A process for the preparation of p-hydroxyphenylacetic acid

Abstract

The present application relates to the technical field of p-hydroxyphenylacetic acid preparation, and particularly relates to a method for preparing p-hydroxyphenylacetic acid. In the first aspect, a strain of Proteus hauseri OMK-101 is provided, which can convert L-tyrosine into p-hydroxyphenylacetaldehyde and a small amount of p-hydroxyphenylacetic acid, and the preservation number is CCTCC NO: M 20252832. In the second aspect, a strain of Bacillus tropicus OMK-102 is provided, which can convert p-hydroxyphenylacetaldehyde into p-hydroxyphenylacetic acid, and the preservation number is CCTCC NO: M 20252833. In the third aspect, the application of OMK-101 or OMK-102 in the preparation of p-hydroxyphenylacetic acid is provided. In the fourth aspect, a method for preparing p-hydroxyphenylacetic acid is provided. By establishing a mixed strain whole-cell catalytic system containing OMK-101 and OMK-102, the present application can efficiently convert L-tyrosine into p-hydroxyphenylacetic acid.

Description

Technical Field

[0001] This invention relates to the field of p-hydroxyphenylacetic acid preparation technology, and more specifically to a method for preparing p-hydroxyphenylacetic acid. Background Technology

[0002] p-Hydroxyphenylacetic acid (molecular formula C8H8O3, CAS number 156-38-7) is an aromatic carboxylic acid compound possessing both phenolic hydroxyl and carboxyl groups. It is a white to pale yellow crystalline powder with a melting point of 147-150℃. It is readily soluble in hot water, ethanol, ether, and other polar solvents, slightly soluble in cold water and benzene, and has a weak aromatic odor. At higher purity, it is not significantly irritating. p-Hydroxyphenylacetic acid is a natural intermediate in human metabolism and possesses certain anti-inflammatory and antioxidant activities. In the biopharmaceutical field, it can be used to prepare antibacterial agents, anti-inflammatory drugs, and raw materials for health products, and has potential auxiliary effects on the regulation of metabolism. As an important organic synthesis intermediate, the bifunctional group in its structure makes it a key bridge connecting the aromatic ring and the aliphatic chain structure, and it is widely used in the synthesis of pharmaceuticals, pesticides, fragrances, and dyes. In the pharmaceutical field, it can be used to synthesize β-receptor blockers and antibiotics; in the pesticide field, it can be used to prepare highly effective and low-toxicity fungicides; and in the fragrance field, it is used to synthesize floral fragrances, demonstrating significant market application value.

[0003] Methods for preparing p-hydroxyphenylacetic acid include chemical methods, fermentation methods, and whole-cell catalysis methods. Chemical methods use anisole, p-cresol, phenol, benzylphenyl ether, or hydroxymandelic acid for synthesis; however, many raw materials used in chemical synthesis are derived from fossil petroleum, making the chemical synthesis route less environmentally friendly, and the process requires high temperature and pressure. Fermentation methods use glucose as a substrate and *E. coli* as a host, with a yield of approximately 25 g / L. Because p-hydroxyphenylacetic acid is toxic to *E. coli*, fermentation time requires more than 90 hours, and the plasmid carried by the strain needs to be maintained with antibiotics; prolonged fermentation easily leads to plasmid loss (Shen YP, Pan Y, Niu FX, et al. Biosensor-assisted evolution for high-level production of 4-hydroxyphenylacetic acid in...). Escherichia coli [J].Metabolicengineering, 2022, 70:1-11.DOI:10.1016 / j.ymben.2021.12.008.).

[0004] Whole-cell catalysis offers shorter reaction times and is less affected by product toxicity. Existing whole-cell catalysis methods, such as Chinese patent application CN101037658A, use p-hydroxyphenylacetonitrile as a substrate, catalyzing its conversion to p-hydroxyphenylacetic acid via nitrile hydrolase. However, the traditional synthesis of p-hydroxyphenylacetonitrile relies on highly toxic reagents such as sodium cyanide and hydrogen cyanide, posing a threat to operator safety and resulting in difficult-to-treat cyanide-containing wastewater, exhibiting significant environmental unfriendliness.

[0005] Therefore, a new whole-cell catalytic method for the preparation of p-hydroxyphenylacetic acid is needed to solve the above problems. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing p-hydroxyphenylacetic acid, which expands the synthetic route of p-hydroxyphenylacetic acid by using a whole-cell catalytic system of mixed strains OMK-101 and OMK-102 to convert the substrate L-tyrosine into p-hydroxyphenylacetic acid.

[0007] Firstly, a *Proteus haureus* strain is provided ( Proteus hauseri The strain OMK-101 can convert L-tyrosine into p-hydroxyphenylacetaldehyde and a small amount of p-hydroxyphenylacetic acid. OMK-101 is from our company's wild-type strain bank and was deposited on December 10, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:M 20252832, located at Wuhan University, Wuhan, China.

[0008] Secondly, a tropical spore-forming bacterium ( Bacillus tropicus The strain OMK-102 can convert p-hydroxyphenylacetaldehyde to p-hydroxyphenylacetic acid. OMK-102 is from our company's wild-type strain library. Bacillus tropicus The original strain was obtained by atmospheric pressure room temperature plasma mutagenesis (ARTP) and was deposited at the China Center for Type Culture Collection on December 10, 2025, with accession number CCTCC NO:M 20252833, at Wuhan University, Wuhan, China.

[0009] Thirdly, the application of OMK-101 described in the first aspect and / or OMK-102 described in the second aspect in the preparation of p-hydroxyphenylacetic acid is provided.

[0010] Fourthly, a method for preparing p-hydroxyphenylacetic acid is provided, comprising the following steps:

[0011] S1: Preparation of small-system resting cells of OMK-101 as described in the first aspect and preparation of small-system resting cells of OMK-102 as described in the second aspect;

[0012] S2: Using resting cells of OMK-101 and resting cells of OMK-102, L-tyrosine is converted into p-hydroxyphenylacetic acid.

[0013] In some embodiments of the fourth aspect, S2 includes:

[0014] S21: Large-scale culture of OMK-101 and OMK-102: The first seed culture in S1 was transferred to secondary culture medium F, and then transferred to a fermenter containing large-scale fermentation medium G. Appropriate stirring speed and dissolved oxygen parameters were set. After fermentation, the bacterial solution was washed to obtain resting OMK-101 cells in the large-scale system. The second seed culture in S1 was transferred to secondary culture medium H, and then transferred to a fermenter containing large-scale fermentation medium I. Appropriate stirring speed and dissolved oxygen parameters were set. After glucose was consumed, glucose was added. After fermentation, the bacterial solution was washed to obtain resting OMK-102 cells in the large-scale system.

[0015] S22: Preparation of p-hydroxyphenylacetic acid in a mixed whole-cell system: Mix buffer, OMK-101 prepared in S21, OMK-102 prepared in S21 and glucose, pour into a fermenter, add L-tyrosine, set a suitable temperature and fermentation speed, and collect the fermenter after the reaction is complete.

[0016] In some embodiments of the fourth aspect, in S1, the preparation of the OMK-101 small system resting cells includes: reviving the OMK-101 and inoculating it with a first seed culture, transferring it to a shake flask culture medium B, setting a suitable temperature, culturing it on a shaker, then centrifuging the strain, washing it multiple times with buffer, and obtaining the OMK-101 small system resting cells.

[0017] In some embodiments of the fourth aspect, in S1, the preparation of the OMK-102 small system resting cells includes: reviving the OMK-102 and inoculating it with a second seed culture, transferring it to a shake flask culture medium E, setting a suitable temperature, culturing it on a shaker, then centrifuging the strain, washing it multiple times with buffer, and obtaining the OMK-102 small system resting cells.

[0018] In some embodiments of the fourth aspect, in S1, the step of reviving and inoculating the OMK-101 with the first seed solution is as follows: take an OMK-101 glycerol tube, streak it on a first revival plate for revival, and inoculate a single colony into 20 mL of the first seed solution for culture. The first seed solution is seed culture medium A, which includes sodium chloride, yeast extract, tryptone, and water. The first revival plate is composed of seed culture medium A and agar.

[0019] In some embodiments of the fourth aspect, in S1, the first seed culture is transferred to shake flask culture medium B at 10 wt%.

[0020] In some embodiments of the fourth aspect, in S1, the steps of resuscitating OMK-102 and inoculating it with the second seed culture are as follows: take an OMK-102 glycerol tube, streak it on a second resuscitation plate for separation, and inoculate a single colony into 20 mL of the second seed culture for cultivation. The second seed culture consists of seed culture medium C, which includes sodium chloride, yeast extract, and tryptone. The second resuscitation plate consists of seed culture medium C and agar.

[0021] In some embodiments of the fourth aspect, in S1, the second seed culture is transferred to shake flask culture medium E at 10 wt%.

[0022] In some embodiments of the fourth aspect, the shake flask culture medium B comprises the following components: glycerol, yeast extract, tryptone, Na2HPO4·2H2O, NaH2PO4·2H2O, sodium oxalate tetraacetate, and urea.

[0023] In some embodiments of the fourth aspect, the shake flask culture medium E comprises the following components: glucose, yeast extract, tryptone, soybean peptone, Na2HPO4·2H2O, NaH2PO4·2H2O, citric acid, magnesium sulfate, ferrous sulfate heptahydrate, and calcium carbonate.

[0024] In some embodiments of the fourth aspect, in S1, the buffer solution is a phosphate buffer, preferably with a concentration of 50-300 mM, more preferably 50 mM, 100 mM, 150 mM, 200 mM or 300 mM.

[0025] In some embodiments of the fourth aspect, the seed culture medium A comprises the following components: 5 g / L yeast extract, 10 g / L sodium chloride, and 10 g / L tryptone, wherein the first resuscitation plate is prepared by adding 1.8% (w / v) agar to the seed culture medium A.

[0026] In some embodiments of the fourth aspect, in S1, the culture temperature of the first resuscitation plate and / or the first seed solution is 30-35°C; and / or the culture time of the first seed solution is 12-16 h.

[0027] In some embodiments of the fourth aspect, in S1, the shaking speed of the small system of resting cells of OMK-101 is 220 rpm; and / or the shaking culture time is 24 h.

[0028] In some embodiments of the fourth aspect, in S1, the seed culture medium C contains the following components: 5 g / L yeast extract, 10 g / L sodium chloride, and 10 g / L tryptone, and the second resuscitation plate is prepared by adding 1.8% (w / v) agar to the seed culture medium C.

[0029] In some embodiments of the fourth aspect, in S1, the culture temperature of the second resuscitation plate and / or the second seed culture is 35-40°C; and / or the shaking speed is 220 rpm; and / or the seed culture time is 14-18 h.

[0030] In some embodiments of the fourth aspect, in S1, the preparation of the small system of resting cells of OMK-102 involves a shaking speed of 200 rpm; and / or a culture temperature of 35-40°C; and / or a culture time of 24-30 h.

[0031] In some embodiments of the fourth aspect, the shake flask culture medium B comprises the following components: glycerol 8-15 g / L, yeast extract 12-18 g / L, tryptone 10-16 g / L, Na2HPO4·2H2O 2.5-12.5 g / L, NaH2PO4·2H2O 2.5-12.5 g / L, sodium oxotetraacetate 50-200 mg / L, and urea 1-4 g / L.

[0032] In some embodiments of the fourth aspect, the shake flask culture medium E comprises the following components: glucose 15-25 g / L, yeast extract 15-30 g / L, tryptone 10-20 g / L, soybean peptone 15-30 g / L, Na2HPO4·2H2O 2.5-12.5 g / L, NaH2PO4·2H2O 2.5-12.5 g / L, citric acid 1-2 g / L, magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 50-100 mg / L, and calcium carbonate 5-10 g / L.

[0033] In some embodiments of the fourth aspect, S2 includes: S20: Preparation of p-hydroxyphenylacetic acid in a mixed whole-cell system: The resting cells of the OMK-101 system and the resting cells of the OMK-102 system are mixed with buffer and L-tyrosine, and the reaction is carried out at a suitable temperature and rotation speed.

[0034] In some embodiments of the fourth aspect, in S20, the buffer solution is a phosphate buffer solution with a concentration of 50-300 mM, preferably 50 mM, 100 mM, 150 mM, 200 mM or 300 mM.

[0035] In some embodiments of the fourth aspect, in S20, OD 600The final concentration of OMK-101 and OMK-102 is calculated to be 10:10-40:40, with 10:10, 10:20, 10:30, 10:40, 20:10, 20:20, 20:30, 20:40, 30:10, 30:20, 30:30, 30:40 or 40:40 being particularly preferred.

[0036] In some embodiments of the fourth aspect, in S20, the pH of the reaction system is 6.5-8.0.

[0037] In some embodiments of the fourth aspect, the reaction temperature in S20 is 25-40°C.

[0038] In some embodiments of the fourth aspect, S2 includes:

[0039] S21: Large-scale culture of OMK-101 and OMK-102: The first seed culture in S1 was transferred to secondary culture medium F, and then transferred to a fermenter containing large-scale fermentation medium G. Appropriate stirring speed and dissolved oxygen parameters were set. After fermentation, the bacterial solution was washed to obtain resting OMK-101 cells in the large-scale system. The second seed culture in S1 was transferred to secondary culture medium H, and then transferred to a fermenter containing large-scale fermentation medium I. Appropriate stirring speed and dissolved oxygen parameters were set. After glucose was consumed, glucose was added. After fermentation, the bacterial solution was washed to obtain resting OMK-102 cells in the large-scale system.

[0040] S22: Preparation of p-hydroxyphenylacetic acid in a mixed whole-cell system: Mix buffer, OMK-101 prepared in S21, OMK-102 prepared in S21 and glucose, pour into a fermenter, add L-tyrosine, set a suitable temperature and fermentation speed, and collect the fermenter after the reaction is complete.

[0041] In some embodiments of the fourth aspect, in S21, the secondary culture medium F comprises the following components: glycerol 10-20 g / L, yeast extract 10-20 g / L, tryptone 10-20 g / L, Na2HPO4·2H2O 2.5-12.5 g / L, NaH2PO4·2H2O 2.5-12.5 g / L, urea 1-10 g / L, niacin 50-200 mg / L, and vitamin B1. 12 200-500 μg / L.

[0042] In some embodiments of the fourth aspect, in S21, the large-scale fermentation medium G contains the following components: glycerol 25-35 g / L, yeast extract 18-24 g / L, tryptone 18-24 g / L, ammonium sulfate 5-10 g / L, Na2HPO4·2H2O 2.5-5 g / L, NaH2PO4·2H2O 2.5-5 g / L, sodium oxotetraacetate 100-500 mg / L, urea 1-5 g / L, magnesium sulfate 0.5-2 g / L, ferrous sulfate 20-100 mg / L, manganese sulfate 5-20 mg / L, cobalt chloride 5-10 mg / L, and nickel chloride 5-10 mg / L.

[0043] In some embodiments of the fourth aspect, in S21, the secondary culture medium H comprises the following components: glucose 20-30 g / L, yeast extract 20-30 g / L, soybean peptone 20-30 g / L, Na2HPO4·2H2O 2.5-5 g / L, NaH2PO4·2H2O 2.5-5 g / L, magnesium sulfate 0.5-2 g / L, and calcium carbonate 5-10 g / L.

[0044] In some embodiments of the fourth aspect, in S21, the large-scale fermentation medium I comprises the following components: glucose 30-50 g / L, yeast extract 24-48 g / L, soybean peptone 24-36 g / L, Na2HPO4·2H2O 2.5-5 g / L, NaH2PO4·2H2O 2.5-5 g / L, magnesium sulfate 0.5-2 g / L, ammonium molybdate 1-5 mg / L, ferrous sulfate 20-100 mg / L, manganese sulfate 5-20 mg / L, zinc sulfate 5-20 mg / L, and nickel chloride 5-10 mg / L.

[0045] In some embodiments of the fourth aspect, in S21, the fermentation temperature of OMK-101 is 28-35°C, for example, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C.

[0046] In some embodiments of the fourth aspect, in S21, the stirring speed is set from an initial speed of 200-1000 rpm, and when the dissolved oxygen drops to 20% or 30% or 40%, the stirring paddles are connected in series, with the minimum speed set to 200 rpm or 300 rpm or 400 rpm.

[0047] In some embodiments of the fourth aspect, in S21, the pH of the fermentation process is controlled at 6.0-8.0, for example, 6.0, 6.5, 7.0, 7.5, 8.0.

[0048] In some embodiments of the fourth aspect, in S21, the fermentation time is controlled at 24-30 h, preferably 24, 26, 28 or 30 h. Generally, a longer culture time will produce more biomass.

[0049] In some embodiments of the fourth aspect, in S21, the fermentation temperature of OMK-102 is 30-40°C, for example 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0050] In some embodiments of the fourth aspect, in S21, the stirring speed is initially set to 300-800 rpm, and when the dissolved oxygen drops to 20%-40%, the stirring paddles are connected in series, with a minimum speed set to 300-500 rpm; preferably, when the dissolved oxygen drops to 20% or 30% or 40%, the stirring paddles are connected in series, with a minimum speed set to 300 rpm or 400 rpm or 500 rpm.

[0051] In some embodiments of the fourth aspect, in S21, after the glucose is consumed, 30-60 g / L of glucose is added once, for example, 30 g / L, 40 g / L, 50 g / L or 60 g / L of glucose is added.

[0052] In some embodiments of the fourth aspect, in S21, the pH of the fermentation process is controlled at 6.5-8.0, for example, 6.0, 6.5, 7.0, 7.5, 8.0.

[0053] In some embodiments of the fourth aspect, in S21, the fermentation time is controlled at 36-48h, preferably 36, 40, 44 or 48h.

[0054] In some embodiments of the fourth aspect, the method of adding the substrate L-tyrosine in S22 greatly affects the reaction process. L-tyrosine is poorly soluble in water, and adding too much at once can easily cause particle overflow, resulting in unnecessary waste. Preferably, the L-tyrosine is added in the following manner: a single addition of 90 g / L, two additions of 45 g / L, and three additions of 30 g / L. The two additions of 45 g / L are at the beginning of the reaction and at 5 hours of reaction, while the three additions of 30 g / L are at the beginning of the reaction, 5 hours of reaction, and 10 hours of reaction.

[0055] In some embodiments of the fourth aspect, in S22, the initial stirring speed is 100 rpm. The speed should not be too high, otherwise the L-tyrosine powder will easily stick to the plate. The initial stirring speed of 100 rpm is stopped after 10 minutes. After 10 minutes, the stirring speed is adjusted to 300-800 rpm.

[0056] In some embodiments of the fourth aspect, in S22, the large-scale fermentation tank has a capacity of 5-100L, preferably 5L, 15L, 50L or 100L.

[0057] In some embodiments of the fourth aspect, in S22, the buffer solution is a phosphate buffer solution with a concentration of 50-300 mM, preferably 50 mM, 100 mM, 150 mM, 200 mM or 300 mM.

[0058] In some embodiments of the fourth aspect, in S22, OD 600 The final concentration of OMK-101 and OMK-102 is calculated to be 10:10-40:40, preferably 10:10, 10:20, 10:30, 10:40, 20:10, 20:20, 20:30, 20:40, 30:10, 30:20, 30:30, 30:40 or 40:40.

[0059] In some embodiments of the fourth aspect, in S22, the pH of the reaction system is 6.5-8.0, preferably 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0. The pH of the fermenter reaction greatly affects the catalytic activity of the enzymes contained in the two bacteria, thus affecting the whole-cell catalytic effect.

[0060] In some embodiments of the fourth aspect, in S22, the reaction temperature is 25-40°C.

[0061] Compared with the prior art, a certain embodiment of the present invention includes at least one of the following beneficial effects:

[0062] (1) By establishing a whole-cell catalytic system containing mixed strains of OMK-101 and OMK-102, L-tyrosine can be efficiently converted into p-hydroxyphenylacetic acid with high yield and high efficiency. The substrate L-tyrosine is cheap and readily available.

[0063] (2) The culture of mixed strains containing OMK-101 and OMK-102 does not require additional antibiotics for maintenance, thus reducing antibiotic costs.

[0064] (3) The system uses whole-cell catalysis, and p-hydroxyphenylacetic acid has little effect on the reaction system, so a high yield can be obtained.

[0065] Terminology Definition

[0066] In the context of this invention, all figures disclosed herein are approximate values, regardless of whether the words "approximately" or "about" are used. Based on the disclosed figures, each figure may vary by less than ±10% or by a difference that is considered reasonable by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0067] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0068] The terms “optional,” “optional,” or “optionally” mean that the events or circumstances described below may, but are not necessarily, occur. For example, “mixing the yeast described in the first aspect with the fermentation medium and optional substrate” means “mixing the yeast described in the first aspect with the fermentation medium and substrate” or “mixing the yeast described in the first aspect with the fermentation medium.”

[0069] The terms "transfer at 10% vol" and "transfer at 10% ratio" both refer to inoculating 10% of the previous culture medium into the new culture medium described below.

[0070] The term "OD" 600 "" refers to the optical density detected at a wavelength of 600 nm.

[0071] The term "wt%" refers to a percentage by mass.

[0072] The term "%vol" refers to a volume percentage.

[0073] The term "large system" refers to fermentation in a 5-100L fermenter; the term "small system" refers to laboratory culture in a 50-500mL shake flask.

[0074] The term "tandem stirring paddles" refers to the fermenter being equipped with a dissolved oxygen-triggered speed linkage regulation mechanism, which can maintain the dissolved oxygen in the fermenter within the target range when the rapid proliferation of bacteria and the significant increase in oxygen consumption cause the dissolved oxygen concentration in the fermentation broth to drop to a certain value.

[0075] The term "sticking" refers to the phenomenon that L-tyrosine powder easily adheres to the walls of the fermenter during stirring because L-tyrosine is poorly soluble in water. Attached Figure Description

[0076] Figure 1 The curves for the synthesis of p-hydroxyphenylacetic acid in a large system are shown. Detailed Implementation

[0077] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0078] The experimental and detection methods involved in the following embodiments are as follows:

[0079] 1. Detection methods for p-hydroxyphenylacetic acid, p-hydroxyphenylacetaldehyde, and p-hydroxyphenylethanol

[0080] After the reaction, the sample was diluted several times, centrifuged at 12000 rpm for 2 min, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm filter membrane for later use. The chromatographic analysis conditions were: Agilent 1260 Infinity liquid chromatograph, ZORBAX Eclipse Plus column, mobile phase methanol-water (85:15, V / V), flow rate 0.8 mL / min, UV detection wavelength 260 nm, column temperature 35℃, and injection volume 10 μl.

[0081] 2. Bacterial cell count (OD) 600 Determination of )

[0082] Using a blank culture medium as a control, take 1 mL of the target bacterial solution and place it in a cuvette. Measure the absorbance at 600 nm. The absorbance should be controlled within the range of 0.5-1.0. If the value exceeds this range, dilution is required for testing.

[0083] 3. Determination of protein content

[0084] First, weigh bovine serum albumin to prepare a 100 mg / L protein standard stock solution. Pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the protein standard solution into different test tubes. Then, add 4 mL of Coomassie Brilliant Blue G-250 reagent to each test tube, and add water to bring the total volume to 5 mL. Mix thoroughly and let stand for 10 min. Measure the absorbance at a wavelength of 595 nm to create a protein standard curve.

[0085] Weigh out the target solution and dilute it several times. Take 1 mL of diluent and 4 mL of Coomassie Brilliant Blue G-250 reagent, mix them thoroughly, let them stand for 10 min, and measure the absorbance at a wavelength of 595 nm. Calculate the protein concentration of the target solution based on the standard curve and sample dilution factor from the previous step.

[0086] 4. Calculation of bacterial cell removal rate

[0087] Crude cell removal rate = (OD before flocculation) 600 - OD after flocculation 600 ) / Pre-flocculation OD 600 ) × 100%

[0088] 5. Calculation of protein removal rate

[0089] Protein removal rate = ((Pre-flocculation protein concentration M1 - Post-flocculation protein concentration M2) / Pre-flocculation protein concentration M1) × 100%

[0090] Raw materials used in the examples:

[0091] The culture media involved in the following examples are as follows:

[0092] The seed culture medium A for OMK-101 consists of 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L NaCl.

[0093] The shake flask culture medium B formula for OMK-101 is 10 g / L glycerol, 12 g / L yeast extract, 12 g / L tryptone, 5 g / L Na2HPO4·2H2O, 12.5 g / L NaH2PO4·2H2O, 100 mg / L sodium oxalate tetraacetate, and 2 g / L urea.

[0094] Bacillus tropicus The seed culture medium C was formulated with yeast extract 5 g / L, tryptone 10 g / L, and NaCl 10 g / L.

[0095] Bacillus tropicus The initial screening medium D consisted of 15 g / L glucose, 24 g / L yeast extract, 12 g / L tryptone, 12 g / L soybean peptone, 4 g / L ammonium sulfate, 5 g / L Na2HPO4·2H2O, 7.5 g / L NaH2PO4·2H2O, 1 g / L citric acid, 1.2 g / L magnesium sulfate, 50 mg / L ferrous sulfate heptahydrate, and 5 g / L calcium carbonate.

[0096] The shake flask culture medium E formula for OMK-102 is as follows: glucose 15 g / L, yeast extract 24 g / L, tryptone 12 g / L, soybean peptone 12 g / L, ammonium sulfate 4 g / L, Na2HPO4·2H2O 5 g / L, NaH2PO4·2H2O 7.5 g / L, citric acid 1 g / L, magnesium sulfate 1.2 g / L, ferrous sulfate heptahydrate 50 mg / L, and calcium carbonate 5 g / L.

[0097] The secondary culture medium F of OMK-101 consisted of 10 g / L glycerol, 12 g / L yeast extract, 12 g / L tryptone, 5 g / L Na2HPO4·2H2O, 12.5 g / L NaH2PO4·2H2O, 2 g / L urea, 200 mg / L nicotinic acid, and 200 μg / L vitamin B12.

[0098] The fermentation medium G for OMK-101 consisted of 25 g / L glycerol, 24 g / L yeast extract, 18 g / L tryptone, 5 g / L ammonium sulfate, 5 g / L Na2HPO4·2H2O, 5 g / L NaH2PO4·2H2O, 200 mg / L sodium oxalate tetraacetate, 2 g / L urea, 1.2 g / L magnesium sulfate, 20 mg / L ferrous sulfate, 5 mg / L manganese sulfate, 5 mg / L cobalt chloride, and 5 mg / L nickel chloride.

[0099] The secondary culture medium H of OMK-102 consists of 20 g / L glucose, 24 g / L yeast extract, 12 g / L soybean peptone, 5 g / L Na2HPO4·2H2O, 7.5 g / L NaH2PO4·2H2O, 1.2 g / L magnesium sulfate, and 5 g / L calcium carbonate.

[0100] The fermentation medium I for OMK-102 consisted of 30 g / L glucose, 36 g / L yeast extract, 12 g / L soybean peptone, 5 g / L Na2HPO4·2H2O, 7.5 g / L NaH2PO4·2H2O, 1.2 g / L magnesium sulfate, 5 mg / L ammonium molybdate, 20 mg / L ferrous sulfate, 5 mg / L manganese sulfate, 5 mg / L zinc sulfate, and 5 mg / L nickel chloride.

[0101] The tryptone and yeast extract were purchased from Oxoid, while the rest of the drugs were purchased from Sinopharm (Shanghai) Chemical Reagent Co., Ltd.

[0102] Example 1: OMK-101 Cultivation and Preparation

[0103] Thaw the glycerol tubes of OMK-101 from the -80℃ freezer, pick up the bacterial suspension with an inoculation loop and streak it on a recovery plate. Place the plate in a 30℃ incubator. When a single small colony appears on the plate, inoculate the single colony into a 100 mL flat-bottomed conical flask containing 20 mL of seed medium A, and incubate at 30℃ and 220 rpm for 16 h on a shaker.

[0104] The OMK-101 seed culture was transferred at 10% vol to a 500 mL Erlenmeyer flask containing 100 mL of shake flask medium B, and cultured at 30℃ and 200 rpm for 24 h. After the culture was completed, the culture was centrifuged (10000 rpm, 20 min, 4℃), the supernatant was discarded, and the precipitated bacterial cells were obtained. The cells were washed twice with 100 mM pH 7.0 phosphate buffer to obtain bacterial sludge.

[0105] Example 2: Whole-cell catalysis of L-tyrosine using OMK-101

[0106] Resuspend the bacterial cells in 200 mM pH 7.0 phosphate buffer, and add approximately 25 mL of OD to the reaction system. 600 OMK-101 with a concentration of 60 was added to a final volume of 50 mL using phosphate buffer of the same concentration. Finally, 40 g / L L-tyrosine was added, and the mixture was incubated at 35°C and 200 rpm for 24 h on a shaker.

[0107] After conversion, the bacterial sludge was centrifuged and discarded to obtain the supernatant. Chitosan was then added at a concentration of 0.1% to remove proteins. The mixture was shaken at 150 rpm for 10 minutes at 35 °C and allowed to stand. After protein removal, the solution was filtered through filter paper, and the resulting filtrate was the reaction solution for L-tyrosine.

[0108] The filtrate was appropriately diluted, and the absorbance was measured at 600 nm. The protein concentration was measured at 595 nm. Unreacted phosphate buffer was used as a blank control. Based on the experimental parameters, the chitosan addition was 0.1%, the cell removal rate was 93%, and the crude protein removal rate was 81%.

[0109] According to high performance liquid chromatography (HPLC) analysis, the contents of p-hydroxyphenylacetaldehyde in the reaction solution of L-tyrosine were 23.5 g / L, p-hydroxyphenylethanol was 1.3 g / L, and p-hydroxyphenylacetic acid was 1.9 g / L.

[0110] Example 3: ARTP Mutagenesis Bacillus tropicus

[0111] Several strains were screened from our strain library that can convert p-hydroxyphenylacetaldehyde to p-hydroxyphenylacetic acid, including wild-type strains. Bacillus tropicus The strain exhibited the strongest catalytic activity and produced the least amount of the byproduct p-hydroxyphenylethanol, thus it was selected as the target strain for further enhancement of catalytic activity through ARTP mutagenesis.

[0112] Strains were taken from a -80°C freezer. Bacillus tropicus Thaw the glycerol tubes, use an inoculation loop to pick up the bacterial suspension and streak it on a recovery plate. Place the plate in a 37°C incubator. When a single small colony appears on the plate, inoculate the single colony into a 100 mL flat-bottomed conical flask containing 20 mL of seed culture medium and incubate at 37°C and 220 rpm for 24 h.

[0113] After completing this process, dilute the bacterial culture to OD. 600 ≈0.5~0.8, take 20 μl of bacterial solution and spread it on the sterile metal pad of ARTP, and then perform ARTP mutagenesis. The ARTP conditions are: ARTP exposure for 60s, 70s, and 80s under helium pressure of 120 MPa, gas flow of 10 SLM, and incident power of 120W. After the process is completed, use sterile water to wash away the bacteria, dilute it several times, take 100 μL of bacterial solution and spread it on a plate. Bacteria that have not been exposed to ARTP are used as controls. The ARTP irradiation time with a lethality rate of more than 90% is selected as the optimal treatment time.

[0114] Based on the CFU values ​​calculated from the plates, 70 seconds of ARTP irradiation was selected as the optimal mutagenesis time. Single colonies of the relevant mutant library were inoculated onto seed medium C and cultured overnight. The next day, they were transferred at a 10% ratio to... Bacillus tropicusThe initial screening medium D was cultured at 37℃ and 200 rpm for 24 h on a shaker. After the culture was completed, the culture was centrifuged (10000 rpm, 20 min, 4℃), the supernatant was discarded, and the precipitated bacterial cells were obtained. The cells were washed twice with 100 mM pH 7.0 phosphate buffer to obtain single-colony bacterial sludge.

[0115] Next step Bacillus tropicus The mutant library's bacterial sludge is based on OD 600 = 20 added to a 50 mL Erlenmeyer flask, followed by the L-tyrosine reaction solution from Example 2, to prepare a 10 mL reaction system with a p-hydroxyphenylacetaldehyde content of 20 g / L. The mixture was incubated at 35 °C and 200 rpm for 5 h on a shaker. After the reaction, the bacterial sludge was discarded by centrifugation, and the supernatant was obtained. (The text abruptly ends here, so the translation stops as well.) Bacillus tropicus The original bacteria served as a control group.

[0116] The supernatant was diluted and filtered through a 0.2 μL filter membrane to remove impurities. The filtrate was then analyzed by liquid chromatography, and single colonies with increased p-hydroxyphenylacetic acid content were selected for data analysis and processing.

[0117] Table 1 Bacillus tropicus Mutation library screening results

[0118]

[0119] From approximately 200 mutant strains, five strains with significantly improved [performance] were selected. Bacillus tropicus Based on the results in Table 1, the mutant strains... Bacillus tropicus The optimal yield was achieved on March 21st, with a 60.8% increase in p-hydroxyphenylacetic acid yield and a 55.4% decrease in the content of the byproduct p-hydroxyphenylethanol. This strain was named OMK-102 and used for the next step of mixed strain reaction.

[0120] Example 4: Cultivation and preparation of OMK-102

[0121] Thaw the glycerol tubes of OMK-102 from Example 3 at -80℃. Use an inoculation loop to pick up the bacterial suspension and streak it onto a resuscitation plate. Place the plate in a 37℃ incubator. Once a single small colony appears on the plate, inoculate the colony into a 100 mL flat-bottomed Erlenmeyer flask containing 20 mL of seed medium C. Incubate for 24 h at 37℃ and 220 rpm on a shaker. The next day, transfer the culture at a 10% ratio to... Bacillus tropicus The culture medium E in shake flasks was incubated at 37 ℃ and 200 rpm for 24 h. After incubation, the culture was centrifuged (10000 rpm, 20 min, 4℃), the supernatant was discarded, and the precipitated bacterial cells were obtained. The cells were washed twice with 100 mM pH 7.0 phosphate buffer to obtain single-colony bacterial sludge.

[0122] Example 5: Small-scale mixed-culture whole-cell reaction

[0123] Adding both OMK-101 and OMK-102 bacterial cells simultaneously can save experimental steps, and the multi-enzyme cascade system composed of different enzymes in the two bacterial cells can accelerate the catalytic rate.

[0124] The initial reaction pH was controlled at 7.0 and the reaction temperature at 35°C. 200 mM phosphate buffer, OMK-101 prepared in Example 1 and OMK-102 prepared in Example 4 were mixed and the total volume of the mixture (50 mL) was poured into a 250 mL Erlenmeyer flask. Finally, 50 g / L L-tyrosine and 10 g / L glucose were added to carry out the reaction, which was completed after 24 h.

[0125] In the first group of reactions, the concentration of strain 1 was OD. 600 =10, strain 2 concentration is OD 600 =10, the concentration of strain 1 in the second group of reactions was OD10. 600 =40, the concentration of strain 2 is OD 600 =20, the concentration of strain 1 in the third group of reactions was OD20. 600 =30, strain 2 concentration is OD 600 =30, the concentration of strain 1 in the fourth group of reactions was OD 600 =20, strain 2 concentration is OD 600 =40, and the remaining volume was made up with phosphate buffer.

[0126] Table 2 Whole-cell reaction of small-system mixed bacteria

[0127]

[0128] The results are shown in Table 2. In the first group, the bacterial cell count was insufficient, resulting in a p-hydroxyphenylacetic acid yield of only 8.41 g / L. In the second group, the proportion of OMK-101 was too high, leading to a large accumulation of the intermediate p-hydroxyphenylacetaldehyde. In the third group, the catalytic efficiency of the two bacteria in a 1:1 ratio was significantly improved, with a yield of 23.81 g / L. In the fourth group, the proportion of strain 2 was increased, and the yield of p-hydroxyphenylacetic acid synthesized was 26.57 g / L, which was 11% higher than that of the third group. Furthermore, the fourth group produced significantly less p-hydroxyphenylethanol as a byproduct. Therefore, the OD of OMK-101 was significantly higher. 600 =20 and OMK-102 OD 600 =40 was selected as the optimal reaction system combination.

[0129] Example 6: OMK-101 fermentation (5L tank)

[0130] Using the seed culture medium A of OMK-101 from Example 1 as the primary seed culture, after the strain culture was completed, it was transferred at a ratio of 10% to the secondary culture medium F of OMK-101. After culturing at 30℃ and 220 rpm for 16 h, it was transferred to a 5 L fermenter containing 1.8 L of fermentation medium G. The fermentation temperature was controlled at 30℃, and the dissolved oxygen relative value was maintained at 30% by adjusting the stirring speed and aeration rate. The pH was controlled by 20% (w / v) ammonia and 50% (w / v) phosphoric acid. Fermentation was carried out under the condition of pH=7.5. When the glycerol was consumed, the dissolved oxygen began to rebound, and the stirring speed began to decrease. The minimum stirring speed was set to 300 rpm. After 24 h of fermentation, the fermentation broth was collected, centrifuged, the supernatant was discarded, and the whole cell catalyst was collected.

[0131] Example 7: OMK-102 fermentation (5L tank)

[0132] Using the seed culture medium C of OMK-102 in Example 4 as the primary seed culture, after the strain culture was completed, it was transferred to the secondary culture medium H of OMK-102 at a ratio of 10%. After culturing at 37°C and 200 rpm for 16 h, it was transferred to a 5 L fermenter containing 1.8 L of fermentation medium I. The fermentation temperature was controlled at 37°C. The dissolved oxygen relative value was maintained at 20% by adjusting the stirring speed and aeration rate. The pH was controlled by 20% (w / v) ammonia and 50% (w / v) phosphoric acid. Fermentation was carried out under the condition of pH=7.

[0133] During fermentation, samples were taken every 2 hours to measure OD. 600 The glucose concentration was adjusted, and when the glucose concentration in the fermentation broth dropped below 3 g / L, glucose was added to a final concentration of 40 g / L to continue fermentation. Fermentation was stopped after 36 h. The fermentation broth was centrifuged, the supernatant was discarded, and the whole-cell catalyst was collected.

[0134] Example 8: Large-scale system reaction

[0135] The OMK-101 and OMK-102 prepared in Examples 6 and 7 were prepared according to OD... 600 Mix 20 and 40 solutions and pour the mixture into a 5L fermenter. Add 200 mM pH 7 phosphate buffer to bring the volume to 2.5 L. Add 90 g / L L-tyrosine powder and 10 g / L glucose. To prevent L-tyrosine from sticking to the plate, adjust the stir bar speed to 100 rpm and premix for 10 min. Increase the stir bar speed to 600 rpm. Maintain the pH of the fermenter at 7.4 and stop the reaction after 24 h.

[0136] like Figure 1As shown, in the initial stage of the reaction, p-hydroxyphenylacetaldehyde is generated in large quantities, reaching a peak at about 6 hours. As the reaction proceeds, OMK-102 can convert it into p-hydroxyphenylacetic acid. After about 24 hours, p-hydroxyphenylacetaldehyde is basically consumed, and the yield of p-hydroxyphenylacetic acid reaches 71.65 g / L. Throughout the entire reaction process, the byproduct p-hydroxyphenylethanol is always controlled at a low level (the content at 26 hours is only 2.56 g / L).

[0137] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0138] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A type of Proteus haureus ( Proteus hauseri The strain is OMK-101, with the accession number CCTCC NO:M20252832.

2. A tropical Bacillus ( Bacillus tropicus The strain OMK-102, with accession number CCTCC NO:M20252833, is used.

3. The use of OMK-101 as described in claim 1 and / or OMK-102 as described in claim 2 in the preparation of p-hydroxyphenylacetic acid.

4. A method for preparing p-hydroxyphenylacetic acid, characterized in that: Includes the following steps: S1: Prepare resting cells of the OMK-101 small system according to claim 1 and the OMK-102 small system according to claim 2; wherein, the small system refers to 50-500mL shake flask culture; S2: Using resting cells from the OMK-101 and OMK-102 small systems, L-tyrosine is converted into p-hydroxyphenylacetic acid.

5. The method according to claim 4, characterized in that: In S1, the preparation of resting cells of the OMK-101 small system includes: resuscitating the OMK-101 and inoculating it with a first seed culture, transferring it to shake flask culture medium B, setting a suitable temperature, culturing it on a shaker, then centrifuging the strain, washing it multiple times with buffer, and obtaining resting cells of the OMK-101 small system; and / or In S1, the preparation of resting cells of the OMK-102 small system includes: reviving the OMK-102 and inoculating it with a second seed culture, transferring it to a shake flask culture medium E, setting a suitable temperature, culturing it on a shaker, then centrifuging the strain, washing it multiple times with buffer, and obtaining resting cells of the OMK-102 small system. in, In S1, the steps for reviving and inoculating the OMK-101 with the first seed culture are as follows: Take an OMK-101 glycerol tube, streak it on a first revival plate for revival, and inoculate a single colony into 20 mL of the first seed culture for cultivation. The first seed culture consists of seed culture medium A, which includes sodium chloride, yeast extract, tryptone, and water. The first revival plate consists of seed culture medium A and agar; and / or In S1, the first seed culture is transferred to shake flask culture medium B at 10 wt%; and / or In S1, the steps for reviving OMK-102 and inoculating it with the second seed culture are as follows: Take an OMK-102 glycerol tube, streak it onto a second revival plate for isolation, and inoculate a single colony into 20 mL of the second seed culture for incubation. The second seed culture consists of seed culture medium C, which includes sodium chloride, yeast extract, and tryptone. The second revival plate consists of seed culture medium C and agar; and / or In S1, the second seed culture was transferred to shake-flask medium E at 10 wt%; and / or The shake flask culture medium B comprises the following components: glycerol, yeast extract, tryptone, Na₂HPO₄·2H₂O, NaH₂PO₄·2H₂O, sodium oxalate tetraacetate, and urea; and / or The shake flask culture medium E comprises the following components: glucose, yeast extract, tryptone, soybean peptone, Na₂HPO₄·2H₂O, NaH₂PO₄·2H₂O, citric acid, magnesium sulfate, ferrous sulfate heptahydrate, and calcium carbonate; and / or In S1, the buffer solution is a phosphate buffer with a concentration of 50-300 mM; and / or In S1, the initial pH of the buffer solution is 6.5-8.

5.

6. The method according to claim 5, characterized in that: The seed culture medium A comprises the following components: 5 g / L yeast extract, 10 g / L sodium chloride, and 10 g / L tryptone; the first resuscitation plate is prepared by adding 1.8% (w / v) agar to the seed culture medium A; and / or In S1, the culture temperature of the first resuscitation plate and / or the first seed culture is 30-35℃; and / or the culture time of the first seed culture is 12-16 h; and / or In S1, during the preparation of resting cells in the OMK-101 small system, the shaking speed was 220 rpm; and / or the shaking culture time was 24 h; and / or In S1, the seed culture medium C comprises the following components: 5 g / L yeast extract, 10 g / L sodium chloride, and 10 g / L tryptone; the second resuscitation plate is prepared by adding 1.8% (w / v) agar to the seed culture medium C; and / or In S1, the culture temperature of the second resuscitation plate and / or the second seed culture is 35-40℃; and / or the shaking speed is 220 rpm; and / or the seed culture culture time is 14-18 h; and / or In S1, during the preparation of resting cells in the OMK-102 small system, the shaking speed was 200 rpm; and / or the culture temperature was 35-40℃; and / or the culture time was 24-30 h; and / or The shake flask culture medium B comprises the following components: glycerol 8-15 g / L, yeast extract 12-18 g / L, tryptone 10-16 g / L, Na₂HPO₄·2H₂O 2.5-12.5 g / L, NaH₂PO₄·2H₂O 2.5-12.5 g / L, sodium oxotetraacetate 50-200 mg / L, and urea 1-4 g / L; and / or The shake flask culture medium E contains the following components: glucose 15-25 g / L, yeast extract 15-30 g / L, tryptone 10-20 g / L, soybean peptone 15-30 g / L, Na2HPO4·2H2O 2.5-12.5 g / L, NaH2PO4·2H2O 2.5-12.5 g / L, citric acid 1-2 g / L, magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 50-100 mg / L, and calcium carbonate 5-10 g / L.

7. The method according to claim 6, characterized in that: S2 includes: S20: Preparation of p-hydroxyphenylacetic acid from mixed whole-cell microsystem: The resting cells of the OMK-101 microsystem and the resting cells of the OMK-102 microsystem are mixed with buffer and L-tyrosine, and the reaction is carried out at a suitable temperature and speed. The buffer solution is a phosphate buffer with a concentration of 50-300 mM; Among them, OD 600 The final concentration of OMK-101 and OMK-102 added is calculated to be 10:10-40:40; The pH of the reaction system is 6.5-8.0; The reaction temperature is 25-40℃.

8. The method according to claim 6, characterized in that: S2 includes: S21: Large-scale culture of OMK-101 and OMK-102: The first seed culture in S1 was transferred to secondary culture medium F, and then transferred to a fermenter containing large-scale fermentation medium G. Appropriate stirring speed and dissolved oxygen parameters were set. After fermentation, the bacterial solution was washed to obtain resting OMK-101 cells in the large-scale system. The second seed culture in S1 was transferred to secondary culture medium H, and then transferred to a fermenter containing large-scale fermentation medium I. Appropriate stirring speed and dissolved oxygen parameters were set. After glucose was consumed, glucose was added. After fermentation, the bacterial solution was washed to obtain resting OMK-102 cells in the large-scale system. The large-scale system was cultured in a 5-100L fermenter. S22: Preparation of p-hydroxyphenylacetic acid in a mixed whole-cell system: Mix buffer, OMK-101 prepared in S21, OMK-102 prepared in S21 and glucose, pour into a fermenter, add L-tyrosine, set a suitable temperature and fermentation speed, and collect the fermenter after the reaction is complete. in, In S21, the secondary culture medium F comprises the following components: 10-20 g / L glycerol, 10-20 g / L yeast extract, 10-20 g / L tryptone, 2.5-12.5 g / L Na2HPO4·2H2O, 2.5-12.5 g / L NaH2PO4·2H2O, 1-10 g / L urea, 50-200 mg / L niacin, and vitamin B1. 12 200-500 μg / L; and / or In S21, the large-scale fermentation medium G contains the following components: glycerol 25-35 g / L, yeast extract 18-24 g / L, tryptone 18-24 g / L, ammonium sulfate 5-10 g / L, Na2HPO4·2H2O 2.5-5 g / L, NaH2PO4·2H2O 2.5-5 g / L, sodium oxotetraacetate 100-500 mg / L, urea 1-5 g / L, magnesium sulfate 0.5-2 g / L, ferrous sulfate 20-100 mg / L, manganese sulfate 5-20 mg / L, cobalt chloride 5-10 mg / L, and nickel chloride 5-10 mg / L; and / or In S21, the secondary culture medium H contains the following components: glucose 20-30 g / L, yeast extract 20-30 g / L, soybean peptone 20-30 g / L, Na2HPO4·2H2O 2.5-5 g / L, NaH2PO4·2H2O 2.5-5 g / L, magnesium sulfate 0.5-2 g / L, and calcium carbonate 5-10 g / L; and / or In S21, the large-scale fermentation medium I contains the following components: glucose 30-50 g / L, yeast extract 24-48 g / L, soybean peptone 24-36 g / L, Na2HPO4·2H2O 2.5-5 g / L, NaH2PO4·2H2O 2.5-5 g / L, magnesium sulfate 0.5-2 g / L, ammonium molybdate 1-5 mg / L, ferrous sulfate 20-100 mg / L, manganese sulfate 5-20 mg / L, zinc sulfate 5-20 mg / L, and nickel chloride 5-10 mg / L; and / or In S21, the fermentation temperature of OMK-101 is 28-35℃; and / or In S21, the stirring speed is initially set to 200-1000 rpm. When dissolved oxygen drops to 20%, 30%, or 40%, the stirring paddles are connected in series, with the minimum speed set to 200, 300, or 400 rpm; and / or In S21, the pH during fermentation is controlled between 6.0 and 8.0; and / or In S21, the fermentation time is controlled at 24-30 h; In S21, the fermentation temperature of OMK-102 is 30-40℃; and / or In S21, the stirring speed is initially set to 300-800 rpm. When the dissolved oxygen level drops to 20%-40%, the stirring paddles are connected in series, with the minimum speed set to 300-500 rpm; and / or In S21, once the glucose is depleted, replenish it with 30-60 g / L glucose; and / or In S21, the pH during fermentation is controlled between 6.5 and 8.0; and / or In S21, the fermentation time is controlled at 36-48 hours; and / or In S21, the stirring paddle is connected in series, indicating that the fermenter is equipped with a dissolved oxygen-triggered speed linkage adjustment mechanism; and / or In S22, L-tyrosine is added in the following ways: a single addition of 90 g / L, two additions of 45 g / L, or three additions of 30 g / L. The two additions of 45 g / L are at the beginning of the reaction and after 5 hours of reaction. The three additions of 30 g / L are at the beginning of the reaction, after 5 hours of reaction, and after 10 hours of reaction; and / or In S22, the initial stirring speed is 100 rpm, and after 10 minutes, the stirring speed is adjusted to 300-800 rpm; and / or In S22, the buffer solution is a phosphate buffer with a concentration of 50-300 mM; and / or In S22, with OD 600 The final concentrations of OMK-101 and OMK-102 added are calculated to be 10:10 to 40:40; and / or In S22, the pH of the reaction system is 6.5-8.0; and / or In S22, the reaction temperature is 25-40℃.

Images