Method for optimizing 7-dehydrocholesterol fermentation by data fitting and fermentation method

Abstract

The invention relates to a method for optimizing 7-dehydrocholesterol fermentation by using data fitting and a fermentation method, and the method comprises the following steps: constructing fermentation influence parameters including at least one of fermentation strain activity, fermentation temperature, inoculum size and culture medium components; adjusting the numerical values of the fermentation influence parameters, obtaining an optimal fermentation effect through an experiment, and taking the fermentation influence parameters with the optimal fermentation effect as optimized 7-dehydrocholesterol fermentation parameters; wherein when the fermentation influence parameters are culture medium components, the importance of each component in the culture medium components is analyzed by adopting a random forest model, and a component importance sequence is obtained; performing multiple linear regression modeling by taking at least two components with the top importance sequence as input to obtain a regression equation, and constructing fermentation culture medium components by using the regression equation to obtain optimized culture medium components; the fermentation titer can be improved, and the production cost can be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and specifically relates to a method for optimizing 7-dehydrocholesterol fermentation by data fitting and a fermentation method. Background Art

[0002] 7-dehydrocholesterol (7-DHC) is a sterol substance with the molecular formula C 27 H 44 O and a relative molecular weight of 384.64. 7-DHC is also known as the precursor of vitamin D3. 7-dehydrocholesterol present in the epidermis and dermis of most people can absorb ultraviolet B (UVB) with wavelengths of 80 - 320 nm in sunlight to generate vitamin D3. Its reaction mechanism is that under the action of UVB (the optimal efficiency is usually 275 - 300 nm), the cyclohexadiene in the 7-DHC molecule opens the ring and transforms into an open-chain conjugated triene structure (i.e., previtamin D3). In the second step, under the action of heat, a σ-H atom on the corresponding angular methyl group in previtamin D3 undergoes a [1,7] migration reaction to obtain vitamin D3. The inactive vitamin D3 precursor molecule is converted into the main circulating form in the body, 25-hydroxyvitamin D3, by the liver, and then into 1α,25-dihydroxyvitamin D3 by the kidney, and various physiological functions are regulated through this active form. In addition to vitamin D3, 7-DHC can also be used to synthesize polyoxysterol derivatives and is the main raw material for preparing cholesteric liquid crystal materials.

[0003] There are many synthesis processes for vitamin D3, but its key precursor is always 7-DHC. At present, the industrial production processes of 7-DHC and vitamin D3 mainly adopt the bromination / debromination / hydrogenation method and the oxidation / reduction / elimination method. The common core raw material for both synthesis routes is NF-grade cholesterol (cholesterol with a purity of more than 95%), and the supply of NF-grade cholesterol limits the production and price of vitamin D3. At the same time, in both synthesis routes, the bromine-containing by-products generated by the bromination / debromination / hydrogenation method are difficult to completely remove, and a small amount of bromine or bromine-containing substances will have an adverse effect on the photochemical ring-opening reaction of 7-DHC, and bromine is harmful to the environment; although the oxidation / reduction / elimination method avoids the environmental pollution caused by bromine, the oxidant (such as zirconium trioxide) still has environmental pollution problems.

[0004] Compared with the chemical synthesis method, the biological synthesis of 7-DHC has the advantages of a mild reaction environment, fast reaction speed, and strong specificity. Industrial strains for efficient biosynthesis of 7-DHC have been obtained by metabolic engineering of Saccharomyces cerevisiae (CN114703077 B, CN 118006571 A), but there are still deficiencies such as low fermentation unit titer and high fermentation cost in the production process. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for optimizing the fermentation of 7-dehydrocholesterol by data fitting and a fermentation method, so as to improve the fermentation titer and reduce the production cost.

[0006] An embodiment of the present invention provides a method for optimizing the fermentation of 7-dehydrocholesterol by data fitting, which includes the following steps: Construct fermentation influence parameters, where the fermentation influence parameters include at least one of the activity of the fermentation strain, fermentation temperature, inoculum size, and medium components; Adjust the values of the fermentation influence parameters, and experimentally obtain the best fermentation effect. Take the fermentation influence parameters at the best fermentation effect as the optimized 7-dehydrocholesterol fermentation parameters; Among them, when the fermentation influence parameter is the medium component, use the random forest model to analyze the importance of each component in the medium component to obtain the component importance ranking; take at least 2 components ranked in the front of the importance ranking as inputs, perform multiple linear regression modeling to obtain a regression equation, and construct the fermentation medium component with the regression equation to obtain the optimized medium component.

[0007] Optionally, the medium components include at least glucose, corn steep liquor powder, yeast extract, beef extract, and peptone.

[0008] Optionally, the index for evaluating the fermentation effect is the total fermentation biomass or the titer of 7-DHC.

[0009] Optionally, adjusting the values of the fermentation influence parameters, experimentally obtaining the best fermentation effect, and taking the fermentation influence parameters at the best fermentation effect as the optimized 7-dehydrocholesterol fermentation parameters includes setting the values of multiple fermentation influence parameters, performing fermentation, calculating the absorbance value of the strain after fermentation, and taking the value of the fermentation influence parameter with the highest absorbance value of the strain as the optimized 7-dehydrocholesterol fermentation parameter; The optimized 7-dehydrocholesterol fermentation parameters are that the fermentation temperature is 30 °C and the inoculum size is 20%.

[0010] Optionally, the regression equation is OD = 0.344 + 3.374 Glu + 4.457 N3, where Glu is the glucose content and N3 is the beef extract content, and the ratio of the glucose content to the beef extract content is controlled to be 1:1.3.

[0011] An embodiment of the present invention provides a fermentation method, which ferments the substrate with the optimized 7-dehydrocholesterol fermentation parameters obtained by using the method for optimizing the fermentation of 7-dehydrocholesterol by data fitting to obtain 7-dehydrocholesterol.

[0012] Optionally, the fermentation strain is Saccharomyces cerevisiaeSaccharomyces cerevisiae The fermentation temperature is 30 °C and the inoculum size is 20%.

[0013] Optionally, the fermentation medium comprises 20 - 50 g / L of glucose and 26 - 65 g / L of beef extract, and the feeding medium for fermentation comprises 200 - 500 g / L of glucose and 260 - 650 g / L of beef extract.

[0014] Optionally, the fermentation medium comprises 40 g / L of glucose and 52 g / L of beef extract, and the feeding medium for fermentation comprises 400 g / L of glucose and 520 g / L of beef extract.

[0015] Optionally, the fermentation medium further comprises 1 ml / L of trace elements, and the trace elements are 2.8 g / L of FeSO4·7H2O and 3.0 g / L of MgCO3.

[0016] The chassis strain for fermentation in the present invention is 7-DHC-5 in Patent CN 114703077 B (which is a known strain and does not need to be preserved), and its genotype is CEN.PK2-1D; MATα; MAL2-8C; SUC2; gal80::KanMX; erg5Δ::DHCR24; erg6Δ::DHCR24; mot3Δ::ERG2,ERG3; nem1-Δ; Ty1Δ::tHMG1, IDI; Ty2Δ::ERG2, ERG3, CTT1, DHCR24.

[0017] The beneficial effects of the present invention are as follows: The present invention uses a random forest model to analyze the importance of each component in the culture medium, and obtains the importance ranking of the components; using at least 2 components with the top importance ranking as inputs, a multiple linear regression model is established to obtain a regression equation, and the culture medium components are constructed based on the regression equation to obtain optimized culture medium components. It can be known through experiments that when fermenting with the culture medium components optimized by the above method, compared with the fermentation method before optimization, the effect is increased from 2.022 g / L to 3.370 g / L, and the titer is increased by 160%. By optimizing the fermentation process using the method of the present invention, the fermentation titer can be significantly improved and the production cost can be reduced.

[0018] The present invention collects multiple factor parameters in the fermentation process of the 7-DHC genetic engineering strain, fits the data model of 7-DHC fermentation production, provides data support for the fermentation process control of the genetically modified strain for the total biosynthesis of 7-DHC, reduces the preparation of trace elements in the culture medium components, simplifies the process flow, optimizes the fermentation effect, and increases the titer of the 7-DHC product to 3.370 g / L. Description of the Drawings

[0019] Figure 1 Results of screening for the activity of fermentation strains.

[0020] Figure 2 Results of screening for fermentation temperature conditions. (a) Results of screening for fermentation temperature conditions with OD600 value as the ordinate, and (b) Results of screening for fermentation temperature conditions with titer as the ordinate.

[0021] Figure 3 Results of screening for fermentation inoculum amount. (a) Results of screening for fermentation inoculum amount with OD600 value as the ordinate, and (b) Results of screening for fermentation inoculum amount with titer as the ordinate.

[0022] Figure 4 Results of analyzing the variable importance by the random forest model.

[0023] Figure 5 Results of fed-batch fermentation. Specific implementation manners

[0024] The culture media, the method for measuring the OD600 value of Saccharomyces cerevisiae, and the method for measuring the 7-DHC content used in each example are as follows.

[0025] 1. Culture media LB culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride. Add 20 g / L agar powder to prepare solid LB culture medium.

[0026] YPD culture medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose. Add 20 g / L agar powder to prepare solid YPD culture medium.

[0027] Trace elements: 2.8 g / L FeSO4·7H2O, 3.0 g / L MgCO3.

[0028] 2. Method for measuring the OD600 value of Saccharomyces cerevisiae Take 1 ml of the bacterial solution, centrifuge at 4000 rpm for 3 min, discard the supernatant, resuspend with PBS, wash twice and then resuspend thoroughly, and dilute 10 - 30 times; take 4 ml of the diluted bacterial solution into a quartz cuvette and detect its OD600 value at a wavelength of 600 nm on a spectrophotometer; multiply the obtained value by the dilution factor to obtain the real-time OD600 value of the fermentation bacterial solution.

[0029] 3. Detection method for 7-DHC content Chromatographic conditions Chromatographic column: Agela promosil C18, 4.6×250mm, 5µm.

[0030] Mobile phase: 0.1% trifluoroacetic acid methanol solution.

[0031] Flow rate: 1.0 ml / min, column temperature: 30 °C, wavelength: 281 nm, injection volume: 10 μl.

[0032] Running time: 30 min.

[0033] Diluent: Mobile phase (methanol).

[0034] Test solution: Prepare 30% potassium hydroxide ethanol solution for standby. Take 1 ml of the fermentation broth and place it in a crushing tube, centrifuge at 13000 rpm for 5 min, discard the supernatant, add 30% potassium hydroxide ethanol solution, shake well, place it in a water bath at 85 °C for 3.5 hours, and mix several times during this period. After the reaction is completed, place the reaction solution in a 5 ml centrifuge tube, add 0.75 ml of sterile water to the centrifuge tube in advance, and then add anhydrous ether to make up to 3.5 ml. Normally, it will be layered, with 2.5 ml in the upper layer and 1 ml in the lower layer. Take the upper organic phase and filter it as the test solution (if the concentration of the upper organic phase is high, dilute it with methanol to a concentration of about 0.1 - 0.5 mg / ml). Reference stock solution: Take about 20 mg of 7-dehydrocholesterol and place it in a 20 ml brown volumetric flask, add mobile phase and dissolve it by ultrasonic wave and dilute to the scale, shake well, and filter.

[0035] Determination: After the baseline is stable, accurately measure the linear standard solution and the test solution respectively, inject them into the chromatograph, and record the chromatogram.

[0036] Result calculation: Taking the reference peak area as the ordinate and the concentration as the abscissa, draw a linear regression equation, and the correlation coefficient R value should not be less than 0.990. Substitute the peak area of the test solution into the linear equation (y = kx + b) to calculate the 7-DHC concentration in the sample.

[0037] Example 1 A method for optimizing 7-dehydrocholesterol fermentation by data fitting, comprising the following steps: Construct fermentation influencing parameters, which include at least one of fermentation strain activity, fermentation temperature, inoculum size, and medium composition; Adjust the values of the fermentation influencing parameters, and experimentally obtain the best fermentation effect. Take the fermentation influencing parameters at the best fermentation effect as the optimized 7-dehydrocholesterol fermentation parameters; Among them, when the fermentation influence parameter is the culture medium composition, a random forest model is used to analyze the importance of each component in the culture medium composition to obtain the component importance ranking; at least two components with the top importance ranking are used as inputs for multiple linear regression modeling to obtain a regression equation, and the optimized culture medium composition is obtained by constructing the fermentation culture medium composition with the regression equation.

[0038] 1. The method for optimizing the activity of the fermentation strain is as follows: Take an appropriate amount of the bacterial liquid from the glycerol tube of the 7-DHC genetic engineering strain, dilute and spread it on the YPD solid medium, and after culturing at 30 °C for 72 h, pick 15 single colonies with an inoculation loop and inoculate them into 5 ml of YPD medium; after culturing at 30 °C and 220 rpm for 24 h, measure the OD600 value of each group.

[0039] The results are as Figure 1 shown. Among them, the OD600 values of G9 and G13 are both greater than 13, which are 13.3 and 13.2 respectively. The growth rate of the G9 strain is relatively fast, and G9 is selected as the chassis strain for optimizing the fermentation process.

[0040] 2. Screening of fermentation temperature conditions Pick a single colony of 7-DHC G9 and inoculate it into 5 ml of YPD medium. After culturing at 30 °C and 220 rpm for 24 h, inoculate it into a 5 L fermenter at 5% v / v and carry out fermentation culture under temperature conditions of 25 °C and 30 °C respectively.

[0041] Fermentation medium: glucose 20 g / L, corn steep liquor dry powder 30 g / L, trace elements 1 ml / L; Feed medium: glucose 700 g / L, corn steep liquor dry powder 200 g / L.

[0042] After 108 h of fermentation culture, the results are as Figure 2 shown.

[0043] Under the condition of 25 °C, the average OD600 of the 7-DHC strain is 106.3, and the average titer is 1.484 g / L. Under the condition of 30 °C, the average OD600 is 115.8, and the average titer is 2.022 g / L. That is, the fermentation result of the 7-DHC strain under the temperature condition of 30 °C is better than that under the temperature condition of 25 °C.

[0044] 3. Screening of fermentation inoculation amount According to the fermentation seed liquid culture method and the fermentation medium composition method under the screening of the above fermentation temperature conditions, inoculate the seed liquid into a 5 L fermenter at 5%, 10%, 15%, and 20% v / v respectively for fermentation culture with different inoculation amounts.

[0045] After 108 h of fermentation culture, the results are as Figure 3 shown.

[0046] At the OD600 value level, there was no significant difference between the inoculation amounts of 5% and 10%, and significant differences existed between both of them and the inoculation amounts of 15% and 20%; while at the product titer level, only the inoculation amounts of 5% and 10% had significant differences with the inoculation amount of 20% (p*<0.05) respectively. The fermentation biomass was positively correlated with the product titer, that is, the best fermentation culture was carried out with an inoculation amount of 20%.

[0047] 4. Screening of fermentation medium components According to the fermentation seed liquid culture method under the screening of the above fermentation temperature conditions, inoculate at an inoculation amount of 5% v / v into 100 ml of fermentation broth, and carry out fermentation culture experiments at 30°C.

[0048] Composition of the fermentation medium: Glu glucose: 20, 30, 40, 50, 60, 70 g / L, a total of 6 factor levels; N1 corn steep liquor dry powder: 10, 20, 30 g / L, a total of 3 factor levels; N2 yeast extract: 10, 30, 50 g / L, a total of 3 factor levels; N3 beef extract: 10, 15, 30 g / L, a total of 3 factor levels; N4 peptone: 10, 20, 25 g / L, a total of 3 factor levels; A total of 59 groups of experiments were carried out, and OD600 value data were collected.

[0049] Call the R packages DALEX and randomForest to perform random forest modeling to analyze the importance of various types of variables. The analysis results are as Figure 4 shown, and determine the importance of each predictive variable to the response variable, that is, the OD600 value, from the score ranking.

[0050] Select the top 3 variables, Glu glucose, N3 beef extract, and N4 peptone, as predictive variable inputs, and the model selects to use stepwise to perform multiple linear regression modeling. The results are shown in Table 1, and a data model with Glu glucose and N3 beef extract as characteristics is obtained. The coefficient significance is all <0.05, and the model R 2 is 0.621, and the goodness of fit is better.

[0051] Table 1 Coefficient and significance test results of the fitted model

[0052] 5. Optimizing the fed-batch culture of 7-DHC using the data model Determine the optimal ratio of the two according to the regression formula (OD = 0.344 + 3.374 Glu + 4.457 N3), that is, prepare the components of the fermentation medium according to the ratio of Glu:N3 = 1:1.3. After single-factor optimization analysis, the optimal concentration of Glu is obtained as 40 g / L, and then the components of the fermentation medium are obtained.

[0053] Fermentation medium: glucose 40 g / L, beef extract 52 g / L, trace elements 1 ml / L; Feed medium: glucose 400 g / L, beef extract 520 g / L.

[0054] According to the fermentation seed liquid culture method under the screening of the above fermentation temperature conditions, inoculate into a 5L fermenter at an inoculation amount of 20% v / v, and carry out fed-batch fermentation at 30°C. The results are as Figure 5 shown.

[0055] After 104h of fermentation culture, the OD600 value of the 7-DHC strain reached the peak at 98h, which was 140.2, and the product titer was 3.370 g / L at this time.

[0056] Those of ordinary skill in the art should understand that: the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; under the idea of this application, the technical features in the above embodiments or different embodiments can also be combined, and the steps can be implemented in any order, and there are many other variations in different aspects of one or more embodiments of this application as described above, and they are not provided in detail for the sake of brevity.

[0057] One or more embodiments of this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of this application shall be included within the scope of protection of this application.

Claims

1. A method for optimizing 7-dehydrocholesterol fermentation using data fitting, characterized in that: The steps include: Constructing fermentation influencing parameters, wherein the fermentation influencing parameters include at least one of fermentation strain activity, fermentation temperature, inoculation amount and culture medium composition; Adjusting the value of the fermentation influencing parameter to obtain the best fermentation effect through experiment, and taking the fermentation influencing parameter at the best fermentation effect as the optimized 7-dehydrocholesterol fermentation parameter; Among them, when the fermentation influencing parameter is the culture medium component, the random forest model is used to analyze the importance of each component in the culture medium to obtain the component importance ranking; at least two components ranked first in importance are used as input to perform multivariate linear regression modeling to obtain a regression equation, and the fermentation culture medium components are constructed using the regression equation to obtain the optimized culture medium components.

2. The method according to claim 1, characterized in that: The culture medium components at least include glucose, corn steep liquor powder, yeast extract, beef extract and peptone.

3. The method according to claim 1, characterized in that: The indicators for evaluating the fermentation effect are the total fermentation biomass or the titer of 7-DHC.

4. The method according to claim 1, characterized in that: Adjusting the value of the fermentation influencing parameter to experimentally obtain the best fermentation effect, and using the fermentation influencing parameter at the best fermentation effect as the optimized 7-dehydrocholesterol fermentation parameter, including setting multiple values ​​of the fermentation influencing parameter, performing fermentation, calculating the absorbance value of the strain after fermentation, and using the value of the fermentation influencing parameter with the highest absorbance value of the strain as the optimized 7-dehydrocholesterol fermentation parameter; The optimized fermentation parameters of 7-dehydrocholesterol were fermentation temperature of 30°C and inoculation amount of 20%.

5. The method according to claim 2, characterized in that: The regression equation is OD = 0.344 + 3.374 Glu + 4.457N3, wherein Glu is the glucose content, and N3 is the beef extract content. The ratio of the glucose content to the beef extract content is controlled to be 1:1.

3.

6. A fermentation method, characterized in that: The substrate is fermented using the optimized 7-dehydrocholesterol fermentation parameters obtained by the method for optimizing 7-dehydrocholesterol fermentation using data fitting as described in any one of claims 1 to 5 to obtain 7-dehydrocholesterol.

7. The fermentation method according to claim 6, characterized in that: The fermented strain is Saccharomyces cerevisiae Saccharomyces cerevisiae , the fermentation temperature is 30℃ and the inoculation amount is 20%.

8. The fermentation method according to claim 6, characterized in that: The fermentation medium comprises 20-50 g / L of glucose and 26-65 g / L of beef extract, and the feed medium for fermentation comprises 200-500 g / L of glucose and 260-650 g / L of beef extract.

9. The fermentation method according to claim 8, characterized in that: The fermentation medium includes 40 g / L of glucose and 52 g / L of beef extract, and the feed medium for fermentation includes 400 g / L of glucose and 520 g / L of beef extract.

10. The fermentation method according to claim 8, characterized in that: The fermentation medium also includes 1 ml / L of trace elements, wherein the trace elements are 2.8 g / L FeSO4·7H2O and 3.0 g / L MgCO3.

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