Escherichia coli with high yield of L-phenylalanine, construction method and application thereof

Abstract

The application discloses a kind of recombinant escherichia coli and its construction method and application, the recombinant escherichia coli can be used for the production of L-phenylalanine.The application utilizes multiple rounds of mutagenesis screening to have high resistance to L-phenylalanine structural analogues, and has tyrosine auxotrophic mutant strain, and is regulated in the form of temperature-sensitive recombinant plasmid to code 3-deoxy-7-phosphoheptanoate synthase and branch acid mutase gene expression in L-phenylalanine synthesis pathway.The recombinant escherichia coli constructed in the application, in 5L fermenter, according to the cell growth stage adopts different temperature control and feeding mode, fermentation time is 56 h, under the condition that L-phenylalanine production is 62 g / L, sugar acid conversion rate is 25.2%, has the prospect and value of industrialization application.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a high-yield L-phenylalanine-producing Escherichia coli, its construction method, and its application. Background Technology

[0002] L-Phenylalanine (L-Phe) is one of the essential amino acids for the human body. As an important biochemical product, L-Phe has wide applications in food additives, nutritional supplements, and pharmaceuticals. For example, L-Phe is the main raw material for aspartame, an important food additive and sweetener, and in the pharmaceutical industry, it is mainly used in the production of amino acid infusions and amino acid-based anticancer drugs. L-Phe can be produced through protein hydrolysis extraction, chemical synthesis, enzymatic methods, and microbial fermentation. The first two methods have been gradually replaced by enzymatic and fermentation methods due to their complex processes, severe pollution, and low product purity. Although enzymatic production offers high product purity and high production capacity, its high cost and limited availability of substrates and enzymes, along with poor enzyme stability during the reaction process, have led to its gradual loss of market competitiveness. Microbial fermentation, which uses inexpensive raw materials such as glucose as substrates and utilizes microbial strains to produce L-Phe, has become the mainstream method for industrial production of L-Phe both domestically and internationally due to its low production cost, ease of scale-up, and minimal environmental pollution.

[0003] Escherichia coli is a typical model microorganism with advantages such as a clear genetic background, simple operation, and short growth cycle, and is commonly used as a production strain for organic acids and amino acids. Mutagenesis breeding is a traditional and commonly used microbial breeding method that can increase the mutation rate and obtain a large number of mutant types in a short time, but it also has problems such as low frequency of beneficial mutants and difficulty in controlling the direction of mutation. Targeted modification using metabolic engineering and synthetic biology techniques can achieve directed and rational breeding based on traditional non-directional mutagenesis breeding. The shikimic acid pathway is the only synthetic pathway for E. coli to produce L-phenylalanine by fermentation using glucose as a carbon source. Increasing the flux of the shikimic acid pathway and reducing the conversion of shikimic acid to other aromatic amino acids can improve the synthesis capacity of L-phenylalanine. From the perspective of microbial growth and metabolism, L-phenylalanine synthesis is theoretically synchronous with microbial growth. In actual fermentation, rationally adjusting the distribution of carbon flux between the L-phenylalanine synthesis pathway and microbial growth is the foundation for realizing the industrial fermentation production of L-phenylalanine. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a recombinant Escherichia coli capable of producing high levels of L-phenylalanine, its construction method and application.

[0005] Technical solution: The present invention provides a strain of Escherichia coli JNYPQ, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 27, 2022, with the accession number GDMCC No. 62245.

[0006] The present invention also includes a recombinant Escherichia coli strain, wherein the recombinant Escherichia coli strain is expressed with the recombinant plasmid pACYC177-CI-aroF-pheA fbr Escherichia coli JNYPQ strain was introduced to obtain E. coli JNY021.

[0007] The present invention also includes a method for constructing the recombinant Escherichia coli strain, wherein the construction method involves expressing the recombinant plasmid pACYC177-CI-aroF-pheA fbr It was obtained by introducing the Escherichia coli JNYPQ strain described above.

[0008] The recombinant plasmid pACYC177-CI-aroF-pheA fbr The CI-aroF-pheA gene fragment is ligated into plasmid pACYC177 to obtain plasmid pACYC177-CI-aroF-pheA, and the pheA gene on plasmid pACYC177-CI-aroF-pheA is then subjected to site-directed mutagenesis.

[0009] The site-directed mutation of the pheA gene involves replacing the 977th nucleotide of the pheA gene with a G instead of a T.

[0010] The recombinant plasmid pACYC177-CI-aroF-pheA fbr The following steps are included in the construction process:

[0011] 1) Using the genome of *E. coli* K12 as a template, the *aroF* and *pheA* genes were amplified by PCR using primer pairs *aroF-F / aroF-R* and *pheA-F / pheA-R*. 2) The CI857 gene containing the λpR promoter was synthesized. The *aroF* gene was ligated to the right side of the λpR promoter, and the λpL promoter sequence was ligated upstream of the *pheA* gene. Homologous recombination was performed downstream of the *aroF* gene at the other end, yielding the homologous recombination ligation product CI-aroF-pheA. 3) The homologous recombination ligation product CI-aroF-pheA was digested and ligated into the pACYC177 plasmid to obtain the expression vector pACYC177-CI-aroF-pheA. 4) Using the pACYC177-CI-aroF-pheA expression plasmid as a template, primer pairs PheA were used to amplify the *pheA* gene.fbr -UF / PheA fbr -UR and PheA fbr -DF / PheA fbr -DR amplifies the upstream and downstream sequences of the pheA gene containing the mutation site, respectively, and then ligates the upstream and downstream sequences through homologous recombination to obtain CI-aroF-pheA. fbr Gene fragment, CI-aroF-pheA fbr The gene fragment was ligated into the pACYC177 plasmid to obtain pACYC177-CI-aroF-pheA fbr Plasmid.

[0012] The primer pair aroF-F / aroF-R sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2, the primer pair pheA-F / pheA-R sequence is shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the primer pair PheA fbr -UF / PheA fbr -UR sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, and the primer pair PheA fbr -DF / PheA fbr -DR sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0013] Among them, the CI-aroF-pheA fbr The gene sequence of the gene fragment is shown in SEQ ID NO: 11.

[0014] The present invention also includes a method for producing L-phenylalanine, wherein the method comprises picking the recombinant E. coli JNY021 strain and inoculating it into a seed culture medium for cultivation, and then transferring it to a fermentation culture medium for further fermentation to obtain a fermentation broth containing L-phenylalanine.

[0015] The seed culture medium consists of the following per liter: 32g tryptone, 20g yeast extract, 5g sodium chloride, 5g glycerol, and 50mg kanamycin.

[0016] The seed culture medium is cultured at a temperature of 33°C for 16 hours and at a rotation speed of 220 rpm.

[0017] The fermentation medium consists of the following components per liter: 22.5g glucose, 2g yeast extract, 1.2g tyrosine, 1g magnesium sulfate, 7g dipotassium hydrogen phosphate, 3g ammonium sulfate, 2g citric acid, 2g fumaric acid, 0.03g ferrous sulfate, 0.01g manganese sulfate, 0.02g calcium chloride, 0.002g cobalt chloride, 0.001g zinc sulfate, 0.005g vitamin B2, 0.01g pyridoxal phosphate, 0.01g niacin, 0.01g thiamine, and 50mg kanamycin.

[0018] The fermentation culture adopts a fed-batch fermentation method. The parameters of the fed-batch fermentation are set as follows: initial fermentation temperature 33℃, two-stage temperature control, when the cells enter the late logarithmic growth stage, the temperature is increased to 38℃; initial pH value 7.0, the pH is controlled at 6.7-7.0 with 20% ammonia water; initial glucose concentration 22.5g / L, 600g / L glucose is added at different rates in the later stage; initial rotation speed and aeration rate are 400rpm and 1.3vvm respectively, the dissolved oxygen is not lower than 20% throughout the fermentation process; fermentation time is 56h.

[0019] The method employs different feeding strategies based on the growth stage of the cells. When the sugar in the fermenter is about to be depleted, glucose is added exponentially. When the cells enter the stationary phase, glucose is added at a linearly decreasing rate.

[0020] During the logarithmic growth phase of the bacterial cells, according to the formula Glucose was added in an exponentially fed manner.

[0021] After the bacteria enter the stationary phase, glucose is added at a linearly decreasing rate according to the formula F(mL / h)=-0.1518t+9.473.

[0022] First, this invention obtains auxotrophic Escherichia coli strains containing tyrosine and tryptophan through ultraviolet mutagenesis. Then, it uses ambient temperature and pressure plasma mutagenesis to screen for mutant strains of E. coli resistant to 5 mg / mL 4-amino-DL-phenylalanine, E. coli JNYPQ(tyr). - trp lea PAP r This strain has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 62245.

[0023] Secondly, construct the recombinant plasmid pACYC177-CI-aroF-pheA fbr Transformed into the JNYPQ strain, the λ phage Cl temperature-sensitive repressor protein and strong promoters pL and pR were used to regulate the key gene pheA in the L-phenylalanine synthesis pathway. fbrThe expression of (encoding 3-deoxy-7-phosphate heptanoate synthase) and aroF (encoding branching acid mutase) yielded a recombinant Escherichia coli strain capable of producing high levels of L-phenylalanine.

[0024] Specifically, the method for producing L-phenylalanine by fermentation of recombinant Escherichia coli includes the following culture steps: picking a loopful of the recombinant E. coli JNY021 colony obtained above from the slant and transferring it to seed culture medium; the seed culture temperature is 33℃, the culture time is 14 hours, and the culture is carried out until OD is reached. 600 At approximately 16°C, the inoculum was transferred from the seed culture to a 5L fermenter at a 15% inoculum rate. The initial fermentation speed was 400 rpm, the initial aeration rate was 1.3 vvm, and the dissolved oxygen level was not lower than 20%. The pH was controlled at 6.7-7.0 using 20% ​​ammonia. During the initial growth phase, the fermentation temperature was set at 33°C. When the logarithmic growth phase began, the fermentation temperature was set at 38°C. The initial glucose concentration was 22.5 g / L. When the glucose in the fermenter was nearly depleted, glucose was added exponentially. Once the cells entered the stationary phase, glucose was added at a linearly decreasing rate to maintain a residual glucose concentration of 0.5-2 g / L during the later stages of fermentation.

[0025] The culture medium for producing L-phenylalanine consists of the following components:

[0026] The composition of each liter of slant culture medium is: 10g sodium chloride, 10g tryptone, 5g yeast extract, 20g agar powder, and 50mg kanamycin;

[0027] The seed culture medium consists of 32g tryptone, 20g yeast extract, 5g sodium chloride, 5g glycerol, and 50mg kanamycin per liter.

[0028] The fermentation medium per liter (shake flask level) consists of: 10g glucose, 5g yeast extract, 2g magnesium sulfate heptahydrate, 3g dipotassium hydrogen phosphate, 5g ammonium sulfate, 2g citric acid, 1g NaCl, 0.03g ferrous sulfate, 0.01g manganese sulfate, 0.02g calcium chloride, 0.002g cobalt chloride, 0.001g zinc sulfate, 0.005g vitamin B2, 0.01g pyridoxal phosphate, 0.01g nicotinic acid, 0.01g thiamine, and 50mg kanamycin.

[0029] The fermentation medium per liter (fermenter level) consists of: glucose 22.5g, yeast extract 2g, tyrosine 1.2g, magnesium sulfate 1g, dipotassium hydrogen phosphate 7g, ammonium sulfate 3g, citric acid 2g, fumaric acid 2g, ferrous sulfate 0.03g, manganese sulfate 0.01g, calcium chloride 0.02g, cobalt chloride 0.002g, zinc sulfate 0.001g, vitamin B2 0.005g, pyridoxal phosphate 0.01g, nicotinic acid 0.01g, thiamine 0.01g, and kanamycin 50mg.

[0030] Beneficial effects: This invention first obtains the auxotrophic Escherichia coli JNYPQ containing tyrosine and tryptophan through ultraviolet mutagenesis, and then constructs the recombinant plasmid pACYC177-CI-aroF-pheA. fbr A recombinant E. coli strain, ENY021, was obtained by transforming the JNYPQ strain to produce high levels of L-phenylalanine. Under specific fermentation conditions, this strain can accumulate 62 g / L of L-phenylalanine in 56 h, with a sugar-acid conversion rate of 25.2%. Therefore, this strain has promising applications in the practical production of L-phenylalanine. Attached Figure Description

[0031] Figure 1 HPLC chromatograms of L-phenylalanine standard sample and fermentation broth sample;

[0032] Figure 2 This is a flowchart illustrating the construction process of expression plasmids for L-phenylalanine synthesis-related genes in this invention.

[0033] Figure 3 The growth of recombinant E. coli JNY021 in seed culture;

[0034] Figure 4 Fermentation of recombinant E. coli JNY021 in shake flasks;

[0035] Figure 5 The growth and acid production of recombinant Escherichia coli in a 5L fermenter. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] Example 1: Screening of L-phenylalanine-producing chassis bacteria using multiple rounds of mutagenesis in Escherichia coli K12

[0038] (1) Screening for tyrosine and tryptophan-deficient strains by ultraviolet mutagenesis

[0039] Screening for strains lacking both tyrosine and tryptophan can disrupt the metabolic pathways of L-phenylalanine and L-tyrosine, aiming to shift the carbon flux from the shikimic acid pathway to L-phenylalanine. A single colony of *E. coli* K12, stored at 4°C, was inoculated into 5 mL of LB medium (10 g peptone, 5 g yeast extract, 10 g NaCl, diluted to 1 L with deionized water, pH adjusted to 7.0) and cultured overnight at 37°C and 220 rpm until the logarithmic growth phase. Cell concentration was measured, cells were collected, and diluted with physiological saline to approximately 10⁻⁶. 8CFU / mL. Pipette 3 mL of the above bacterial suspension into a 6 cm diameter petri dish to form a thin layer. Irradiate the dish with the UV lamp at a distance of 28.5 cm for 1 min (determined based on the initial 70% lethality). Wrap the dish in aluminum foil and add 3 mL of LB liquid medium. Incubate at 37°C for 18 h in the dark. Wash 5 mL of the bacterial suspension with an equal volume of physiological saline to remove the medium. Add 0.1 mL of the bacterial suspension to 5 mL of nitrogen-free medium (containing 20 g glucose, 10 g dipotassium hydrogen phosphate, 2 g sodium citrate, and 0.2 g magnesium sulfate heptahydrate per liter). Incubate at 37°C for 12 h. Add 10 mL of nitrogen-free medium containing 2 g / L ammonium chloride, and simultaneously add penicillin to bring the final concentration of the culture medium to approximately 1000 U / mL. Incubate for 18 h. Spread 0.1 mL of the bacterial suspension onto an LB plate and incubate at 37°C for 36 h.

[0040] Colonies were picked up from LB medium using a toothpick and inoculated separately into M9 medium (containing 4g glucose, 6.78g Na2HPO4, 3g KH2PO4, 0.5g NaCl, 1g NH4Cl, 0.493g MgSO4·7H2O, and 0.011g CaCl2 per liter) and LB medium, respectively, and incubated for 12 hours. Colonies that did not grow on M9 medium but grew on LB medium were preliminarily identified as auxotrophs. These colonies were then inoculated into liquid LB medium and incubated at 37°C for 14-16 hours, followed by washing away the medium with sterile physiological saline. 0.1mL of the bacterial culture was inoculated into M9 liquid test tubes containing only tyrosine, only tryptophan, and both tyrosine and tryptophan. Colonies that did not grow on the first two media but grew on the third medium were selected: JNYP25, JNYP33, and JNYP92. The three strains lacking both tyrosine and tryptophan were inoculated into test tube seed culture medium and cultured overnight at 37°C. Then, they were transferred at a 10% inoculation rate to 500 mL shake flasks containing 30 mL of fermentation medium and cultured at 33°C and 220 rpm for 24 h. The L-phenylalanine yields of strains JNYP25, JNYP33, and JNYP92 were 1.92 g / L, 1.56 g / L, and 1.98 g / L, respectively.

[0041] (2) Screening for L-phenylalanine structural analog resistance mutants by plasma mutagenesis

[0042] Selecting L-phenylalanine structural analogue-resistant mutants aims to alleviate the feedback inhibition and repression of specific synthetic pathways by L-phenylalanine to some extent, thereby further improving the acid-producing capacity of the strain. After LB culture in test tubes, strain JNYP92 was diluted with physiological saline to approximately 10... 8CFU / mL, 10 μL of bacterial suspension was placed in an ARTP mutagenesis breeding instrument. The mutagenesis conditions were: temperature 20℃, power 100W, helium flow rate 10L / min, and treatment time 180s. The mutagenized cells were spread on LB medium containing 5 mg / mL 4-amino-DL-phenylalanine (PAP). Rapidly growing strains with large single colonies were selected and inoculated into 48-well plates containing 1 mL of fermentation medium. After 48 h of incubation, the L-phenylalanine content was detected using an amino acid analyzer for initial screening. Mutants with high L-phenylalanine production, JNYP92-12, JNYPQ, JNYP92-25, JNYP92-26, and JNYP92-31, were subjected to shake-flask fermentation experiments. The results showed that JNYP92-17 (tyr) was the most effective mutant. - trp lea pAP r The highest L-phenylalanine yield was 4.12 g / L. The strain was named Escherichia coli JNYPQ and deposited at the Guangdong Provincial Microbial Culture Collection Center on January 27, 2022, with accession number GDMCC No. 62245.

[0043] Example 2: Detection method for L-phenylalanine

[0044] L-phenylalanine was detected using an amino acid derivatization method. An Agilent ZORBAX SB-Aq column was used, with aqueous phase A: 0.01 mol / L KH₂PO₄ and organic phase B: acetonitrile:methanol:aqueous phase A = 5:3:1 (volume ratio). 8 μL of sample was mixed with 4 μL of phthalaldehyde as a derivatizing agent and injected online for derivatization. A two-phase gradient elution was performed from 0 to 25 min (0 min, 20% A phase, 80% B phase; 5 min, 65% A phase, 35% B phase; 10 min, 35% A phase, 65% B phase; 15 min, 30% A phase, 70% B phase; 20 min, 50% A phase, 50% B phase; 23 min, 80% A phase, 20% B phase). The column temperature was 35℃, and the flow rate was 1 mL / min. Detection was performed using a fluorescence detector; the excitation (Ex) and emission (Em) wavelengths were 330 nm and 465 nm, respectively. Figure 1 The peak elution time of both the L-phenylalanine standard and the fermentation broth was 13.837 min.

[0045] Example 3: Expression of key genes in the L-phenylalanine synthesis pathway in *Bacteroides edulis*

[0046] like Figure 2 As shown, using pACYC177 (purchased from the Miaoling plasmid platform) as the backbone plasmid, a temperature-sensitive repressor protein from λ phage Cl was used to construct a temperature-controlled switch, with the key gene pheA in the L-phenylalanine synthesis pathway as the target. fbrThe target genes are (encoding 3-deoxy-7-phosphate heptanoate synthase) and aroF (encoding branched acid mutase), which regulate the expression of the L-phenylalanine synthesis pathway.

[0047] Using the genome of *E. coli* K12 as a template, the *aroF* and *pheA* genes were amplified by PCR using primer pairs *aroF-R / aroF-F* (SEQ ID NO: 1–2) and *pheA-R / pheA-F* (SEQ ID NO: 3–4). The PCR system for the *aroF* gene was as follows: 25 μL PrimeSTAR MAX Premix (2×), 1.5 μL each of *aroF-R* and *aroF-F*, 2 μL template, and 2 μL ddH2O. The PCR conditions were: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 10 s, 57℃ annealing for 5 s, 72℃ extension for 60 s, for 29 cycles, followed by a final extension at 72℃ for 2 min, to obtain the *aroF* gene fragment. The PCR annealing temperature for the *pheA* gene was 50℃, and all other conditions were the same as the *aroF* gene PCR system and amplification conditions, resulting in the *pheA* gene fragment. To achieve the regulation of L-phenylalanine synthesis-related gene expression by the λ phage Cl thermosensitive regulatory element, it is necessary to synthesize the CI repressor protein, λpR, and λpL promoter-related sequences. The C1857 repressor protein encoding gene was BLASTed in NCBI, and the Bacteriophage sp. isolate 528 genome sequence was obtained. Using this as a template, C1857 and its upstream and downstream sequences (including the λpR promoter) were synthesized (the gene sequence is shown in SEQ ID NO: 9). Based on the principle of homologous recombination, the aroF gene fragment obtained by PCR was ligated to the right side of the λpR promoter. The homologous recombination ligation system was as follows: 6 μL of pheA gene fragment obtained by PCR, 2 μL of synthesized C1857-λpR sequence, and 2 μL of homologous recombination enzyme (purchased from ABM), incubated on ice for 30 min for ligation. Similarly, in order to achieve C1857 regulation of pheA expression, the synthesized λpL promoter sequence (its gene sequence is shown in SEQ ID NO: 10) was linked upstream of pheA, and the other end was homologously recombined downstream of the aroF gene to obtain the homologous recombination ligation product CI-aroF-pheA.

[0048] The above-mentioned multi-fragment homologous recombination ligation product CI-aroF-pheA was digested and ligated into the pACYC177 plasmid treated with BamHI and PstI to obtain the expression vector pACYC177-CI-aroF-pheA. Using the pACYC177-CI-aroF-pheA plasmid as a template, primers were used to express the expression vector pACYC177-CI-aroF-pheA. fbr -UF / PheA fbr-UR (shown in SEQ ID NO: 5 and SEQ ID NO: 6) and primer pair PheA fbr -DF / PheA fbr -DR (shown in SEQ ID NO: 7 and SEQ ID NO: 8) amplified the upstream and downstream sequences of the pheA gene containing the mutation site (the T substitution G at nucleotide 977 of the pheA gene). The PCR of the upstream sequence of the pheA gene containing the mutation site was performed with an annealing temperature of 55℃ and an extension temperature of 72℃ for 5 seconds. Other conditions and amplification methods were the same as those for the aroF gene PCR. fbr The upstream fragment of the gene; the downstream sequence of the pheA gene with the mutation site. The annealing temperature for PCR was 55℃ annealing, 72℃ extension for 50s, and other conditions were the same as those for the aroF gene PCR system. fbr Downstream fragments of the gene. Based on homologous sequences, pheA... fbr upstream gene fragments and pheA fbr The downstream segments of the gene are linked together to obtain pheA fbr The gene fragment, homologous recombination ligation system is: pheA fbr 5 μL of upstream fragment, pheA fbr Ligation was performed with 3 μL of the downstream fragment and 2 μL of homologous recombinase (purchased from ABM), incubated on ice for 30 min. The plasmid pACYC177-CI-aroF-pheA was digested with BamHI and EcoRI, and then ligated with the -pheA fragment containing the mutation site. fbr The gene fragments were ligated using T4 ligase to obtain pACYC177-CI-aroF-pheA fbr plasmid, CI-aroF-pheA fbr See SEQ ID NO: 11. pACYC177-CI-aroF-pheA fbr The plasmid was transformed into Escherichia coli JNYPQ to obtain recombinant E. coli JNY021.

[0049] SEQ ID NO: 1: aroF-R:

[0050] SEQ ID NO: 2: aroF-F:

[0051] SEQ ID NO: 3: pheA-R: 5'- cgcggatcc tcaggttggatcaacag-3'

[0052] SEQ ID NO: 4: pheA-F: 5'- ccgccggaattcctcccaaatcgggg-3'

[0053] SEQ ID NO: 5: PheA fbr -UF:5'- cgcggatcc tcaggttggatcaacaggc-3'

[0054] SEQ ID NO: 6: PheA fbr -UR:

[0055] SEQ ID NO: 7: PheA fbr -DF:

[0056] SEQ ID NO: 8: PheA fbr -DR:5'- ccggaattc ctcccaaatcgggg-3'.

[0057] Underlined restriction enzyme sites and protective bases; italicized text indicates homologous fragments linked to the target gene; bold text indicates pheA point mutation bases.

[0058] Example 4: Determination of acid production during growth and fermentation of recombinant E. coli JNY021 in shake flasks.

[0059] Recombinant *E. coli* strain JNY021, stored at -80℃, was inoculated into slant agar and cultured for two generations at 37℃, each generation lasting 12 hours. The strain from the second generation slant was then inoculated into a 500mL round-bottom flask containing 30mL of seed culture medium and cultured at 37℃ and 220rpm for 24 hours. The growth curve of recombinant *E. coli* strain JNY021 in seed culture medium is shown below. Figure 3 As shown, the strain enters the logarithmic growth phase at approximately 10 hours and the stationary phase at approximately 24 hours. Between 13 and 14 hours, the OD600 is in the range of 12.33-16.85, meeting the inoculation requirements, and it can be inoculated into fermentation medium for further cultivation. The strain's final OD600 reaches 30.05, indicating that this recombinant *E. coli* has strong growth capacity. The slant culture medium consists of: 10g sodium chloride, 10g tryptone, 5g yeast extract, 20g agar powder, and 50mg kanamycin per liter; the seed culture medium consists of: 32g tryptone, 20g yeast extract, 5g sodium chloride, 5g glycerol, and 50mg kanamycin per liter. The culture media are sterilized at 121℃ for 20 minutes. When the temperature drops to approximately 65℃, the kanamycin stock solution is added and the mixture is shaken well before use.

[0060] In addition, the fermentation of L-phenylalanine by recombinant *E. coli* JNY021 at the shake-flask level was investigated. The seed culture of recombinant *E. coli* JNY021, cultured for 10–12 h as described above, was transferred at a 10% inoculum to 500 mL round-bottom flasks containing 30 mL of fermentation medium and incubated at 33 °C and 220 rpm for 24 h. Figure 4 As shown, the yield of phenylalanine can reach 8.21 g / L. Phenol red with a final mass concentration of 8 mg / L was added to the fermentation medium as an acid-base indicator. During the culture process, ammonia water needs to be manually added according to the color change of the medium to maintain the pH of the medium at about 7.0. When the glucose is exhausted, the medium turns red and does not change color. Then, 0.5 mL of 600 g / L glucose solution is added at one time using a pipette.

[0061] Example 5: L-Phenylalanine Production from Recombinant Escherichia coli JNY021 in a 5L Fermenter

[0062] Recombinant *E. coli* strain JNY021, stored at -80℃, was inoculated into slant agar and cultured for two generations at 37℃, each generation lasting 12 hours. The strain from the second-generation slant was then inoculated into a 500mL round-bottom flask containing 30mL of seed culture medium and cultured at 33℃ and 220rpm for 14 hours, shaking until the OD reached approximately 600 to 15. A 15% inoculum was then added to a 5L fermenter with a 2.4L volume for fed-batch fermentation. The fermentation medium consisted of: 22.5g glucose, 2g yeast extract, 1.2g tyrosine, 1g magnesium sulfate, 7g dipotassium hydrogen phosphate, 3g ammonium sulfate, 2g citric acid, 2g fumaric acid, 0.03g ferrous sulfate, 0.01g manganese sulfate, 0.02g calcium chloride, 0.002g cobalt chloride, 0.001g zinc sulfate, 0.005g vitamin B2, 0.01g pyridoxal phosphate, 0.01g nicotinic acid, 0.01g thiamine, and 50mg kanamycin, with a pH of 7.0. The added glucose concentration (SO) was 600g / L. The initial fermentation speed was 400 rpm, and the initial aeration rate was 1.3 vvm. Dissolved oxygen decreased automatically in the early stage, and the aeration rate and speed were adjusted in the later stage to maintain dissolved oxygen at 30%-45%. The pH was automatically adjusted to 6.7-7.0 using 20% ​​ammonia water.

[0063] Different feeding strategies were adopted according to the different growth stages of the cells, with 600 g / L glucose added at different rates. At 16 hours, the glucose concentration in the fermenter dropped to 0.9 g / L, indicating that the carbon source was almost depleted, and feeding control was initiated. During the logarithmic growth phase of the cells (16-32 hours), each phase lasted 4 hours, and feeding was initiated according to the formula... Glucose was added in an exponential fed-batch manner. The initial cell concentration (x0) and culture medium volume (v0) were 43.31 g / L and 2.4 L, respectively; the cell glucose yield (Y) was 2.0; the initial glucose concentration (S0) was 600 g / L; t was the fermentation time; and F was the glucose feeding rate. The μset was set to 0.18 h. -1 0.15h -1 0.12h -1 0.09h -1 0.06h -1 After the cells enter the stationary phase (36h-52h), glucose is added linearly at a decreasing rate according to the formula F(mL / h)=-0.1518t+9.473, where F is the glucose feeding rate and t is the fermentation time.

[0064] The growth, acid production, and sugar consumption of the recombinant E. coli JNY021 throughout the fermentation process are as follows: Figure 5 As shown in the figure, the growth temperature of the recombinant *E. coli* JNY021 was controlled at 33℃ during the initial growth stage. After 4 hours, the cell growth accelerated, and by 8 hours it had entered the logarithmic growth phase with an OD600 of 14.3. In the mid-to-late logarithmic growth phase (28 hours, OD600 118.62), the fermentation temperature was increased to 38℃. After entering the stationary phase, the biomass remained at 130-140. The increase in L-phenylalanine production lagged behind the increase in biomass. After 16 hours of fermentation, the L-phenylalanine production began to increase rapidly, reaching 62 g / L by the end of fermentation (56 hours). The initial glucose concentration was 22.5 g / L. The glucose consumption rate was relatively fast in the early stages of fermentation. At 16 hours, the glucose concentration in the fermenter dropped to 0.9 g / L, and 600 g / L glucose was added at different rates. The total sugar consumption during fermentation was 268.47 g / L, and the yield of L-phenylalanine to glucose was 25.2% (molar ratio). sequence list <110> Yangzhou University, Jiangnan University <120> A high-L-phenylalanine-producing Escherichia coli, its construction method and application <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 32 <212> DNA <213> aroF-R (Artificial Sequence) <400> 1 gttttttata tgaatttaag ccacgcgagc cg 32 <210> 2 <211> 36 <212> DNA <213> aroF-F(Artificial Sequence) <400> 2 agatctttag ctgtcgagga tcaactatcg caaacg 36 <210> 3 <211> 26 <212> DNA <213> pheA-R(Artificial Sequence) <400> 3 cgcggatcct caggttggat caacag 26 <210> 4 <211> 23 <212> DNA <213> pheA-F(Artificial Sequence) <400> 4 ccggaattcc tcccaaatcg ggg 23 <210> 5 <211> 28 <212> DNA <213> PheAfbr-U-F(Artificial Sequence) <400> 5 cgcggatcct caggttggat caacaggc 28 <210> 6 <211> 19 <212> DNA <213> PheAfbr-U-R(Artificial Sequence) <400> 6 tctgattatg ccccgtctg 19 <210> 7 <211> 19 <212> DNA <213> PheAfbr-DF(Artificial Sequence) <400> 7 cagacggggc ataatcaga 19 <210> 8 <211> 23 <212> DNA <213> PheAfbr-DR(Artificial Sequence) <400> 8 ccggaatttcc tcccaatcg ggg 23 <210> 9 <211> 1128 <212> DNA <213> CI857-pR(Artificial Sequence) <400> 9 gatctttagc tgtcttggtt tgccaaagc gcattgcata atctttcagg gttatgcgtt 60 gttccataca acctccttag tacatgcaac cattatcacc gccagaggta aaatagtcaa 120 cacgcacggt gttagatatt tatcccttgc ggtgatagat ttaacgtag agcacaaaaa 180 agaaaccatt aacacaagag cagcttgagg acgcacgtcg ccttaaagca atttatgaaa 240 aaaagaaaaa tgaacttggc ttacccagg aatctgtcgc agacagatg gggatggggc 300 agtcaggcgt tggtgcttta tttaatgca tcaatgcatt aaatgcttat aacgccgcat 360 tgcttacaaa aattctcaaa gttagcgttg aagaatttag cccttcaatc gccagagaaa 420 tctacgagat gtatgaagcg gttagtatgc agccgtcact tagaagtgag tatgagtacc 480 ctgtttttc tcatgttcag gcagggatgt tctcacctaa gcttagaacc tttaccaaag 540 gtgatgcgga gagatgggta agcacaacca aaaaagccag tgattctgca ttctggcttg 600 aggttgaagg taattccatg accgcaccaa caggctccaa gccaagcttt cctgacggaa 660 tgttaattct cgttgaccct gagcaggctg ttgagccagg tgatttctgc atagccagac 720 ttgggggtga tgagtttacc ttcaagaaac tgatcaggga tagcggtcag gtgtttttac 780 aaccactaaa cccacagtac ccaatgatcc catgcaatga gagttgttcc gttgtgggga 840 aagttatcgc tagtcagtgg cctgaagaga cgtttggctg atcggcaagg tgttctggtc 900 ggcgcatagc tgataacaat tgagcaagaa tcttcatcga attaggggaa ttttcactcc 960 cctcagaaca taacatagta aatggattga attatgaaga atggttttta tgcgacttac 1020 cgcagcaaaa ataaagggaa agataagcgc tcaataaacc tgtctgtttt ccttaattct 1080 ctgctggctg ataatcatca cctgcagcaa ccaatgcatt ggatgcat 1128 <210> 10 <211> 222 <212> DNA <213> pL(Artificial Sequence) <400> 10 gattgggag gaattccaat gcttcgtttc gtatcacaca ccccaaagcc ttctgctttg 60 aatgctgcccc ttcttcaggg cttaattttt aagagcgtca ccttcatggt ggtcagtgcg 120 tcctgctgat gtgctcagta tcaccgccag tggtatttat gtcaacaccg ccagagataa 180 tttatcaccg cagatggtta tctgtatgtt ttttatatga at 222 <210> 11 <211> 3643 <212> DNA <213> CI-aroF-pheAfbr(Artificial Sequence) <400> 11 ggatcctcag gttggatcaa caggcactac gttctcactt gggtaacagc ccaatacctt 60 cattgaacgg gtgatttccc ctaactcttt caatgctttt tgcatttccg ctgattcaag 120 attggcctga atatccagat agaacatctc ttcccatgga ttaccgtgaa tcgggcgtga 180 ttccagacgg ggcataatca gattgtggtt gcgcagtacc agcaacgctt caaccagcgc 240 accggcttgt tgcccggtcg ccattaacaa cgtggttttc gccggaacct gatcagacac 300 gttaatggct ttacgcgcca acaccacaaa tcgggtgaag ttttgtcgct gatttgcttc 360 aatacgctcc agtacctgca aaccgtacaa agtgccgcca gcttcgcttc ccaacgcagc 420 aacatgcggt gattttgcct gtgcaacctt ttccattgcc gcagacgtac tttcggtata 480 ttcaatcttc cagtgcggat aacgattaag gaatttgctg cattgctgga atggctgcgg 540 atggctgtag accgtattga tggtggataa atcagtagtg ccggagacca acaaacaatg 600 gtcgatagtt aacgtcatct cgccaacaat cgacaagctg gtatgttgca gcagatcgta 660 aacgtcgttt atggcaccgg agctggtatt ttcaatcggt acgacggcat agtcggcctg 720 gccggtttcc acctgattaa aaatatcggc aaatttggcg cagccacttt caatgaattg 780 ctcaaagtga cgggcagcat actggcgcgc cgcaagatgg gaataagaac ctttggggcc 840 gagaaaagcg atgcgtgctg agtgcggatt aattttattg agatgttgtt ggagcaaagc 900 ctgctgagtt aatacggaat cttcaatgat gagctggaac aggcgagtaa tgtaatgggc 960 gtccagatgg tgcgctttac cgagcgtaat taatctttcc agcaaatcgc gttcacgatc 1020 aatatcacgt accgggcgat gcgagagcag tttggctttt cccacctcga cggccagttc 1080 gcgccgttct gccagtaacg ctaataattt ttcatccagc gcgctgattt tctctcgcag 1140 cgccagtaac gggttttccg atgtcatagt gttgcctttt tgttatcaat aaaaaaggcc 1200 ccccgatttg ggaggaattc caatgcttcg tttcgtatca cacaccccaa agccttctgc 1260 tttgaatgct gcccttcttc agggcttaat ttttaagagc gtcaccttca tggtggtcag 1320 tgcgtcctgc tgatgtgctc agtatcaccg ccagtggtat ttatgtcaac accgccagag 1380 ataatttatc accgcagatg gttatctgta tgttttttat atgaatttaa gccacgcgag 1440 ccgtcagctg cccgttcaga tcctgatgaa tttcacgcag caaggcatcg gtcatttccc 1500 agctaatgca ggcatcggtt acggatacac cgtatttcat ttcactgcgc ggttgctcgg 1560 aagactgatt gccctcgtgg atattacttt cgatcatcag accaataatt gagcgattgc 1620 catctttgat ttgagcaacc acggattctg ccaccgcagg ctgacggcga taatctttat 1680 tggaattacc gtggctgcaa tctaccatca gagacgggcg cagtcccgcc tgttccatct 1740 ctttttcaca ttgcgcaaca tccgcagggc tatagttcgg cgctttacca ccgcgcagga 1800 tcacatggcc gtccggattc ccctgagttt gtagcaacgc aacctgccct gcctggttaa 1860 tgccaacaaa acggtgcggc tgggcggcgg cgcgcatagc gttaattgct gttgccagac 1920 tgccgtcggt gccgttttta aaaccaaccg gcatggaaag cccggaggcc atttcacggt 1980 gagtttgcga ttccgttgta cgagcaccaa ttgctgacca gctaaacaga tcgcccaggt 2040 attgcgggct attcggatct aacgcttccg tcgccagtgg cagtcccata ttcaccagct 2100 caagcagcaa tttacgcgcg atctgcagcc cggcttctac atcaaaagag ccatccatat 2160 ggggatcgtt aattaaccct ttccagccga cagtggtacg gggtttttca aatagacgc 2220 gcattaccag atagaggcta tcgctgacct ctgcggcaag ggctttaaat cgacgagcat 2280 attccagagc agtttccgga tcatgaatgg aacaaggacc acatactacc agcagacgag 2340 gatcgcgccc ggcgataata tctgaaatgc tttacgcga gtcagcaatc tgggcttctt 2400 gttgcaggct caatggaaaa gcggccttca gttgttccgg agtcattaaa acctgttcgt 2460 cggtatgtac gttattcagc gcgtcttttt gcatgatggc gatcctgttt atgctcgttt 2520 gcgatagttg atcctcagat ctttagctgt cttggtttgc ccaaagcgca ttgcataatc 2580 tttcagggtt atgcgttgtt ccatacaacc tccttagtac atgcaaccat tatcaccgcc 2640 agaggtaaaa tagtcaacac gcacggtgtt agatatttat cccttgcggt gatagattta 2700 acgtatgagc acaaaaaaga aaccattaac acaagagcag cttgaggacg cacgtcgcct 2760 taaagcaatt tatgaaaaaa agaaaaatga acttggctta tcccaggaat ctgtcgcaga 2820 caagatgggg atggggcagt caggcgttgg tgctttattt aatggcatca atgcattaaa 2880 tgcttataac gccgcattgc ttacaaaaat tctcaaagtt agcgttgaag aatttagccc 2940 ttcaatcgcc agagaaatct acgagatgta tgaagcggtt agtatgcagc cgtcacttag 3000 aagtgagtat gagtaccctg ttttttctca tgttcaggca gggatgttct cacctaagct 3060 tagaaccttt accaaaggtg atgcggagag atgggtaagc acaaccaaaa aagccagtga 3120 ttctgcattc tggcttgagg ttgaaggtaa ttccatgacc gcaccaacag gctccaagcc 3180 aagctttcct gacggaatgt taattctcgt tgaccctgag caggctgttg agccaggtga 3240 tttctgcata gccagacttg ggggtgatga gtttaccttc aagaaactga tcagggatag 3300 cggtcaggtg tttttacaac cactaaaccc acagtaccca atgatcccat caatgagag 3360 ttgttccgtt gtggggaaag ttatcgctag tcagtggcct gaagagacgt ttggctgatc 3420 ggcaaggtgt tctggtcggc catagctga taacaattga caagaatct tcatcgaatt 3480 agggaattt tcactcccct cagaacataa catagtaat ggattgaatt atgaagaatg 3540 gtttttatgc gacttaccgc agcaaaaata aagggaaaga taagcgctca ataaacctgt 3600 ctgttttcct taattctctg ctggctgata atcatcacct gca 3643

Claims

1. A recombinant strain of Escherichia coli, characterized in that, The recombinant E. coli strain expressed using the recombinant plasmid pACYC177-CI-aroF-pheA fbr Introducing E. coli ( Escherichia coli Obtained from JNYPQ strain E. coli JNY021, the Escherichia coli ( Escherichia coli JNYPQ, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 27, 2022, with accession number GDMCC No. 62245, contains the recombinant plasmid pACYC177-CI-aroF-pheA. fbr The CI-aroF-pheA gene fragment was ligated into plasmid pACYC177 to obtain plasmid pACYC177-CI-aroF-pheA, and the plasmid pACYC177-CI-aroF-pheA was then processed. pheA The gene was obtained through site-directed mutagenesis. pheA Site-directed mutation of genes is... pheA The 977th nucleotide of the gene is replaced by a G, and the resulting fragment is pheA. fbr Among them, pACYC177-CI-aroF-pheA fbr CI-aroF-pheA fbr The gene sequence of the gene fragment is shown in SEQ ID NO:

11.

2. The method for constructing a recombinant E. coli bacterium according to claim 1, characterized in that, The construction method involves expressing the recombinant plasmid pACYC177-CI-aroF-pheA. fbr Introducing the *Escherichia coli* as described in claim 1 (…) Escherichia coli Obtained from JNYPQ strain E. coli JNY021, the recombinant plasmid pACYC177-CI-aroF-pheA fbr The CI-aroF-pheA gene fragment was ligated into plasmid pACYC177 to obtain plasmid pACYC177-CI-aroF-pheA, and the plasmid pACYC177-CI-aroF-pheA was then processed. pheA The gene was obtained through site-directed mutagenesis. pheA Site-directed mutation of genes is... pheA The 977th nucleotide of the gene is replaced by a G, and the resulting fragment is pheA. fbr Among them, pACYC177-CI-aroF-pheA fbr CI-aroF-pheA fbr The gene sequence of the gene fragment is shown in SEQ ID NO:

11.

3. The method for constructing a recombinant bacterium of Escherichia coli according to claim 2, characterized by, The recombinant plasmid pACYC177-CI-aroF-pheA fbr was constructed by the following steps: 1) The genes were amplified by PCR using the primer pairs aroF-F / aroF-R and pheA-F / pheA-R with the genome of E. coli K12 as template aroF and pheA the genes 2) synthesis of CI857 gene containing λpR promoter, and aroF the λpL promoter sequence is ligated to the upstream of the pheA gene, and the other end is homologously recombined to the downstream of the aroF gene, to obtain the homologous recombination ligation product CI-aroF-pheA; 3) The homologous recombination ligation product CI-aroF-pheA was digested and ligated into the pACYC177 plasmid to obtain the expression vector pACYC177-CI-aroF-pheA; 4) Using the pACYC177-CI-aroF-pheA expression plasmid as a template, primer pair PheA was used. fbr -UF / PheA fbr -UR and PheA fbr -DF / PheA fbr -DR amplifies the structures containing mutation sites respectively. pheA The upstream and downstream sequences of the gene were joined together via homologous recombination to obtain CI-aroF-pheA. fbr Gene fragment, CI-aroF-pheA fbr The gene fragment was ligated into the pACYC177 plasmid to obtain pACYC177-CI-aroF-pheA fbr The plasmid, the primer pair aroF-F / aroF-R sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2, the primer pair pheA-F / pheA-R sequence is shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the primer pair PheA fbr -UF / PheA fbr -UR sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, and the primer pair PheA fbr -DF / PheA fbr -DR sequences are shown in SEQ ID NO: 7 and SEQ ID NO:

8.

4. A method for producing L-phenylalanine, characterized by, The method involves selecting the recombinant Escherichia coli as described in claim 1. E. coli JNY021 was inoculated into seed culture medium and then transferred to fermentation medium for further fermentation to obtain a fermentation broth containing L-phenylalanine.

5. The L-phenylalanine production method according to claim 4, characterized by, The fermentation culture adopts a fed-batch fermentation method. The parameters of the fed-batch fermentation are set as follows: initial fermentation temperature 33.5±0.5℃, two-stage temperature control, when the cells enter the late logarithmic growth stage, the temperature is increased to 38.5±0.5℃; initial pH value 7.0±0.2℃, the pH is controlled at 6.7-7.0 with 20% ammonia water; initial glucose concentration 20-30 g / L, when the sugar in the fermenter is about to be exhausted, 600 g / L glucose is added at different rates; initial rotation speed and aeration rate are 400 rpm and 1.3 vvm, respectively, and dissolved oxygen is not lower than 20% throughout the fermentation process; fermentation time is 56 h.

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