CN111676202B

CN111676202B - Fermentation process for coexpression of hydroxysteroid dehydrogenase

Info

Publication number: CN111676202B
Application number: CN202010779952.3A
Authority: CN
Inventors: 赵志斌; 程雷; 王丹丹; 丁峰; 张苏敏; 罗德彬
Original assignee: Chongqing Jize Biotechnology Co ltd
Current assignee: Chongqing Jize Biotechnology Co ltd
Priority date: 2020-08-05
Filing date: 2020-08-05
Publication date: 2023-04-25
Anticipated expiration: 2040-08-05
Also published as: CN111676202A

Abstract

The invention relates to the field of biological medicine, in particular to an industrial fermentation process of hydroxysteroid dehydrogenase, which comprises the following steps of induction fermentation culture: the engineering bacteria to be expressed sequentially pass through a culture stage, a feeding stage and a lactose induction stage to express hydroxysteroid dehydrogenase; a high-density fermentation medium is used in the culture stage, wherein the high-density fermentation medium comprises basic components and trace element components, and the trace element components comprise calcium, divalent manganese and trivalent iron. The method is used for solving the technical problems of low proliferation speed of engineering bacteria and low expression quantity and activity of target proteins when lactose is used as an inducer to induce the engineering bacteria to express the target proteins. The scheme can be used for industrial preparation of tauroursodeoxycholic acid, and is used for improving the production efficiency of tauroursodeoxycholic acid and reducing the production cost.

Description

Fermentation process for coexpression of hydroxysteroid dehydrogenase

Technical Field

The invention relates to the field of biological medicine, in particular to an industrial fermentation process of hydroxysteroid dehydrogenase.

Background

Tauroursodeoxycholic acid (TUDCA) is a main component contained in rare traditional Chinese medicine bear bile. The fowl bile does not contain TUDCA, but contains chenodeoxycholic acid (TCDCA) as main ingredient. TUDCA and TCDCA are structurally epimers of the hydroxyl group at position 7, and 7α -hydroxysteroid dehydrogenase and 7β -hydroxysteroid dehydrogenase can catalyze the conversion of TCDCA to TCDCA. At present, the 7 alpha-hydroxysteroid dehydrogenase and the 7 beta-hydroxysteroid dehydrogenase are prepared by large-scale high-density fermentation of recombinant engineering bacteria. However, in the prior art, the preparation of two enzymes, 7. Alpha. -hydroxysteroid dehydrogenase and 7. Beta. -hydroxysteroid dehydrogenase, has the following problems: lactose operon is an E.coli operon which has been studied more recently, has been widely used in the construction of prokaryotic expression vectors, and the expression of genes for 7α -hydroxysteroid dehydrogenase and 7β -hydroxysteroid dehydrogenase is mostly controlled by lactose operon. Lactose operon is typically initiated by the inducer IPTG (isopropyl- β -D thiogalactoside), i.e.: in the production of both the 7α -hydroxysteroid dehydrogenase and 7β -hydroxysteroid dehydrogenase enzymes using recombinant engineering bacteria, the inducer IPTG is required to initiate the lactose operon during the induction expression phase of the fermentation process. IPTG, while a highly potent inducer, has long been shown to be potentially toxic to humans and is expensive to manufacture, and therefore is only suitable for use in laboratory small-scale protein expression and not for use in industrial large-scale production. The enzyme 7 alpha-hydroxysteroid dehydrogenase and 7 beta-hydroxysteroid dehydrogenase can be used as an important raw material and auxiliary material in the field of food and medicine production and processing, and the production and processing processes of the two enzymes also need to be safe and nontoxic so as to meet the requirements of practical application. Although attempts have been made to induce expression of engineering bacteria using lactose instead of IPTG in the prior art, the addition of lactose causes retardation of bacterial growth, reduction of the expression level of the target protein, and reduction of the activity of the target protein. In particular, in industrial production, the production scale is often in ton-scale units, the fermentation expression system is huge, and when lactose is used as an inducer, the proliferation of engineering bacteria and the efficiency of expressing target proteins are severely inhibited. There is a need to find a method which can not only avoid toxicity caused by IPTG residues, but also effectively promote the expression and activity of target proteins, and the method can be applied to industrial scale-up production methods.

Disclosure of Invention

The invention aims to provide a fermentation process for co-expressing hydroxysteroid dehydrogenase, which is used for solving the technical problems of low proliferation speed of engineering bacteria and low expression quantity and activity of target proteins when lactose is used as an inducer to induce the engineering bacteria to express the target proteins.

In order to achieve the above purpose, the invention adopts the following technical scheme:

an industrial fermentation process of hydroxysteroid dehydrogenase, comprising an induction fermentation culture step using lactose as an inducer, the induction fermentation culture step comprising a culture stage of: and culturing the engineering bacteria to be expressed by using a high-density fermentation culture medium, wherein the high-density fermentation culture medium comprises basic components and trace element components, and the trace element components comprise calcium, divalent manganese and trivalent iron.

The principle and the advantages of the scheme are as follows: the engineering bacteria capable of expressing the hydroxysteroid dehydrogenase are cultured by using a high-density fermentation medium, and are fully amplified through passage and continuous feed supplement, so that a large number of thalli with high expression activity are obtained, and then the hydroxysteroid dehydrogenase is efficiently expressed through low-temperature induction. The trace element components are added in the scheme, so that the high-density fermentation medium is formed, the propagation of engineering bacteria can be effectively promoted, and the bacteria with high expression activity can be obtained. And then supplementing other culture mediums to supplement nutrient substances required by cell proliferation and expression, and finally using lactose as an inducer to induce engineering bacteria to express hydroxysteroid dehydrogenase so as to obtain a large amount of high-activity target product hydroxysteroid dehydrogenase. The engineering bacteria capable of expressing the hydroxysteroid dehydrogenase can be obtained by integrating the hydroxysteroid dehydrogenase gene on an expression vector by the existing molecular cloning technology, then entering the escherichia coli through transgenosis, and utilizing a protein expression system of the escherichia coli to produce the hydroxysteroid dehydrogenase in a large quantity.

In the production of artificial bear gall powder, it is necessary to convert taurochenodeoxycholic acid into tauroursodeoxycholic acid by using 7 alpha-hydroxysteroid dehydrogenase and 7 beta-hydroxysteroid dehydrogenase, while the natural yield of hydroxysteroid dehydrogenase is low, it is necessary to artificially construct engineering bacteria capable of expressing hydroxysteroid dehydrogenase and induce the expression of the target product. Because of the characteristics of low toxicity and low price of lactose, the inventor selects lactose as an inducer to induce the expression of engineering bacteria, but the addition of lactose can inhibit the proliferation of the engineering bacteria to a certain extent, the protein expression efficiency of the engineering bacteria is affected, and a large number of misfolded inclusion bodies without enzyme activity can be obtained. This phenomenon is more serious in the industrial expansion production, and various parameter conditions become complicated and uncontrollable due to the expansion of the system, further reducing the production efficiency of hydroxysteroid dehydrogenase. The inventors tried various methods to reduce the negative effect of lactose on the production efficiency, and found that the use of a specific culture medium (containing trace element components of calcium ions, divalent manganese ions and trivalent iron ions) for culturing engineering bacteria in the culturing stage can increase the proliferation rate of the engineering bacteria, and in the subsequent lactose induction process, the engineering bacteria can be quickly adapted to lactose environment, the expression efficiency of hydroxysteroid dehydrogenase of the engineering bacteria is improved, and the production amount of inclusion bodies is reduced. The three microelements can stimulate the growth of engineering bacteria and adapt to the fermentation environment containing lactose, and the reason for analyzing the phenomenon is that the three microelements can assist the engineering bacteria to prepare for resisting stress and protein expression.

In sum, the beneficial effect of this scheme lies in:

(1) The method can be used for large-scale expression of the hydroxysteroid dehydrogenase in a ton-scale fermentation system, and has high expression efficiency and activity and small inclusion body production. Recombinant escherichia coli is an expression system with the highest production speed and highest yield of the accepted recombinant enzyme in the world, and at present, the fermentation volume of hydroxysteroid dehydrogenase engineering bacteria is generally 50L-200L, and the enzyme-containing thalli which can be collected in a single batch is less than 20kg. By adopting the method, the single-batch fermentation volume can be increased to 30000L, and the single-batch enzyme-containing thallus yield can be increased to 1500kg.

(2) The proposal does not use toxic substance IPTG, uses lactose which can be used as food to induce the expression of hydroxysteroid dehydrogenase, and widens the application of producing hydroxysteroid dehydrogenase by engineering bacteria (used in the processing industries of food, health care products, medicines and the like). The hydroxysteroid dehydrogenase is used for preparing artificial bear gall powder, if IPTG is used for inducing engineering bacteria to express the hydroxysteroid dehydrogenase, the obtained enzyme contains a certain amount of toxic impurity components, so that the hydroxysteroid dehydrogenase prepared by the method cannot be used for processing foods, medicines and the like. And lactose has the advantage of low price compared with IPTG, and can further reduce the production cost. In addition, antibiotics such as ampicillin (kanamycin sulfate is used only in a small amount in the seed preparation stage) are not added in the induced fermentation culture, so that the safety of food is improved.

(3) The process can improve the expression efficiency of the hydroxysteroid dehydrogenase, reduce the generation amount of inclusion bodies, and solve the problems of poor growth condition and low protein expression activity of engineering bacteria when lactose is used as an inducer.

Further, the base component comprises: 1.75-4.00g/L of yeast extract, 3.5-8.0g/L of peptone, 1.5-2.5g/L of magnesium sulfate heptahydrate, 2.5-4.0g/L of potassium dihydrogen phosphate, 4.5-8.0g/L of ammonium chloride, 10.0-15.0g/L of disodium hydrogen phosphate dodecahydrate and 12.5-25.0g/L of glycerin; the trace element components comprise: 0.01-0.08g/L of calcium chloride hexahydrate, 0.01-0.10g/L of manganous chloride tetrahydrate and 0.01-0.10g/L of ferric chloride hexahydrate.

By adopting the scheme, the basic components of the scheme are different from the prior art, the content of the organic nitrogen source is reduced, the content of the inorganic nitrogen source is improved, the proliferation efficiency can be improved, and the expression of a target product is further promoted.

Calcium chloride hexahydrate is used for providing divalent calcium, manganous chloride tetrahydrate is used for providing divalent manganese, ferric chloride hexahydrate is used for providing divalent iron, and the three salts can be replaced by other salts containing divalent calcium, divalent manganese or trivalent iron and being used as additives of a culture medium according to actual conditions and common knowledge. The trace elements are kept in the range, so that the growth of engineering bacteria can be stimulated well, and toxic effects on the engineering bacteria due to excessive trace elements can be avoided.

Further, in the culture stage, the engineering bacteria amplified by fermentation are inoculated in a high-density fermentation medium to obtain a fermentation system, and the culture conditions are as follows: culturing at 37deg.C with stirring speed of 100-200rpm, aeration rate of 5000-30000L/min, pH of 6.0-7.0, and dissolved oxygen of 20% -80%, culturing for 3-5 hr until OD of fermentation broth ₆₀₀ The value is 10-15, and a fermentation system I is obtained.

By adopting the scheme, a certain number of engineering bacteria can be obtained by culture, and the engineering bacteria are prepared for the subsequent expression of target proteins in terms of substances and energy.

Further, the volume of the fermentation system is denoted by V in L; the culture phase is followed by a feed phase: the initial culture conditions were: continuously feeding a feed medium into a fermentation system I at a speed of 6.0-7.5V ml/h, wherein the culture temperature is 37 ℃, the stirring speed is 100-200rpm, the aeration rate is 5000-30000L/min, the pH is 6.0-7.0, and the dissolved oxygen is 5-30%, and culturing until the OD of the fermentation liquid is reached ₆₀₀ A value of 15-25; then adjusting the flow acceleration of the feed medium to 4.5V-7.5V ml/h, and adjusting the culture temperature to 20-25deg.C to culture to OD of the fermentation broth ₆₀₀ The value is 25-35, and a fermentation system II is obtained.

By adopting the scheme, nutrient substances are supplemented, the proliferation of engineering bacteria is further promoted, the temperature is reduced after the engineering bacteria proliferate to a certain extent, a better temperature environment is provided for the induction expression of the target protein, and the activity of the target protein is ensured. The method is suitable for cooling, the flow acceleration of the feed supplement culture medium is correspondingly reduced, the growth speed of engineering bacteria is reduced after cooling, consumed nutrition is properly reduced, and required nutrition supply is correspondingly reduced.

Further, the feeding phase is followed by a lactose induction phase: feeding a feed medium into a fermentation system II at a speed of 4.5-7.5V ml/h, and maintaining the lactose concentration at 8-15g/L; the culture conditions for the lactose induction phase were: culturing at 20-30deg.C with stirring speed of 100-200rpm, aeration rate of 5000-30000L/min, pH of 6.0-7.0, and dissolved oxygen of 5-30%; induction for 20h, OD ₆₀₀ The value reaches 100-150, and the fermentation is stopped.

By adopting the scheme, lactose is added into the system in the lactose culture stage, and the lactose concentration is maintained to be 8-15g/L, and the lactose with the concentration (and the concentration maintained at the level) can induce the engineering bacteria to express the target protein. The addition of the feed supplement culture medium can provide enough energy for the growth of engineering bacteria, prevent the engineering bacteria from consuming a large amount of lactose, avoid the loss of inducers and ensure the induction efficiency. The culture temperature is 20-30 ℃ to provide a better temperature environment for the induced expression of the target protein, and ensure the activity of the target protein.

Further, in the lactose induction stage, a lactose inducer is fed to the fermentation system II at a rate of 50-500 g/h, and the lactose concentration is brought to 8-15g/L in 5 hours, and then maintained at 8-15g/L.

By adopting the scheme, the concentration of lactose is gradually adjusted to 8-15g/L, so that engineering bacteria have a gradual adaptation process to lactose, and the inhibition effect of lactose on the growth of the engineering bacteria by adding a large amount of lactose in one step is avoided.

Further, the feed medium comprises the following components: 1.75-5.00g/L yeast extract, 3.75-10.0g/L peptone, 200-400g/L glycerol, 0.25-1.00g/L magnesium sulfate heptahydrate; the lactose concentration in the lactose inducer is 200g/L.

By adopting the scheme, the feed supplement culture medium can effectively promote the growth of engineering bacteria and maintain the expression activity of the engineering bacteria.

Further, in the step of induction fermentation culture, the volume of the fermentation system is 10000L-30000L.

By adopting the scheme, the hydroxysteroid dehydrogenase can be expressed in a large scale in a ton-scale fermentation system, the expression efficiency and activity of the hydroxysteroid dehydrogenase are high, the generation amount of inclusion bodies is small, and the method has great industrial application value.

Further, the method also comprises a fermentation amplification step for obtaining engineering bacteria to be expressed before the induced fermentation culture step, wherein the fermentation amplification step comprises primary fermentation culture, secondary fermentation culture and tertiary fermentation culture; in the three-stage fermentation culture, the culture medium comprises 5g/L of yeast extract, 10g/L of peptone, 6g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, 1.5g/L of ammonium chloride, 8.5g/L of disodium hydrogen phosphate dodecahydrate and 1.5g/L of glycerin.

By adopting the scheme, the engineering bacteria are subjected to three-step fermentation amplification before the step of induced fermentation culture, so that the number of the engineering bacteria can be increased, and the engineering bacteria are prepared for adapting to the culture medium conditions in the step of fermentation culture. The three-stage fermentation culture medium can slowly improve the osmotic pressure of the culture medium and reduce the adaptation period of engineering bacteria in the fermentation process.

Further, the method comprises the step of preparing shake flask seeds before the fermentation amplification step: and culturing the recombinase strain twice by using an LB culture medium to obtain the engineering bacteria to be amplified for the fermentation amplification step.

By adopting the scheme, the recombinase strain is initially amplified to obtain seeds, and then fermentation amplification is carried out. Gradually increasing the number of engineering bacteria and creating conditions for subsequent induced expression. The recombinant enzyme strain is subjected to resistance screening and contains escherichia coli engineering bacteria for stably expressing target proteins, and the preparation process of the recombinant enzyme strain is shown in example 1.

Detailed Description

Example 1: preparation of engineering bacteria

E.coli expression codon optimization is carried out on 7 alpha-hydroxysteroid dehydrogenase gene S1-a-1, and total gene synthesis is carried out. The optimized 7α -hydroxysteroid dehydrogenase gene S1-a-1 is abbreviated herein as 7α -steroid dehydrogenase gene, designated 7α -HSDH (DNA sequence: SEQ ID NO:1, encoded protein sequence: SEQ ID NO: 2).

The 7α -HSDH was amplified by PCR using primer pairs 5'-TTCCCCTCTAGAATGGGCAGCAGCCATCATCA-3' (SEQ ID NO: 3) and 5'-GCGCGCAAGCTTTTAACGGCTGCGCTCCATCAT-3' (SEQ ID NO: 4), digested with XbaI and HindIII, and digested with DpnI enzyme (methylate template digesting enzyme). The pET29a vector was digested with XbaI and HindIII. The 7 alpha-steroid dehydrogenase gene fragment and the pET29a vector were ligated with a ligase to obtain a ligation product. DH 5. Alpha. Was transformed with the ligation product and plated on LB plates with kanamycin resistance for selection. After colony formation, the monoclonal was selected and inoculated into 5mL of LB for overnight culture. The thalli were collected, plasmids were extracted with the root plasmid extraction kit and sent to sequencing. The plasmid pET29a-7α -HSDH with correct sequence was obtained by preserving the plasmid whose gene expression operon was lactose operon.

pET29a-7α -HSDH is transformed into competent cells of escherichia coli BL21 (DE 3) to obtain engineering bacteria capable of expressing 7α -HSDH. Spread on a screening plate containing 50. Mu.g/ml kanamycin sulfate, cultured overnight at 37℃to give single colonies, inoculated into 500ml shake flask, 100ml LB medium containing 50. Mu.g/ml kanamycin sulfate, and cultured in a shaker at 37℃at 150rpm to OD ₆₀₀ The value is 0.6-0.8, the culture time is about 8-12h, and the culture is added according to the final concentration of 10%Adding glycerol protectant, packaging into 50 μl each tube, and storing at-80deg.C to obtain original seed. Taking 1 tube of original seeds, inoculating into 500ml shake flask, culturing in 100ml LB medium containing 50mg/ml kanamycin sulfate at 37deg.C and 150rpm in shake flask to OD ₆₀₀ The value is 0.6-0.8, the culture time is about 4-6h, the glycerol protectant is added according to the final concentration of 10%, and the mixture is divided into 50 μl of each tube and stored at minus 80 ℃ to be used as main seeds. Taking 1 tube of main seeds, and subpackaging and preserving the working seeds, wherein the method is the same as the preparation method of the main seeds. During the establishment of the seed bank, the upper limit of the culture time and OD are reached ₆₀₀ When the value still does not reach the normal interval, the abnormal growth speed of the strain in the batch is indicated, and the seed should be prepared again. To ensure the stability of the batch seeds, working seeds were used as recombinant enzyme strain (for expression of 7α -HSDH).

Example 2:

the expression codon of 7 beta-hydroxysteroid dehydrogenase gene pHsh-7 beta-Y1-b-1 is optimized, and total gene synthesis is carried out. The optimized 7β -hydroxysteroid dehydrogenase gene Y1-b-1 is abbreviated herein as 7β -steroid dehydrogenase gene, designated 7β -HSDH (DNA sequence: SEQ ID NO:5, encoded protein sequence: SEQ ID NO: 6).

The 7β -HSDH was amplified by PCR using primer pairs 5'-TTCCCCTCTAGAATGAATCTAAGG-3' (SEQ ID NO: 7) and 5'-GCGCGCAAGCTTTTAATCACGATAGAAGC-3' (SEQ ID NO: 8), digested with XbaI and HindIII, and digested with DpnI enzyme (methylation template digestion enzyme). The pET29a vector was digested with XbaI and HindIII. The 7 alpha-steroid dehydrogenase gene fragment and the pET29a vector were ligated with a ligase to obtain a ligation product. DH 5. Alpha. Was transformed with the ligation product and plated on LB plates with kanamycin resistance for selection. After colony formation, the monoclonal was selected and inoculated into 5mL of LB for overnight culture. The thalli were collected, plasmids were extracted with the root plasmid extraction kit and sent to sequencing. The plasmid pET29a-7β -HSDH with correct sequence was obtained by preserving the plasmid whose gene expression operon was lactose operon.

A recombinant enzyme strain (for expressing 7β -HSDH) was obtained by the method described in example 1. This example is basically the same as example 1, except that the 7β -hydroxysteroid dehydrogenase gene Y1-b-1 is used instead of the 7α -hydroxysteroid dehydrogenase gene S1-a-1.

Example 3: engineering bacteria fermentation

Shaking bottle seed production

1 tube of working seed (recombinant enzyme strain prepared in example 1) was inoculated into 200ml of LB medium and cultured in a shaker at 37℃at 150rpm to OD ₆₀₀ The value is 2.0-3.0, and the culture time is about 5-8 hours, so as to obtain the first-stage shake flask seeds. At OD ₆₀₀ The value is a qualified index, and if the OD value exceeds the upper time limit, the batch of seeds is discarded. Inoculating first-stage shake flask seeds into 1L LB culture medium, culturing at 37deg.C and 150rpm in shaking table to OD ₆₀₀ The value is 2.0-3.0, the culture time is about 2-4h, and OD is used ₆₀₀ And obtaining the second-stage shake flask seeds by taking the value as a qualified index.

In this scheme, the steps specifically include: inoculating 1 tube of recombinase strain into 200ml LB culture medium, culturing at 37deg.C and 150rpm in shaking table for about 6 hr to OD ₆₀₀ The value is 2.5, and the first-stage shake flask seeds are obtained. Inoculating the first-stage shake flask seeds into 1L LB medium, culturing at 37deg.C and 150rpm in a shaker for 3 hr to OD ₆₀₀ The value was 2.5, and a secondary shake flask seed was obtained.

Wherein OD ₆₀₀ The measuring method of the value comprises the following steps: 1ml of the fermentation broth (medium in which bacteria were uniformly dispersed) was thoroughly mixed with 9ml of tap water, and the dilution ratio was N1. 2ml of the mixture was added to a cuvette and zeroed at 600nm with another cuvette containing tap water. Zeroing OD ₆₀₀ After the end of the values, the reading of the cuvette with the mixed liquor is taken at a wavelength of 600 nm. If the reading exceeds 1, the previous dilution is taken for re-dilution, the subsequent dilution is counted as N2, and so on, until the reading of the dilution at a wavelength of 600nm is in the range of 0.200-1.000. Bacterial concentration OD of fermentation broth ₆₀₀ Uv reading x dilution N1 x dilution N2 x dilution N3, subsequent OD ₆₀₀ The values were measured using the method described above.

Primary fermentation culture:

inoculating the cultured secondary shake flask seed (prepared in example 1) into a medium containing 20L LB medium by differential pressure inoculationInoculating in a ratio of 1:20-1:10, setting temperature at 37deg.C, stirring at 100-250rpm, aeration rate of 10-20L/min, pH of 6.5-7.0, and dissolved oxygen of 20-80%, and culturing to OD ₆₀₀ The value is 2.5-4.0, and the culture is continued for 3-5h, and OD is used ₆₀₀ And obtaining the primary fermentation culture solution by taking the value as a qualified index.

In this scheme, the steps specifically include: inoculating at 1:11 ratio, setting temperature 37 deg.C, stirring speed 150rpm, aeration rate 10-20L/min, pH 6.5-7.0, and dissolved oxygen 20-80% (aeration rate, pH value and dissolved oxygen degree in the maintaining process are in the above range), culturing for 4 hr, and OD ₆₀₀ The value is about 3.0, and the primary fermentation culture solution is obtained.

Secondary fermentation culture:

inoculating the cultured primary fermentation culture solution to a secondary fermentation tank filled with 200L LB culture medium through a seed transfer pipeline, inoculating according to the proportion of 1:20-1:10, setting the temperature to 37 ℃, stirring at 100-250rpm, introducing air at 50-150L/min, pH at 6.0-7.0 and dissolved oxygen at 20-80%, and culturing until OD ₆₀₀ The value is 2.5-4.0, and the culture is continued for 3-5h, and OD is used ₆₀₀ The value is a qualified index, and the secondary fermentation culture solution.

In this scheme, the steps specifically include: inoculating at 1:11 ratio, setting temperature 37 deg.C, stirring speed 150rpm, aeration rate 50-150L/min, pH6.0-7.0, and dissolved oxygen 20-80% (aeration rate, pH value and dissolved oxygen degree in the maintaining process are in the above range), culturing for 4 hr, and OD ₆₀₀ The value is about 3.0, and the secondary fermentation culture solution is obtained.

And (3) three-stage fermentation culture:

inoculating the cultured secondary fermentation culture solution to a tertiary fermentation tank filled with 2000L of tertiary fermentation culture medium through a seed transfer pipeline, wherein the tertiary fermentation culture medium comprises 5g/L of yeast extract, 10g/L of peptone, 6g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, 1.5g/L of ammonium chloride, 8.5g/L of disodium hydrogen phosphate dodecahydrate and 1.5g/L of glycerol, and the secondary fermentation culture solution aims to slowly increase the osmotic pressure of the culture medium, reduce the adaptation period of escherichia coli in a quaternary culture tank, inoculate according to the proportion of 1:20-1:10, set the temperature to 37 ℃, the stirring speed to 100-250rpm, the aeration quantity to 500-1500L/min, the pH to 6.0-7.0 and the dissolved oxygen to 20 percent-80%, culture to OD ₆₀₀ The value is 2.5-4.0, and the culture is continued for 3-5h.

In this scheme, the steps specifically include: inoculating at 1:11 ratio, setting temperature 37 deg.C, stirring at 150rpm, aeration rate 500-1500L/min, pH6.0-7.0, and dissolved oxygen 20-80% (aeration rate, pH value and dissolved oxygen degree in the maintaining process are in the above range), and continuously culturing for 4 hr.

Four-stage fermentation culture (also called induced fermentation culture):

(1) Culturing:

the cultured tertiary fermentation broth was inoculated to a 30T fermenter containing 18000L of a high-density fermentation medium through a transfer line to obtain 20000L of a fermentation system (the volume of the fermentation system is represented by V in L). The high density fermentation culture medium comprises yeast extract 1.75-4.00g/L, peptone 3.5-8.0g/L, magnesium sulfate heptahydrate 1.5-2.5g/L, potassium dihydrogen phosphate 2.5-4.0g/L, ammonium chloride 4.5-8.0g/L, disodium hydrogen phosphate dodecahydrate 10.0-15.0g/L, glycerol 12.5-25.0g/L, microelements (calcium chloride hexahydrate 0.01-0.08g/L, manganous chloride tetrahydrate 0.01-0.10g/L, ferric chloride hexahydrate 0.01-0.10 g/L), inoculating at a ratio of 1:20-1:10, setting temperature 37 ℃, stirring speed 100-200rpm, aeration amount 5000-30000L/min, pH6.0-7.0, dissolved oxygen 20% -80%, culturing to OD ₆₀₀ The value is 10-15, and the culture is continued for 3-5 hours, so as to obtain a fermentation system I.

In this scheme, the steps specifically include: inoculating the cultured 2000L three-stage fermentation culture solution to a 30T fermentation tank filled with 18000L high-density fermentation culture medium (namely, the fermentation volume is 20000L, the inoculation ratio is 1:10) through a seed transfer pipeline, setting the temperature to 37 ℃, stirring at 150rpm, the aeration rate to be 5000-30000L/min, the pH to be 6.0-7.0, and the dissolved oxygen to be 20-80% (the aeration rate, the pH value and the dissolved oxygen in the maintaining process are in the ranges), continuously culturing for 4h, and starting to enter a feeding stage when the dissolved oxygen is changed suddenly (suddenly rises to a range of 90-100%). The components of the high-density fermentation medium of this example are shown in table 1, wherein the trace elements comprise the following components: calcium chloride hexahydrate 0.022g/L, manganous chloride tetrahydrate 0.015g/L, ferric chloride hexahydrate 0.02g/L. Calcium chloride hexahydrate is used for providing divalent calcium, manganous chloride tetrahydrate is used for providing divalent manganese, ferric chloride hexahydrate is used for providing divalent iron, and the three salts can be replaced by other salts containing divalent calcium, divalent manganese or trivalent iron and being used as additives of a culture medium according to actual conditions and common knowledge.

(2) And (3) a material supplementing stage:

the common batch feeding mode is abandoned, batch feeding control is adopted, and the components of the feeding culture medium are as follows: yeast extract 1.75-5.00g/L, peptone 3.75-10.0g/L, glycerol 200-400g/L, magnesium sulfate heptahydrate 0.25-1.00g/L, initial feed medium flow acceleration of 6.0V-7.5V ml/h (V represents volume of fermentation system, unit is L, and constant (i.e. means 6.0-7.5) unit is L) ^-1 ) Culturing at 37deg.C, stirring speed of 100-200rpm, aeration rate of 5000-30000L/min, pH of 6.0-7.0, and dissolved oxygen of 5% -30% to OD ₆₀₀ The value is between 15 and 25. Then the feed medium flow acceleration is set to be 4.5V-7.5V ml/h (V represents the volume of the fermentation system, the unit is L, and the unit of the constant (namely 4.5-7.5) is L) ^-1 ) The temperature is 20-30 ℃, and other parameters are unchanged. Culturing until the OD600 value of the fermentation liquid is 25-35 to obtain a fermentation system II.

In this scheme, the steps specifically include: the initial feed medium flow acceleration is 7.0V ml/h, aeration rate is maintained at 5000-30000L/min, pH is maintained at 6.0-7.0, dissolved oxygen is maintained at 5% -30%, stirring speed is 150rpm, and culturing is carried out until OD ₆₀₀ About 25, then the feed medium flow acceleration was maintained at 6.0Vml/h and the temperature was adjusted to 25 ℃. The feed medium comprises 2.5g/L yeast extract, 5.0g/L peptone, 300g/L glycerol and 0.5g/L magnesium sulfate heptahydrate.

(3) Lactose induction phase:

OD ₆₀₀ when the value is 25-35, the culture temperature is 20-30 ℃, lactose inducer (lactose inducer solution concentration is 200 g/L) is independently added, the flow acceleration of lactose inducer is set to be 50-500L/h, and the lactose concentration reaches 8-15g/L within 5h. Other parameters were set to feed medium flow acceleration of 4.5V-7.5V ml/h (V denotes volume of fermentation system in L, constant (i.e. 4.5-7.5) in L) ^-1 ) The temperature is 20-30 ℃, the stirring speed is 100-200rpm, the aeration rate is 5000-30000L/min, the pH is 6.0-7.0, and the dissolved oxygen is 5-30%. Sampling and detecting OD at intervals of 0.5h ₆₀₀ The value, the expression quantity of the recombinase and the lactose content of the culture solution are adjusted within the range of 0-500L/h according to the lactose consumption, so that the final concentration of lactose is ensured to be 8-15g/L. Timing is started after the inducer is added, fermentation is stopped after 20h of induction (OD is required to be ensured) ₆₀₀ The value reaches 100-150), and the enzyme-containing thalli is collected by centrifugation, and indexes such as total enzyme activity and the like are detected.

In this scheme, the steps specifically include: OD (optical density) ₆₀₀ When the value is about 35, the culture temperature is kept at 25 ℃, the lactose inducer is independently added, the flow acceleration of the lactose inducer is maintained at 300-500L/h, and the lactose concentration reaches 12.5g/L within 5h. The feed medium flow acceleration was maintained at 6.0V ml/h throughout the lactose induction phase. Stirring speed 150rpm, aeration rate 5000-30000L/min, pH6.0-7.0, and dissolved oxygen 5% -30%. Sampling and detecting OD at intervals of 0.5h ₆₀₀ The value, the expression quantity of the recombinase and the lactose content of the culture solution are adjusted according to the lactose consumption, the flow acceleration of the lactose inducer is adjusted (but the flow acceleration is required to be ensured to be less than 30L/h), and the final concentration of lactose is ensured to be maintained at about 12.5g/L. And (3) starting timing after the addition of the inducer, stopping fermentation after 20 hours, starting centrifugal collection of enzyme-containing thalli, and detecting indexes such as total enzyme activity.

Example 4-example 5: substantially the same as in example 3, the differences are shown in Table 1. And in example 4 the trace element components of the high density medium were: 0.01g/L of calcium chloride hexahydrate, 0.01g/L of manganous chloride tetrahydrate and 0.01g/L of ferric chloride hexahydrate; the trace element components of the high density medium in example 5 were: 0.08g/L of calcium chloride hexahydrate, 0.10g/L of manganous chloride tetrahydrate and 0.10g/L of ferric chloride hexahydrate.

Example 6 was basically the same as example 3, except that the recombinant enzyme strain of example 2 was used in this example, and after completion of fermentation, enzyme-containing cells were collected and subjected to index detection such as total enzyme activity.

Example 7-example 14 was essentially the same as example 3, except that the differences are shown in tables 1 and 2. In example 7, the feed medium had the following composition: 1.75g/L of yeast extract, 3.75g/L of peptone, 200g/L of glycerol and 0.25g/L of magnesium sulfate heptahydrate; in example 8, the feed medium had the following composition: yeast extract 5.00g/L, peptone 10.0g/L, glycerin 400g/L, magnesium sulfate heptahydrate 1.00g/L.

Comparative example 1 was essentially the same as example 3 except that the high-density fermentation medium of example 1 was not used in the stage of the four-stage fermentation culture (1), and the high-density fermentation medium contained no trace elements, and the other parameter settings were detailed in table 1. After the fermentation is completed, the enzyme-containing thalli are collected and the indexes such as total enzyme activity and the like are detected. Comparative example 2 basically the same as in example 3 except that the trace elements of the high-density fermentation medium were 0.1g/L of calcium chloride hexahydrate, 0.2g/L of manganous chloride tetrahydrate and 0.2g/L of ferric chloride hexahydrate, the trace elements were used in a higher content than in experimental example 3. Comparative example 3 essentially the same as example 3, except that the high density fermentation medium was of conventional configuration, namely: 5.0g/L of yeast extract, 10.0g/L of peptone, 1.0g/L of magnesium sulfate, 2.5g/L of monopotassium phosphate, 2.5g/L of ammonium chloride, 7.5g/L of disodium hydrogen phosphate and 10.0g/L of glycerol, and does not contain trace elements. In comparative example 14, a conventional high-density medium (same as in comparative example 3) was used, and trace elements (kind and amount in accordance with example 3) as in example 3 were added. Comparative example 4 was basically the same as example 3, except that the feed medium flow acceleration exceeded the upper limit. Comparative example 5 was essentially the same as example 3, except that the feed medium flow acceleration was below the lower limit. Comparative example 6 is basically the same as example 3 except that the lactose concentration is higher than the upper limit. Comparative example 7 was essentially the same as example 3, except that the lactose concentration was below the lower limit. Comparative example 8 was basically the same as example 3, except that the culture temperature was too high. Comparative example 9 was basically the same as example 3, except that the culture temperature was too low. Comparative example 10 substantially the same as in example 3, except that LB medium was directly used as the tertiary fermentation medium. Comparative examples 11-13 used IPTG inducer. The specific parameter settings for comparative examples 1-14 are shown in tables 2 and 3.

In the examples and the comparative examples, when the volume of the fermentation system is 200L, the aeration rate in the induced fermentation culture step is required to be maintained at 50-150L/min, so that the proliferation rate of engineering bacteria is proper; when the volume of the fermentation system is 2000L, the aeration rate in the induced fermentation culture step is required to be maintained at 500-1500L/min, so that the proliferation rate of engineering bacteria is proper.

Experimental example 1:

the enzyme-containing cells obtained in example 3-example 14, comparative example 1-comparative example 14 were subjected to various parameter tests to determine the quality of the product, and the test results are shown in tables 1-3.

OD ₆₀₀ See measurement methods recorded in example 3.

The inclusion body proportion was measured by polyacrylamide gel electrophoresis (SDS-PAGE), specifically: 5g of the thalli collected by centrifugation is taken, 50ml of 10mM Tis-HCl is added, the mixture is stirred uniformly and then is crushed by ultrasonic waves for 3min, 2 parts of 30 μl of the same sample is taken and placed in a centrifuge tube, and the number of the centrifuge tube is tube 1 and tube 2. To tube 1, 30. Mu.l of staining solution was added. Tube 1 was designated as the recombinase sample. Tube 2 was centrifuged at 12000r/min for 10min, 30. Mu.l of staining solution and 30. Mu.l of aqueous solution were added to the pellet, and the pellet was re-mixed, and tube 2 was designated as a sample of recombinant enzyme inclusion body. The supernatant was transferred to tube 3, an equal volume of staining solution was added, and the mixture was re-mixed, and tube 3 was designated as a soluble recombinase sample. Tubes 1,2,3 were boiled for 10 min. Mu.l of the sample was taken and subjected to SDS-PAGE. After SDS-PAGE profile scanning, total expression amount of the recombinant enzyme, inclusion bodies and respective ratios of soluble enzymes were analyzed by BandScan software.

The method for measuring the enzyme activity of the hydroxycholesterol dehydrogenase comprises the following steps: taking 7 alpha-steroid dehydrogenase as an example: with taurochenodeoxycholic acid as a substrate, 2.97ml of 100mM phosphate buffer with pH8.0, final concentration of 0.5mM taurochenodeoxycholic acid, 10 μl of diluted cholesterol dehydrogenase cell dilution and final concentration of 0.5mM NADP+ are added into a 3ml reaction system, reacted for 1min at 25 ℃, absorbance values are measured at 340nm wavelength, and enzyme activity is calculated by comparison with a standard curve.

The lactose concentration determination method adopts an enzyme colorimetric method, and specifically comprises the following steps: taking 1ml of culture solution, placing the culture solution in a 10ml colorimetric tube, adding 0.2ml of citric acid buffer solution and 0.05ml of beta-GLS- (NH) ₄ ) ₂ SO ₄ The suspension was shaken and then subjected to water bath at 36℃for 15min, 1.0ml of NADP+ -ATP-TEA buffer solution and 0.05ml of HK-G6PDH- (NH) were added ₄ ) ₂ SO ₄ The ammonium suspension was shaken and diluted with water to 5ml in a 36℃water bath for 60min, allowed to stand for 5min, absorbance was measured at 340nm, and lactose concentration was calculated by comparison with a standard curve.

Table 1: example 3-example 12 parameter settings and test results

Table 2: examples 13 and 14, comparative example 1-comparative example 7 parameter settings and test results

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Table 3: comparative example 8-comparative example 14 parameter settings and test results

The fermentation process of the invention can be scaled up, and the data of examples 3-12 show that the fermentation process is suitable for a fermentation system of 200-30000L, while the fermentation process of the prior art can only reach the level of hundred liters, and the fermentation volume can reach the ton level by adopting the fermentation process, and the fermentation process also has stable target protein yield and higher activity of the target protein. The use of IPTG as an inducer in comparative examples 11-13 is a conventional mode of operation in the prior art, which effectively induces the expression of the target protein, but for large-volume fermentation expression, the production cost is too high due to the high price of IPTG, and potential safety risks also exist. By using the fermentation process, lactose with low price is used as an inducer, so that a good induced expression effect can be obtained, the enzyme activity is obviously improved, and the production cost is reduced.

Comparative example 1 without adding microelement in high density fermentation medium, resulting in late growth debilitation, OD of engineering bacteria ₆₀₀ The lower value indicates the importance of trace element addition in the (1) culture stage of the four-stage fermentation culture (also called induction fermentation culture). The three microelements can stimulate the growth of engineering bacteria and adapt to the fermentation environment containing lactose, and the reason for analyzing the phenomenon is that the three microelements can assist the engineering bacteria to prepare for resisting stress and protein expression. Before the engineering bacteria are induced to express, a culture medium containing trace elements is added in the stage of culturing the engineering bacteria, so that the engineering bacteria are prepared for expressing a large amount of target proteins and resisting inhibition caused by lactose in advance. After the inducer is replaced by lactose from IPTG (comparative example 1 and comparative examples 11-13), the lactose can produce a certain inhibition effect on engineering bacteria, so that the growth speed of the engineering bacteria is reduced, and OD is reduced ₆₀₀ The value is low. The inventor adopts various means to relieve the adverse effects, and finally discovers that the problems can be well solved by adding trace elements into a high-density fermentation medium, and the problems of reduced protein induction expression efficiency and reduced protein quality caused by the replacement of an inducer are solved. The too many trace elements added in comparative example 2 have an inhibiting effect on the growth of engineering bacteria, and the final result of protein expression is not ideal. In comparative example 3, a conventional high-density medium without trace elements was used, namely, 5.0g/L of yeast extract, 10.0g/L of peptone, 1.0g/L of magnesium sulfate, 2.5g/L of potassium dihydrogen phosphate, 2.5g/L of ammonium chloride, 7.5g/L of disodium hydrogen phosphate and 10.0g/L of glycerol, and the proliferation of engineering bacteria during fermentation was poor due to the absence of trace elements. In comparative example 14, a conventional high-density medium was used, and trace elements (the kind and amount are the same as those in example 3) as in example 3 were added, so that the proliferation of the engineering bacteria during fermentation was better than in comparative example 3, but the engineering bacteria were still at a distance from the effect in example. The arrangement of the high inorganic nitrogen source and the low organic nitrogen source of the proposal shows a great amount of high quality for the induced engineering bacteriaThe target protein has promoting effect.

In comparative example 4, too high feed medium flow acceleration resulted in too much nutrition in the system and too high engineering bacteria density resulted in the production of a large number of inclusion bodies. The feed medium flow acceleration in comparative example 5 was too low, resulting in insufficient nutrition and too low engineering bacteria density. The lactose concentration in comparative example 6 is too high, which inhibits the growth of engineering bacteria; the lactose concentration in comparative example 7 was too low to effectively induce the engineering bacteria to express the target protein. The temperature of the induced expression in comparative example 8 was too high, resulting in an increase in inclusion body content; the induction expression temperature in comparative example 9 was too low, resulting in too low a density of engineering bacteria. In comparative example 10, LB medium was used in the tertiary fermentation culture instead of the tertiary fermentation medium group as in example 3, resulting in that the engineering bacteria could not be well adapted to the subsequent large-volume fermentation process, and the induction expression effect of the target protein was not ideal.

The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

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SEQUENCE LISTING

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<120> a fermentation process for coexpression of hydroxysteroid dehydrogenase

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Claims

1. An industrial fermentation process of hydroxysteroid dehydrogenase, comprising the following steps in sequence:

the preparation step of shake flask seeds: culturing the recombinase strain twice by using an LB culture medium to obtain engineering bacteria to be amplified for a fermentation amplification step; the recombinant enzyme strain is engineering bacteria expressing 7 alpha-HSDH or engineering bacteria expressing 7 beta-HSDH; the protein sequence of 7alpha-HSDH is shown as SEQ ID NO. 2, and the protein sequence of 7beta-HSDH is shown as SEQ ID NO. 6;

fermentation and amplification steps: comprises primary fermentation culture, secondary fermentation culture and tertiary fermentation culture; in the three-stage fermentation culture, the culture medium comprises 5g/L of yeast extract, 10g/L of peptone, 6g/L of sodium chloride, 0.5g/L of magnesium sulfate heptahydrate, 1.5g/L of ammonium chloride, 8.5g/L of disodium hydrogen phosphate dodecahydrate and 1.5g/L of glycerol;

culturing: inoculating the engineering bacteria subjected to fermentation amplification into a high-density fermentation culture medium to obtain a fermentation system with the volume of 10000L-30000L, wherein the culture conditions are as follows: culturing at 37deg.C with stirring speed of 100-200rpm, aeration rate of 5000-30000L/min, pH of 6.0-7.0, and dissolved oxygen of 20% -80%, culturing for 3-5 hr until OD of fermentation broth ₆₀₀ The value is 10-15, and a fermentation system I is obtained; the high-density fermentation medium comprises basic components and trace element components; the basic components comprise: 1.75-4.00g/L of yeast extract, 3.5-8.0g/L of peptone, 1.5-2.5g/L of magnesium sulfate heptahydrate, 2.5-4.0g/L of potassium dihydrogen phosphate, 4.5-8.0g/L of ammonium chloride, 10.0-15.0g/L of disodium hydrogen phosphate dodecahydrate and 12.5-25.0g/L of glycerin; the trace element components comprise: 0.01-0.08g/L of calcium chloride hexahydrate, 0.01-0.10g/L of manganous chloride tetrahydrate and 0.01-0.10g/L of ferric chloride hexahydrate;

and (3) a material supplementing stage: the volume of the fermentation system is represented by V, and the unit is L; the initial culture conditions were: continuously feeding a feed medium into a fermentation system I at a speed of 6.0V-7.5Vml/h, wherein the culture temperature is 37 ℃, the stirring speed is 100-200rpm, the aeration rate is 5000-30000L/min, the pH is 6.0-7.0, and the dissolved oxygen is 5-30%, and culturing until the OD of the fermentation liquid is reached ₆₀₀ A value of 15-25; then adjusting the flow acceleration of the feed medium to 4.5V-7.5V ml/h, and adjusting the culture temperature to 20-25deg.C to culture to OD of the fermentation broth ₆₀₀ The value is 25-35, and a fermentation system II is obtained;

lactose induction phase: into fermentation system IIFeeding the feed medium at a rate of 4.5V-7.5V ml/h and maintaining lactose concentration at 8-15g/L; the culture conditions for the lactose induction phase were: culturing at 20-30deg.C with stirring speed of 100-200rpm, aeration rate of 5000-30000L/min, pH of 6.0-7.0, and dissolved oxygen of 5-30%; induction for 20h, OD ₆₀₀ The value reaches 100-150, and the fermentation is stopped; feeding lactose inducer into fermentation system II at a speed of 50-500L/h, and keeping lactose concentration at 8-15g/L after lactose concentration reaches 8-15g/L in 5 h; the feed medium comprises the following components: 1.75-5.00g/L yeast extract, 3.75-10.0g/L peptone, 200-400g/L glycerol, 0.25-1.00g/L magnesium sulfate heptahydrate; the lactose concentration in the lactose inducer is 200g/L.