Engineering strain for producing (S)-higenamine, method and application

Abstract

The invention relates to an engineering strain for producing (S)-higenamine, a method and application. The engineering strain is a recombinant pichia stipitis engineering strain containing coding genes of exogenous tyrosine hydroxylase mutants TyrHW13L and F309L, a dopa decarboxylase DODC and a higenamine synthase NCS. The recombinant pichia stipitis engineering strain is a recombinant pichia stipitis engineering strain containing coding genes of the exogenous tyrosine hydroxylase mutants TyrHW13L and F309L. According to the engineering strain, (S)-higenamine can be synthesized through a whole-cell catalytic reaction one-step method, exogenous coenzyme and exogenous reducing power are not needed, and the endogenous reducing power of the strain is utilized; the reaction path is short, products can be quickly accumulated in a short time, and the catalytic efficiency is high; the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to an engineered strain, a method and an application for producing (S)-demethylcoclaurine. Background Art

[0002] (S)-Demethylcoclaurine is an alkaloid with important pharmacological activities. Traditional production methods rely on plant extraction or chemical synthesis. The plant extraction method is limited by unstable raw material supply and high separation and purification costs; the chemical synthesis method is mainly based on the Pictet-Spengler reaction, which is a cyclization condensation reaction of β-arylethylamine with aldehyde or ketone under acid-catalyzed conditions. Although there are currently enantioselective catalytic synthesis methods, large-scale preparation requires the use of a large amount of organic solvents and metal catalysts, which is not conducive to the recovery of solvents and metals and limits its industrial application. In related technologies, there is little research on the microbial synthesis of (S)-demethylcoclaurine, and there are problems such as complex reaction steps and dependence on exogenous coenzyme addition. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems in the related technologies to some extent. For this reason, an object of the present invention is to provide an engineered strain, a method and an application for producing (S)-demethylcoclaurine, to solve the problems of low production efficiency, complex process and high cost of (S)-demethylcoclaurine in the prior art, and to provide a production method of (S)-demethylcoclaurine that does not require exogenous coenzyme, has a short reaction path and a high yield.

[0004] For this reason, in one aspect of the present invention, the present invention provides an engineered strain for producing (S)-demethylcoclaurine, and the engineered strain is a recombinant Pichia stipitis engineering bacterium containing the coding genes of exogenous tyrosine hydroxylase mutant TyrH W13L，F309L , dopa decarboxylase DODC and norcoclaurine synthase NCS.

[0005] According to an engineered strain for producing (S)-demethylcoclaurine of the present invention, the engineered strain can synthesize (S)-demethylcoclaurine by a one-step whole-cell catalytic reaction, without exogenous addition of coenzyme and exogenous addition of reducing power, and utilizes the endogenous reducing power in the bacteria; the reaction path is short, the product can be rapidly accumulated in a short time, and the catalytic efficiency is high; the production cost is reduced by more than 50% compared with the traditional method.

[0006] In addition, according to the engineered strain for producing (S)-demethylcoclaurine proposed in the above embodiments of the present invention, the following additional technical features may also be provided:

[0007] Optionally, using the genome of Pichia stipitis as a template, and using OLE1p-F shown in SEQ ID NO:9 and OLE1p-R shown in SEQ ID NO:10 as primers, GLN1t-F shown in SEQ ID NO:11 and GLN1t-R shown in SEQ ID NO:12 as primers, sGLN1p-F shown in SEQ ID NO:13 and sGLN1p-R shown in SEQ ID NO:14 as primers, sTDH2t-F shown in SEQ ID NO:15 and sTDH2t-R shown in SEQ ID NO:16 as primers, sPGK1p-F shown in SEQ ID NO:17 and sPGK1p-R shown in SEQ ID NO:18 as primers, sOLE1t-F shown in SEQ ID NO:19 and sOLE1t-R shown in SEQ ID NO:20 as primers, perform PCR amplification on OLE1p, GLN1t, sGLN1p, sTDH2t, sPGK1p, sOLE1t fragments;

[0008] Using the TyrH directed synthesis gene as a template, and using TyrH-F shown in SEQ ID NO:21 and TyrH-R shown in SEQ ID NO:22 as primers, perform PCR amplification on the TyrH W13L，F309L gene; using the DODC directed synthesis gene as a template, and using DODC-F shown in SEQ ID NO:23 and DODC-R shown in SEQ ID NO:24 as primers, perform PCR amplification on the DODC gene; using the NCS directed synthesis gene as a template, and using NCS-F shown in SEQ ID NO:25 and NCS-R shown in SEQ ID NO:26 as primers, perform PCR amplification on the NCS gene;

[0009] Obtain the OLE1p-TyrH W13L,F309L -GLN1t, sGLN1p-DODC-sTDH2t and sPGK1p-NCS-sOLE1t fragments by overlap PCR;

[0010] Connect the said OLE1p-TyrH W13L,F309L -GLN1t, sGLN1p-DODC-sTDH2t and sPGK1p-NCS-sOLE1t fragments to obtain the TDN fragment by overlap PCR;

[0011] Connect the said TDN fragment with the plasmid pRS414CEN-750 to obtain the plasmid pRS414CEN750-TDN;

[0012] The plasmid pRS414CEN750-TDN was transferred into the competent cells of Pichia stipitis to obtain the engineered strain for producing (S)-demethylcoclaurine.

[0013] Furthermore, using the Pichia stipitis genome as a template, with ARS2-F shown in SEQ ID NO:1 and ARS2-R shown in SEQ ID NO:2 as primers, URA-F shown in SEQ ID NO:3 and URA-R shown in SEQ ID NO:4 as primers, and CEN-F shown in SEQ ID NO:5 and CEN-R shown in SEQ ID NO:6 as primers, the ARS, Ura, and CEN-750 genes were amplified by PCR.

[0014] The ARS, Ura, and CEN-750 genes were ligated by overlap PCR to obtain an overlap fragment.

[0015] The overlap fragment was ligated with the PRS414 vector to obtain the plasmid pRS414CEN-750.

[0016] Optionally, the nucleotide sequence of the coding gene of the tyrosine hydroxylase mutant TyrH W13L，F309L is shown in SEQ ID NO:27; the nucleotide sequence of the coding gene of the dopa decarboxylase DODC is shown in SEQ ID NO:28; the nucleotide sequence of the coding gene of the norcoclaurine synthase NCS is shown in SEQ ID NO:29.

[0017] In the second aspect of the present invention, the present invention provides a method for producing (S)-demethylcoclaurine, comprising:

[0018] Using the above-mentioned engineered strain as a whole-cell catalyst, with L-tyrosine as a substrate, a whole-cell catalytic reaction was carried out to obtain (S)-demethylcoclaurine.

[0019] According to a method for producing (S)-demethylcoclaurine of the present invention, this method uses L-tyrosine as a substrate and the constructed recombinant Pichia stipitis engineering bacteria as a whole-cell catalyst to achieve the whole-cell catalytic production of (S)-demethylcoclaurine; the outstanding advantage of this method is that it does not require coenzymes and exogenous addition of reducing power, thereby simplifying the production steps and reducing the cost; at the same time, the reaction path is short, and the product can be rapidly accumulated in a short time; the cost is low and the process is simple.

[0020] Optionally, the pH of the whole-cell catalytic reaction is 5.9 - 7.3; the reaction temperature is 25 - 35 °C; the reaction rotation speed is 200 - 300 rpm; the addition amount of the whole-cell catalyst concentration is OD 600is 20 - 40; the concentration of the substrate L - tyrosine is 1 - 4 g / L; the concentration of the buffer solution is 0.05 - 0.2 mol / L.

[0021] In the third aspect of the present invention, the present invention provides the application of the above - mentioned engineered strain in the preparation of (S) - demethylcoclaurine.

[0022] The additional aspects and advantages of the present invention will be partly given in the following description, partly become obvious from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0023] Figure 1 is the plasmid map of the recombinant Pichia stipitis strain TDN according to the embodiment of the present invention;

[0024] Figure 2 is the schematic flow chart of whole - cell catalysis according to the embodiment of the present invention;

[0025] Figure 3 is the result of the effect of different pH values on the synthesis of (S) - demethylcoclaurine by whole - cell catalysis of L - tyrosine according to the embodiment of the present invention;

[0026] Figure 4 is the result of the effect of different temperatures on the synthesis of (S) - demethylcoclaurine by whole - cell catalysis of L - tyrosine according to the embodiment of the present invention;

[0027] Figure 5 is the result of the effect of different rotation speeds on the synthesis of (S) - demethylcoclaurine by whole - cell catalysis of L - tyrosine according to the embodiment of the present invention;

[0028] Figure 6 is the result of the effect of different whole - cell catalyst concentrations on the synthesis of (S) - demethylcoclaurine by whole - cell catalysis of L - tyrosine according to the embodiment of the present invention;

[0029] Figure 7 is the result of the effect of different L - tyrosine concentrations on the synthesis of (S) - demethylcoclaurine by whole - cell catalysis according to the embodiment of the present invention;

[0030] Figure 8 is the result of the effect of different buffer solution concentrations on the synthesis of (S) - demethylcoclaurine by whole - cell catalysis according to the embodiment of the present invention. Detailed Embodiments

[0031] The technical solution of the present invention will be described below through specific specific examples. It should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combined steps or that other method steps can be inserted between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise specified, the numbers of the method steps are only convenient tools for identifying the method steps, rather than limiting the arrangement order of the method steps or the scope in which the present invention can be implemented. The change or adjustment of their relative relationship, without substantial change in the technical content, should also be regarded as the scope in which the present invention can be implemented.

[0032] To better understand the above technical solution, the exemplary embodiments of the present invention will be described in more detail below. Although the exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0033] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market; the experiments involved are all conventional experimental methods unless otherwise specified.

[0034] Pichia stipitis is disclosed in the following literature:

[0035] [1] Lu P, Davis BP, Hendrick J, Jeffries TW. Cloning and disruption of the beta-isopropylmalate dehydrogenase gene (LEU2) of Pichia stipitis with URA3 and recovery of the double auxotroph. Appl Microbiol Biotechnol. 1998, 49: 141-146.

[0036] [2] Cao M, Seetharam AS, Severin AJ, Shao Z. Rapid Isolation of Centromeres from Scheffersomyces stipitis. ACS Synth Biol. 2017, 6(11): 2028-2034.

[0037] The composition of the SC-URA liquid medium is: 20 g / L glucose, 8 g / L SC-URA (containing yeast nitrogen base YNB, ammonium sulfate, various amino acids and nucleotide molecules).

[0038] The SC-URA medium is as follows: purchased from Xiamen Lambolide Biotechnology Co., Ltd., with the product number Y408102.

[0039] In the following examples, the nucleotide sequences of the primers are shown in Table 1.

[0040]

[0041]

[0042] The present invention will be described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way.

[0043] Example 1 Construction of a recombinant Pichia stipitis strain

[0044] The pRS414 yeast general vector was used as the expression plasmid for modification. The genomic DNA of Pichia stipitis was extracted (extracted according to the instructions of the TIANamp Yeast DNA Kit (TIANGEN-DP307) yeast genomic DNA extraction kit) as the template. The target gene ARS was obtained by PCR amplification using ARS2-F shown in SEQ ID NO:1 and ARS2-R shown in SEQ ID NO:2 as primers; the key gene Ura for uracil synthesis was obtained by PCR amplification using URA-F shown in SEQ ID NO:3 and URA-R shown in SEQ ID NO:4 as primers; CEN-750 was obtained by PCR amplification using CEN-F shown in SEQ ID NO:5 and CEN-R shown in SEQ ID NO:6 as primers. The overlap fragment was obtained by overlap PCR ligation.

[0045] The overlap fragment and the PRS414 vector were double-digested with the restriction enzymes XhoⅠ and SacⅠ. After purification, they were ligated overnight using the DNA Ligation Kit Ver.2.1 ligase from Takara, and the ligation product was transformed into E. coli DH5α competent cells (refer to the usage method of Shanghai Sangon Biotech's DH5α competent cells (B528413)). The plasmid was extracted from the single colonies grown on the LB ampicillin-resistant plate, and the plasmid was verified by sequencing using the primers F1 / R1 shown in SEQ ID NO:7 and SEQ ID NO:8. The sequencing results were correctly aligned, indicating that the plasmid pRS414CEN-750 with uracil screening function was successfully constructed.

[0046] Based on plasmid pRS414CEN-750, the restriction endonucleases XhoⅠ and ClaⅠ were selected for double digestion to obtain a linearized vector. Using the genome of Pichia stipitis as a template, with OLE1p-F shown in SEQ ID NO:9 and OLE1p-R shown in SEQ ID NO:10 as primers, GLN1t-F shown in SEQ ID NO:11 and GLN1t-R shown in SEQ ID NO:12 as primers, sGLN1p-F shown in SEQ ID NO:13 and sGLN1p-R shown in SEQ ID NO:14 as primers, sTDH2t-F shown in SEQ ID NO:15 and sTDH2t-R shown in SEQ ID NO:16 as primers, sPGK1p-F shown in SEQ ID NO:17 and sPGK1p-R shown in SEQ ID NO:18 as primers, sOLE1t-F shown in SEQ ID NO:19 and sOLE1t-R shown in SEQ ID NO:20 as primers, the fragments of OLE1p, GLN1t, sGLN1p, sTDH2t, sPGK1p, and sOLE1t were amplified by PCR. Using the TyrH gene synthesized by directed synthesis as a template, with TyrH-F shown in SEQ ID NO:21 and TyrH-R shown in SEQ ID NO:22 as primers, the TyrH gene was amplified by PCR. W13L，F309L Heterologous synthase gene (such as that shown in SEQ ID NO:27); using the DODC gene synthesized by directed synthesis as a template, with DODC-F shown in SEQ ID NO:23 and DODC-R shown in SEQ ID NO:24 as primers, the heterologous synthase gene of DODC (such as that shown in SEQ ID NO:28) was amplified by PCR; using the NCS gene synthesized by directed synthesis as a template, with NCS-F shown in SEQ ID NO:25 and NCS-R shown in SEQ ID NO:26 as primers, the heterologous synthase gene of NCS (such as that shown in SEQ ID NO:29) was amplified by PCR. Further, OLE1p-TyrH W13L,F309L -GLN1t, sGLN1p-DODC-sTDH2t, and sPGK1p-NCS-sOLE1t fragments were obtained by overlap PCR. The above three fragments were again subjected to overlap PCR to obtain the TDN fragment. Finally, after Gibson assembly of the linearized vector and the TDN fragment and transformation into competent E. coli DH5α cells, plasmids were extracted from the single colonies grown on the LB ampicillin-resistant plate, and the plasmids were sequenced and verified using the primers F1 / R2 shown in SEQ ID NO:7 and SEQ ID NO:30. The correct results indicated the successful construction of plasmid pRS414CEN750-TDN carrying the (S)-demethylcoclaurine synthesis pathway (the plasmid map is as Figure 1As shown in the figure, the plasmid pRS414CEN750-TDN was transferred into the competent cells of Pichia stipitis by cell electroporation, and screened through the SC-URA uracil auxotrophic function to obtain the strain TDN of the (S)-demethylcoclaurine synthesis pathway, that is, the recombinant Pichia stipitis strain.

[0047] The high-fidelity enzyme used in the above PCR amplification was 2×Phanta Master Mix (Vazyme-P510), and the reaction system was as shown in Table 2, and the reaction program was shown in Table 3.

[0048] Table 2 PCR reaction system

[0049]

[0050]

[0051] Table 3 PCR amplification program of 2×Phanta Master Mix (Vazyme-P510) high-fidelity enzyme

[0052]

[0053] Since multiple PCR fragments are involved in the construction of the pathway, when necessary, Overlap extension PCR is used to amplify multiple fragments into one fragment. The experimental conditions of Overlap extension PCR are as follows:

[0054] Table 4 1st PCR reaction system

[0055]

[0056] Note: The addition amount of each fragment needs to ensure the same molar concentration of the fragment.

[0057] Table 5 1st PCR reaction program

[0058]

[0059] The overlap fragment obtained from the 1st PCR reaction was used as a template for large-scale amplification.

[0060] Table 6 2nd PCR reaction system

[0061]

[0062] Table 7 2nd PCR reaction program

[0063]

[0064] Example 2

[0065] Obtaining the whole-cell catalyst: Streak the recombinant Pichia stipitis TDN (obtained in Example 1) on the SC-URA screening plate, activate it in the SC-URA liquid medium, transfer it into 200 mL of the SC-URA medium, and continuously shake-culture at 30 °C and 250 rpm until the OD 600 is close to 30. Centrifuge the cells at 4 °C and 8000 rpm for 10 min to collect the cells. Wash the cells twice with 0.1 mol / L potassium phosphate buffer (pH 7.0). After centrifuging to collect the cells, use them as the whole-cell catalyst.

[0066] Relationship between the yields of (S)-demethylcoclaurine catalyzed by the whole-cell catalyst under different whole-cell catalytic conditions: Using the above whole-cell catalyst, (S)-demethylcoclaurine is synthesized in one step using L-tyrosine as the substrate (as Figure 2 shown); the pH of the catalytic reaction is 5.9 - 7.3; the reaction temperature is 25 - 35 °C; the reaction rotation speed is 200 - 300 rpm; the added amount of the whole-cell catalyst concentration OD 600 is 20 - 40; the concentration of the substrate L-tyrosine is 1 - 4 g / L; the buffer concentration is 0.05 - 0.2 mol / L. Appropriately extract 100 μL of the reaction solution at an appropriate time, add 400 μL of methanol and 400 μL of acetonitrile, mix well, vortex for 30 s, and ultrasonically treat in an ice bath for 10 min. After incubating at -20 °C for 1 h, centrifuge at 13000 rpm and 4 °C for 15 min. Take 800 μL of the supernatant, concentrate and dry it by low-temperature centrifugation, and store it at -80 °C or directly measure the sample for liquid chromatography-tandem mass spectrometry analysis and detection. Liquid chromatography-tandem mass spectrometry detection method: Use Waters Acquity I-class UPLC tandem AB SCIEX QTRAP6500 + . Chromatographic column: BEH C18 (2.1×100 mm, 1.7 μm). The procedure is as follows: Flow rate: 0.3 mL / min; Mobile phase A: Aqueous solution containing 0.1% formic acid; Mobile phase B: Acetonitrile containing 0.1% formic acid; Column temperature: 40 °C; Injection volume: 5 μL; Gradient elution: Keep 95% A for the first 6 min, drop to 10% A at 7.5 min, rise to 95% A at 7.6 min, and keep it until 10 min; Analysis time is 10 min; Electrospray positive ion mode: Fragmentation voltage: 130 V, Collision energy: 33 V, Mass-to-charge ratio (m / z): 272.1; Fragment: 107.1.

[0067] As Figure 3As shown in the figure, the effects of potassium phosphate buffers with pH values of 5.9, 6.3, and 7.3 on the synthesis of (S)-demethylcoclaurine by whole-cell catalysis of L-tyrosine were investigated respectively. The yield of (S)-demethylcoclaurine decreased with the increase of pH. When the pH was 5.9, the yield was the highest, indicating that in the whole-cell catalysis system of recombinant Pichia stipitis, a slightly acidic environment was beneficial to the catalysis of L-tyrosine to synthesize (S)-demethylcoclaurine. After 20 h of the catalytic reaction, the highest yield of (S)-demethylcoclaurine reached 4.36 μg / L, and the yield also decreased with the increase of time. At this time, the drastic change of the catalytic system due to the extension of the reaction time indicated that the stability of the catalytic system had a great impact on the enzyme activity and the effective accumulation of (S)-demethylcoclaurine. Therefore, the potassium phosphate buffer with pH 5.9 was determined as the reaction buffer.

[0068] As Figure 4 shown, the effects of reaction conditions at temperatures of 25 °C, 30 °C, and 35 °C on the synthesis of (S)-demethylcoclaurine by whole-cell catalysis of L-tyrosine were investigated respectively. When the temperature was 30 °C, the whole-cell catalysis showed relatively high activity. Therefore, 30 °C was selected as the catalytic temperature for the whole-cell catalysis of recombinant Pichia stipitis to synthesize (S)-demethylcoclaurine from L-tyrosine.

[0069] As Figure 5 shown, the effects of different rotation speeds (200 rpm, 250 rpm, and 300 rpm) on the synthesis of (S)-demethylcoclaurine by whole-cell catalysis of L-tyrosine were investigated respectively. When the rotation speed was 250 rpm, the whole-cell catalysis showed relatively high activity. Therefore, 250 rpm was selected as the catalytic reaction rotation speed for the whole-cell catalysis of L-tyrosine to synthesize (S)-demethylcoclaurine.

[0070] As Figure 6 shown, the effects of different addition amounts of whole-cell catalysts (OD 600 of 20, 30, and 40) on the synthesis of (S)-demethylcoclaurine by whole-cell catalysis of L-tyrosine were investigated respectively. When the addition concentration of the whole-cell catalyst OD 600 was 30, the enzyme catalytic reaction rate was relatively fast. Therefore, the whole-cell catalyst concentration with OD 600 of 30 was selected as the addition amount of the whole-cell catalyst for the whole-cell catalysis of L-tyrosine to synthesize (S)-demethylcoclaurine.

[0071] As Figure 7As shown, the effects of different L-tyrosine substrate concentrations (1 g / L, 2 g / L, and 4 g / L) on the whole-cell catalyzed synthesis of (S)-demethylcoclaurine from L-tyrosine were investigated. When the L-tyrosine concentration was 2 g / L, the whole-cell catalysis of recombinant Pichia stipitis showed good substrate utilization. Therefore, an L-tyrosine concentration of 2 g / L was selected as the substrate concentration for the whole-cell catalyzed synthesis of (S)-demethylcoclaurine from L-tyrosine.

[0072] As Figure 8 shown, the effects of different potassium phosphate buffer concentrations (0.05 mol / L, 0.1 mol / L, and 0.2 mol / L) on the whole-cell catalyzed synthesis of (S)-demethylcoclaurine from L-tyrosine were investigated. When the potassium phosphate buffer concentration increased from 0.05 mol / L to 0.2 mol / L, the whole-cell catalysis rate accelerated, and the highest yield of (S)-demethylcoclaurine reached 4.78 μg / L at 30 h. This indicates that the higher the buffer concentration, the better the effect of maintaining stability in the whole-cell catalysis system, and the corresponding synthetic product (S)-demethylcoclaurine can be continuously and effectively accumulated. However, too high a buffer concentration may lead to too high an osmotic pressure, forcing the bacterial cells to rupture. Therefore, a potassium phosphate buffer concentration of 0.2 mol / L was selected as the buffer concentration for the whole-cell catalyzed synthesis of (S)-demethylcoclaurine from L-tyrosine.

[0073] In summary, when the optimized catalytic reaction pH was 5.9; the reaction temperature was 30 °C; the reaction rotation speed was 250 rpm; the addition amount of the whole-cell catalyst concentration OD 600 was 30; the substrate L-tyrosine concentration was 2 g / L; and the buffer concentration was 0.2 mol / L. Through whole-cell catalysis, the catalytic yield of (S)-demethylcoclaurine was 4.2 - 5.6 μg / L.

[0074] Thus, according to the embodiments of the present invention, a recombinant Pichia stipitis engineering bacterium containing the encoding genes of exogenous tyrosine hydroxylase mutant TyrH W13L，F309L , dopa decarboxylase DODC, and norcoclaurine synthase NCS was constructed; using L-tyrosine as the substrate, the constructed recombinant Pichia stipitis engineering bacterium was used as a whole-cell catalyst to further realize the whole-cell catalytic production of (S)-demethylcoclaurine; the prominent advantage of this method is that it does not require coenzymes and exogenous addition of reducing power, thereby simplifying the production steps and reducing the cost; at the same time, the reaction path is short, and the product can be rapidly accumulated in a short time; the cost is low, and the process is simple.

[0075] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc., mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic descriptions of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification.

[0076] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An engineered strain for producing (S)-demethylcoclaurine, characterized in that, The engineered strain contains an exogenous tyrosine hydroxylase mutant TyrH W13L，F309L , DOPA decarboxylase DODC and higenamine synthase NCS encoding genes of recombinant Pichia stipitis engineering bacteria.

2. The engineered strain for producing (S)-demethylcoclaurine according to claim 1, characterized in that, Using the Pichia stipitis genome as a template, PCR amplified OLE1p, GLN1t, sGLN1p, sTDH2t, sPGK1p, and sOLE1t fragments using OLE1p-F shown in SEQ ID NO:9 and OLE1p-R shown in SEQ ID NO:10, GLN1t-F shown in SEQ ID NO:11 and GLN1t-R shown in SEQ ID NO:12, sGLN1p-F shown in SEQ ID NO:13 and sGLN1p-R shown in SEQ ID NO:14, sTDH2t-F shown in SEQ ID NO:15 and sTDH2t-R shown in SEQ ID NO:16, sPGK1p-F shown in SEQ ID NO:17 and sPGK1p-R shown in SEQ ID NO:18, and sOLE1t-F shown in SEQ ID NO:19 and sOLE1t-R shown in SEQ ID NO:

20. Using the TyrH directed synthesis gene as a template, and using TyrH-F shown in SEQ ID NO:21 and TyrH-R shown in SEQ ID NO:22 as primers, PCR amplify the TyrH gene; W13L，F309L Using the DODC directed synthesis gene as a template, and using DODC-F shown in SEQ ID NO:23 and DODC-R shown in SEQ ID NO:24 as primers, PCR amplify the DODC gene; The NCS gene was amplified by PCR using the NCS directed synthetic gene as a template and NCS-F shown in SEQ ID NO: 25 and NCS-R shown in SEQ ID NO: 26 as primers; Obtaining OLE1p-TyrH by overlap PCR W13L,F309L -GLN1t, sGLN1p-DODC-sTDH2t and sPGK 1p-NCS-sOLE1t fragments; The OLE1p-TyrH W13L,F309L -GLN1t, sGLN1p-DODC-sTDH2t and sPGK1p-NCS-sOLE1t fragments were used to obtain the TDN fragment by overlap PCR; The TDN fragment was ligated with plasmid pRS414CEN-750 to obtain plasmid pRS414CEN750-TDN; The plasmid pRS414CEN750-TDN is transformed into Pichia stipitis competent cells to obtain the engineered strain producing (S)-higenamine.

3. The engineered strain for producing (S)-higenamine according to claim 2, wherein Using the Pichia stipitis genome as a template, PCR amplified the ARS, Ura, and CEN-750 genes using ARS2-F shown in SEQ ID NO: 1 and ARS2-R shown in SEQ ID NO: 2 as primers, URA-F shown in SEQ ID NO: 3 and URA-R shown in SEQ ID NO: 4 as primers, and CEN-F shown in SEQ ID NO: 5 and CEN-R shown in SEQ ID NO: 6 as primers; The ARS, Ura, and CEN-750 genes were connected by overlap PCR to obtain overlap fragments; The overlap fragment was ligated with the pRS414 vector to obtain the plasmid pRS414CEN-750.

4. The engineered strain for producing (S)-demethylcoclaurine according to claim 1, wherein, The tyrosine hydroxylase mutant TyrH W13L，F309L The nucleotide sequence of the gene encoding the enzyme is shown in SEQ ID NO: 27; the nucleotide sequence of the gene encoding the enzyme dopa decarboxylase DODC is shown in SEQ ID NO: 28; the nucleotide sequence of the gene encoding the enzyme higenamine synthase NCS is shown in SEQ ID NO:

29.

5. A method for producing (S)-demethylcoclaurine, characterized in that, include: The engineered strain according to any one of claims 1 to 4 is used as a whole-cell catalyst, L-tyrosine is used as a substrate, and a whole-cell catalytic reaction is carried out to obtain (S)-higenamine.

6. The method according to claim 5, characterized in that, The pH of the whole-cell catalytic reaction is 5.9-7.3; the reaction temperature is 25-35°C; the reaction speed is 200-300 rpm; the whole-cell catalyst concentration is OD 600 is 20-40; the concentration of substrate L-tyrosine is 1-4 g / L; and the concentration of buffer is 0.05-0.2 mol / L.

7. Use of the engineered strain according to any one of claims 1 to 4 in the preparation of (S)-higenamine.

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