A culture medium for fermentation of terminal deoxynucleotidyl transferase and its application

Abstract

This invention relates to the field of microbial fermentation technology, specifically disclosing a culture medium for fermenting terminal deoxynucleotidyl transferase (TDLT) and its applications. The invention provides a culture medium suitable for fermenting TDLT, comprising a basal medium and a fed medium. The basal medium includes a vitamin composition containing vitamin B3 and inositol, and a trace element composition containing cobalt and calcium. The fed medium includes a supplemented carbon source medium and a supplemented nitrogen source medium. This invention creatively discovers that adding vitamin B3 and inositol, along with the trace elements cobalt and calcium, during fermentation can significantly increase the yield of the target product, TDLT. Based on this culture medium, this invention further optimizes key parameters in the fermentation process, such as the amount and frequency of inducer addition and the induction temperature, providing a fermentation process for TDLT with significantly increased TdT yield and significantly reduced production costs.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and specifically discloses a culture medium for terminal deoxynucleotidyl transferase and its application. Background Technology

[0002] In recent years, synthetic biology has developed rapidly, and DNA synthesis is one of the most important supporting technologies in the field. DNA synthesis technologies mainly include chemical methods and enzymatic methods. Given the advantages of enzymatic DNA synthesis, such as a mild synthesis environment and environmental friendliness, it has attracted increasing attention.

[0003] Terminal deoxynucleotidyl transferase (TdT), also known as deoxyribonucleotidyl extranucleotide transferase (DNTT) or terminal transferase, is a template-free DNA polymerase that randomly adds deoxyribonucleotides (dNTPs) to the ends of primers, elongating them to produce high-molecular-weight DNA. It has been reported that TdT catalyzes the sequential addition of dNTPs to the (3'-OH) ends of single-stranded DNA (ssDNA) in the absence of a DNA template, generating random sequence DNA, or synthesizing DNA with specific sequences under controlled substrate conditions. Therefore, TdT is widely used in the synthesis of single-stranded nucleotides.

[0004] High-density expression of TdT is a key technology for its widespread application. However, there are few reports on high-density fermentation of TdT and its fermentation process in existing technologies. Conventional fermentation processes for preparing TdT generally suffer from low enzyme yield and high production costs. Summary of the Invention

[0005] To address the shortcomings of existing fermentation technologies for TdT synthesis, this invention provides a culture medium suitable for high-density TdT production through optimization of components such as vitamins and trace elements. Furthermore, it provides a fermentation process for a terminal deoxynucleotidyl transferase that significantly increases the yield of the target enzyme.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a culture medium for fermenting terminal deoxynucleotidyl transferase, which includes a basal culture medium and a fed culture medium.

[0008] The basal culture medium includes a vitamin composition and a trace element composition;

[0009] The vitamin composition includes vitamin B3 and inositol;

[0010] The trace element composition includes cobalt and calcium;

[0011] The fed culture medium includes a fed carbon source culture medium and a fed nitrogen source culture medium.

[0012] For example, the inositol is meso-inositol.

[0013] This invention provides a culture medium suitable for high-density fermentation of terminal deoxynucleotidyl transferase in recombinant Escherichia coli by optimizing the components of the culture medium. Under essentially the same culture conditions, this invention creatively discovers that adding vitamin B3 (such as nicotinamide) and inositol, as well as cobalt and calcium, during fermentation can significantly increase the yield of the target product, terminal deoxynucleotidyl transferase.

[0014] Preferably, the vitamin composition further includes vitamin B7, vitamin B5, vitamin B9, vitamin B6, vitamin B1, and vitamin B2.

[0015] Preferably, the trace element composition further includes manganese, zinc, molybdenum, copper, boron, and iron.

[0016] For example, the trace element composition comprises the following elements at concentrations: manganese 0.65 mg / L-0.98 mg / L, zinc 0.48 mg / L-0.96 mg / L, cobalt 0.20 mg / L-0.42 mg / L, molybdenum 0.35 mg / L-0.72 mg / L, calcium 0.27 mg / L-0.55 mg / L, copper 0.12 mg / L-0.25 mg / L, boron 0.17 mg / L-0.35 mg / L, and iron 4.0 mg / L-6.0 mg / L.

[0017] More preferably, the vitamin composition includes biotin, D-calcium pantothenate, folic acid, pyridoxal hydrochloride, thiamine hydrochloride, and riboflavin.

[0018] More preferably, the trace element composition includes manganese sulfate, zinc chloride, cobalt sulfate, ammonium molybdate, calcium chloride, copper sulfate, boric acid, and ferrous sulfate.

[0019] More preferably, the vitamin composition comprises the following components in mass concentrations: biotin 0.0005 mg / L-0.0015 mg / L, choline chloride 0.0005 mg / L-0.0015 mg / L, D-calcium pantothenate 0.0005 mg / L-0.0015 mg / L, folic acid 0.0005 mg / L-0.0015 mg / L, nicotinamide 0.0005 mg / L-0.0015 mg / L, pyridoxal hydrochloride 0.0005 mg / L-0.0015 mg / L, riboflavin 0.00001 mg / L-0.00002 mg / L, thiamine hydrochloride 0.0005 mg / L-0.0015 mg / L, and inositol 0.001 mg / L-0.003 mg / L.

[0020] The trace element composition comprises the following components in mass concentrations: manganese sulfate monohydrate 2 mg / L-3 mg / L, zinc chloride 1 mg / L-2 mg / L, cobalt sulfate heptahydrate 1 mg / L-2 mg / L, ammonium molybdate tetrahydrate 1 mg / L-2 mg / L, calcium chloride dihydrate 1 mg / L-2 mg / L, copper sulfate pentahydrate 0.5 mg / L-1 mg / L, boric acid 1 mg / L-2 mg / L, and ferrous sulfate heptahydrate 20 mg / L-30 mg / L.

[0021] Preferably, the basal culture medium also includes basic nutrients such as glycerol, yeast extract, peptone, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, and sodium citrate.

[0022] For example, the basal culture medium further includes the following basic nutrients at concentrations: glycerol 18 g / L-22 g / L, yeast extract 25 g / L-35 g / L, peptone 15 g / L-25 g / L, anhydrous dipotassium hydrogen phosphate 9.0 g / L-10 g / L, anhydrous sodium dihydrogen phosphate 2.0 g / L-3.0 g / L, anhydrous magnesium sulfate 1.0 g / L-2.0 g / L, ammonium sulfate 0.5 g / L-1.5 g / L, sodium citrate dihydrate 1.0 g / L-3.0 g / L, antifoaming agent 0.05 mL / L-0.30 mL / L, and kanamycin sulfate 0.05 g / L-0.20 g / L.

[0023] Preferably, the supplemental carbon source culture medium includes glycerol and magnesium sulfate.

[0024] For example, the supplemental carbon source culture medium also includes kanamycin sulfate.

[0025] Preferably, the supplemental nitrogen source culture medium includes yeast extract, peptone, dipotassium hydrogen phosphate, and sodium dihydrogen phosphate.

[0026] For example, the supplemental carbon source culture medium comprises the following components at the following concentrations: glycerol 600-1000 g / L, anhydrous magnesium sulfate 1.0-1.5 g / L, and kanamycin sulfate 0.05-0.20 g / L.

[0027] The supplemental nitrogen source culture medium comprises the following components at the following concentrations: yeast extract 300 g / L-500 g / L, peptone 100 g / L-300 g / L, anhydrous dipotassium hydrogen phosphate 100 g / L-150 g / L, and anhydrous sodium dihydrogen phosphate 50 g / L-70 g / L.

[0028] Secondly, the present invention provides the application of the culture medium of the above-mentioned terminal deoxynucleotidyl transferase in the high-density fermentation of terminal deoxynucleotidyl transferase in recombinant Escherichia coli.

[0029] Thirdly, the present invention provides a fermentation process for a terminal deoxynucleotidyl transferase, the fermentation process comprising the following steps:

[0030] S1. The TdT-producing recombinant Escherichia coli is subjected to initial fermentation in the basic culture described in the first aspect;

[0031] S2. After the initial fermentation is completed, the supplemented carbon source culture medium and the supplemented nitrogen source culture medium described in the first aspect are added in sequence to carry out supplemented fermentation;

[0032] S3. After the fed-batch fermentation is completed, isopropyl-β-D-thiogalactoside (IPTG) is used to induce expression for the first and second time at a temperature of 27.5 ℃-28.5 ℃ to obtain terminal deoxynucleotidyl transferase.

[0033] The fermentation process for terminal deoxynucleotidyl transferase provided by this invention not only conforms to the actual growth conditions of the cells, enabling the cells to grow and reproduce rapidly during the exponential phase and shorten the stagnation phase, but also extends the enzyme production cycle of the cells and promotes the accumulation of TdT, thereby achieving the goal of significantly increasing the enzyme production of TdT-producing recombinant Escherichia coli and reducing production costs.

[0034] Preferably, step S1 specifically includes the following steps:

[0035] Recombinant Escherichia coli seed culture producing TdT was inoculated into the basal medium per 1 L of basal medium and fermented for 6 h to 8 h at 35 ℃-42 ℃, pH 6.5-7.5, 180 rpm-220 rpm and aeration rate of 0.7 L / min-2.9 L / min, with the rotation speed correlated with dissolved oxygen.

[0036] In this invention, ventilation volume is the flow rate of sterile air introduced per unit time.

[0037] Preferably, during the initial fermentation stage, the dissolved oxygen level is maintained at 30%-70%.

[0038] During the initial fermentation stage, the dissolved oxygen level is monitored in real time. When the dissolved oxygen level is below 30%, oxygen supply is increased by increasing the rotation speed and aeration.

[0039] For example, the OD of the TdT-producing recombinant Escherichia coli seed culture 600 The dosage is 2-5, and the inoculation amount is 2%-4%.

[0040] Preferably, step S2 specifically includes the following steps:

[0041] S21. After the initial fermentation is completed, the feed carbon source medium is added at a feed rate of 14.2 g / h-20 g / h, based on 1 L of basal medium.

[0042] S22. After adding the supplemental carbon source medium per 1 L of basal medium, ferment at 35 ℃-42 ℃ until OD. 600 When the temperature reaches 55-65, add 62.8 mL-80 mL of the supplemental nitrogen source culture medium.

[0043] S23. After adding the supplemental nitrogen source culture medium, ferment at 35 ℃-42 ℃ until OD reaches [the desired growth rate]. 600 Once the concentration reaches 80% or higher, stop the feeding and fermentation process.

[0044] Preferably, step S3 specifically includes the following steps:

[0045] S31. Based on 1L of basal medium, after the fed-batch fermentation is completed, add 62.8mL-80mL of the fed-batch nitrogen source medium and 1.14mmol-2.57mmol of isopropyl-β-D-thiogalactoside, and induce expression for the first time for 6h-8h at 27.5℃-28.5℃ and a feeding rate of 12.8 g / h-15.7 g / h for the fed-batch carbon source medium.

[0046] S32. After the first induction expression was completed, 1.14 mmol-2.57 mmol of isopropyl-β-D-thiogalactoside was added per 1L of basal medium. The expression was induced for a second time at 27.5 ℃-28.5 ℃ for 8 h-10 h. The mixture was then transferred to a fermentation tank, the fermentation broth was harvested, the bacterial sludge was collected, the cells were broken up, centrifuged, filtered, and the protein was purified to obtain terminal deoxynucleotidyl transferase.

[0047] This invention develops a fermentation process for terminal deoxynucleotidyl transferase (TdT) by optimizing the composition of trace elements and vitamins in the culture medium, as well as key parameters such as the amount and frequency of inducer addition and induction temperature during fermentation. This fermentation process can significantly shorten the lag phase of TdT-producing recombinant E. coli, increase cell density, and significantly prolong its exponential and stationary phases, thus promoting the accumulation of the target product TdT, increasing the TdT enzyme yield, and reducing the production cost of TdT. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 This is a comparison diagram of cell growth curves in the fermentation process provided in Example 1 and Comparative Example 1 of the present invention;

[0050] Figure 2 This is a protein peak diagram of the target protein TdT in Example 1 of this invention;

[0051] Figure 3 The protein peak diagram of the target protein TdT in Comparative Example 4 was determined according to the present invention;

[0052] Figure 4 The protein peak diagram of the target protein TdT in Comparative Example 5 is shown in the figure. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0054] The TdT-producing recombinant Escherichia coli used in this invention is the pET-28a(+)-TdT strain constructed as described below, using Escherichia coli... E. coli BL21(DE3) was used as the host bacterial species. The inserted TdT gene was extracted from the white-throated finch (Zonotrichia albicollis), whose accession number is U53366.1. A recombinant E. coli expression system was constructed using the pET-28a plasmid to achieve TdT expression. The method for constructing this recombinant E. coli is disclosed in Chinese Patent CN115725675A, entitled: A method for biosynthesis of a long-fragment gene and its application.

[0055] Example 1

[0056] This invention provides a culture medium for fermenting terminal deoxynucleotidyl transferase, which includes a basal culture medium and a fed culture medium;

[0057] The basal culture medium includes basic nutrients, a vitamin composition, and a trace element composition, wherein the basic nutrients are specifically as follows:

[0058] The formula consists of 20 g / L glycerol, 30 g / L yeast extract, 20 g / L peptone, 9.4 g / L anhydrous dipotassium hydrogen phosphate, 2.2 g / L anhydrous sodium dihydrogen phosphate, 1.5 g / L anhydrous magnesium sulfate, 1 g / L ammonium sulfate, 2 g / L sodium citrate dihydrate, 0.1 mL / L defoamer, and 0.1 g / L kanamycin sulfate, with the remainder being water.

[0059] The specific vitamin composition is as follows:

[0060] Biotin 0.001 mg / L, choline chloride 0.001 mg / L, D-calcium pantothenate 0.001 mg / L, folic acid 0.001 mg / L, nicotinamide 0.001 mg / L, pyridoxal hydrochloride 0.001 mg / L, riboflavin 0.00001 mg / L, thiamine hydrochloride 0.001 mg / L, and meso-inositol 0.002 mg / L, with the balance being water;

[0061] The specific composition of trace elements is as follows:

[0062] The mixture contained 27 mg / L ferrous sulfate heptahydrate, 2.43 mg / L manganese sulfate monohydrate, 1.26 mg / L zinc chloride, 1.15 mg / L cobalt sulfate heptahydrate, 1.07 mg / L ammonium molybdate tetrahydrate, 1.46 mg / L calcium chloride dihydrate, 0.9 mg / L copper sulfate pentahydrate, and 1.21 mg / L boric acid, with the balance being water.

[0063] The fed culture medium includes a fed carbon source culture medium and a fed nitrogen source culture medium;

[0064] The supplemental carbon source culture medium included: 800 g / L glycerol, 1.2 g / L anhydrous magnesium sulfate, and 0.10 g / L kanamycin sulfate;

[0065] The supplemental nitrogen source culture medium includes: yeast extract 400 g / L, peptone 200 g / L, anhydrous dipotassium hydrogen phosphate 120 g / L and anhydrous sodium dihydrogen phosphate 60 g / L.

[0066] This invention also provides a fermentation process for terminal deoxynucleotidyl transferase, the fermentation process comprising the following steps:

[0067] S1. Initial fermentation stage

[0068] S11. Add 100 μL kanamycin sulfate (100 g / L) to LB medium. Inoculate the TdT-producing recombinant Escherichia coli strain preserved in glycerol tubes into the above medium at an inoculum volume of 0.02%, and culture at 33 ℃ and 180 rpm until OD. 600 The result was 4, yielding a recombinant Escherichia coli seed solution that produced TdT.

[0069] S12. Prepare 3.5L of basic culture medium according to the above-preset components of the basic culture medium and place it in a fermenter;

[0070] 100 mL of TdT-producing recombinant Escherichia coli seed culture was inoculated into 3.5 L of the above-mentioned basal medium and fermented for 6 h at 37 ℃, pH 6.90, 200 rpm, and an aeration rate of 2.5 L / min-10 L / min.

[0071] During the initial fermentation stage, the dissolved oxygen level in the culture medium is monitored in real time. When the dissolved oxygen level is below 30%, the oxygen supply is increased by increasing the rotation speed and aeration rate. During this stage, the dissolved oxygen level is maintained between 30% and 70%.

[0072] S2. Fed-up Fermentation Stage

[0073] S21. After the initial fermentation is completed, the above-mentioned feed carbon source medium is added at a feed rate of 60 g / h;

[0074] S22. After adding the prepared carbon source medium, fermentation was carried out at 37 ℃, 200 rpm, and an aeration rate of 2.5 L / min-10 L / min until OD reached. 600 When the concentration reaches 60, add 250 mL of supplemental nitrogen source culture medium at once;

[0075] S23. After adding supplemental nitrogen source to the culture medium, ferment at 37 ℃, 200 rpm, and appropriate aeration rate until OD reaches [the desired growth rate]. 600 Once the temperature reaches 80, the feeding and fermentation process is complete.

[0076] S3. Induction of Expression Phase

[0077] S31. After the fed-batch fermentation was completed, the temperature of the culture medium was lowered to 28 ℃, the feeding rate of the fed-batch carbon source culture medium was reduced to 48 g / h, 250 mL of fed-batch nitrogen source culture medium and 7 mL of isopropyl-β-D-thiogalactoside (IPTG) at a concentration of 1 mol / L were added, and the first induction expression was carried out at 28 ℃, 200 rpm and appropriate aeration for 7 h.

[0078] S32. After the first induction expression was completed, 7 mL of 1 mol / L IPTG was added, and the second induction expression was carried out for 9 h under the same culture conditions as the first induction expression. The culture was then transferred to a tank, the fermentation broth was harvested, the bacterial sludge was collected, the cells were broken up, centrifuged, filtered, and the protein was purified to obtain terminal deoxynucleotidyl transferase.

[0079] Throughout the fermentation process, the dissolved oxygen level in the culture medium should be maintained at no less than 30%.

[0080] Example 2

[0081] This invention provides a culture medium for fermenting terminal deoxynucleotidyl transferase, which includes a basal culture medium and a fed culture medium;

[0082] The basal culture medium includes basic nutrients, a vitamin composition, and a trace element composition, wherein the basic nutrients are specifically as follows:

[0083] The formula contains 18 g / L glycerol, 35 g / L yeast extract, 25 g / L peptone, 9 g / L anhydrous dipotassium hydrogen phosphate, 2 g / L anhydrous sodium dihydrogen phosphate, 1 g / L anhydrous magnesium sulfate, 0.5 g / L ammonium sulfate, 3 g / L sodium citrate dihydrate, 0.1 mL / L defoamer, and 0.1 g / L kanamycin sulfate, with the balance being water.

[0084] The specific vitamin composition is as follows:

[0085] Biotin 0.0015 mg / L, choline chloride 0.0015 mg / L, D-calcium pantothenate 0.0015 mg / L, folic acid 0.0015 mg / L, nicotinamide 0.0015 mg / L, pyridoxal hydrochloride 0.0015 mg / L, riboflavin 0.000015 mg / L, thiamine hydrochloride 0.0015 mg / L, and meso-inositol 0.003 mg / L, with the balance being water;

[0086] The specific composition of trace elements is as follows:

[0087] The mixture contains 20 mg / L ferrous sulfate heptahydrate, 2 mg / L manganese sulfate monohydrate, 2 mg / L zinc chloride, 1 mg / L cobalt sulfate heptahydrate, 2 mg / L ammonium molybdate tetrahydrate, 2 mg / L calcium chloride dihydrate, 1 mg / L copper sulfate pentahydrate, and 2 mg / L boric acid, with the remainder being water.

[0088] The fed culture medium includes a fed carbon source culture medium and a fed nitrogen source culture medium;

[0089] The supplemental carbon source culture medium included: 600 g / L glycerol, 1.5 g / L anhydrous magnesium sulfate, and 0.05 g / L kanamycin sulfate;

[0090] The supplemental nitrogen source culture medium includes: yeast extract 300 g / L, peptone 300 g / L, anhydrous dipotassium hydrogen phosphate 100 g / L and anhydrous sodium dihydrogen phosphate 70 g / L.

[0091] This invention also provides a fermentation process for terminal deoxynucleotidyl transferase, the fermentation process comprising the following steps:

[0092] S1. Initial fermentation stage

[0093] S11. Using the same method as described in Example 1, a seed culture of recombinant Escherichia coli producing TdT was prepared.

[0094] S12. Prepare 3.5 L of basic culture medium according to the above-preset components of the basic culture medium and place it in a fermenter;

[0095] 100 mL of the TdT-producing recombinant Escherichia coli seed culture was inoculated into the above 3.5 L basal medium and fermented for 8 h at 35 °C, pH 6.50, 180 rpm, and an aeration rate of 2.5 L / min-10 L / min.

[0096] During the initial fermentation stage, the dissolved oxygen level in the culture medium is monitored in real time. When the dissolved oxygen level is below 30%, the oxygen supply is increased by increasing the rotation speed and aeration rate. During this stage, the dissolved oxygen level is maintained between 30% and 70%.

[0097] S2. Fed-up Fermentation Stage

[0098] S21. After the initial fermentation is completed, the above-mentioned feed carbon source medium is added at a feed rate of 50 g / h;

[0099] S22. After adding the prepared carbon source medium, ferment at 35°C, 180 rpm, and 2.5 L / min aeration rate until OD is reached. 600 When the concentration reaches 55, add 220 mL of supplemental nitrogen source culture medium at once;

[0100] S23. After adding supplemental nitrogen source to the culture medium, ferment at 35 ℃, pH 6.50, 180 rpm, and appropriate aeration rate until OD reaches [the desired growth rate]. 600 Once the temperature reaches 80, the feeding and fermentation process is complete.

[0101] S3. Induction of Expression Phase

[0102] S31. After the fed-batch fermentation was completed, the temperature of the culture medium was lowered to 27.5 ℃, the feeding rate of the fed-batch carbon source culture medium was reduced to 45 g / h, 220 mL of fed-batch nitrogen source culture medium and 4 mL of 1 mol / L IPTG were added, and the first induction expression was carried out for 6 h at 27.5 ℃, 180 rpm and appropriate aeration rate.

[0103] S32. After the first induction expression was completed, 9 mL of 1 mol / L IPTG was added, and the second induction expression was carried out for 10 h under the same culture conditions as the first induction expression. The culture was then transferred to a tank, the fermentation broth was harvested, the bacterial sludge was collected, the cells were broken up, centrifuged, filtered, and the protein was purified to obtain terminal deoxynucleotidyl transferase.

[0104] Throughout the fermentation process, the dissolved oxygen level in the culture medium should be maintained at no less than 30%.

[0105] Example 3

[0106] This invention provides a culture medium for fermenting terminal deoxynucleotidyl transferase, which includes a basal culture medium and a fed culture medium;

[0107] The basal culture medium includes basic nutrients, a vitamin composition, and a trace element composition, wherein the basic nutrients are specifically as follows:

[0108] The formula consists of 22 g / L glycerol, 25 g / L yeast extract, 15 g / L peptone, 10 g / L anhydrous dipotassium hydrogen phosphate, 3 g / L anhydrous sodium dihydrogen phosphate, 2 g / L anhydrous magnesium sulfate, 1.5 g / L ammonium sulfate, 1 g / L sodium citrate dihydrate, 0.1 mL / L defoamer, and 0.1 g / L kanamycin sulfate, with the remainder being water.

[0109] The specific vitamin composition is as follows:

[0110] Biotin 0.0005 mg / L, choline chloride 0.0005 mg / L, D-calcium pantothenate 0.0005 mg / L, folic acid 0.0005 mg / L, nicotinamide 0.0005 mg / L, pyridoxal hydrochloride 0.0005 mg / L, riboflavin 0.00002 mg / L, thiamine hydrochloride 0.0005 mg / L, and meso-inositol 0.001 mg / L, with the balance being water;

[0111] The specific composition of trace elements is as follows:

[0112] The mixture contains 30 mg / L ferrous sulfate heptahydrate, 3 mg / L manganese sulfate monohydrate, 1 mg / L zinc chloride, 2 mg / L cobalt sulfate heptahydrate, 1 mg / L ammonium molybdate tetrahydrate, 1 mg / L calcium chloride dihydrate, 0.5 mg / L copper sulfate pentahydrate, and 1 mg / L boric acid, with the remainder being water.

[0113] The fed culture medium includes a fed carbon source culture medium and a fed nitrogen source culture medium;

[0114] The supplemental carbon source culture medium includes: 1000 g / L glycerol, 1 g / L anhydrous magnesium sulfate, and 0.2 g / L kanamycin sulfate;

[0115] The supplemental nitrogen source culture medium includes: yeast extract 500 g / L, peptone 100 g / L, anhydrous dipotassium hydrogen phosphate 150 g / L and anhydrous sodium dihydrogen phosphate 50 g / L.

[0116] This invention also provides a fermentation process for terminal deoxynucleotidyl transferase, the fermentation process comprising the following steps:

[0117] S1. Initial fermentation stage

[0118] S11. Using the same method as described in Example 1, a seed culture of recombinant Escherichia coli producing TdT was prepared.

[0119] S12. Prepare 3.5L of basic culture medium according to the above-preset components of the basic culture medium and place it in a fermenter;

[0120] 100 mL of the TdT-producing recombinant Escherichia coli seed culture was inoculated into the above 3.5 L basal medium and fermented for 7 h at 42 °C, pH 7.50, 220 rpm, and an aeration rate of 2.5 L / min-10 L / min.

[0121] During the initial fermentation stage, the dissolved oxygen level in the culture medium is monitored in real time. When the dissolved oxygen level is below 30%, the oxygen supply is increased by increasing the rotation speed and aeration rate. During this stage, the dissolved oxygen level is maintained between 30% and 70%.

[0122] S2. Fed-up Fermentation Stage

[0123] S21. After the initial fermentation is completed, the above-mentioned fed carbon source medium is added at a feeding rate of 70 g / h;

[0124] S22. After adding the prepared carbon source medium, fermentation is carried out at 42℃, 220 rpm, and an aeration rate of 2.5 L / min-10 L / min until OD reaches the target value. 600 When the temperature reaches 65, add 280 mL of supplemental nitrogen source culture medium at once;

[0125] S23. After adding supplemental nitrogen source to the culture medium, ferment at 42 ℃, 220 rpm, and appropriate aeration rate until OD reaches [the desired growth rate]. 600 Once the temperature reaches 80, the feeding and fermentation process is complete.

[0126] S3. Induction of Expression Phase

[0127] S31. After the fed-batch fermentation was completed, the temperature of the culture medium was lowered to 28.5 ℃, the feeding rate of the fed-batch carbon source culture medium was reduced to 45 g / h, 280 mL of fed-batch nitrogen source culture medium and 9 mL of 1 mol / L IPTG were added, and the first induction expression was carried out at 28.5 ℃, 220 rpm and appropriate aeration for 8 h.

[0128] S32. After the first induction expression was completed, 4 mL of 1 mol / L IPTG was added, and the second induction expression was carried out for 8 h under the same culture conditions as the first induction expression. The culture was then transferred to a tank, the fermentation broth was harvested, the bacterial sludge was collected, the cells were broken up, centrifuged, filtered, and the protein was purified to obtain terminal deoxynucleotidyl transferase.

[0129] Throughout the fermentation process, the dissolved oxygen level in the culture medium should be maintained at no less than 30%.

[0130] Comparative Example 1

[0131] The present invention provides a comparative example of a fermentation process for terminal deoxynucleotidyl transferase. The fermentation process is basically the same as that in Example 1, except that the basic culture medium in Example 1 is replaced with the conventional culture medium for high-density culture of Escherichia coli - TB medium. The components, amounts and fermentation process parameters of the other culture media are the same as those in Example 1.

[0132] Comparative Example 2

[0133] This invention provides a comparative example of a fermentation process for terminal deoxynucleotidyl transferase. The fermentation process is essentially the same as in Example 1, except that the amount of inducer added in step S3 of Example 1 is reduced and only one induction culture is performed. However, the total induction culture time remains unchanged, and the culture medium composition, dosage, and other fermentation process parameters are the same as in Example 1. Step S3 is specifically replaced with:

[0134] After the fed-batch fermentation was completed, the culture medium temperature was lowered to 28 ℃, the feeding rate of the fed-batch carbon source medium was reduced to 48 g / h, 250 mL of fed-batch nitrogen source medium and 7 mL of 1 mol / L IPTG were added, and expression was induced for 16 h at 28 ℃, 200 rpm and appropriate aeration. The culture was then transferred to a tank, the fermentation broth was harvested, the bacterial sludge was collected, the cells were broken up, centrifuged, filtered and the protein was purified to obtain terminal deoxynucleotidyl transferase.

[0135] Comparative Example 3

[0136] The present invention provides a comparative example of a fermentation process for terminal deoxynucleotidyl transferase. The fermentation process is basically the same as that in Example 1, except that the induction culture temperature in step S3 of Example 1 is replaced with 25°C instead of 28°C. The composition and amount of the culture medium and the remaining fermentation process parameters are the same as those in Example 1.

[0137] Comparative Example 4

[0138] The present invention provides a comparative example of a fermentation process for terminal deoxynucleotidyl transferase. The fermentation process is basically the same as that in Example 1, except that: based on the basic culture medium provided in Example 1, cobalt and calcium elements in the trace element composition are not added, that is, cobalt sulfate heptahydrate and calcium chloride dihydrate are not added. The composition, amount and fermentation process parameters of the other culture media are the same as those in Example 1.

[0139] Comparative Example 5

[0140] The present invention provides a comparative example of a fermentation process for terminal deoxynucleotidyl transferase, which is basically the same as that in Example 1, except that: based on the basic culture medium provided in Example 1, nicotinamide and meso-inositol in the vitamin composition are not added, and the components, amounts and fermentation process parameters of the other culture media are the same as those in Example 1.

[0141] Example 1

[0142] This invention detects OD at different fermentation times. 600 This reflects the cell growth status. In the fermentation processes of terminal deoxynucleotidyl transferases provided in Examples 1-3 and Comparative Example 1, cell OD was measured every 2 hours. 600 The absorbance was measured at a wavelength of 600 nm using a spectrophotometer. Different dilutions were performed to maintain the absorbance between 0.2 and 0.8. OD 600 The calculation method is absorbance value × dilution factor. This invention uses the cell growth curves in the fermentation process of Example 1 and Comparative Example 1 as examples for illustration. A comparison of the cell growth curves in the fermentation process provided in Example 1 and Comparative Example 1 is shown below. Figure 1 As shown, Example 1 is referred to as the experimental group, and Comparative Example 1 is referred to as the control group 1.

[0143] Depend on Figure 1 It can be seen that in the early stage of fermentation, as the culture time increases, the cell OD... 600 The growth rate gradually increased. The experimental group experienced a lag phase of 0-2 h, an exponential phase of 2 h-18 h, and a stationary phase of 18 h-30 h. For control group 1, the lag phase was 0-4 h, the exponential phase was 4 h-12 h, the stationary phase was 12 h-18 h, and the decline phase was 18 h-20 h. From the above comparison, it can be seen that, compared to TB medium, the basal medium used in Example 1 of this invention significantly shortened the lag phase of TdT-producing recombinant E. coli, significantly prolonged the exponential phase, allowing the strain to grow sufficiently and significantly increasing cell density. Furthermore, Example 1 of this invention also significantly prolonged the stationary phase of TdT-producing recombinant E. coli.

[0144] In summary, the fermentation process of terminal deoxynucleotidyl transferase provided by this invention can significantly shorten the lag phase of TdT-producing recombinant Escherichia coli, increase cell density, and significantly prolong its exponential and stationary phases, which helps the accumulation of the target product TdT enzyme. This can improve the yield of the target product and reduce production costs in the fermentation process.

[0145] Example 2

[0146] After obtaining the fermentation broth in this invention, the terminal deoxynucleotidyl transferase is purified and the enzyme yield of different embodiments or comparative examples is measured through the following steps:

[0147] Take the same volume of fermentation broth from different groups (2L in this invention) for cell disruption, centrifugation, filtration, protein purification, loading onto the machine, and calculating the target enzyme yield.

[0148] (1) Cell disruption: The bacterial precipitate obtained in the above examples or comparative examples was disrupted twice using a high-pressure low-temperature disruptor at a pressure of 1200 bar and a temperature of 4°C. After centrifugation at 4°C and 10000 r / min for 45 min, the precipitate and supernatant were collected for sample preparation.

[0149] (2) Purification: The supernatant was filtered through a 0.45 mm microporous membrane and purified by nickel affinity chromatography. The specific steps are as follows:

[0150] Step 1: Column Balancing

[0151] Before attaching the column, wash with ddH2O for 2 column volumes, then equilibrate the Ni affinity chromatography column with protein buffer for 1 column volume.

[0152] Step 2: Sample loading

[0153] The supernatant was slowly passed through the Ni affinity chromatography column at a flow rate of 0.5 mL / min, and this process was repeated once more.

[0154] Step 3: Elution of impurities

[0155] Wash one column volume with protein buffer, then elute strongly bound contaminating proteins with 50 mL of protein buffer containing 50 mM imidazole. Take the first 5-8 drops to flow through the sample and prepare the sample.

[0156] Step 4: Elute the target protein

[0157] The target protein was eluted with 20 mL of 500 mM imidazole protein buffer. The first 5-8 drops were used to flow through the sample for sample preparation and detection by 12% SDS-PAGE.

[0158] (3) Concentration and liquid replacement

[0159] The collected target protein was concentrated by centrifugation (4℃, 3400 r / min) using a 50 mL Amicon ultrafiltration tube (30 kDa, Millipore) to a final volume of 1 mL. 10 mL of protein buffer was added, and the concentration was repeated once to obtain purified protein TdT.

[0160] (4) On-machine testing

[0161] The concentrations of purified protein TdT in the above-mentioned different groups were determined using a protein purification instrument at a wavelength of 280 nm.

[0162] For example, the protein peak diagram of the target protein TdT in Example 1 is shown below. Figure 2 As shown, the protein peak diagram of the target protein TdT in Comparative Example 4 is as follows. Figure 3 As shown, the protein peak diagram of the target protein TdT in Comparative Example 5 is as follows. Figure 4 As shown.

[0163] Table 1 shows the peak area statistics of the target protein TdT in different groups.

[0164] Table 1

[0165]

[0166] Note: The same letter on the shoulder label indicates that the difference is not significant, while different letters indicate that the difference is significant.

[0167] As shown in Table 1, under the same fermentation broth conditions, the peak area of ​​TdT obtained by the fermentation process of terminal deoxynucleotidyl transferase provided in Examples 1-3 of this invention is significantly higher than that of Comparative Examples 1-5.

[0168] Specifically, the yield of the target enzyme TdT in Example 1 was 1.64 times that of Comparative Example 1. Compared to TB medium, the basal medium provided in Example 1 has a positive impact on increasing the target enzyme yield and reducing production costs.

[0169] In Example 1, the yield of the target enzyme TdT was 2.31 times that of Comparative Example 2 and 1.66 times that of Comparative Example 3. This indicates that the appropriate induction temperature, the number of IPTG inductions, and the amount added have a significant impact on promoting the expression of the target enzyme TdT.

[0170] In Example 1, the yield of the target enzyme TdT was 1.34 times that of Comparative Example 4 and 1.25 times that of Comparative Example 5. This indicates that vitamin B3 (such as nicotinamide) and inositol, as well as cobalt and calcium, play a positive role in promoting the expression of the target enzyme TdT.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A medium for recombinant Escherichia coli high-density fermentation of endo-deoxyribonucleotidyl transferase, characterized in that, consists of a basal medium and a feed medium; the basal medium consists of basal nutrients, a vitamin composition and a trace element composition; the vitamin composition consists of vitamin B3, myo-inositol, vitamin B7, vitamin B5, vitamin B9, vitamin B6, vitamin B1, vitamin B2 and choline chloride; the vitamin composition consists of the following components with the following mass concentrations: biotin 0.0005 mg / L-0.0015 mg / L, choline chloride 0.0005 mg / L-0.0015 mg / L, D-calcium pantothenate 0.0005 mg / L-0.0015 mg / L, folic acid 0.0005 mg / L-0.0015 mg / L, nicotinamide 0.0005 mg / L-0.0015 mg / L, pyridoxal hydrochloride 0.0005 mg / L-0.0015 mg / L, riboflavin 0.00001 mg / L-0.00002 mg / L, thiamine hydrochloride 0.0005 mg / L-0.0015 mg / L, myo-inositol 0.001 mg / L-0.003 mg / L; the trace element composition consists of the following elements with the following concentrations: manganese element 0.65 mg / L-0.98 mg / L, zinc element 0.48 mg / L-0.96 mg / L, cobalt element 0.20 mg / L-0.42 mg / L, molybdenum element 0.35 mg / L-0.72 mg / L, calcium element 0.27 mg / L-0.55 mg / L, copper element 0.12 mg / L-0.25 mg / L, boron element 0.17 mg / L-0.35 mg / L and iron element 4.0 mg / L-6.0 mg / L; the feed medium comprises a feed carbon source medium and a feed nitrogen source medium.

2. The medium for reconstituting endogenous deoxynucleotidyl transferase of E. coli for high-density fermentation of claim 1, characterized by, the trace element composition is manganese sulfate, zinc chloride, cobalt sulfate, ammonium molybdate, calcium chloride, copper sulfate, boric acid and ferrous sulfate.

3. The medium for reconstituting endogenous deoxynucleotidyl transferase of E. coli for high-density fermentation according to claim 1, characterized by, the trace element composition consists of the following components with the following mass concentrations: manganese sulfate monohydrate 2 mg / L-3 mg / L, zinc chloride 1 mg / L-2 mg / L, cobalt sulfate heptahydrate 1 mg / L-2 mg / L, ammonium molybdate tetrahydrate 1 mg / L-2 mg / L, calcium chloride dihydrate 1 mg / L-2 mg / L, copper sulfate pentahydrate 0.5 mg / L-1 mg / L, boric acid 1 mg / L-2 mg / L and ferrous sulfate heptahydrate 20 mg / L-30 mg / L.

4. The medium for reconstituting endogenous deoxynucleotidyl transferase of E. coli for high-density fermentation according to claim 1, characterized by, the basal nutrients include glycerol, yeast extract, peptone, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, ammonium sulfate and sodium citrate; and / or the feed carbon source medium comprises glycerol and magnesium sulfate; and / or the feed nitrogen source medium comprises yeast extract, peptone, dipotassium hydrogen phosphate and sodium dihydrogen phosphate.

5. Use of the medium for fermenting terminal deoxynucleotidyl transferase according to any one of claims 1-4 in the high-density fermentation of recombinant Escherichia coli for terminal deoxynucleotidyl transferase.

6. A fermentation process for terminal deoxynucleotidyl transferase, characterized in that, the fermentation process comprises the following steps: S1. carrying out initial fermentation of the recombinant Escherichia coli producing TdT in the base medium described in any one of claims 1-4; S2. after the initial fermentation is completed, sequentially adding the feed carbon source medium and the feed nitrogen source medium described in any one of claims 1-4 to carry out fed-batch fermentation; S3. after the fed-batch fermentation is completed, carrying out twice induction expression at 27.5 ℃-28.5 ℃ using isopropyl-β-D-thiogalactoside to obtain terminal deoxynucleotidyl transferase.

7. The fermentation process of terminal deoxynucleotidyl transferase as claimed in claim 6, wherein, Step S1 specifically comprises the following steps: Inoculating the seed liquid of the recombinant Escherichia coli producing TdT into the base medium at a ratio of 1 L of the base medium per 1 L of the seed liquid, and carrying out fermentation at 35 ℃-42 ℃, pH 6.5-7.5, 180 rpm-220 rpm, and 0.7 L / min-2.9 L / min of aeration rate for 6 h-8 h; and / or During the initial fermentation stage, the dissolved oxygen level is maintained at 30%-70%.

8. The fermentation process of terminal deoxynucleotidyl transferase as claimed in claim 6, wherein, Step S2 specifically comprises the following steps: S21. after the initial fermentation is completed, adding the feed carbon source medium at a feed rate of 14.2 g / h-20 g / h per 1 L of the base medium; S22. After the addition of the feed carbon source medium, the fermentation is carried out at 35-42°C to an OD 600 of 55-65, 62.8 mL-80 mL of the feed nitrogen source medium is added; S23. After the addition of the feed nitrogen source medium, fermentation is carried out at 35-42°C until OD 600 above 80, the feed fermentation is terminated.

9. The fermentation process of terminal deoxynucleotidyl transferase according to any one of claims 6 to 8, characterized in that, Step S3 specifically comprises the following steps: S31. after the fed-batch fermentation is completed, adding 62.8 mL-80 mL of the feed nitrogen source medium, 1.14 mmol-2.57 mmol of isopropyl-β-D-thiogalactoside per 1 L of the base medium, and carrying out first induction expression at 27.5 ℃-28.5 ℃ for 6 h-8 h at a feed rate of 12.8 g / h-15.7 g / h of the feed carbon source medium; S32. after the first induction expression is completed, adding 1.14 mmol-2.57 mmol of isopropyl-β-D-thiogalactoside per 1 L of the base medium, and carrying out second induction expression at 27.5 ℃-28.5 ℃ for 8 h-10 h to obtain terminal deoxynucleotidyl transferase.

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