CN109971817B - Preparation of baodanone by sequential transformation of Arthrobacter simplex and genetically engineered yeast strain - Google Patents
Preparation of baodanone by sequential transformation of Arthrobacter simplex and genetically engineered yeast strain Download PDFInfo
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Abstract
The invention relates to a production method of steroid drugs, in particular to a method for preparing a baodanone steroid compound by utilizing the sequential fermentation of Arthrobacter simplex and a genetically engineered yeast strain. The invention firstly provides a Pichia pastoris genetic engineering strain capable of efficiently expressing 17 beta carbonyl reductase, and secondly provides a method for preparing a compound BD by sequentially converting a fermentation compound AD by utilizing the Pichia pastoris genetic engineering strain.
Description
Technical field:
the invention relates to a production method of steroid drugs, in particular to a method for preparing a baodanone steroid compound by utilizing the sequential fermentation of Arthrobacter simplex and a genetically engineered yeast strain.
The background technology is as follows:
baodanone (BD), also known as Bodenone, is a white or off-white crystalline powder of formula C 19 H 26 O 2 . Baodarone is a derivative of testosterone, thus inheriting most of the properties of testosterone, and has androgenic and protein synthesis capabilities. The baodano can keep the nitrogen balance state of the myofibroblasts to be forward at any time, so that the myofibroblasts are accelerated to synthesize protein, and the myofibroblasts are expanded and inflated, and the baodano has great efficacy on strengthening muscles and endurance of physical exercisers. For a long time, raw material production enterprises of baodano ketone are not more in China, and most of the baodano ketone is synthesized by a chemical method. The chemical method is unfavorable for industrial mass production because of complicated production steps, use of toxic and harmful reagents, high pollution and the like. Therefore, how to replace chemical synthesis methods by biosynthetic pathways and establish efficient biosynthetic strategies is a critical issue that is currently in need of solution.
In recent years, researches on the synthesis of baodarone by a microbial transformation method are focused on catalysis by a single strain, and the problems of low substrate feeding concentration, insufficient transformation efficiency and the like still exist in the catalysis process. Although microorganisms such as Mucor racemosus, nostocmuscorum, arihrobacteroxydans and the like can produce BD by 17 beta carbonyl reduction using androsta-1, 4-diene-3, 17-dione (ADD) as a substrate, few of them can directly synthesize BD using relatively inexpensive androsta-4-ene-3, 17-dione (AD) as a substrate. AD is a precursor for synthesizing ADD, and AD can be synthesized into ADD through C1, 2-dehydrogenation of 3-sterone-delta 1-dehydrogenase (KsdD), and then through 17 beta carbonyl reduction, the target product BD is generated. Although research has been made on the exogenous expression of fungal-derived KsdD protein in Pichia pastoris to obtain the product BD, the substrate dosage and BD yield of Pichia pastoris are not high because the 17 beta carbonyl reduction of Pichia pastoris itself is not strong enough.
So far, microorganisms have not been found to have both strong C1,2 dehydrogenation and strong 17 beta carbonyl reduction. Arthrobacter simplicissimus (Arthrobacter simplex) has become a widely used steroid C1,2 dehydrogenation reaction strain in industrial production because of the advantages of high conversion efficiency, high reaction rate and the like; and the 17 beta carbonyl reductase from yeast has stronger 17 beta carbonyl reduction effect. Currently, there is no study on the production of BD by combined fermentation using Arthrobacter simplex and yeast strains.
The invention comprises the following steps:
in order to solve the problems, save the cost and improve the product yield, the invention firstly provides a Pichia pastoris gene engineering strain capable of efficiently expressing 17 beta carbonyl reductase, and secondly provides a method for preparing the compound BD by sequentially converting the compound AD by microorganisms by utilizing the Pichia pastoris gene engineering strain.
The pichia pastoris gene engineering strain is prepared by over-expressing 17 beta carbonyl reductase coding genes in pichia pastoris host cells;
preferably, the pichia host cell is pichia pastoris GS115;
preferably, the 17 beta carbonyl reductase (GenBank accession number: KZV 10489.1) encoding Gene is the ayr1 Gene, gene ID:854682;
preferably, the Pichia pastoris genetically engineered strain is Pichia pastoris (P.pastoris) GS115AYR1 S.c Is obtained by expressing the ayr1 Gene shown in Gene ID 854682 in Pichia pastoris GS115 by means of plasmid pPIC3.5K;
the method for preparing the compound BD by sequentially converting the fermentation compound AD by the microorganism comprises the following steps:
in a bioconversion system containing a substrate AD concentration of 3g/L to 10g/L, a dehydrogenation catalyst having C1,2 with a biomass of 4 to 6g/L (wet weight) is addedThe method comprises the steps of inactivating a reaction system after the quantity of an intermediate product ADD reaches the highest, and passing through a Pichia pastoris genetic engineering strain (Pichia pastoris) GS115AYR1 with the weight of 25-200g/L S.c Preparing a compound baodanone by sequential fermentation;
further, the bioconversion system was 50mM PBS buffer solution (pH 7.2);
further, the fermentation time of the Arthrobacter simplex is 25-50h, and the fermentation conditions are as follows: 20-40 ℃ and 150-250r/min;
further, the inactivation conditions of the reaction system are as follows: 121 ℃ for 20min;
further, adding pichia pastoris and glucose with the final concentration of 0-100g/L into the system at the same time;
preferably, the glucose concentration is 75g/L;
further, the fermentation time of the Pichia pastoris gene engineering strain is 2-4h, and the fermentation conditions are as follows: 20-40 ℃ and 150-250r/min;
preferably, the Arthrobacter simplicissimus is Arthrobacter simplicissimus (Arthrobacter simplex) TCCC 11037;
preferably, hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is used as a substrate cosolvent in the bioconversion system, and the molar ratio of the hydroxypropyl-beta-cyclodextrin to the substrate AD is 0-2:1;
preferably, HP-beta-CD is used as a substrate cosolvent in a molar ratio of 1:1 to substrate AD;
after the reaction is finished, the yield of BD in a fermentation system can reach 1.68-7.7g/L, and the yield reaches 56% -83%.
The beneficial effects are that:
(1) The method for preparing the compound BD by utilizing the microbial combined fermentation and transformation AD is realized for the first time, the final yield of BD reaches 83%, the purpose of improving the yield of the compound BD is achieved, and the method has good application value and popularization prospect.
(2) The invention can improve the utilization rate of coenzyme in the reaction system, reduce the oxidation in the system and improve the final conversion rate of BD.
(3) The microorganism combined fermentation is simple and convenient, is convenient to realize and has obvious effect.
Description of the drawings:
FIG. 1 PCR analysis of recombinant plasmid pPIC3.5K-ayr1
Wherein, M is DL5000DNA Marker;1,2:pPIC3.5K-ayr1 S.c A PCR product;
FIG. 2 double cleavage of the recombinant plasmid pPIC3.5K-ayr1 by BamHI and EcoRI
Wherein M is 1kbDNA Marker;1,2:pPIC3.5K-ayr1 S.c Double enzyme cutting products;
FIG. 3 PCR verification of P.pastoris GS115 Gene engineering Strain containing recombinant expression vector pPIC3.5K-ayr1
Wherein, M is DL5000DNA Marker;1,2: pPICC 3.5K-ayr1 S.c Recombinant p.pastoris GS115 genome PCR product;
FIG. 4 simultaneous transformation of AD with Arthrobacter simplex and Pichia pastoris recombinant;
FIG. 5 transformation of AD and ADD by recombinant Pichia;
FIG. 6 effect of inactivated Arthrobacter simplex on BD production.
The specific embodiment is as follows:
the invention will now be further illustrated by the following examples, which are intended to be illustrative only and not limiting in any way.
The Arthrobacter simplicissimus (Arthrobacter simplex) TCCC 11037 and Pichia pastoris (P.pastoris) GS115AYR1 S.c The wet bacterial cells for biotransformation were obtained by conventional slant culture and seed culture and fermentation culture, and the wet bacterial cells used in the following examples of the present invention were obtained by the following methods, unless otherwise specified:
arthrobacter simplex (Arthrobacter simplex) TCCC 11037:
(1) Slant culture:
slant culture medium: 1% glucose, 1% yeast extract, 2% agar, and adjusting pH to 7.2;
culture conditions: 32 ℃ for 3-5 days;
(2) Seed culture:
seed culture medium: 1% glucose, 1% corn steep liquor (supernatant), 0.5% peptone, 0.25% potassium dihydrogen phosphate, and adjusting pH to 7.2;
culture conditions: 32 ℃,160r/min and 18 hours;
(3) Fermentation culture:
fermentation medium: 1% glucose, 1% corn steep liquor (supernatant), 0.5% peptone, 0.25% potassium dihydrogen phosphate, adjusting pH to an inoculum size of 7.2, inoculating the seed solution into a fermentation medium, adding ethanol-dissolved substrate AD at 32 deg.C and 200r/min for 16 hours to make the substrate final concentration of the fermentation broth be 0.1%, inducing simple arthrobacter cells to produce dehydrogenase, and culturing for 8 hours.
After the fermentation culture was completed, the fermentation broth was centrifuged at 5000 Xg for 10min at 4℃to discard the supernatant, and the centrifuged cells were washed 2 times with 0.05mol/L PBS buffer (pH 7.2) and centrifuged at 5000 Xg for 10min to collect the cells to obtain wet cells.
Pichia pastoris (P.pastoris) GS115AYR1 S.c ：
(1) Slant culture:
slant culture medium: 1% of yeast extract, 2% of tryptone, 2% of glucose and 2% of agar powder;
culture conditions: 30 ℃ for 3-5 days;
(2) Seed culture:
seed culture medium: 1% yeast extract, 2% tryptone, 2% glucose;
culture conditions: 30 ℃,200r/min and 20 hours;
(3) Fermentation culture
Fermentation medium: 1% Yeast extract, 2% tryptone, 100mmol/L Potassium phosphate (pH 6.0), 1.34% YNB (filter degerming), 4X 10 -5 Biotin (filter sterilization), 0.5% methanol (filter sterilization);
culture conditions: culturing at 30deg.C for 4 days at 200r/min, adding 0.5% methanol every 24h, and inducing Pichia pastoris to produce carbonyl reductase. After the fermentation culture was completed, the fermentation broth was centrifuged at 6000r/min at 4℃for 10min, the supernatant was discarded, the centrifuged cells were washed 2 times with 0.05mol/L PBS buffer (pH 7.2), centrifuged for 10min, and the cells were collected to obtain wet cells.
Laboratory preliminary studies show that the presence of the more potent 17-hydroxydehydrogenase in Arthrobacter simplex can seriously affect the production of the end product BD. The technical difficulty and key point of the combined reforming is therefore how to determine the proper catalytic sequence and catalytic manner to obtain the maximum yield of the product BD. Compared with the method for carrying out dehydrogenation and hydroxylation reactions by using the two strains singly or simultaneously, the method for preparing the steroid compound BD by using the two strains through sequential fermentation has the advantages that the total yield of the product is greatly improved, the energy consumption and the production period are reduced, the production cost and the labor force are reduced, and the requirements of scientific production are met.
The invention will be further explained by means of specific embodiments.
EXAMPLE 1 acquisition of 17 beta carbonyl reductase Gene and construction of Pichia Gene engineering Strain
(1) Acquisition of 17 beta carbonyl reductase gene ayr1
The gene engineering strain containing the target gene is obtained by amplifying 17 beta carbonyl reductase (GenBank number: KZV 10489.1) encoding gene ayr1 (GeneID: 854682) by using Saccharomyces cerevisiae W303 genome as a template, designing a primer pair as shown below, recovering PCR products by using Gel Extraction Kit (Omega, USA), connecting the recovered fragments to a vector pPICC 3.5K, constructing a recombinant plasmid pPICC 3.5K-ayr1, and transforming E.coli JM 109. The bacterial liquid PCR verification and the double enzyme digestion verification show that the recombinant plasmid pPIC3.5K-ayr1 is successfully constructed (shown in figures 1 and 2). And sequencing the cloned product, and comparing the sequenced product with the obtained ayr1 sequence, wherein the sequencing result completely accords with the target gene sequence.
An upstream primer: CGCGGATCCATGTCGGAGTTACAGTCACAACCTAA;
a downstream primer: CCGGAATTCCTAATCGTCCTTATTCTTCTGTTTCG.
(2) Construction and screening of 17 beta carbonyl reductase pichia pastoris gene engineering strain
(1) Preparation of pichia pastoris competent cells:
(1) pichia pastoris GS115 was cultured in a 50mL centrifuge tube containing 5mL of YPD at 30℃overnight;
(2) taking 0.1-0.5mL of overnight culture, inoculating 2L shake flask containing 500mL of fresh culture medium, and growing overnight until OD600 = 1.3-1.5;
(3) cells were collected by centrifugation at 1500g for 5min at 4 ℃, and suspended with 500mL of pre-chilled sterile water;
(4) centrifuging as above, suspending the cells with 250mL of pre-chilled sterile water;
(5) centrifugation as above, suspending cells with 20mL of precooled 1mol/L sorbitol;
(6) cells were suspended by centrifugation as above with 1mL of pre-chilled 1mol/L sorbitol to a final volume of about 1.5mL.
(2) Electrotransformation of pichia pastoris competent cells:
linearizing the extracted recombinant expression plasmid pPIC3.5K-ayr1 by SacI, extracting by phenol and chloroform, precipitating by ethanol, and purifying the linearization product.
(1) Mixing 80 μL of the cells with 5-20 μg of linearization DNA, and transferring into a precooled 0.2cm electric rotating cup;
(2) placing on ice for 5min;
(3) performing electric shock according to Saccharomyces cerevisiae parameters recommended by the used device;
(4) immediately add 1mL of pre-chilled 1mol/L sorbitol to the cup and transfer the contents to a sterile centrifuge tube;
(5) dividing into 200-600 mu L equal parts, and coating on an MD plate;
(6) plates were incubated at 30℃until clone production.
The construction of Pichia pastoris genetic engineering strain (P pastoris) GS115AYR1 for expressing the ayr1 gene is determined by carrying out genome PCR identification on Pichia pastoris containing recombinant expression vector pPIC3.5K-ayr1 S.c . (as shown in FIG. 3)
EXAMPLE 2 establishment of a double-bacterial sequential catalytic System
The effect of the Pichia pastoris gene engineering strain on AD or ADD, the effect of simultaneous transformation and sequential transformation of the Arthrobacter simplex and Pichia pastoris gene engineering strain on BD synthesis, and the effect of inactivation of the Arthrobacter simplex reaction system on BD synthesis were examined in the examples below, respectively.
Example 2-1
Picking Pichia pastoris GS115AYR1 as genetic engineering strain S.c Monoclonal, inoculated into 30mL BMGY, 30 ℃,200r/min, and shaken to OD 600 =5-6; centrifuging at 1500g-3000g for 5min at room temperature, collecting cells, removing supernatant, and re-suspending cells with 30mL BMMY for induced expression; every 24h, methanol was added to a final concentration of 0.5% to continue induction for 4d; and respectively adding 5g/L of substrate AD (or ADD) and HP-beta-CD with the molar ratio of the substrate to the substrate of 1:1 into the fermentation broth after 4d induction, continuously converting for 4d at 30 ℃ at 200r/min, sampling 1mL, ultrasonically extracting by using ethyl acetate with equal volume, and analyzing by HPLC. As shown in FIG. 5, the Pichia pastoris GS115AYR1 strain S.c After transformation of substrates AD and ADD by growing cells, the yields of the corresponding products TS (testosterone) and BD were 59% and 69%, respectively. This suggests that ADD is more suitable for use as a substrate for recombinant Pichia 17. Beta. Carbonyl reduction.
Example 2-2
The Arthrobacter simplex and Pichia pastoris gene engineering strain P.pastoris GS115AYR1 S.c Simultaneously adding the wet thalli of the Arthrobacter simplex into a 50mM PBS buffer solution (pH 7.2) reaction system, so that the final concentration of the Arthrobacter simplex is 5g/L (wet weight) and the final concentration of the Pichia pastoris gene engineering strain is 175g/L (wet weight), adding a substrate AD with the final concentration of 5g/L and HP-beta-CD with the molar ratio of the substrate to the substrate of 1:1 into the reaction system, and continuously converting for 30 hours at the temperature of 30 ℃ at the speed of 200 r/min. Samples were taken every 6h during the conversion process, the equivalent volume of ethyl acetate was extracted by sonication, and the BD yield was 36% as shown in fig. 4 by HPLC analysis. However, the generated BD cannot exist in the system stably, and then the generation rate of BD is obviously reduced; in contrast, the accumulation of ADD continues to rise to more than about 80% of the total system. The reason for this is probably the action of the endogenous 17 beta hydroxyl oxidase present in the simple arthrobacter of the bipartite catalytic system, whereupon the newly formed BD is again continued to be oxidized to ADD.
Examples 2 to 3
The prepared wet Arthrobacter simplex is suspended to a concentration of 5g/L by 30mL of 50mM PBS (pH 7.2) buffer solution, 5g/L of substrate AD and HP-beta-CD with a molar ratio of 1:1 are added at the same time, after the conversion for 30h at 34 ℃ and 200r/min, the final product is directly added into the reaction systemPichia pastoris gene engineering strain GS115AYR1 with concentration of 175g/L S.c The wet cells were transformed at 30℃and 200r/min for 12 hours. Sampling is carried out regularly in the conversion process, the ultrasonic extraction is carried out by using the ethyl acetate with the same volume, and the BD yield can reach 62.4 percent by HPLC analysis.
Examples 2 to 4
The prepared wet Arthrobacter simplex was resuspended to a concentration of 5g/L in 30mL of PBS (pH 7.2) buffer, and 5g/L of substrate AD and HP-beta-CD in a molar ratio of 1:1 were added at the same time, and after 30h of conversion at 34℃at 200r/min, the conversion system was subjected to inactivation treatment at 121℃for 20 min. Then adding 175g/L Pichia pastoris gene engineering strain GS115AYR1 into the inactivated reaction system S.c The wet cells were transformed at 30℃and 200r/min for 12 hours. Sampling is carried out regularly in the conversion process, the ultrasonic extraction is carried out by using the ethyl acetate with equal volume, and the BD yield can reach 73% after HPLC analysis.
In FIG. 6, "control" is the transformation procedure of examples 2-3 and "inactivation" is the transformation procedure of examples 2-4. Comparison shows that inactivation of Arthrobacter simplex helps to increase BD yield.
Examples 2 to 5
The prepared wet Arthrobacter simplex is resuspended to a concentration of 5g/L by 30mL PBS (pH 7.2) buffer solution, 5g/L of substrate AD and HP-beta-CD with a molar ratio of 1:1 with the substrate are simultaneously added, after the conversion is carried out for 30h at 34 ℃ and 200r/min, the conversion system is subjected to inactivation treatment by 121 ℃ for 20 min. Then adding 175g/L (wet weight) of Pichia pastoris gene engineering strain GS115AYR1 into the inactivated reaction system S.c The wet cells and 75g/L glucose were further transformed at 30℃for 12 hours at 200 r/min. Sampling is carried out regularly in the conversion process, the ultrasonic extraction is carried out by using the ethyl acetate with the same volume, and the BD yield can reach 83% after HPLC analysis.
Example 2 illustrates that the effect of sequentially transforming and synthesizing BD by using Arthrobacter simplex and Pichia pastoris genetic engineering strains is better than that of simultaneous transformation by double bacteria and single bacteria; when the method is used for sequential transformation, the effect of adding a sterilization program after the simple arthrobacter finishes transformation is better than that of directly carrying out pichia pastoris transformation without sterilization; the effect of adding glucose into the reaction system is better than that of adding glucose without adding glucose.
EXAMPLE 3 method for preparing Compound BD by microbial sequential conversion of fermentation Compound AD
Adding HP-beta-CD with a molar ratio of 1:1 with a substrate into a bioconversion system containing 50mM PBS buffer solution (pH 7.2) with a substrate AD concentration of 3g/L, adding wet thallus of Arthrobacter simplex TCCC 11037 with a biomass of 5g/L, reacting at 20 ℃ for 30 hours at 150r/min until the quantity of an intermediate product ADD reaches the highest, inactivating the reaction system at 121 ℃ for 20 minutes, and then treating the mixture with Pichia pastoris GS115AYR1 with a wet weight of 25g/L S.c Sequentially fermenting for 2 hours at 25 ℃ and 150r/min to prepare the compound baodanone;
after the reaction is finished, the yield of BD in a fermentation system can reach 1.68g/L, and the yield reaches 56%.
EXAMPLE 4 method for preparing Compound BD by microbial sequential conversion of fermentation Compound AD
Adding HP-beta-CD with a molar ratio of 1:1 with a substrate into a bioconversion system containing 50mM PBS buffer solution (pH 7.2) with a substrate AD concentration of 10g/L, adding wet thallus of Arthrobacter simplex TCCC 11037 with a biomass of 5g/L, reacting at 35 ℃ for 50h until the quantity of an intermediate product ADD reaches the highest, inactivating the reaction system at 121 ℃ for 20min, and adding Pichia pastoris GS115AYR1 with a final concentration of 200g/L S.c Sequentially fermenting wet thalli for 4 hours at 37 ℃ under the condition of 250r/min to prepare a compound baodanone, adding yeast and adding glucose with the final concentration of 100g/L of the system;
after the reaction is finished, the yield of BD in a fermentation system can reach 7.7g/L, and the yield reaches 77%.
Claims (1)
1. A method for preparing a compound Baodarone (BD) by sequentially converting and fermenting a compound androstane-4-ene-3, 17-dione (AD) by microorganisms is characterized in that the method comprises the steps of fermenting AD by simple arthrobacter, inactivating a reaction system, and sequentially fermenting by a pichia pastoris genetic engineering strain to prepare the compound baodarone;
the pichia pastoris gene engineering strain is obtained by over-expressing 17 beta carbonyl reductase coding genes in pichia pastoris host cells;
the method comprises the following steps: in the presence ofAdding Arthrobacter simplex with C1,2 dehydrogenation capability with biomass of 5g/L into a bioconversion system with substrate AD concentration of 3g/L-10g/L, inactivating the reaction system after the quantity of intermediate product androstane-1, 4-diene-3, 17-dione (ADD) reaches the highest, and adding 175-200 g/L of Pichia pastoris genetic engineering strainPichia pastoris）GS115 AYR1 S.c Preparing a compound baodanone by sequential fermentation;
the 17 beta carbonyl reductase GenBank number is: KZV10489.1; the host cell is Pichia pastoris GS115, and the Pichia pastoris gene engineering strain is preparedPichia pastoris) For GS115AYR1 S.c ；
The Arthrobacter simplicissimus is Arthrobacter simplicissimus @Arthrobacter simplex）TCCC 11037；
Adding pichia pastoris and glucose 75g/L into the transformation system;
hydroxypropyl-beta-cyclodextrin is adopted as a substrate cosolvent in the bioconversion system, and the molar ratio of the hydroxypropyl-beta-cyclodextrin to the substrate AD is 1:1-2:1.
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