A high-L-tryptophan-producing evolved strain, its construction method and application

Abstract

This invention discloses a high-yield L-tryptophan-producing evolved strain, its construction method, and its applications. This invention modifies and optimizes an L-tryptophan biosensor to obtain a growth-production coupled strain with excellent L-tryptophan response performance. Through mutagenesis and evolution, a mutant strain X01-T21 was obtained, exhibiting increased L-tryptophan yield and sugar-acid conversion rate of 11.5% and 21.9%, respectively. Whole-genome sequencing and alignment analysis were used to infer gene... rrlH , lptD and suffer It may have a promoting effect on L-tryptophan synthesis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a high-yield L-tryptophan-producing evolved strain, its construction method, and its application. Background Technology

[0002] L-Tryptophan is an essential limiting amino acid for vital physiological activities such as growth, development, and metabolism in humans and animals, and is widely used in the pharmaceutical, food, and feed industries. With the continuous development of the feed and pharmaceutical industries both domestically and internationally, L-Tryptophan has become the third largest feed amino acid after methionine and lysine, with stable market demand and huge growth potential. However, due to its complex production process and high cost, there is a significant market gap for L-Tryptophan, and its high price limits its widespread application in areas such as soybean meal as a feed substitute. Improving the conversion rate of L-Tryptophan engineered strains can not only reduce costs and meet market demand but also reduce by-products and waste emissions, thus reducing environmental pollution. Furthermore, L-Tryptophan is a precursor to bioactive substances such as serotonin and melatonin, and the development of high-yield strains can also contribute to the development of fields such as medical aesthetics, health products, and agriculture.

[0003] Early studies used random mutations to screen for high-yield L-tryptophan-producing strains, but this method could introduce unfavorable mutations and interfere with metabolic analysis and further strain improvement. With the development of synthetic biology techniques, metabolic engineering has greatly facilitated the development of high-yield engineered strains. However, the rational design of L-tryptophan engineered strains faces challenges such as complex metabolic pathways, feedback inhibition, product burden, and poor strain stability, making further improvements in conversion efficiency extremely difficult.

[0004] In the prior art, for example, invention patent CN120775888A discloses a method for improving L-tryptophan synthesis in Escherichia coli through high-throughput screening based on ARTP mutagenesis, obtaining a mutant strain YB-2 with increased L-tryptophan yield and sugar-acid conversion rate by 11.4% and 10.7%, respectively. Another example is invention patent CN116376852A, which discloses a dipeptide-tripeptide permease mutant and its application in L-tryptophan production. This invention obtained a high-tryptophan-producing strain through mutagenesis and obtained a dipeptide-tripeptide permease mutant that increases L-tryptophan yield. By expressing the mutant in E. coli, tryptophan production was increased.

[0005] Therefore, there is an urgent need to further develop modification strategies for high-yield L-tryptophan engineered bacteria in order to obtain L-tryptophan strains that meet industrial needs. Summary of the Invention

[0006] This invention addresses the problem of low yield and conversion rate of L-tryptophan engineered strains. The purpose of this invention is to overcome the shortcomings of existing technologies by combining genetic engineering and adaptive evolution. Therefore, it provides a method for constructing a high-yield L-tryptophan-producing evolved engineered strain, which exhibits good stability, high yield, and high conversion rate.

[0007] The technical solution adopted in this invention is as follows: To construct a growth-production coupled strain, the L-tryptophan biosensor based on the natural leader peptide TnaC is first systematically optimized to obtain a constitutively expressed L-tryptophan biosensor element (J23111-TanC) with good response performance. D21S The promoter of an essential gene in an engineered strain was replaced with this gene to obtain a growth-deficient strain with a dose-dependent relationship to L-tryptophan. Simultaneously, to screen the evolved strains, a biosensor carrying a resistance gene was inserted into the genome of the engineered strain. Subsequently, the growth-production coupled strains were subjected to mutagenesis and adaptive evolution, resulting in the enhancement and enrichment of mutants with increased L-tryptophan production capacity during the evolutionary process. Finally, based on resistance plates, high-yield traits could be rapidly screened to obtain evolved strains with improved L-tryptophan synthesis performance. Based on the whole-genome sequencing results of the dominant strains, comparative analysis was performed on gene loci that might have a promoting effect.

[0008] The first technical solution provided by this invention is to construct a growth-production coupled strain based on the L-tryptophan biosensor of the natural precursor peptide TnaC. The growth-production coupling is achieved by regulating the expression of essential genes of the engineered strain through L-tryptophan. After mutagenesis and evolution, if the L-tryptophan synthesis capacity of the strain is enhanced, the expression level of essential genes will increase, and the growth of the strain will be restored.

[0009] Specifically, this invention provides a method for constructing a high-L-tryptophan-producing evolved strain, comprising the following steps: (1) Provide L-tryptophan-producing chassis cells, said chassis cells being knocked out of the starting strain tnaAB Gene, aroF Gene, aroG Genes and aroH Genes, and overexpress mutants aroG Genes and trpEDCBA Obtained by the manipulator; (2) Construct a biosensor element based on the leader peptide TnaC mutant that can characterize the concentration of L-tryptophan in cells; (3) Replace the natural promoter of an essential gene in the chassis cell obtained in step (1) with the biosensor element constructed in step (2) to construct a growth-production coupled strain whose growth depends on L-tryptophan synthesis. (4) Connect the biosensor element constructed in step (2) with an exogenous resistance gene to construct a resistance expression unit, and insert it into the genome of the growth-production coupled strain obtained in step (3) to obtain a resistance-production coupled strain whose resistance expression also depends on L-tryptophan synthesis. (5) The resistant-production coupled strain obtained in step (4) is subjected to mutagenesis to obtain a mutant library; (6) Perform automatic continuous evolution on the mutant library obtained in step (5); (7) Based on the resistance level, the evolved strains with higher L-tryptophan production than the chassis cells are screened from the bacterial community evolved in step (6).

[0010] In some embodiments, in step (1), the starting strain is Escherichia coli; further, the Escherichia coli is Escherichia coli W3110 or Escherichia coli DY330; In some implementations, in step (1), the tnaAB Gene, aroF Gene, aroG Genes and aroH The nucleotide sequences of the gene are shown sequentially as SEQ ID NO.1-SEQ ID NO.4, and the mutant... aroG The nucleotide sequence of the gene is shown in SEQ ID NO.5. trpEDCBA The nucleotide sequence of the operon is shown in SEQ ID NO.6; In some embodiments, in step (2), the biosensor element includes a constitutive promoter J23111 and an encoding of the leader peptide TnaC mutant. tnaC D21S The gene, wherein the nucleotide sequence of the constitutive promoter J23111 is shown in SEQ ID NO.7, is... tnaC D21S The nucleotide sequence of the gene is shown in SEQ ID NO.8; In some implementations, in step (3), the essential gene is folA The gene, with its nucleotide sequence shown in SEQ ID NO. 9; In some embodiments, step (4) involves inserting a biosensor linked to a resistance gene into the genome of an engineered strain to obtain an L-tryptophan-dependent resistant strain in which resistance gene expression is coupled to L-tryptophan production. The strain's ability to synthesize L-tryptophan is reflected by the intensity of resistance expression. Further, the exogenous resistance gene is streptomycin resistance. str The gene, with its nucleotide sequence shown in SEQ ID NO.10.

[0011] Furthermore, step (5) involves the construction of a mutant library based on random mutagenesis and automated continuous evolution. To provide the driving force for evolution, the growth-production coupled strain is mutagenized to obtain a mutant library, which is then transferred to an automated continuous evolution device. Under growth pressure, the high-yield trait evolves and is enhanced and enriched during automated passaging.

[0012] In some embodiments, the mutagenesis treatment is physical mutagenesis, chemical mutagenesis, or biological mutagenesis; Furthermore, the mutagenesis methods include ARTP, diethyl sulfate, and mutagenic plasmids.

[0013] In step (6), the automatic continuous evolution screening uses an automatic continuous evolution system to carry out adaptive evolution culture until the strain growth recovers; the automatic continuous evolution system is a constant temperature culture system that can realize growth curve detection, automatic feeding and automatic waste liquid discharge. The automated continuous evolution system includes a chemostat culture device for culturing strains, a feeding device, a discharging device, a peristaltic pump, a cell density monitoring device, and a controller. The chemostatic culture device and the feeding device are connected through a feed pipe, and the chemostatic culture device and the discharge device are connected through a discharge pipe. Both the feed pipe and the discharge pipe are equipped with peristaltic pumps. The cell density monitoring device is used to measure the cell density in the chemostat culture device. The bacterial density monitoring device and each peristaltic pump are all connected to the controller.

[0014] The chemostat culture device is equipped with a stirrer and sensors for monitoring temperature, stirring speed, oxygen, and pH.

[0015] The constant chemistry culture device, feeding device, and discharging device are all equipped with vents, and the vents are also equipped with valves and sterile filters for controlling the opening and closing of the vents.

[0016] The controller is used to determine the bacterial density data (OD) returned by the bacterial density monitoring device. 600 Whether the value is higher than the first preset threshold (set to OD in this embodiment of the invention) 600 =1.5), if so, the controller controls the peristaltic pumps of the feed pipe and the discharge pipe to open, so as to introduce fresh culture medium into the constant chemistry culture device and discharge waste culture medium, thereby diluting the cell density.

[0017] The controller is also used to determine the bacterial density data (OD) returned by the bacterial density monitoring device. 600 Whether the value is lower than the second preset threshold (set to OD in this embodiment of the invention) 600=0.5), if so, the controller controls the peristaltic pumps of the feed pipe and the discharge pipe to shut off, so as to stop the flow of fresh culture medium into the constant chemistry culture device and the discharge of waste culture medium, thereby stopping the dilution of cell density and starting a new round of culture.

[0018] In step (7), a rapid plate screening method based on resistance is used. The evolved strains are screened on plates with gradually increasing antibiotic concentrations to obtain strains with improved growth rate and resistance, which are potential high-yield L-tryptophan strains.

[0019] The screening process involves plating the evolved strains onto M9 resistance plates containing streptomycin for culture and screening.

[0020] This invention also provides an evolved strain that produces high levels of L-tryptophan, which was obtained through screening using the aforementioned method; the evolved strain is *Escherichia coli* (…). Escherichia coli X01-T21, with accession number CGMCCNo.37257.

[0021] The present invention also provides the application of the aforementioned evolved strain in the production of L-tryptophan.

[0022] The present invention also provides a method for preparing L-tryptophan, wherein the selected evolved strain is cultured overnight at a certain temperature, then transferred to a seed culture medium and cultured to the mid-to-late logarithmic phase, and then inoculated into a fermentation medium in a certain proportion. After fermentation, the yield of L-tryptophan in the fermentation broth is detected.

[0023] The specific method includes the following steps: S1. Cultivate the aforementioned evolved strain; S2. Transfer the bacterial culture from step S1 to seed culture medium and culture until OD200. 600 Reaching 8-12; S3. Inoculate the seed culture obtained in step S2 into the fermentation medium for fermentation culture; S4. Collect the fermentation broth from step S3 to obtain L-tryptophan.

[0024] Furthermore, the culture temperature in step S1 is 30-40℃; preferably 37℃. The volume percentage of transfer in step S2 is 2%-10%, preferably 5%; the OD of the culture target 600 It is 10; The inoculum volume percentage in step S3 is 2%-10%, preferably 10%; the fermentation time is 30-60 hours, preferably 42 hours.

[0025] The present invention also provides a method for screening key genes for L-tryptophan synthesis in Escherichia coli. The method involves comparing and analyzing the genomes of the strains screened by the method or the evolved strains with those of the starting strain and wild-type Escherichia coli to obtain key genes for L-tryptophan synthesis.

[0026] Furthermore, key genes for L-tryptophan synthesis include genes rrlH ,Gene lptD and genes trpE The Gene IDs are 944900, 945011, and 945846, respectively.

[0027] This invention also provides genes rrlH ,Gene lptD or genes trpE The application of key mutation sites in promoting L-tryptophan synthesis in Escherichia coli, the gene rrlH ,Gene lptD and genes trpE The gene IDs are 944900, 945011, and 945846, respectively. The gene... rrlH The mutations include mutations in the non-coding region upstream of its start codon; The gene lptD The mutations include those that cause the amino acid at position 703 of the encoded protein to change from tyrosine to aspartic acid. The gene trpE Mutations include those that change the amino acid at position 63 of the encoded protein from valine to alanine, the amino acid at position 71 from glutamine to lysine, and / or the amino acid at position 94 from serine to asparagine.

[0028] In this invention, compared to the L-tryptophan-producing chassis cell strain X01, the evolved strain *Escherichia coli* (… Escherichia coli Genes in X01-T21 rrlH ,Gene lptD and genes trpE A mutation occurred; specifically, the mutated gene... rrlH ,Gene lptD and genes trpE The nucleotide sequences are shown in SEQ ID NO.11~13 respectively.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention modifies and optimizes an L-tryptophan biosensor to obtain a growth-production coupled strain with excellent L-tryptophan response. Through mutagenesis and evolution, a mutant strain X01-T21 was obtained, exhibiting increased L-tryptophan yield and sugar-acid conversion rate of 11.5% and 21.9%, respectively. Whole-genome sequencing and alignment analysis were used to infer gene...rrlH , lptD and trpE It may have a promoting effect on L-tryptophan synthesis. Attached Figure Description

[0030] Figure 1 To verify the response of the growth-production coupled strain to L-tryptophan.

[0031] Figure 2 pN20-ΔP folA :: tnaC Plasmid map.

[0032] Figure 3 pN20-Δ yijV :: str Plasmid map.

[0033] Figure 4 This is a map of the pEcCas plasmid.

[0034] Figure 5 pIN- aroG mut -trpEDCBA Plasmid map.

[0035] Figure 6 To determine the lethality of ARTP-induced mutagenesis.

[0036] Figure 7 This is a schematic diagram of an automatic continuous evolution system.

[0037] Figure 8 The graph shows the L-tryptophan yield and conversion rate curves during the 42-hour fermentation of X01-T21 and the initial strain X01. Detailed Implementation

[0038] The present invention will be further illustrated below with specific examples, but these examples do not constitute a limitation thereof.

[0039] Example 1 Constructing growth-production coupled strains based on L-tryptophan biosensors.

[0040] The leader peptide TnaC is a naturally occurring L-tryptophan-responsive ribosome switch. When the L-tryptophan concentration is low, ribosome translation completes and releases the TnaC peptide chain, which dissociates from the mRNA. The Rho factor approaches and terminates RNA polymerase transcription, leading to the inhibition of downstream gene expression. When the intracellular L-tryptophan concentration increases, L-tryptophan interacts with the TnaC peptide chain and the channel wall, preventing the TnaC peptide chain from being hydrolyzed and released. This causes the ribosome to arrest at the stop codon of the tnaC gene, allowing downstream genes to be transcribed and expressed. First, single-amino acid saturation mutagenesis was performed on key amino acids of the TnaC peptide chain. Flow cytometry screening was conducted based on "low background, high magnification" fluorescence signal intensity, resulting in 17 mutants with a dynamic range increase greater than 2-fold. Subsequently, the promoter of the tnaC gene was replaced with promoters of different intensities, yielding a series of ribosomal switches with significantly different responses to L-tryptophan range and expression intensity. Among these, the mutant TnaC, with J23111 as the promoter (nucleotide sequence shown in SEQ ID NO. 7) and a mutation of aspartic acid at position 21 to serine, was identified. D21S (The nucleotide sequence is shown in SEQ ID NO. 8) As a combination of tryptophan-responsive elements, an L-tryptophan-responsive ribosomal switch J23111- with optimal dynamic range and moderate response strength was obtained. tnaC D21S (The nucleotide sequence is shown in SEQ ID NO.15), which forms the basis for constructing the L-tryptophan biosensor.

[0041] The starting strain was wild-type Escherichia coli W3110, which was first removed tnaAB Gene (nucleotide sequence as shown in SEQ ID NO. 1), using primers tnaAB -M1 / tnaAB -M2 (Table 1) amplified the selection marker (kanamycin resistance gene) and the reverse selection marker (M2) from the template plasmid pJLK (nucleotide sequence as shown in SEQ ID NO. 18). rpsL Genes. Homologous recombination will... tnaAB Genes were replaced with marker genes to obtain recombinant strains. tnaAB::rpsL -Kan. Select recombinants that are resistant to kanamycin (Kan) but not to streptomycin (Sm), and use primers. tnaAB -C1 / tnaAB -C2 (Table 1) was confirmed by colony PCR. Simultaneously, primer pairs were used... tnaAB -C1 / tnaAB -C2 was amplified from the genome of Escherichia coli W3110 by... tnaAB DNA fragments consisting of genes and their upstream and downstream DNA U- tnaAB-D (nucleotide sequence as shown in SEQ ID NO.19). Subsequently, the U- fragment... tnaAB -D was inserted into the pJET1.2 vector (nucleotide sequence as shown in SEQ ID NO.20) to obtain plasmid pJET-U. tnaAB D. By using primers tnaAB -del_F / tnaAB -del_R amplifies the plasmid from plasmid pJET-U tnaAB Remove from D tnaAB Gene. After phosphorylation, ligation, transformation, and culture, plasmid pJET-UΔ was obtained. tnaAB D. Then, using primers tnaAB-C1 / tnaAB-C2 from pJET-UΔ tnaAB UΔ amplified in D tnaAB The D DNA fragment was transformed into the recombinant strain. tnaAB:: In rpsL-Kan, the resistance marker is removed. This results in knockout. tnaAB The recombinant strains were then knocked out sequentially using the corresponding primers (Table 1) following a similar workflow. aroF , aroG and aroH Gene (nucleotide sequence as shown in SEQ ID NO.2-4). Finally, the mutant gene was added back. aroG Gene (nucleotide sequence as shown in SEQ ID NO.5) and trpEDCBA The free plasmid pIN (nucleotide sequence shown in SEQ ID NO. 21) containing the operon (nucleotide sequence shown in SEQ ID NO. 6) was used to obtain L-tryptophan-producing chassis cells, named X01. To propel evolution in a direction favorable to L-tryptophan production, L-tryptophan synthesis needs to be coupled with host growth. Essential growth genes of X01 were... folA As a regulatory target, folA promoter P folA (The nucleotide sequence shown in SEQ ID NO.16) is replaced with a modified L-tryptophan-responsive ribosome switch (J23111-). tnaC D21S This process yields a growth-production coupled strain. The specific steps are as follows: First, using the genomic DNA of Escherichia coli W3110 as a template, J23111- tnaC -LF & J23111- tnaC -LR primer pairs (Table 1) amplify J23111- tnaC D21S Gene fragments; using P folA -up-LF & P folA -up-LR and P folA-Down-LF & P folA -Down-LR primer pairs (Table 1) amplify respectively folA Upstream and downstream gene fragments of the promoter; Using pN20 plasmid (nucleotide sequence as shown in SEQ ID NO.17) as a template, P folA -N20-LF & P folA -N20-LR and pN20- tnaC -LF & pN20- tna C-LR primer pairs (Table 1) were used to amplify the gRNA and linear vector fragments; ligation was then performed according to the conditions in Table 2. Subsequently, 10 µL of the ligation mixture was transformed into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected and sequenced for verification. The successfully constructed plasmid for knockout was named pN20-ΔP. folA :: tnaC (See plasmid map) Figure 2 ).

[0042] Subsequently, using CRISP-cas9 gene editing technology, pN20-ΔP folA :: tna C and helper plasmid pEcCas (plasmid map see...) Figure 4 The antibodies were co-introduced into X01 competent cells and plated on LB agar plates containing ampicillin (Amp) and kanamycin (Kan). Positive clones were selected for PCR verification and sequencing. The verification primers were P. folA -YZ-LF and P folA -YZ-LR (Table 1), the correct strain is the growth-production coupled strain X01-D1. The effect of exogenous L-tryptophan addition on the growth of the coupled strain was observed. It was found that as the concentration of exogenous L-tryptophan increased, the growth rate of the strain gradually recovered. Figure 1 ).

[0043] Simultaneously, an L-tryptophan-regulated resistance gene expression system was introduced, inserting an L-tryptophan-responsive ribosomal switch (J23111-) into the genome of the growth-production coupled strain X01-D1. [[ID=A9]]tnaC D21S Streptomycin resistance str The gene (nucleotide sequence shown in SEQ ID NO.10) and the operational steps are similar to those for constructing a growth-production coupled strain, as follows: Neutral sites on the genome of strain X01-D1 were selected. yijV The gene (gene sequence as shown in SEQ ID NO.7) is used as the insertion site, first with Escherichia coli Using W3110 genomic DNA as a template, and J23111- tnaC-LF &J23111- tnaC -LR primer pairs (Table 1) amplify J23111- tnaC D21S Gene fragments, using yijV -up-LF & yijV -up-LR and yijV -Down-LF & yijV -Down-LR primer pairs (Table 1) amplify respectively yijV Upstream and downstream gene segments; Using pBBR1 plasmid as a template, str -LF & str -LR primer pairs (Table 1) amplification str Genes, using pN20 plasmid as a template, were... yijV -N20-LF & yijV -N20-LR and pN20- str -LF & pN20- str -LR primer pairs (Table 1) were used to amplify gRNA and linear vector fragments; ligation was performed according to the conditions in Table 3.

[0044] The 10 µL ligation system was then transferred into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected and sequenced for verification. The successfully constructed knock-in plasmid was named pN20-Δ. yijV :: str (See plasmid map) Figure 3 ).

[0045] Then pN20-Δ yijV :: str And the helper plasmid pEcCas (see plasmid map) Figure 4 The enzyme was co-introduced into growth-production coupled competent cells X01-D1, plated on LB agar plates containing ampicillin (Amp) and kanamycin (Kan), and positive clones were selected for PCR verification and sequencing. The verification primers were: yijV -YZ-LF and yijV -YZ-LR (Table 1), the strain that is verified to be correct is the strain that simultaneously possesses growth-production coupling and resistance-production coupling, named X01-D2. In a medium containing the same antibiotic, the higher the concentration of L-tryptophan, the better the growth status of the strain.

[0046] Plasmid elimination: 1) Eliminate pN20-gRNA plasmid, pick the verified colonies in fermentation medium (containing 1M rhamnose 1:100 and 50 µg / mL Kan), incubate overnight at 37°C and 220 rpm; streak the colonies in three zones onto solid LB plates containing Kan (50 µg / mL), incubate at 37°C until single colonies are formed, pick several single colonies and copy them onto LB plates containing Kan and Amp (incubate overnight at 37°C), the single colonies with Kan resistance and no Amp resistance indicate that the pN20-gRNA plasmid has been eliminated.

[0047] 2) Eliminate the pEcCas plasmid. Inoculate single colonies into liquid fermentation medium containing glucose (5 g / L) and incubate overnight (37°C, 220 rpm). Streak the colonies in three zones onto LB agar plates containing glucose (5 g / L) and sucrose (10 g / L), and incubate at 37°C until single colonies form. Then, randomly select several single colonies and inoculate them onto Kan and antibiotic-free LB agar plates, respectively. Single colonies without Kan resistance indicate that the pEcCas plasmid has been lost.

[0048] Table 1. Primer list used in Example 1 Table 2. Construction of pN20-ΔP folA :: tnaC plasmid enzyme ligation system Table 3. Construction of pN20- yijV :: str plasmid enzyme ligation system Example 2 Mutant libraries were constructed and automated continuous evolution was performed based on ARTP mutagenesis.

[0049] Random mutagenesis strategy: Natural evolution of microorganisms is inefficient and cannot meet the need for rapid screening of high-yield L-tryptophan-producing strains. To accelerate this process, various mutagenesis methods can be introduced to construct mutant libraries of engineered strains, providing a driving force for adaptive evolution. Library construction through random mutagenesis includes: physical mutagenesis using ultraviolet light (UV) and ambient pressure room temperature plasma (ARTP); chemical mutagenesis using diethyl sulfate (DES) and sodium azide (NaN3); and biological mutagenesis by introducing mutagenic plasmids and transposase plasmids. Taking ARTP mutagenesis as an example, the ARTP mutagenesis breeding instrument from Tianmu Biotechnology was used to mutagenesis the constructed growth-production coupled strain X01-D2. To determine the optimal mutagenesis time for ARTP, this study measured the lethality of strain X01-D2 under gradient irradiation times of 10, 30, 50, 70, and 90 s. As the irradiation time increased, the lethality of the strain gradually increased. When the irradiation time reached 70 seconds, the lethality was about 90%, and at 90 seconds, the lethality was close to 100%, with almost no viable bacteria remaining. Figure 6 When the lethality reaches approximately 90%, a high mutation rate and a certain survival rate are achieved, ensuring the acquisition of a suitable mutant library. Therefore, an irradiation time of 70 s was selected as the optimal treatment time for ARTP mutagenesis.

[0050] Automated Continuous Evolution System: All mutant strains are transferred to an automated continuous evolution instrument capable of detecting cell growth curves. For example... Figure 7 As shown, the automated continuous evolution system includes a peristaltic pump, a feeding device, a discharging device, a chemostat culture device, and a cell density OD (dose distribution) system. 600 Online monitoring device and controller. The entire automated continuous culture process is as follows: the bacterial mutant library is inoculated into a chemostat, cultured by controlling a constant temperature and stirring speed, and the changes in bacterial cell density are detected online and recorded in a computer; when OD... 600 Once the preset upper limit is exceeded, the computer will sequentially activate the peristaltic pumps on the inlet and outlet pipes to pump fresh culture medium and waste culture medium into and out of the chemostat, respectively, to dilute the cell density. The cell density is checked after each dilution until the OD value is reached. 600 If the cell density falls below the preset lower limit, dilution is stopped, and one passage is completed. This cycle is repeated to achieve automated continuous culture. During evolution, the cell density (OD) of the culture medium is checked every 20 minutes. 600 Value), setting OD 600 When the concentration reaches 1.5, turn on the inlet and outlet pumps to dilute the culture medium to OD0.05. 600 When the concentration reaches 0.5, stop dilution, start a new round of culture, and record the growth curve of the strain.

[0051] Building upon this, a mutagenesis device can be introduced to simultaneously induce mutagenesis and evolution, ensuring the strain remains in an optimal state for both processes. As the evolutionary process continues, the growth of the defective growth-production coupled strain recovers, indicating a potential enhancement in its L-tryptophan production capacity.

[0052] Example 3 Based on resistance plate screening of evolutionary strains and L-tryptophan production.

[0053] The strain library that recovered its growth after evolution in Example 2 was diluted and plated on M9 plates containing gradually increasing antibiotic concentrations. Strains that preferentially grew on high-concentration plates were selected and the tryptophan overexpression plasmid pIN- was reintroduced. aroG - trpEDCBA (See plasmid map) Figure + The inoculum was inoculated into 48-well plates containing seed culture medium (Table 4) and cultured overnight at 37°C and 800 rpm in a Microscreen incubator until the logarithmic growth phase. Then, the inoculum was transferred at a 10% inoculum to 48-well plates containing fermentation medium (Table 5), and fermented for 24 hours. L-tryptophan production was measured, and a second screening was performed to obtain the most significantly improved L-tryptophan production strain, which was named *Escherichia coli*. Escherichia coli X01-T21 was deposited on December 31, 2025, at the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37257. Subsequently, continuous sugar-fed fermentation was carried out in a 1.5L fermenter.

[0054] Table 4. Seed culture medium for L-tryptophan strains Table 5. Fermentation medium for L-tryptophan strains Dasgip fermentation process: The X01-T21 glycerol strain was brought to room temperature and streaked onto solid LB medium containing tetracycline (50 g / mL) using the streak plating method. It was then incubated overnight at 37°C. Single colonies with good growth were picked and inoculated into shake flasks containing LB medium. Tetracycline (50 g / mL) was added, and the mixture was incubated at 37°C and 220 rpm in a constant-temperature shaker until the logarithmic growth phase. The logarithmic growth phase culture was then inoculated at 5% into a 1.5 L fermenter containing 400 mL of seed culture medium. Dissolved oxygen was controlled at 30%, pH 6.8, and the culture was incubated at 37°C until the OD reached the target growth phase. 600The yield was approximately 10%. The seed culture was inoculated at a ratio of 10% into a 1.5 L fermenter containing 400 mL of fermentation medium. The dissolved oxygen was controlled at 30%, pH=6.8, and the culture was carried out at 37℃. The residual sugar in the medium was manually added to ensure it did not exceed 1 g / L. The fermentation culture lasted for about 42 hours, and the L-tryptophan yield was measured and the conversion rate was calculated.

[0055] Cell concentration determination: After diluting the fermentation broth appropriately, the cell density was measured and recorded using a spectrophotometer at a wavelength of 600 nm (OD200 after dilution). 600 (Measurements between 0.2 and 0.8 are more accurate). Repeat the measurement three times and take the average value to reduce error. Cell concentration = absorbance at 600 nm × dilution factor.

[0056] Glucose concentration determination: Centrifuge 1 mL of fermentation broth at 12000 rpm for 2 min, collect the supernatant, and dilute to an appropriate factor (after dilution, the sample concentration should be between 0-1 g / L for accurate measurement). Detect the glucose concentration using a biochemical analyzer. After successful standard calibration, repeat the test 3 times and take the average value to minimize error. Residual sugar value = test value × dilution factor.

[0057] L-Tryptophan Detection Method: L-Tryptophan is detected using the p-dimethylaminobenzaldehyde colorimetric method. The fermentation supernatant is diluted appropriately. 1 mL of the diluted supernatant is added to 9 mL of p-dimethylaminobenzaldehyde solution, mixed by inversion, and then boiled in a water bath for 2 min. Afterward, 2% sodium nitrite solution is added, mixed by inversion, and then boiled in a water bath for 3 min. The mixture is then allowed to return to room temperature, and the concentration is measured using a spectrophotometer at a detection wavelength of 600 nm. A standard curve is plotted based on the L-Tryptophan standard detection results. The L-Tryptophan concentration is calculated by substituting the sample detection results into the curve. L-Tryptophan concentration = test value × dilution factor.

[0058] The results are shown in Table 6 and Figure 8 As shown, throughout the fermentation cycle, the L-tryptophan concentration in X01-T21 showed a steady upward trend, reaching a final yield of 48.23 g / L at 42 h. The glucose-to-L-tryptophan conversion rate was 0.195 g / g. Compared with the control X01, the L-tryptophan yield increased by 11.5%, and the glucose-to-acid conversion rate increased by 21.9%. X01's carbon source consumption was mainly used for biomass accumulation, which fiercely competed with L-tryptophan synthesis. This resulted in its L-tryptophan concentration being slightly better than X01-T21 in the early stage, but its glucose-to-acid conversion rate was consistently lower than that of X01-T21. The latter, with its controlled growth rate and low cell density in the stationary phase, showed that more carbon flow was directed to the L-tryptophan synthesis pathway, indicating that the evolved strain had a significant carbon flow reprogramming effect.

[0059] Table 6. Fermentation L-tryptophan (Trp) yield Example 4 Based on whole-genome sequencing and comparative analysis of the starting strain and high-yield evolved strains, key mutant genes that promote L-tryptophan synthesis were identified.

[0060] Whole-genome sequencing was performed on the dominant mutant strain X01-T21 and strain X01 obtained in Example 3. Compared with strain X01, strain X01-T21 showed genetic differences. rrlH (Gene ID 944900, encoding 23S rRNA, a core component of the 50S large subunit, involved in peptide bond formation and binding to tRNA and translation factors) One point mutation was added at [location missing], and multiple point mutations appeared upstream of the start codon. The mutated gene... rrlH As shown in SEQ ID NO.11, this indicates that its transcriptional level may have changed; in the gene lptD A point mutation was added to the gene (Gene ID 945011, encoding a lipopolysaccharide transporter on the outer membrane), changing the amino acid at position 703 from tyrosine to aspartic acid. The mutated gene... lptD As shown in SEQ ID NO.12, this indicates that the activity of the LptD protein may have been altered, thereby affecting cell membrane permeability, etc. (In the gene...) trpE A total of 15 point mutations were found in the gene (GeneID 945846, encoding component I of anthranilate synthase), including 11 synonymous mutations and 3 mutations that altered the amino acid composition: V63A, Q71K, and S94N. The mutated gene... trpE As shown in SEQ ID NO.13, these point mutations may help eliminate the feedback inhibition of L-tryptophan and improve its catalytic efficiency and enzyme stability for shikimic acid, thereby increasing the flow of substrate to the tryptophan synthesis pathway.

Claims

1. A high-L-tryptophan-producing evolved strain, characterized in that, The evolved strain is Escherichia coli (Escherichia coli) Escherichia coli The plant number is X01-T21, and the preservation number is CGMCC No.37257.

2. The application of the evolved strain according to claim 1 in the production of L-tryptophan.

3. A method for preparing L-tryptophan, characterized in that, Includes the following steps: S1. Cultivate the evolved strain according to claim 1; S2. Transfer the bacterial culture from step S1 to seed culture medium and culture until OD200. 600 Reaching 8-12; S3. Inoculate the seed culture obtained in step S2 into the fermentation medium for fermentation culture; S4. Collect the fermentation broth from step S3 to obtain L-tryptophan.

4. The method according to claim 3, characterized in that, The incubation temperature in step S1 is 30-40℃; The transfer volume percentage in step S2 is 2%-10%, and the culture target OD 600 It is 10; The inoculum volume percentage in step S3 is 2%-10%, and the fermentation time is 30-60 hours.

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