CN111454871B - Recombinant mycobacterium with high androstenedione yield, construction method and application - Google Patents
Recombinant mycobacterium with high androstenedione yield, construction method and application Download PDFInfo
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Abstract
The invention discloses a recombinant mycobacterium with high androstenedione yield, a construction method and application thereof, wherein the construction method comprises the following steps: introducing cholesterol oxidase gene ChoM2, sterone C27 monooxygenase gene Smo2 carrying RBS fragment and 17 beta-hydroxysteroid dehydrogenase gene Hsd4A carrying RBS fragment into Mycobacterium neoformans (Mycobacterium sp.) CICC21097 to obtain recombinant Mycobacterium MN with high androstenedione yieldCSH(ii) a Obtained by the method of the inventionThe main fermentation product of the recombinant strain is 4-androstenedione, the yield is over 90 percent, the selectivity is over 90 percent, the generation of byproducts is reduced, and the androstenedione yield is improved. Compared with the original strain, the recombinant strain has better catalytic conversion performance and can improve the production efficiency. The process method is simple and convenient, and the efficiency is high.
Description
Technical Field
The invention belongs to the fields of biotechnology and biochemical industry, and particularly relates to a recombinant mycobacterium with high androstenedione yield, a construction method and application.
Background
The steroid hormone medicine refers to a hormone medicine containing a steroid structure in a molecular structure, is a clinically important medicine and mainly comprises adrenocortical hormone and sex hormone. Steroid hormones have many pharmacological actions, such as anti-infection, anti-allergy, anti-virus and anti-shock, and are widely used for treating various diseases, such as rheumatism, cardiovascular diseases, cancer, skin diseases and endocrine disorders. After decades of research and development, steroid hormone drugs form a large class of steroid drugs with various types, wide clinical application and vigorous demand, and become the second largest drug next to antibiotics.
Androst-4-ene-3,17-dione (English name: androstene-4-ene-3, 17-dione, or 4-AD) has an appearance of almost white crystal powder, has no odor, no toxicity, and is insoluble in water. Androstenedione is a steroid compound with androgenic action, which is originally extracted from testis or urine, and is found to play an important role in regulating the body through research. Androstenedione is an irreplaceable intermediate for preparing steroid hormone medicaments, almost all steroid hormone medicaments can be produced by taking 4-AD as a raw material, can be used for producing sex hormones, progestational hormones and protein assimilation hormones, and can also be used for preparing more than 100 medicaments such as hydrocortisone, prednisone oxide, progesterone and the like.
The global market for androstenedione is around $ 800 million and grows at a rate of 20% per year. The annual androstenedione demand of pharmaceutical enterprises in China is about 6000 tons, but the annual production capacity of androstenedione in China is only about 1000 tons, and the gap between the annual production capacity and the market demand is large.
At present, the production of androstenedione in China mainly has two ways, namely a diosgenin way and a microbial transformation way, and the androstenedione produced by the two ways in the market approximately accounts for half of the two ways. However, the diosgenin pathway (separation of saponin from dioscorea (yellow ginger) to produce androstenedione) has complicated production process, high cost and serious pollution, and production is prohibited in many places. The microbial transformation approach (the microbial transformation of phytosterol or cholesterol to produce androstenedione) has the advantages of low production cost, simplified production steps and the like, and is more and more concerned and more emphasized by wide manufacturers. With the further development of biotechnology, the proportion of androstenedione produced by microbial conversion in the global market will rapidly increase, and the market prospect is very attractive.
Due to the relative lag of some microbial steroid manufacturing processes and the introduction of efficient strains and production lines, a huge expense is required, so that most enterprises depend on the existing traditional production mode for a long time. In recent years, as some enterprises begin to vigorously introduce excellent steroid-converting microorganisms and fermentation processes, the production mode of hormone drugs has been greatly improved. Since 1998, the production of androstenedione by microbial transformation of phytosterols has begun, and the technology for producing hormones by microbial fermentation has been a long-term endeavor of technicians in this field, and improvements and developments in microbial transformation of steroids have been a long way.
At present, the microbial transformation of phytosterol to produce androstenedione has the main problems that: the strain has poor conversion capability, the steroid is easy to degrade in the conversion engineering, byproducts are generated, and the like, thereby seriously affecting the production benefit.
Disclosure of Invention
The invention aims to provide a construction method of a recombinant mycobacterium with high androstenedione yield, which can reduce the generation of byproducts and improve the conversion efficiency of preparing androstenedione from phytosterol.
The second purpose of the invention is to provide a recombinant mycobacterium with high androstenedione yield.
The third purpose of the invention is to provide an application of the recombinant mycobacterium with high androstenedione yield in fermentation production of androstenedione.
The technical scheme of the invention is summarized as follows:
a recombinant mycobacterium with high androstenedione yield is constructed by the following method: introducing cholesterol oxidase gene ChoM2, sterone C27 monooxygenase gene Smo2 carrying RBS fragment and 17 beta-hydroxysteroid dehydrogenase gene Hsd4A carrying RBS fragment into Mycobacterium neoformans (Mycobacterium sp.) CICC21097 to obtain recombinant Mycobacterium MN with high androstenedione yieldCSH；
The nucleotide sequence of the cholesterol oxidase gene ChoM2 is shown in SEQ ID NO. 1;
the nucleotide sequence of the RBS fragment is shown as SEQ ID NO. 2;
the nucleotide sequence of the ketosteroid C27 monooxygenase gene Smo2 is shown as SEQ ID NO. 3;
the nucleotide sequence of the 17 beta-hydroxysteroid dehydrogenase gene Hsd4A is shown in SEQ ID No. 4.
A construction method of recombinant mycobacteria with high androstenedione yield comprises the following steps: carrying out enzyme digestion and connection on the cholesterol oxidase gene ChoM2 and the plasmid pMV261 to construct a single-gene over-expression recombinant plasmid pMV261-ChoM 2; carrying out enzyme digestion and connection on a sterone C27 monooxygenase gene Smo2 carrying an RBS fragment and a recombinant plasmid pMV261-ChoM2 to construct a double-gene over-expression recombinant plasmid pMV261-ChoM2-Smo 2; carrying 17 beta-hydroxysteroid dehydrogenase gene Hsd4A of RBS fragment and double-gene over-expression recombinant plasmid pMV261-ChoM2-Smo2 through enzyme digestion and connection to construct three-gene over-expression recombinant plasmid pMv261-ChoM2-Smo2-Hsd 4A;
introducing the three-gene over-expression recombinant plasmid pMV261-ChoM2-Smo2-Hsd4A into susceptible cells of Mycobacterium neoplasma to obtain a positive transformant, namely the recombinant mycobacterium with high androstenedione yield, which is named as MNCSH；
The construction of three genes of the three-gene over-expression recombinant plasmid pMV261-ChoM2-Smo2-Hsd4A, namely ChoM2, a sterone C27 monooxygenase gene Smo2 carrying an RBS fragment and a 17 beta-hydroxysteroid dehydrogenase gene Hsd4A carrying the RBS fragment are sequentially interchanged.
The application of the recombinant mycobacterium with high androstenedione yield in fermentation production of androstenedione is provided.
Experiments prove that the main product of the phytosterol converted by the recombinant mycobacterium with high androstenedione yield is 4-androstenedione, the yield is over 90%, and the selectivity is over 90%. Compared with the original strain, the recombinant strain has better catalytic conversion performance and can improve the production efficiency. The process is simple and efficient.
Drawings
FIG. 1 is a schematic representation of recombinant plasmid pMv261-ChoM2-Smo2-Hsd 4A;
FIG. 2 recombinant strain MN for high-yield androstenedione productionCSHConstructing;
FIG. 3 is a liquid chromatography (HPLC) chart of detection of extracted fermentation broth after 168 hours of fermentation conversion, in FIG. 3, (A) is an original strain (B) is a recombinant strain;
FIG. 4 Positive transformants MN of the original and recombinant strainsCSH4-AD kinetic curves are generated through conversion;
Detailed Description
The host cell is preferably Mycobacterium neogold (Mycobacterium sp), which is purchased from China Industrial microbial cultures Collection management center (CICC21097 for short) and has the number of CICC 21097;
9 and 20 months in 2018; http:// www.china-cicc
pMV261 (commercially available)
The invention is further illustrated with reference to specific examples. It should be understood that the embodiments described herein are illustrative only and are not limiting, and that various equivalent substitutions and modifications may be made without departing from the spirit and scope of the invention.
Example 1
A construction method of recombinant mycobacteria with high androstenedione yield comprises the following steps:
(1) constructing a three-gene overexpression recombinant plasmid pMV261-ChoM2-Smo2-Hsd 4A:
synthesizing a target gene, namely, adding enzyme cutting sites BamH I/EcoR I to both ends of a gene ChoM2 sequence, adding enzyme cutting sites EcoR I to both ends of a gene Smo2 carrying an RBS fragment, adding enzyme cutting sites HindIII to both ends of a gene Smo Hsd4A carrying an RBS fragment, and carrying out gene synthesis by using a cholesterol oxidase gene ChoM2(SEQ ID No.1), a sterone C27 monooxygenase gene Smo2 carrying an RBS fragment (the RBS fragment is connected to the 5 'end of the gene Smo2, the sequence of the RBS fragment is shown in SEQ ID No.2, and the sequence of the gene Smo2 is shown in SEQ ID No. 3) and a nucleotide sequence of 17 beta-hydroxysteroid dehydrogenase Hsd4A carrying an RBS fragment (the RBS fragment is connected to the 5' end of the gene Hsd4A, and the sequence of the gene Hsd4A is shown in SEQ ID No. 4), and carrying an RBS fragment;
secondly, a target gene fragment ChoM2, Smo2 carrying an RBS fragment and Hsd4A gene carrying the RBS fragment, which are obtained in the PCR amplification step;
thirdly, the target gene fragment ChoM2 and the plasmid pMV261 obtained in the second step are respectively subjected to double enzyme digestion by BamH I/EcoR I, purified and connected to obtain a single-gene over-expression recombinant plasmid pMV261-ChoM 2;
carrying out enzyme digestion on the target gene fragment Smo2 carrying the RBS fragment and the single-gene over-expression recombinant plasmid pMV261-ChoM2 obtained in the step II by using EcoRI respectively, purifying, and connecting to obtain a double-gene over-expression recombinant plasmid pMV261-ChoM2-Smo 2;
the target gene segment Hsd4A carrying the RBS segment and the double-gene over-expression recombinant plasmid pMV261-ChoM2-Smo2 obtained in the step II are respectively digested by Hind III, purified and connected to obtain a three-gene over-expression recombinant plasmid pMV261-ChoM2-Smo2-Hsd4A, as shown in figure 1.
TABLE 1 primers required for obtaining overexpressed genes by PCR
(2) Construction of a recombinant Mycobacterium for high-yielding androstenedione, named MNCSH：
Preparation of competent cells of Mycobacterium (CICC 21097):
culturing the primary seeds until the OD600 is about 1.0, and transferring the seeds into a seed culture medium according to the inoculation amount of 10% to perform secondary seed culture; after 24h, 2% glycine was added and the culture was continued for 24 h. Centrifuging to collect thallus, washing suspended thallus with 10% precooled glycerol for four times, centrifuging, adding glycerol to suspend thallus, and subpackaging for preservation;
seed culture medium composition: 0.5g/L K2HPO4，0.5g/LMgSO4·7H2O, 0.05g/L ferric ammonium citrate, 2.0g/L citric acid, 3.5g/L diammonium phosphate, 20g/L glycerol and the balance of water, and the pH value is 7.5.
Electrical conversion: 10 mu L of the three-gene overexpression recombinant plasmid obtained in the step (1) is added into 100 mu L of the competent bacteria to be placed for 10min, and the three-gene overexpression recombinant plasmid is electrically transformed twice under the condition of 1.5kV, and each time lasts for 5 ms;
screening and verifying recombinants: adding the electrotransformation product into a seed culture medium for resuscitation and culture for 2h, coating the electrotransformation product on a seed culture medium plate containing 50mg/L kanamycin, standing and culturing for 7d at 30 ℃, selecting a single colony to the seed culture medium for culture, performing bacterial liquid PCR verification by using primers ChoM2-f and Hsd4A-r, wherein the bacterial liquid PCR generates a band with the size of 3840bp, and a positive transformant which is verified to be correct by the bacterial liquid PCR is the recombinant mycobacterium with high androstenedione yield, which is named as MNC transformantSHAs shown in fig. 2.
Example 2
Recombinant mycobacterium with high androstenedione yield, named MNCSHMethod for preparing androstenedione by fermentation (recombinant strain for short)
Inoculating the recombinant Mycobacterium MN with an inoculum size of 1% (v/v)CSHThe strain was inoculated into 5mL of seed medium, cultured with shaking (200rpm) at 30 ℃ for 48 hours, and then inoculated into 200mL of fermentation transformation medium at 37 ℃ and 200rpm in a 10% inoculum size, and transformed for 168 hours.
Fermentation ofThe composition of the transformation medium was 0.5g/L K2HPO4，0.5g/L MgSO4·7H2O, 0.05g/L ferric ammonium citrate, 2.0g/L citric acid, 3.5g/L diammonium hydrogen phosphate, 20g/L phytosterol, 60g/L methylated-beta-cyclodextrin and the balance of water, wherein the pH value is 8.0.
Taking 1mL of fermentation liquor every 24h, extracting with 2 times volume of ethyl acetate, and detecting the androstenedione yield by high performance liquid chromatography. As shown in figure 4, compared with the yields of androstenedione generated by the conversion of the original strain and the recombinant strain at different fermentation times, the yield of androstenedione of the recombinant strain is increased by 31.9% compared with that of the original strain (CICC21097) after 168 hours of fermentation conversion, and the concentration of androstenedione reaches 11.1 g/L.
After 168 hours of fermentation, fermentation broth of the recombinant strain was taken, extracted with 2 volumes of ethyl acetate and the product content was measured by high performance liquid chromatography, the results of which are shown in fig. 3 (B). The same process conditions were used for extraction and product testing after 168 hours of fermentation of the original strain, and the results are shown in fig. 3 (a). The comparison shows that the main products of fermentation transformation of the recombinant strain and the original strain are both 4-AD, and the 4-AD yield of the recombinant strain is far higher than that of the original strain; in addition, the content of the byproduct 2 (21-hydroxy-20-methylpregna-4-en-3-one) in the fermentation liquor of the recombinant strain is far lower than that of the original strain, and the yield and the selectivity of the recombinant strain are far higher than those of the original strain. Compared with the original strain, the recombinant strain has better performance, and the production efficiency can be improved by adopting the recombinant strain for conversion.
Sequence listing
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<210> 9
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aagcttaaga aggagatata atgaacgaca acccg 35
<210> 10
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aagctttcaa gaacccatga gctcag 26
Claims (3)
1. A recombinant mycobacterium producing androstenedione, characterized by being constructed by the following method: introducing cholesterol oxidase gene ChoM2, sterone C27 monooxygenase gene Smo2 carrying RBS fragment and 17 beta-hydroxysteroid dehydrogenase gene Hsd4A carrying RBS fragment into Mycobacterium neoformans (Mycobacterium sp.) CICC21097 to obtain recombinant Mycobacterium MN capable of producing androstenedioneCSH；
The nucleotide sequence of the cholesterol oxidase gene ChoM2 is shown in SEQ ID NO. 1;
the nucleotide sequence of the RBS fragment is shown as SEQ ID NO. 2;
the nucleotide sequence of the ketosteroid C27 monooxygenase gene Smo2 is shown as SEQ ID NO. 3;
the nucleotide sequence of the 17 beta-hydroxysteroid dehydrogenase gene Hsd4A is shown in SEQ ID NO. 4;
the construction method of the androstenedione-producing recombinant mycobacterium comprises the following steps: carrying out enzyme digestion and connection on the cholesterol oxidase gene ChoM2 and the plasmid pMV261 to construct a single-gene over-expression recombinant plasmid pMV261-ChoM 2; carrying a sterone C27 monooxygenase gene Smo2 of an RBS fragment and a recombinant plasmid pMV261-ChoM2, and constructing a double-gene over-expression recombinant plasmid pMV261-ChoM2-Smo2 through enzyme digestion and connection; carrying 17 beta-hydroxysteroid dehydrogenase gene Hsd4A of RBS fragment and double-gene over-expression recombinant plasmid pMV261-ChoM2-Smo2 through enzyme digestion and connection to construct three-gene over-expression recombinant plasmid pMv261-ChoM2-Smo2-Hsd 4A;
introducing the three-gene over-expression recombinant plasmid pMV261-ChoM2-Smo2-Hsd4A into susceptible cells of Mycobacterium neoplasma to obtain a positive transformant, namely the recombinant mycobacterium capable of producing androstenedione, which is named as MNCSH。
2. The recombinant mycobacterium capable of producing androstenedione according to claim 1, wherein the three genes over-expression recombinant plasmid pMV261-ChoM2-Smo2-Hsd4A comprises three genes of ChoM2, sterone C27 monooxygenase gene Smo2 carrying a RBS fragment and 17 β -hydroxysteroid dehydrogenase gene Hsd4A carrying a RBS fragment, which are constructed in sequence.
3. Use of a recombinant mycobacterium as claimed in claim 1 or claim 2 for the fermentative production of androstenedione.
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| CN115029368B (en) * | 2022-06-24 | 2023-10-31 | 中国科学院上海高等研究院 | Genetically engineered bacterium for producing bisnoralcohol and application thereof |
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