medium described in the following examples contains the following components by mass percentage: 1% peptone, 0.5% yeast extract, 1% sodium chloride, and the balance being water.

[0026] The LB solid culture medium described in the following examples contains the following components by weight percentage: 1% peptone, 0.5% yeast extract, 1% sodium chloride, 2% agar powder, and the remainder is water.

[0027] The evolutionary screening medium described in the following examples contains the following components by mass percentage: 1.71% disodium hydrogen phosphate dodecahydrate, 0.3% potassium dihydrogen phosphate, 0.3% sodium chloride, 0.05% yeast extract, 0.1% ammonium chloride, 0.0493% magnesium sulfate heptahydrate, 0.00147% calcium chloride dihydrate, 2.0% glycerol, 0.2% arabinose, and the balance being water.

[0028] The seed culture medium described in the following examples contains the following components by mass percentage: 2.16% dipotassium hydrogen phosphate, 0.864% potassium dihydrogen phosphate, 1.368% yeast extract, 0.45% ammonium sulfate, with the balance being water, and a pH of 7.15; and a sugar supplement solution (3.0% glucose monohydrate and 0.1% magnesium sulfate heptahydrate) is added at a volume ratio of 10%.

[0029] The fermentation medium described in the following examples contains the following components by mass percentage: 0.32% potassium dihydrogen phosphate, 0.48% ammonium sulfate, 0.00605% ferrous sulfate, 0.16% citric acid, 3.2% MOPS, with the balance being water, and a pH of 7.15; and a sugar supplement solution (2.5% glucose monohydrate and 0.3% magnesium sulfate heptahydrate) is added at a volume ratio of 10%.

[0030] Example 1: Based on tnaC Optimized L-tryptophan biosensor construction This embodiment uses endogenous E. coli. tnaC An L-tryptophan biosensor was constructed using regulatory elements as its core (the sensor validation strain is referred to as: Escherichia coli BW25113-). ΔtrpR-ΔtnaAB The specific steps are as follows: 1. Description of Experimental Materials 1.1 Host strain: Escherichia coli BW25113, which is a well-known and commonly used type strain in this field. It can be routinely obtained through the China General Microbiological Culture Collection Center (CGMCC), the American Type Culture Collection (ATCC), or commercial channels. Its genome sequence is publicly available and searchable.

[0031] 1.2 Editing system: pEc-Cas9 vector (containing Cas9 protein expression cassette and resistance selection marker; note: preserved in this laboratory, can be prepared by conventional methods or obtained through commercial channels).

[0032] 1.3 Target gene sequence: The gene knocked out in this invention trpR Gene, tnaAB The genes are all derived from the genome of Escherichia coli BW25113, and their nucleotide sequences are as follows: trpR Gene: Its nucleotide sequence is based on the standard genome sequence of E. coli BW25113. trpRThe gene nucleotide sequence is shown in SEQ ID NO.1; tnaAB Gene: tnaA The gene nucleotide sequence is based on the standard genome sequence of E. coli BW25113. tnaA The gene nucleotide sequence is shown in SEQ ID NO.2; tnaB The gene nucleotide sequence is based on the standard genome sequence of E. coli BW25113. tnaB The gene nucleotide sequence is shown in SEQ ID NO.3.

[0033] 2. Based on the pEcCas9 system trpR, tnaAB Gene knockout steps (1) Preparation of electrocompetent cells: pEc-Cas9 (kanamycin resistant) plasmid was transformed into Escherichia coli BW25113, spread on LB solid medium plates containing 50 mg / L kanamycin, and cultured overnight at 37°C to obtain BW25113 / pEc-Cas9; a single colony of BW25113 / pEc-Cas9 was picked and inoculated into LB liquid medium containing kanamycin, and cultured overnight at 37°C with shaking; 1% inoculum was transferred to 100 mL of LB liquid medium containing kanamycin, and cultured for another 0.5 h, followed by induction with 0.2% arabinose, and cultured at 30°C until OD200. 600 =0.55-0.6, incubate on ice for 30 min, centrifuge to collect bacterial cells, wash 3 times with ice-cold 10% glycerol, and finally resuspend in 0.5 mL of glycerol. Dispense into 100 μL / tube for use to obtain electrotransfer competent cells.

[0034] (2) Construction of knockout fragment and gRNA vector: PCR amplification was performed using primers with nucleotide sequences as shown in SEQ ID NO.4-7 (as shown in Table 1). trpR The gene sequence has approximately 500 bp upstream and downstream homologous arms; PCR amplification was performed using primers with nucleotide sequences as shown in SEQ ID NO. 8-11 (as shown in Table 1). tnaAB The upstream and downstream homologous arms of the gene sequence are each about 500 bp; the target gene fragment (knockout fragment) was recovered using a gel extraction kit (purchased from Omega); the knockout fragment was ligated using overlap PCR amplification, and the target gene fragment was recovered using a gel extraction kit (purchased from Omega); Using the CRISPy-web website, 20 bp specific gRNAs for trpR and tnaAB were designed, and pEc-gRNA-trpR (nucleotide sequence shown in SEQ ID NO.12) and pEc-gRNA-tnaAB (nucleotide sequence shown in SEQ ID NO.13) were constructed.

[0035] Table 1

[0036] (3) Electroporation and screening: Take 100 μL of the electroporated competent cells obtained in step (1), add ≥500 ng of the knockout fragment obtained in step (2) and ≥300 ng of the pEc-gRNA-trpR vector obtained in step (2), revive after electroporation for 45-60 min, spread on LB solid medium plates containing kanamycin and streptomycin (50 mg / L each), and culture overnight at 37°C to obtain BW25113-ΔtrpR.

[0037] (4) Verification and plasmid elimination: The trpR / tnaAB gene in the BW25113-ΔtrpR strain obtained in step (3) was verified by colony PCR (the positive clone band was smaller than the wild type band). The positive clone could eliminate pEc-gRNA-trpR / pEc-gRNA-tnaAB by 10 mmol / L rhamnose, and then eliminate pEc-Cas9 by LB solid medium plates containing 10 g / L sucrose. (5) Based on the BW25113-ΔtrpR obtained above, continue to construct BW25113- according to steps (1)-(4). ΔtrpR-ΔtnaAB .

[0038] 3. Based on the genome of Escherichia coli BW25113 tnaC The upstream and downstream sequences of the gene were segmented and optimized using primers with nucleotide sequences shown in SEQ ID NO. 14-15 to obtain the tnaC sensing regulatory fragment by PCR. The optimized tnaC regulatory element was fused and tandem with the chloramphenicol resistance reporter gene, and inserted into the pLB1s-eGFP plasmid vector backbone by enzyme digestion and ligation to construct the recombinant L-tryptophan biosensor plasmid (the nucleotide sequence of which is shown in SEQ ID NO. 16).

[0039] 4. The BW25113- plasmid containing the recombinant L-tryptophan biosensor obtained above was then processed. ΔtrpR-ΔtnaAB The strain was inoculated into 50 mL shaker tubes containing 5 mL of LB liquid medium. The culture temperature was 37℃, and the shaking speed was 200 rpm for 12 h. Then, 1% of the culture was inoculated into 96-well plates, with 200 µL of evolutionary selection medium containing 12 mg / L chloramphenicol added to each well. Gradient experimental groups of 0, 0.25, 0.5, 0.75, 1, 1.5, and 2 g / L L-tryptophan were set up. The culture temperature was 30℃, and the shaking speed was 300-800 rpm. OD was measured hourly using a multi-mode microplate reader. 600 Continuous monitoring.

[0040] like Figure 1 As shown, the growth of the sensor-validated strain (Escherichia coli BW25113-ΔtrpR-ΔtnaAB) obtained above showed a dose-dependent increase with increasing L-tryptophan concentration. The sensor can effectively respond to the level of intracellular L-tryptophan, enabling growth-coupled screening of high-yielding strains.

[0041] Example 2: Preparation of recombinant Escherichia coli with high L-tryptophan production The Escherichia coli BW25113-Δ obtained in Example 1 trpR -Δ tnaAB As the starting strain, recombinant Escherichia coli with high L-tryptophan production was obtained through screening by combining MP6 whole-genome mutation with the tnaC biosensor. The specific steps are as follows: (1) Constructing the initial strain for evolutionary screening With Escherichia coli BW25113-Δ trpR -Δ tnaAB As the starting strain, introduce the basic trpEDCBA Expression plasmids were used to obtain basic engineered strains capable of synthesizing L-tryptophan, and the above optimizations were then applied. tnaC The biosensor and the MP6 whole-genome random mutant plasmid were co-transformed into the basic engineered strain to construct the initial strain for evolutionary screening.

[0042] (2) MP6 whole-genome mutation binding tnaC Biosensor-coupled screening The initial strain obtained in step (1) was inoculated into a 50 mL shaker containing LB medium (5 mL). The culture was incubated at 37 °C and 200 rpm for 12 h. The bacterial culture was then inoculated into another 50 mL shaker containing 1% of the screening medium (12 mg / L chloramphenicol) at 30 °C and 200 rpm. The culture was then incubated until the bacterial culture OD... 600 When the concentration reaches 0.5-1, the bacterial culture is streaked onto LB solid medium plates to isolate single clones, completing the first generation of evolutionary screening. Simultaneously, the bacterial culture is transferred to fresh evolutionary screening medium at a 1% inoculum rate for a new round of evolutionary screening. During multiple rounds of induced mutation and resistance pressure screening, the proportion of high-yield L-tryptophan strains gradually increases due to their growth advantage. After each round of screening, the isolated single clones are numbered and preserved. The above steps are repeated for 10 consecutive rounds to complete whole-genome evolutionary screening and strain preservation, obtaining the initial screening strains for subsequent fermentation screening and verification of high L-tryptophan production performance.

[0043] (3) Validation of initial screening strains The pre-screened strains obtained in step (2) were inoculated onto LB agar plates for culture. Using an inoculation loop, the inoculation was transferred to 50 mL shaker tubes containing 3 mL of seed culture medium. The culture temperature was 37°C, and the shaking speed was 200 rpm. The culture was continued until the bacterial culture OD... 600 When the bacterial culture reaches 5-6, the bacterial culture is inoculated at 10% into 50 mL shaker tubes containing fermentation medium (3 mL). The culture temperature is 37℃, the rotation speed is 150 rpm, and the culture is carried out for 18-20 h. The L-tryptophan content in the fermentation broth is determined by liquid chromatography. Single colonies with significantly increased L-tryptophan production compared to the initial strain in evolutionary screening are obtained through initial screening and are referred to as: initial screening and verification strains.

[0044] (4) Validation of secondary screening strains The initial screening and verification strains obtained in step (3) were inoculated onto LB solid medium plates for culture. Using an inoculation loop, the inoculation was transferred to a 250 mL baffle bottle containing seed culture medium (15 mL). The culture temperature was 37°C, and the rotation speed was 200 rpm. The culture was carried out until the OD of the bacterial culture was measured. 600 When the bacterial culture reaches 5-6, the bacterial culture is inoculated at 10% into a 250 mL baffle bottle containing fermentation medium (15 mL). The culture temperature is 37℃, the rotation speed is 150 rpm, and the culture time is 18-20 h. The L-tryptophan content in the fermentation broth is determined by liquid chromatography. Finally, a high-yield L-tryptophan-producing evolved strain (referred to as recombinant Escherichia coli TrpPC01, accession number CGMCC No. 38624) is obtained from the tenth generation strain. The above high-yield recombinant Escherichia coli and the original chassis strain are transformed into the basic trpEDCBA expression plasmid and compared in 250 mL shake flask fermentation.

[0045] like Figure 2 As shown, under the background of the basic plasmid, the L-tryptophan production of the evolved strain reached 0.96 g / L, which was 3.4 times higher than that of the original chassis strain; indicating that whole-genome evolution combined with biosensor screening can significantly improve the strain's synthesis capacity.

[0046] Example 3: Application of recombinant Escherichia coli in the fermentation production of L-tryptophan The recombinant Escherichia coli TrpPC01 obtained in Example 2 and the original chassis strain were respectively transformed into the optimization strain. trpEDCBA The expression plasmid was compared between 250 mL shake flask fermentation and 5 L fermenter fermentation.

[0047] 1. The specific steps for 250 mL shake flask fermentation are as follows: Carrying optimization trpEDCBAThe recombinant *E. coli* strain containing the plasmid and the original chassis strain were transferred to LB agar plates for culture. Using an inoculation loop, the inoculation was transferred to a 250 mL baffle bottle containing 15 mL of seed culture medium. The culture temperature was 37°C, and the rotation speed was 200 rpm. The culture was continued until the OD of the bacterial culture was measured. 600 When the bacterial culture reaches 5-6, the bacterial culture is inoculated at 10% into a 250 mL baffle bottle containing fermentation medium, with a liquid volume of 15 mL. The culture temperature is 37℃, the rotation speed is 150 rpm, and the culture is carried out for 18-20 h to obtain the fermentation broth. The L-tryptophan content in the fermentation broth is determined by liquid chromatography.

[0048] like Figure 3 As shown, under the optimized plasmid background, the recombinant Escherichia coli provided by this invention achieves an L-tryptophan yield of 3.69 g / L, with the yield of the evolved strain carrying the optimized plasmid being 84% higher than that of the chassis strain carrying the same optimized plasmid. Although the increase is lower than that under the basic plasmid background, the evolved strain still maintains the ability to produce high levels of L-tryptophan, indicating a difference in adaptability between its genomic evolutionary background and the exogenous optimized plasmid.

[0049] The specific steps for fermentation in a 2.5 L fermenter are as follows: (1) Preparation of culture medium (all components are expressed as mass percentages) Primary seed culture medium (LB medium): 0.1% tryptone, 0.05% yeast extract, 0.1% sodium chloride, deionized water to the required volume, pH adjusted to 7.0-7.2, sterilized at 121℃ and 0.1MPa for 20 min; after sterilization and cooling, add 1000× antibiotics to a final concentration of 1‰ (v / v) for resistance screening.

[0050] Secondary seed culture medium (inorganic salt formula): 0.17% citric acid, 1.4% potassium dihydrogen phosphate, 0.4% diammonium hydrogen phosphate, diluted to 90% volume with deionized water, then adjusted to pH 7.4 with ammonia water, and finally diluted to 100% volume; dispensed into 500mL shake flasks, 100mL per flask, and sterilized at 121℃ for 30min; after sterilization and cooling (before inoculation), add pre-prepared 50% (mass percentage) glucose stock solution (sterilized at 115℃ for 20min) to make the final glucose concentration 0.5%, and simultaneously add 1000× antibiotic to make the final concentration 1‰ (v / v).

[0051] Fermentation tank culture medium (inorganic salt formula): citric acid 0.17%, potassium dihydrogen phosphate 1.4%, diammonium hydrogen phosphate 0.4%, polyether defoamer 0.1‰ (v / v). When making up to volume with deionized water, reserve the volume for the seed culture and subsequent added components (e.g., if the volume of 3L solution needs to be made up to about 2.7L). Sterilize at 121℃ and 0.1MPa for 30min. After sterilization and cooling, add the following components in sequence (all are final concentrations): 100×trace element ① 1% (v / v), 1000×vitamin B1 0.1‰ (v / v, final concentration 0.0009%), glucose 2.0% (add glucose stock solution that has been sterilized at 115℃ for 20min beforehand), magnesium sulfate heptahydrate 0.06% (can be mixed with glucose stock solution for sterilization).

[0052] Feed-on culture medium: 60.0% glucose, 0.2% anhydrous magnesium sulfate, diluted to volume with deionized water and sterilized at 115℃ for 20 min; after sterilization and cooling, add 100×1% (v / v) of trace element ②, for glucose feeding only.

[0053] 100× Trace Elements ①: EDTA 0.084%, cobalt chloride hexahydrate 0.025%, manganese chloride tetrahydrate 0.15%, copper chloride dihydrate 0.015%, boric acid 0.03%, sodium molybdate dihydrate 0.025%, zinc acetate dihydrate 0.13%, ferric citrate (Fe(Ⅲ) citrate) 1.0%.

[0054] 100× Trace Elements ②: EDTA 0.13%, cobalt chloride hexahydrate 0.04%, manganese chloride tetrahydrate 0.235%, copper chloride dihydrate 0.025%, boric acid 0.05%, sodium molybdate dihydrate 0.04%, zinc acetate dihydrate 0.16%, ferric citrate (Fe(Ⅲ) citrate) 0.4%.

[0055] (2) Seed culture and inoculation Primary seed culture: Seeds stored at -80℃ were used for optimized culture. trpEDCBA The recombinant E. coli plasmid and the original chassis strain were aseptically picked from a glycerol tube and inoculated into an Erlenmeyer flask containing 100 mL of primary seed culture medium (containing the corresponding resistance). The flask was then incubated overnight at 37°C with shaking at 220 rpm to obtain the primary seed culture.

[0056] Secondary seed culture: Inoculate the primary seed culture at a 5% (v / v) inoculation rate into a 500mL baffled shake flask containing 100mL of secondary seed culture medium (containing the corresponding resistance). Incubate at 37℃ and 250 r / min with shaking for 6-8 hours until OD reaches the target level. 600 When the value is ≥3.0, it is considered a qualified secondary seed liquid and is used for inoculation in the upper tank.

[0057] Inoculation in the fermenter: The initial temperature of the fermenter is controlled at 37℃. The pH of the fermentation system is maintained at 7.0±0.2 by automatically adding ammonia. Inoculate the fermentation system into a 5L bioreactor containing 3L of fermentation medium at a 10% (v / v) inoculation rate using aseptic operation. Dissolved oxygen (DO) is controlled using a cascade strategy to maintain DO>30%. Dissolved oxygen is maintained primarily by automatically increasing the stirring speed. The speed only increases and does not decrease. When the speed reaches the upper limit of the equipment, the aeration rate is increased to 3vvm (the volume of air introduced per unit volume of culture medium per hour).

[0058] Basic control of the fermentation process: A sharp increase in dissolved oxygen (DO) or pH is used as a signal of glucose depletion, which then triggers the subsequent feeding process; samples are taken regularly throughout the fermentation process, and standard analytical methods are used to quantitatively determine biomass (cell concentration), substrate consumption, and the concentration of the target product (L-tryptophan); the fermentation termination criterion is OD. 600 Once the value remains stable for 3 consecutive hours, stop fermentation and transfer the product to a container.

[0059] Feeding initiation and rate control: Once a glucose depletion signal (dissolved oxygen or pH increase) is detected, the continuous feeding operation is immediately initiated, and only the pre-prepared feed culture medium (containing only glucose, magnesium sulfate and trace elements ②) is fed.

[0060] like Figure 4 As shown, during scale-up cultivation under 5L fermenter conditions, the original starting strain exhibited a certain OD value in the bacterial cells. 600 When the value only increased to around 30, the fermentation system showed abnormal dissolved oxygen levels and a mismatch between the oxygen consumption of the bacterial strain and the dissolved oxygen supply in the tank. The growth of the bacteria and the synthesis and metabolism of the products were inhibited prematurely, and the final L-tryptophan yield was only 3.4 g / L.

[0061] The recombinant Escherichia coli provided by this invention exhibits significantly better cell proliferation performance than the original starting strain, and can grow normally to OD. 600 The value is around 70; the recombinant Escherichia coli only exhibits the problem of limited dissolved oxygen during fermentation after the bacterial cells reach a high biomass level. Even if constrained by the existing dissolved oxygen conditions of the tank and the fermentation process fails to fully realize its synthetic potential, its final L-tryptophan yield can still reach 11.0 g / L.

[0062] The comparison shows that, under the same scaled-up fermentation conditions, the recombinant Escherichia coli provided by this invention has superior cell growth ability, environmental adaptability and L-tryptophan synthesis and accumulation ability compared with the original starting strain, and has outstanding potential for industrial application.

Claims

1. A recombinant Escherichia coli strain that produces high levels of L-tryptophan, characterized in that, The recombinant Escherichia coli was named TrpPC01, with accession number CGMCC No. 38624, and classified as Escherichia coli. Escherichia Coli It is deposited at the China General Microbiological Culture Collection Center, with a deposit date of May 11, 2026.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The screening method for the recombinant Escherichia coli is as follows: using knockout... trpR, tnaAB The gene was introduced from the *E. coli* strain. trpEDCBA Simultaneously constructing a basic plasmid and an MP6 mutant plasmid based on... tnaC A tryptophan biosensor was coupled with chloramphenicol resistance, and recombinant Escherichia coli with high L-tryptophan production was obtained under continuous induced mutation and resistance screening conditions. The trpR The nucleotide sequence of the gene is shown in SEQ ID NO.

1. tnaA The nucleotide sequence of the gene is shown in SEQ ID NO.

2. tnaB The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. The use of the recombinant Escherichia coli according to any one of claims 1 to 2 in the fermentation production of L-tryptophan.

4. A method for producing L-tryptophan by fermentation, characterized in that, The method comprises the following steps: inoculating the recombinant Escherichia coli according to any one of claims 1 to 2 into a seed culture medium, and incubating it with shaking at 37°C and 200 rpm for 4-6 h until the OD reaches 100%. 600 Reaching level 5-6 yields seed liquid; The seed culture was inoculated into a fermentation medium for fermentation, and L-tryptophan was obtained by detection after fermentation.

5. The method according to claim 4, characterized in that, The inoculation amount of the seed solution is 10%.

6. The method according to claim 4, characterized in that, The seed culture medium consists of: 21.6 g / L dipotassium hydrogen phosphate, 8.64 g / L potassium dihydrogen phosphate, 13.68 g / L yeast extract, 4.5 g / L ammonium sulfate, with the remainder being water, and a pH of 7.

15.

7. The method according to claim 6, characterized in that, The seed culture medium is supplemented with a sugar solution at a volume ratio of 10%, wherein the sugar solution is 30 g / L glucose monohydrate and 1 g / L magnesium sulfate heptahydrate.

8. The method according to claim 4, characterized in that, The fermentation medium consisted of: 3.2 g / L potassium dihydrogen phosphate, 4.8 g / L ammonium sulfate, 0.0605 g / L ferrous sulfate, 1.6 g / L citric acid, 0.8 mL trace elements, 32 g / L MOPS, 3.2 mL phenol red solution, with the remainder being water, and a pH of 7.

15.

9. The method according to claim 8, characterized in that, The fermentation medium is supplemented with a sugar solution at a volume ratio of 10%, wherein the sugar solution is 25 g / L glucose monohydrate and 3 g / L magnesium sulfate heptahydrate.

10. The method according to claim 4, characterized in that, The method for producing L-tryptophan by fermentation is shake flask fermentation or fermenter fermentation.

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