Recombinant escherichia coli engineering strain for producing levodopa and application

Abstract

The invention discloses a recombinant escherichia coli engineering strain for producing levodopa and application, and belongs to the technical field of genetic engineering and bioengineering. Escherichia coli WSH-Z06 is used as a starting strain, a levodopa synthesis route is constructed, and supply of cofactors is enhanced by introducing glucose dehydrogenase BmgdH; and the catalytic efficiency of the HpaB is greatly improved by modifying the entrance of the HpaB substrate tunnel. In addition, by optimizing the fermentation pH and induction time, the accumulation amount of the levodopa in the fermentation liquor reaches 60.7 g / L, the production intensity reaches 1.26 g / L / h, a foundation is laid for industrial production of the levodopa, and potential value and significance are achieved for development of synthetic biology.

Description

TECHNICAL FIELD

[0001] The present application relates to a recombinant Escherichia coli engineering strain for producing L-dopa and application thereof, and belongs to the technical field of genetic engineering and bioengineering. BACKGROUND

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease in the world, and its incidence is significantly positively correlated with age. Epidemiological data shows that the prevalence rate of people over 65 years old worldwide has reached 1-2%, and the total number of patients is expected to exceed 12 million by 2040. As a key drug for treating PD, L-dopa has been approved since 1970, and the global annual demand has exceeded 250 tons, so there is an urgent need for an efficient and sustainable production method.

[0003] The common production methods of L-dopa on the market at present include chemical synthesis method, plant extraction method, enzyme catalysis method and microbial synthesis method. The chemical synthesis method is complicated, heavily dependent on expensive metal catalysts and will produce toxic by-products in the synthesis process, which will increase potential environmental pollution. The plant extraction method mainly extracts L-dopa from legume plants such as Vicia faba and Thermopsis lanceolata, but the extraction rate is only 9.2%, and it is limited by planting cycle and regional climate, so it is difficult to achieve large-scale supply. The enzyme catalysis method includes tyrosinase (TH) and tyrosine phenol-lyase (TPL), but TH needs to rely on the continuous addition of exogenous reducing agents (such as ascorbic acid) to maintain the stability of the product, and the substrate L-tyrosine (L-tyr) and the product L-dopa are highly similar in structure, which significantly increases the complexity of the downstream separation and purification process; although TPL can efficiently catalyze the synthesis of L-dopa by using catechol, ammonia and pyruvic acid, the raw material toxicity and cost limit the industrial scale production. Compared with the above methods, the microbial synthesis method by introducing 4-hydroxyphenylacetate 3-hydroxylase (HpaBC) to construct a cell factory and directly synthesizing L-dopa from cheap substrates such as glucose shows its dual advantages of environmental protection and economy, so it is necessary to develop a method for producing L-dopa by microbial fermentation and improving the yield of L-dopa. SUMMARY

[0004] The present application provides a 4-hydroxyphenylacetate 3-hydroxylase mutant, which is based on the parent and the threonine at position 292 is mutated to alanine.

[0005] In one embodiment, the amino acid sequence of the parent is shown in SEQ ID NO. 5.

[0006] The present application also provides a gene encoding the 4-hydroxyphenylacetate 3-hydroxylase mutant.

[0007] In one embodiment, the nucleotide sequence encoding the 4-hydroxyphenylacetic acid 3-hydroxylase mutant is shown in SEQ ID NO. 6.

[0008] The present invention also provides a recombinant plasmid carrying the said gene.

[0009] The present invention also provides recombinant microbial cells expressing the 4-hydroxyphenylacetic acid 3-hydroxylase mutant.

[0010] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli.

[0011] This invention also provides an engineered Escherichia coli strain for the efficient synthesis of L-DOPA, which is based on the original strain and includes at least one of the following improvements:

[0012] (1) Expression of the 3-deoxy-D-arabinohepenolate-7-phosphate synthase mutant aroG, which relieves feedback inhibition of aromatic amino acids. fbr Expressing the bifunctional enzyme mutant tyrA, which is a branched acid mutase / prephenyl acid dehydrogenase. fbr This mutant relieves the feedback inhibition of branching acid mutase and prephenyl acid dehydrogenase caused by excessive accumulation of L-tyr; it expresses the endogenous HpaBC gene from Escherichia coli, further converting L-tyr into L-DOPA;

[0013] (2) Knock out the endogenous pykF gene in Escherichia coli to increase the supply of phosphoenolpyruvate (PEP), a precursor of the shikimic acid pathway; and express the endogenous ppsA and tktA genes in Escherichia coli to further guide carbon metabolic flux to the shikimic acid pathway, thereby increasing the metabolic flux of L-DOPA.

[0014] (3) Express glucose dehydrogenase (BmgdH) derived from Bacillus megaterium to increase the supply of FADH2, the cofactor required for HpaBC catalysis;

[0015] (4) Express the HpaB mutant, which accelerates the enzyme’s catalytic efficiency by expanding the tunnel entrance of the substrate L-tyr.

[0016] In one embodiment, the starting strain is based on *Escherichia coli* WSH-Z06, with the genes TyrR (encoding a global transcriptional regulatory protein of the endogenous aromatic amino acid pathway) and PheA (encoding a shikimate mutase / prephenylate dehydratase) knocked out. *E. coli* WSH-Z06 has been published in the paper "Enhanced L-phenylalanine biosynthesis by co-expression of pheA". fbr and aroF wt "middle.

[0017] In an embodiment, the nucleotide sequence of the gene TyrR is as shown in Gene ID: 945879; and the nucleotide sequence of the gene PheA is as shown in Gene ID: 947081.

[0018] In an embodiment, the E. coli engineering bacteria expresses 3-deoxy-D- arabinohexulosonate-7-phosphate synthase aroG fbr and chorismate mutase / prephenate dehydrogenase tyrA fbr .

[0019] In an embodiment, the plasmid pAP-B is a backbone of pACYC177 and contains pPL450, p15A ori and PRPL promoter, which has been disclosed in the paper “Enhanced L-phenylalanine biosynthesis by co-expression of pheA fbr and aroF wt ”.

[0020] In an embodiment, the nucleotide sequence of the gene encoding 3-deoxy-D- arabinohexulosonate-7-phosphate synthase aroG fbr and chorismate mutase / prephenate dehydrogenase tyrA fbr is as shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0021] In an embodiment, the E. coli engineering bacteria expresses the 4-hydroxyphenylacetic acid-3-monooxygenase mutant and glucose dehydrogenase BmgdH using PAP-B as a vector.

[0022] In an embodiment, the nucleotide sequence of the gene encoding 4-hydroxyphenylacetic acid-3-monooxygenase HpaBC is as shown in SEQ ID NO. 3.

[0023] In an embodiment, the nucleotide sequence of the gene pykF is as shown in Gene ID: 946179.

[0024] In an embodiment, the E. coli engineering bacteria expresses phosphoketolase ppsA at the genomic dadx site.

[0025] In an embodiment, the E. coli engineering bacteria expresses transketolase tktA at the genomic ynci site.

[0026] In an embodiment, the nucleotide sequence of the gene ppsA is as shown in Gene ID: 946209; the nucleotide sequence of the gene tktA is as shown in Gene ID: 947420; and the nucleotide sequence of the gene encoding glucose dehydrogenase BmgdH is as shown in SEQ ID NO. 4.

[0027] The present application also provides a method for improving the L-DOPA production capacity of E. coli, which comprises one or more of the following improvements:

[0028] (1) knocking out the coding gene TyrR of the global transcriptional regulator of the endogenous aromatic amino acid pathway of E. coli, and / or the related gene PheA of L-phenylalanine synthesis;

[0029] (2) expressing a mutant of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase, and a mutant of chorismate mutase / prephenate dehydrogenase bifunctional enzyme;

[0030] (3) expressing the gene HpaBC of 4-hydroxyphenylacetate 3-hydroxylase or a mutant of the 4-hydroxyphenylacetate 3-hydroxylase;

[0031] (4) knocking out the endogenous pykF gene of E. coli, and expressing the endogenous ppsA and tktA genes of E. coli;

[0032] (5) expressing glucose dehydrogenase from B. megaterium.

[0033] In an embodiment, the method further comprises fermenting the E. coli engineering strain in a medium with a pH of 6.5±0.1 to the logarithmic phase, and then reducing the pH of the fermentation environment to 6.0±0.1 for continued fermentation.

[0034] The present application also provides a method for preparing L-DOPA by fermentation using the E. coli engineering strain, which comprises fermenting the E. coli engineering strain in a medium at 33-38°C for at least 24 hours.

[0035] In an embodiment, the E. coli engineering strain is fermented in a medium at 33-38°C and 220 rpm for 24-48 hours.

[0036] In an embodiment, the culture medium contains peptone 4 g / L, yeast powder 2 g / L, glucose 25 g / L, sodium citrate 1.5 g / L, vitamin B1 0.075 g / L, ascorbic acid 5 g / L, MgSO4·7H2O 3 g / L, FeSO4·7H2O 0.1125 g / L, CaCl2·2H2O 0.015 g / L, KH2PO4 3 g / L, NaCl 1 g / L, (NH4)2SO4 5 g / L, Al2(SO4)3·18H2O 8 mg / L, CoSO4·7H2O 3 mg / L, CuSO4·5H2O 10 mg / L, H3BO3 2 mg / L, MnSO4·H2O 96 mg / L, NiSO4·6H2O 10 mg / L, ZnSO4·7H2O 60 mg / L, HCl 0.2 ml / L.

[0037] In an embodiment, the method is to ferment the E. coli in a culture medium with pH of 6.5±0.1 to logarithmic phase first, and then to continue the fermentation by lowering the pH in the fermentation environment to 6.0±0.1.

[0038] In an embodiment, the method is to ferment the E. coli in a culture medium with pH of 6.5±0.1 to logarithmic phase first, and then to continue the fermentation by lowering the pH in the fermentation environment to 6.0±0.1 and increasing the temperature to 37-40°C.

[0039] In an embodiment, the fermentation process further comprises feeding.

[0040] In an embodiment, the feeding is to supplement glucose to control the glucose concentration in the fermentation system at 1-1.5 g / L.

[0041] The application provides the use of the E. coli engineering strain in the production of levodopa or products containing levodopa.

[0042] Advantages:

[0043] (1) The application uses E. coli WSH-Z06 as the starting strain, knocks out the tyrR gene, overexpresses the coding gene AroG of 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase fbr , the coding gene TyrA of shikimate mutase and prephenate dehydrogenase fbrThe feedback inhibition of the shikimic acid pathway flux by relieving the excess accumulation of L-tyrosine is eliminated, the diversion of the L-tyrosine pathway by the competing pathway is reduced by knocking out the pheA gene, and the overall metabolic flux of the shikimic acid pathway is increased by knocking out pykF, overexpressing phosphoenolpyruvate synthase ppsA and transketolase tktA, so that the constructed E. coli engineering bacteria accumulate 4.6g / L of levodopa in a shake flask after 48h fermentation.

[0044] (2) The present application enhances the supply of coenzymes by introducing glucose dehydrogenase BmgdH, so that the conversion rate of L-tyrosine is greatly improved, and the catalytic efficiency of the mutant HpaB is greatly improved by modifying the substrate tunnel entrance of HpaB, and the yield of levodopa produced by the strain expressing the mutant in a shake flask fermentation reaches 6.2g / L.

[0045] (3) The present application slows down the oxidation of levodopa by optimizing the pH value of fermentation, and balances the balance between cell growth and product production by optimizing the induction time, and the accumulation of levodopa in the fermentation broth reaches 60.7g / L and the production intensity reaches 1.26g / L / h after 48h induction fermentation of the E. coli engineering strain obtained by the method of the present application, which provides a new idea for industrial production of levodopa. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The metabolic pathway for synthesizing levodopa in E. coli is shown in the figure.

[0047] Figure 2 The L-tyr, L-dopa yield and OD 600 .

[0048] Figure 3 The docking results of HpaB enzyme and L-tyr and FAD are shown in the tunnel diagram.

[0049] Figure 4 The L-tyr, L-dopa yield and OD 600 .

[0050] Figure 5 The fermentation results of wild HpaB and T292A mutant and the liquid chromatography comparison diagram of L-tyr and L-dopa standard are shown in the figure.

[0051] Figure 6 The fermentation process diagram of strain D12 under different optimization fermentation conditions in a 5L fermenter is shown in the figure. DETAILED DESCRIPTION

[0052] (I) Culture medium

[0053] LB medium: Tryptone 10 g / L, Yeast extract 5 g / L, NaCl 10 g / L. Add 20 g / L agar powder to prepare LB solid medium.

[0054] Fermentation medium: Ingredients (g / L) -glucose 20, MgS04-7H20 3, FeS04-7H20 0.1125, Vitamin B1 0.075, CaCl2-2H20 0.015, KH2P04 3, NaCl 1, (NH4)2S04 5, Trisodium citrate 1.5, Tryptone 4, Yeast extract 2, L-Phenylalanine 1, Ascorbic acid 0.45.

[0055] Trace elements: Al2(S04)3-18H20 2.0 g-L -1 , CoS04-7H20 0.75 g-L -1 , CuS04-5H20 2.5 g-L -1 , H3B03 0.5 g-L -1 , MnS04-H20 24 g-L -1 , NiS04-6H20 2.5 g-L -1 , ZnS04-7H20 15 g-L -1 , HCL 50 mL-L -1 .

[0056] (ii) Transformation of E. coli: Thaw the E. coli competent cells on ice; take 10-20 μL of the recombinant product (plasmid 50 ng) and add to 100 μL of the competent cells, mix gently and incubate on ice for 30 min; heat shock in a 42°C water bath for 90 s, and incubate on ice for 3 min; add 1 mL of LB medium, shake at 37°C, 220 rpm for 60 min; centrifuge at 4500 rpm for 2 min, remove the supernatant, resuspend the bacterial cells with the remaining medium, and spread on a resistant plate.

[0057] (III) Detection Methods: L-DOPA and L-tyrosine were determined by HPLC. The specific steps were as follows: L-tyrosine and L-DOPA were quantitatively detected using a C18 column (4.6 mm × 250 mm) via HPLC (Shimadzu LC-20A, Japan). The fermentation sample was diluted with 3 mol / L dilute hydrochloric acid, and the product concentration was controlled within the standard curve range (0.1-1 g / L). The diluted sample was centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm membrane for HPLC analysis. The mobile phase was 0.1 mol / L sodium acetate and pure methanol, with a flow rate of 1 ml / min and a mobile phase ratio of 9:1. The column temperature was set at 30℃ for 10 min, and L-tyrosine and L-DOPA were quantitatively detected at 280 nm.

[0058] (iv) The strain information is shown in Table 1. Among them, Escherichia coli WSH-Z06 is disclosed in "Enhanced L-phenylalanine biosynthesis by co-expression of pheA(fbr) and aroF(wt)".

[0059] Table 1. Strains involved in this invention

[0060]

[0061]

[0062] (v) Primer sequences are shown in Table 2.

[0063] Table 2 Primer sequences

[0064]

[0065]

[0066]

[0067] Example 1: Knockout of TyrR, a global transcriptional regulatory protein gene

[0068] Escherichia coli WSH-Z06 (published in the paper "Enhanced L-phenylalanine biosynthesis by co-expression of pheA") fbr and aroF wt Using strain (as described in the original text) as the starting strain, the pyruvate dehydrogenase gene tyrR (Gene ID: 945879) was knocked out using CRISPR-Cas9-mediated gene editing technology to reduce its feedback inhibition on L-tyr synthesis. The specific steps are as follows:

[0069] (1) Constructing pTarget-tyrR plasmid: Using pTarget plasmid as template, and SG-TYRR-F / SG-TYRR-R as primers, the first 20bp of the PAM site is introduced into plasmid pTarget as a guide signal for sgRNA. The constructed pTarget-tyrR is sent for sequencing verification, and plasmids with correct sequencing are reserved for subsequent operations.

[0070] (2) Construction of tyrR knockout fusion homologous arms: Using E. coli MG1655 as a template, the upstream and downstream homologous arms of the tyrR gene were amplified by PCR using two pairs of primers (UP-TYRR-F / UP-TYRR-R, DN-TYRR-F / DN-TYRR-R). Using the upstream and downstream homologous arms as templates, the two homologous arms were connected by overlap extension PCR using UP-TYRR-F / DN-TYRR-R primers. Agarose gel electrophoresis was then performed for verification, and PCR products with correct band sizes were purified and recovered.

[0071] (3) The pCas plasmid was introduced into Escherichia coli WSH-Z06, and a single clone was transferred into an LB tube containing 0.05 mg / L kanamycin and cultured overnight at 37°C. Then, a 1% (v / v) inoculum was added to a 250 mL Erlenmeyer flask containing 50 mL of LB medium, along with 500 μL of 1 mol / L arabinose. The flask was incubated at 220 rpm at 37°C until the OD600 reached 0.4-0.6, and then another 500 μL of 1 mol / L arabinose was added. The strain was then collected by centrifugation at 5000 rpm at 4°C, washed three times with 10% glycerol, and aliquoted to prepare electrocompetent cells.

[0072] (4) Take 500 ng of the pTarget-tyrR plasmid constructed in step (1) and the tyrR knockout fusion homologous arm constructed in step (2) and add it to 100 μL of electroporation competent cells. Transfer the cells to a pre-cooled 1 mm electroporation cuvette and perform electroporation transformation. Immediately after electroporation, add 1 mL of LB medium, gently pipette and mix, then transfer to a 1.5 mL centrifuge tube and incubate at 220 rpm and 37°C for 1.5-2 h. Then incubate on an LB plate containing 0.05 mg / L kanamycin and streptomycin at 37°C for 16 h. Perform colony PCR using UP-TYRR-F / DN-TYRR-R as primers. If the band size is 1000 bp, it indicates that the tyrR gene knockout was successful.

[0073] (5) Plasmid removal: Verified colonies were transferred to LB medium containing 1 mM rhamnose and 0.05 mg / L kanamycin and cultured overnight to remove plasmid pTarget-tyrR. After overnight culture, the colonies were streaked onto kanamycin-resistant plates and cultured overnight. Once positive colonies had grown, they were streaked onto kanamycin-resistant and streptomycin-resistant plates and marked. Bacteria that could grow on kanamycin-resistant plates but not on streptomycin-resistant plates indicated that plasmid pTarget-tyrR had been removed. The colonies with pTarget-tyrR removed were then transferred to LB medium containing 0.01 mol / L sucrose to remove pCas9 plasmid. After overnight incubation, the bacteria were streaked onto antibiotic-free plates and incubated overnight. Once positive colonies had grown, they were streaked onto antibiotic-free plates, kanamycin-resistant plates, and streptomycin-resistant plates, and marked accordingly. Bacteria that could grow on antibiotic-free plates but not on kanamycin-resistant or streptomycin-resistant plates indicated that plasmids pCas9 and pTarget had been removed. The strain obtained was named D1.

[0074] Example 2: Knockout of shikimate mutase / prephenylate dehydratase PheA

[0075] L-Phenylalanine, as a competing pathway for L-tyrosine, diverts some of the carbon flux of L-tyrosine. Therefore, in this example, the shikimate mutase / prephenylate dehydratase PheA (Gene ID: 947081) was knocked out to prevent its diversion of L-tyrosine. The pTarget-pheA plasmid was constructed according to step (1) of Example 1, and the pheA knockout homologous arm was constructed according to step (2) of Example 1. The pCas and pTarget-PheA plasmids, along with the pheA knockout homologous arm, were introduced into strain D1 constructed in Example 1 according to steps (3) to (5) of Example 1 to construct pheA-knockout positive colonies, followed by plasmid elimination. The resulting strain was named strain D2.

[0076] Example 3: Construction of the L-DOPA Synthetic Pathway

[0077] 3-Deoxy-D-arabinohepenolate-7-phosphate synthase AroG and branched acid mutase / prephenylate dehydrogenase TyrA are key rate-limiting enzymes in the l-tyrosine synthesis pathway. Their expression levels and activities are subject to feedback inhibition by aromatic amino acids. Therefore, the mutant gene AroG, which relieves this feedback inhibition, was selected for introduction. fbr (nucleotide sequence as shown in SEQ ID NO.1) and TyrA fbr(The nucleotide sequence is shown in SEQ ID NO.2), and it uses the method described in the paper "Enhanced L-phenylalanine biosynthesis by co-expression of pheA". fbr and aroF wt The temperature-responsive vector PAP-B from [the original text is missing] was expressed. The specific steps were as follows:

[0078] (1) Using PA-F / PA-R as primers and PAP-B as template, the PAP vector framework fragment was amplified. Using PL-F / PL-R as primers, the temperature-responsive promoter PL (nucleotide sequence shown in SEQ ID NO. 6) fragment was amplified. Using AROG-F / AROG-R as primers, the AroG... fbr The fragment was amplified using TYRA-F / TYRA-R primers to produce TyrA. fbr The fragments were purified using a DNA purification kit, and then ligated using a Gibson assembly kit to obtain the recombinant vector PAP-AroG. fbr -TyrA fbr The recombinant vector was transferred into strain D2 constructed in Example 2 to obtain strain D3.

[0079] (2) Using an inoculation loop, collect bacterial culture from the glycerol tube and streak it on a plate. Incubate overnight at 37°C. Then, pick a single colony and inoculate it into a shake tube containing 2-3 mL of LB medium. Incubate at 37°C and 220 rpm for 12 h. Take 500 μL of the seed culture and inoculate it into a 250 mL Erlenmeyer flask containing 25 mL of shake flask medium. Set up three replicates for each strain. Incubate at 33°C and 220 rpm for 4 h, then raise the temperature to 38°C and continue fermentation for 44 h. After fermentation, take 1 mL of fermentation broth to determine the OD. 600 Take another 1 mL of fermentation broth, centrifuge at 12000 rpm for 2 min, dilute the supernatant 5 times with 3M dilute hydrochloric acid, and analyze by HPLC. The L-tyr yield and OD of strain D3 were also analyzed. 600 See Figure 2 As shown in the figure, the release of anti-feedback inhibition and the knockout of competitive pathways resulted in an L-tyrosine production of 4.25 g / L, effectively increasing the metabolic flux of L-tyrosine.

[0080] Using PA-F2 / PA-R2 as primers, the recombinant vector PAP-AroG constructed above was... fbr -TyrA fbrUsing PL-F2 / PL-R2 as a template, a fragment of the PAP vector framework was amplified. Using PL-F2 / PL-R2 as primers, a fragment of the temperature-responsive promoter PL was amplified (as shown in SEQ ID NO. 7). Using HPABC-F / HPABC-R as primers, a fragment of HPABC was amplified (as shown in SEQ ID NO. 3). The recombinant vector PAP-AroG was constructed according to step (1) of this embodiment. fbr -TyrA fbr -HpaBC. The recombinant vector PAP-AroG... fbr -TyrA fbr -HpaBC was transferred into strain D2 to obtain strain D4. Strain D4 was then subjected to shake-flask fermentation according to step (2) of this embodiment. The yields of L-Dopa and L-tyr of strain D4 and the strain OD... 600 See Figure 2 The yields of L-Dopa and L-tyr in strain D4 were 2.51 g / L and 0.83 g / L, respectively, indicating that the overexpression of HpaBC successfully achieved the transformation from L-tyr to L-dopa.

[0081] Example 4: Modification of the glycolysis and pentose phosphate pathways to increase the metabolic flux of the target pathway.

[0082] Since the key precursors of the shikimic acid pathway are PEP and E4P, this embodiment performed a knockout of the pykF gene (Gene ID: 946179) and integration of ppsA (Gene ID: 946209) at the dadx site (Gene ID: 945754) to further increase the metabolic flux of shikimic acid, thereby reducing the flow of PEP to pyruvate and increasing the supply of precursor PEP. Furthermore, since tktA is a rate-limiting key gene in the pentose phosphate pathway, this embodiment further integrated tktA (Gene ID: 947420) at the ynci site (Gene ID: 945121) to increase the supply of precursor E4P. The specific steps are as follows:

[0083] (1) Construction of strain D5: The pTarget-pykF plasmid was constructed according to step (1) of Example 1. The pykF knockout homologous arm was constructed according to step (2) of Example 1. The pCas and pTarget-pykF plasmids and the pykF knockout homologous arm were introduced into strain D2 constructed in Example 2 according to steps (3) to (5) of Example 1 to construct pykF knockout positive colonies and perform plasmid elimination. The recombinant vector PAP-AroG constructed in Example 3 was transformed into this strain. fbr -TyrA fbr -HpaBC, and name the constructed strain D5.

[0084] (2) Construction of strain D5-0: Strain D5 was cultured overnight in antibiotic-free LB medium to remove plasmid PAP-AroG. fbr -TyrA fbr -HpaBC. After overnight incubation, streak the culture onto antibiotic-free plates and incubate overnight. Once colonies have grown, transfer them to kanamycin-resistant plates and label them. Bacteria that grow on antibiotic-free plates but not on kanamycin-resistant plates indicate plasmid PAP-AroG. fbr -TyrA fbr -HpaBC has been removed, and the strain is named D5-0.

[0085] (3) Construction of strain D6: The pTarget-DADX plasmid was constructed according to step (1) of Example 1. The dadx-integrated ppsA homologous arm was constructed: using Escherichia coli MG1655 as a template, upstream and downstream homologous arms were obtained by PCR amplification using two pairs of primers (UP-DADX-F / UP-DADX-R, DN-DADX-F / DN-DADX-R), and the ppsA gene was obtained by amplification using PPSA-F / PPSA-R. Using the upstream and downstream homologous arms and the synthesized ppsA gene as templates, the upstream and downstream homologous arms and the target gene were connected by overlap extension PCR using UP-DADX-F / DN-DADX-R primers. Afterwards, agarose gel electrophoresis was performed for verification, and the PCR product was purified and recovered for those with correct band size. Following the methods in steps (4) to (5) of Example 1, the pCas and pTarget-DADX plasmids, along with the ppsA integration homologous arm, were introduced into the vector-free strain D5-0 constructed in step (2) of this example to construct positive colonies integrating ppsA, followed by plasmid elimination. The recombinant vector PAP-AroG constructed in Example 3 was then used... fbr -TyrA fbr -HpaBC was transformed into this strain, and the constructed strain was named strain D6. Following the method described in step (2) above, strain D6-0 with the plasmid eliminated was constructed.

[0086] (4) Construction of strain D7: Following the method in step (1) of Example 1, the pTarget-ynci plasmid was constructed. Using *E. coli* MG1655 as a template, upstream and downstream homologous arms were amplified by PCR using two pairs of primers (UP-YNCI-F / UP-YNCI-R, DN-YNCI-F / DN-YNCI-R). The tktA gene was amplified using TKTA-F / TKTA-R. Using the upstream and downstream homologous arms and the synthesized tktA gene as templates, and with UP-YNCI-F / DN-YNCI-R as primers, the upstream and downstream homologous arms and the target gene were connected by overlap extension PCR to obtain the ynci upstream homologous arm-tktA-ynci downstream homologous arm fragment. Agarose gel electrophoresis was then performed for verification, and PCR products with correct band sizes were purified and recovered. Following the method in step (3) of Example 1, the pCas and pTarget-YNCI plasmids and the tktA integration homologous arm were introduced into the vector-free strain D6-0. Positive colonies integrating tktA were constructed following steps (4) to (5) of Example 1, and plasmid elimination was performed. The recombinant vector PAP-AroG constructed in Example 3 was then used. fbr -TyrA fbr -HpaBC was transformed into this strain, and the constructed strain was named strain D7. The plasmid-free strain D7-0 was constructed by referring to the method in step (2) above.

[0087] Using strain D4 constructed in Example 3 as a control, strains D5, D6, and D7 were subjected to shake-flask fermentation verification according to step (2) of Example 3. The results showed that ( Figure 2 The L-Dopa yields of strains D5, D6, and D7 were 3.22 g / L, 3.57 g / L, and 4.63 g / L, respectively, representing increases of 28%, 42%, and 84% compared to the control D4. This demonstrates that enhanced supply of PEP and E4P significantly promotes L-Dopa production.

[0088] Example 5: Enhancement of cofactor supply

[0089] Since the conversion of L-tyrosine to L-DOPA requires the participation of cofactors NADH and FADH2, and the fermentation results of the strain constructed in Example 4 showed significant residual L-tyrosine, this may indicate an insufficient supply of cofactors. Therefore, the glucose dehydrogenase BmGDH (nucleotide sequence shown in SEQ ID NO.4) from Bacillus megaterium was introduced to enhance the supply of cofactors. The specific steps were as follows: using PA-F3 / PA-R3 as primers, and the vector PAP-AroG constructed in Example 3... fbr -TyrA fbr- Using HpaBC as a template, PAP-AroG was amplified. fbr -TyrAfbr- The HpaBC vector framework fragment was amplified using GDH-F / GDH-R primers to produce the BmGDH fragment. The recombinant vector PAP-AroG was then constructed following step (1) of Example 3. fbr -TyrA fbr -HpaBC-GDH. The recombinant vector was transformed into the vector-free strain D7-0 constructed in Example 4 to obtain strain D8. The plasmid-free strain D8-0 was constructed according to step (2) of Example 4.

[0090] The strain D8 was subjected to shake-flask fermentation according to step (2) of Example 3. The results are as follows: Figure 2 The yields of L-Dopa and L-tyr in strain D4 were 5.6 g / L and 0.2 g / L, respectively, and the residual amount of L-tyr decreased by 76% compared with strain D7. This shows that enhanced supply of cofactors can significantly improve the conversion rate of L-tyrosine.

[0091] Example 6: Modification of the HpaB Substrate Tunnel Entrance

[0092] To improve the catalytic efficiency of HpaB, L-tyr, FAD, and HpaB were molecularly docked. Structural analysis showed that the tunnel entrance was relatively narrow. Figure 3 Therefore, its main channel parameters are calculated: bottleneck radius. length curvature The throughput (0.807) was analyzed, and it was found that the tunnel entrance may have restricted the effective entry of L-tyr, thus limiting the enzymatic reaction rate. Therefore, the amino acid residues near the tunnel entrance were mutated to amino acids with smaller side chains to expand the substrate pocket for L-tyr entry. The specific steps were as follows: using 210A-F / 210A-R, 210G-F / 210G-R, 211G-F / 211G-R, 292A-F / 292A-R, 292G-F / 292G-R, 467A-F / 467A-R, 467G-F / 467G-R, 471A-F / 471A-R, 471G-F / 471G-R, and 474A-F / 474A-R as primers, respectively, with the vector PAP-AroG constructed in Example 5. fbr -TyrA fbr-Using HpaBC-GDH as a template, a recombinant plasmid with the introduced mutation was circularly amplified. 1-2 μL of the PCR product was transformed into competent *E. coli* JM109 cells, and subsequent sequencing verified the presence of the mutated vector. The mutated recombinant vector was then transformed into the vector-free strain D8-0 constructed in Example 5, yielding strains D9-D19. Strains D9-D19 were subjected to shake-flask fermentation according to step (2) of Example 3. The results are as follows... Figure 4 As shown, the strain with the T292A mutation showed the most significant increase in yield, with a yield of 6.2 g / L of levodopa and only 25 mg / L of L-tyrosine residue.

[0093] Example 7: Fermentation production of L-DOPA in a 5L fermenter

[0094] The L-DOPA production capacity of strain D12 constructed in Example 6 was verified in a 5-L fermenter. Single colonies were picked from plates and inoculated into 2 mL of LB broth, incubated at 37°C for 12 h to prepare a primary seed culture. A secondary seed culture was then prepared by inoculating 50 mL of LB broth at a 2% v / v inoculation rate and incubating at 37°C for 10–12 h. This secondary seed culture was then inoculated into a 5L fermenter containing 2.5 L of LB broth for pilot-scale fed-batch fermentation. At the end of inoculation, the temperature was maintained at 33°C, dissolved oxygen at 20%, and the agitator speed between 300–1000 rpm. The pH of the fermentation broth was maintained at 6.4–6.6 using ammonia, and the gas flow rate was maintained at 4 L / min. OD... 600 When the temperature rises to 20°C, the temperature is increased to 38°C for induction, while the pH is controlled at 6.4–6.6. Throughout the fermentation process, a high-concentration glucose solution of 700 g / L is continuously fed to maintain the glucose concentration in the fermentation system at 1–1.5 g / L. After 48 hours of fermentation, the L-DOPA titer reaches 46.8 g / L. Figure 6 A).

[0095] Example 8: Optimization of conditions for the production of L-DOPA by fermentation in a 5L fermenter

[0096] (I) Staged pH Regulation

[0097] Since the fermentation broth gradually turned black during the cultivation process in Example 7, it was speculated that this was caused by the oxidative degradation of L-DOPA, which may reduce the product yield. To reduce oxidative losses, a staged pH control strategy was adopted: the pH was maintained at 6.5 before induction to maximize biomass accumulation, and after induction, the pH was adjusted to 6.0 or 5.5 respectively to slow down product oxidation. Other fermentation parameters were the same as in Example 7. The results are as follows: Figure 6As shown in Figure C, oxidation was reduced when the pH was lowered to 6.0, and the L-DOPA accumulation rate significantly increased during fermentation from 18 to 30 hours, resulting in a final yield of 52.13 g / L. However, at pH 5.5, cell growth and metabolic activity were severely impaired, leading to a significantly reduced yield of only 32.59 g / L. Figure 6 B).

[0098] (II) Optimizing the timing of temperature induction

[0099] Fermentation was carried out according to the method of Example 7, except that a staged pH control strategy was adopted. The pH was maintained at 6.5 before induction and then adjusted to 6.0 after induction. Temperature induction was carried out under conditions of OD600 of 20, 30, and 40, respectively, to determine the optimal balance point between the growth and production stages. Figure 6 C, 6E, and 6D correspond to OD respectively. 600 The effects of induction at temperatures of 20, 30, and 40°C, respectively, followed by heating to 38°C, were observed. The results showed that OD... 600 The optimal induction time was 30 hours: the substrate conversion rate increased rapidly after induction, and the levodopa yield reached a peak of 60.7 g / L at 48 hours, with a production intensity of 1.26 g / L / h. These results indicate that appropriately delayed induction can improve biosynthetic efficiency by coordinating resource allocation between cell growth and product synthesis.

[0100] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1,4-Hydroxyphenylacetic acid 3-hydroxylase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.5, the threonine at position 292 was mutated to alanine.

2. A biomaterial, characterized in that, Including at least one of (a) to (c): (a) The gene encoding the 4-hydroxyphenylacetic acid 3-hydroxylase mutant of claim 1; (b) A recombinant plasmid carrying the gene described in (a); (c) Recombinant microbial cells expressing the 4-hydroxyphenylacetic acid 3-hydroxylase mutant of claim 1.

3. An engineered Escherichia coli strain for synthesizing L-DOPA, characterized in that, Based on the original strain, at least one of the following improvements were made: (1) Expression of the 3-deoxy-D-arabinohepenolate-7-phosphate synthase mutant aroG fbr Expressing the bifunctional enzyme mutant tyrA, which is a branched acid mutase / prephenyl acid dehydrogenase. fbr And express the HpaBC gene or the 4-hydroxyphenylacetic acid 3-hydroxylase mutant as described in claim 1; (2) Knock out the endogenous pykF gene in Escherichia coli and express the endogenous ppsA and tktA genes in Escherichia coli; (3) Express glucose dehydrogenase derived from Bacillus megaterium.

4. The engineered Escherichia coli strain according to claim 3, characterized in that, The starting strain was a strain based on Escherichia coli WSH-Z06 with the TyrR gene and the PheA gene of shikimate mutase / prebenzoic acid dehydratase knocked out.

5. The engineered Escherichia coli strain according to claim 3 or 4, characterized in that, Using PAP-B, derived from the promoters pACYC177, pPL450, p15A_ori, and PRPL, as a vector, 3-deoxy-D-arabinohepenolate-7-phosphate synthase aroG was expressed. fbr tyrA, a branched acid mutase / prebenzoic acid dehydrogenase fbr 4-hydroxyphenylacetic acid-3-monooxygenase mutant and glucose dehydrogenase BmgdH.

6. The engineered Escherichia coli according to any one of claims 3 to 5, characterized in that, Phosphoenolpyruvate synthase ppsA is integrated and expressed at the dadx site of the genome, and / or transketolase tktA is integrated and expressed at the ynci site of the genome.

7. A method for improving the production capacity of L-DOPA in Escherichia coli, characterized in that, One or more improvements were made to Escherichia coli, such as those in (1) to (5): (1) The gene TyrR, which encodes a global transcriptional regulatory protein of the endogenous aromatic amino acid pathway in Escherichia coli, and / or the gene PheA, which is related to the synthesis of L-phenylalanine, was knocked out. (2) Mutants expressing 3-deoxy-D-arabinohepenolate-7-phosphate synthase; and mutants of branched acid mutase / prephenyl acid dehydrogenase bifunctional enzymes; (3) Express the 4-hydroxyphenylacetic acid 3-hydroxylase gene HpaBC or the 4-hydroxyphenylacetic acid 3-hydroxylase mutant; (4) Knock out the endogenous pykF gene in E. coli and express the endogenous ppsA and tktA genes in E. coli; (5) Expressing glucose dehydrogenase derived from Bacillus megaterium; and / or The engineered Escherichia coli was fermented in a medium with a pH of 6.5 ± 0.1 to the logarithmic phase, and the pH of the fermentation environment was lowered to 6.0 ± 0.1 to continue fermentation.

8. A method for preparing L-DOPA by fermentation, characterized in that, The engineered Escherichia coli strain according to any one of claims 3 to 7 is fermented in a culture medium at 33°C to 38°C for at least 24 hours.

9. The method according to claim 8, characterized in that, The *Escherichia coli* was first fermented in a culture medium with a pH of 6.5 ± 0.1 at 32–34 °C to the logarithmic phase. Then, the pH of the fermentation environment was lowered to 6.0 ± 0.1, and the temperature was raised to 37–40 °C to continue fermentation.

10. The use of the engineered Escherichia coli strain according to any one of claims 3 to 7 in the production of levodopa or products containing levodopa.

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