CN118147027A - Method, strain and application for improving steroid precursor production by enhancing intracellular ATP metabolism - Google Patents
Method, strain and application for improving steroid precursor production by enhancing intracellular ATP metabolism Download PDFInfo
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- CN118147027A CN118147027A CN202410225366.2A CN202410225366A CN118147027A CN 118147027 A CN118147027 A CN 118147027A CN 202410225366 A CN202410225366 A CN 202410225366A CN 118147027 A CN118147027 A CN 118147027A
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- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
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- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
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- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- 235000015500 sitosterol Nutrition 0.000 description 1
- NLQLSVXGSXCXFE-UHFFFAOYSA-N sitosterol Natural products CC=C(/CCC(C)C1CC2C3=CCC4C(C)C(O)CCC4(C)C3CCC2(C)C1)C(C)C NLQLSVXGSXCXFE-UHFFFAOYSA-N 0.000 description 1
- VSIVTUIKYVGDCX-UHFFFAOYSA-M sodium;4-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].COC1=CC([N+]([O-])=O)=CC=C1[N+]1=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=NN1C1=CC=C([N+]([O-])=O)C=C1 VSIVTUIKYVGDCX-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 229960002256 spironolactone Drugs 0.000 description 1
- LXMSZDCAJNLERA-ZHYRCANASA-N spironolactone Chemical compound C([C@@H]1[C@]2(C)CC[C@@H]3[C@@]4(C)CCC(=O)C=C4C[C@H]([C@@H]13)SC(=O)C)C[C@@]21CCC(=O)O1 LXMSZDCAJNLERA-ZHYRCANASA-N 0.000 description 1
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Abstract
The invention belongs to the technical field of biocatalysis, and discloses a method, a strain and application for improving steroid precursor production by enhancing intracellular ATP metabolism, wherein the strain is obtained by constructing genes for expressing ATP enzyme subunits singly or in combination in host bacteria. The invention obtains the strain by over-expressing ATPase subunit gene from escherichia coli in sterol conversion strain MNR, and the highest androstenedione conversion rate reaches 89.1% in the fermentation process, which is improved by 12.4% compared with that of the synchronous starting strain MNR; the MNR-ATPase MNR is obtained by over-expressing an ATPase subunit gene derived from the new Mycobacterium aureum in MNR, the highest androstenedione conversion efficiency reaches 93.2% in the fermentation process, the androstenedione conversion efficiency is improved by 16.5% compared with that of the MNR of the contemporaneous starting strain, and the highest conversion rate is 2.2 times of that of the starting strain.
Description
Technical Field
The invention belongs to the technical field of biocatalysis, and in particular relates to a method, a strain and application for improving steroid precursor production by enhancing intracellular ATP metabolism.
Background
Steroid compounds are essential for the normal functioning of the organism and when the organism itself is under-produced, active intake of such steroid compounds is required to supplement the normal needs of the organism. Up to hundreds of steroid drugs are currently marketed, which are widely used clinically as diuretics, anti-inflammatory agents, steroid hormone immunogens and cardiovascular drugs, which lead to a great demand for precursors of such drugs.
The industrial production of steroid medicine precursor mainly utilizes microbial conversion method to make cheap and easily-obtained raw materials of plant sterol, beta sitosterol, ergosterol and cholesterol undergo the process of microbial metabolism, separation and purification so as to obtain the invented multifunctional high-value steroid medicine precursor. The phytosterol is taken as a raw material, and the producible steroid precursor mainly comprises two major categories of C19-steroid (AD, ADD, 9-OHAD) and C22-steroid (20-carboxyl-pregn-4-en-3-one, 4-BNC, 20-hydroxymethyl-pregn-4-en-3-one and 4-BNA). Wherein, androstane-4-alkene-3, 17-diketone (Androstenedione, AD) can be used for producing androgen, protein assimilation hormone, spirolactone and other medicaments; androstane-1, 4-di-ene-3, 17-dione (Androsta-diene-dione, ADD) produced by dehydrogenation of AD at C1, 2-position can be used to synthesize 19-nor-steroid estrogens, such as Estrone (Estrone), norethindrone (Norethisterone), progesterone (Progestin), and the like. In addition to synthetic sex hormones, the introduction of corticosteroid side chains on the keto groups of AD may also enable the use of them in the production of corticoids, or by hydroxylation at different sites, more types of steroid intermediates such as 9α -OH-AD, 5α -OH-AD, 11α -OH-AD, etc. are produced.
In the process of generating androstenedione by sterol metabolism, a molecule of beta sitosterol can be subjected to preliminary oxidation and side chain degradation by microorganisms, and the complete degradation of a molecule of sitosterol can generate about 10mol of FADH 2 and 21mol of NADH, and finally about 80mol of ATP can be generated, so that the ATP in a bacterium body is excessively accumulated, and cytotoxicity is generated. The accumulation of ATP can inhibit activities of key metabolic enzymes such as pyruvate dehydrogenase and the like related to life, even the main energy metabolism pathway of the strain is in reverse circulation, and the activity of the strain is influenced, so that the yield of androstenedione is limited, and the industrial production cost is increased.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method, a strain and application for improving the production of steroid precursors by enhancing intracellular ATP metabolism.
The technical scheme adopted for solving the technical problems is as follows:
a genetic engineering strain for efficiently converting sterols is characterized in that: the strain is obtained by constructing a gene for expressing an ATPase subunit singly or in combination in a host bacterium, and the metabolic capacity of the strain on ATP is enhanced by enhancing the expression of the gene in the sterol conversion strain, so that the sterol conversion rate is improved.
Further, the host bacteria of the genetically engineered strain are bacteria or fungi with sterol conversion capability;
or the steroid precursor is obtained after the genetically engineered strain converts sterols, and comprises hydroxylated derivatives and A-ring degradation products;
or the ATPase subunit is expressed by a vector.
Further, the bacterium or fungus having sterol conversion ability is a mycobacterium microorganism or rhodococcus microorganism;
The hydroxylated derivatives and A-ring degradation products include androsta-4-ene-3, 17-dione (androst-4-ene-3, 17-dione, AD), androsta-1, 4-diene-3,17-dione (Androst-1, 4-diene-3,17-dione, ADD), 9 alpha-hydroxyandrosta-4-ene-3, 17-dione (9 alpha-hydroxyandrost-4-ene-3, 17-dione,9 alpha-OH-AD);
the expression vector is pMV261.
Further, the Mycobacterium microorganism is selected from the group consisting of Mycobacterium (Mycobacterium sp.) NRRLB-3683, mycobacterium (Mycobacterium sp.) NRRLB-3805, mycobacterium smegmatis (Mycobacterium smegmatism), mycobacterium fortuitum (Mycobacterium fortuitum), mycobacterium microbacterium (Mycobacterium gilvum), mycobacterium neogolden (Mycobacterium neoaurum), mycobacterium phlei (Mycobacterium Phlei), mycobacterium avium (Mycobacterium avium);
The genetically engineered strain expresses an ATPase subunit in the novel Mycobacterium aurum MNR via pMV 261.
Further, the Mycobacterium microorganism is Mycobacterium neogold (Mycobacterium sp.) mnrm3Δ KsdD.
Further, the ATPase subunits are respectively derived from escherichia coli MG1655 and MNR, and the amino acid sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
or coding genes E.coliATPase-F1, MNRATPASE-F1 of the ATPase subunit, and the nucleotide sequence is SEQ ID NO.3 and SEQ ID NO.4.
The application of the genetically engineered strain in the production of steroid precursors.
A method for improving the production of steroid precursor by enhancing intracellular ATP metabolism includes such steps as preparing genetically engineered strain by single over-expression or combined expression of ATP enzyme subunit genes in host bacteria, and enhancing the expression of said genes in sterol transformed strain to increase ATP metabolism of said strain and increase sterol conversion rate.
A method for producing androstane-4-alkene-3, 17-dione by using the genetic engineering strain comprises the following steps of;
Transferring seed culture solution of the genetically engineered bacteria into a fermentation culture medium according to the inoculum size of 2% -12%, and culturing for 48-168 hours under the conditions of 25-37 ℃ and 140-220 rpm; the molar conversion rate of androstenedione can reach 50% -99%.
Further, the seed culture medium comprises the following components: k 2HPO40.5 g/L,MgSO4, 0.5, g/L, ferric ammonium citrate, 0.05g/L, citric acid, 2g/L, ammonium nitrate, 20g/L glycerol, 5g/L glucose, caCO 3, 1, g/L and water as a solvent, wherein the pH value is 7.2;
the composition of the fermentation culture is as follows: (NH 4)2HPO40.1～4g/L,K2HPO40.1～3g/L,MgSO4 -0.1 g/L, ferric ammonium citrate 0.01-0.2 g/L, citric acid 1-5 g/L, reducing sugar 5-50 g/L, sterol 1-50 g/L, water as solvent, pH 6.0-7.5, and sterilizing with high pressure steam.
The invention has the advantages and positive effects that:
1. The invention obtains the strain MNR-ATPase E.coli by over-expressing an ATPase subunit gene derived from escherichia coli in a sterol conversion strain MNR, and the highest androstenedione conversion rate reaches 89.1% in the fermentation process, which is improved by 12.4% compared with that of a synchronous starting strain MNR; the MNR-ATPase MNR is obtained by over-expressing an ATPase subunit gene derived from the new Mycobacterium aureum in MNR, the highest androstenedione conversion efficiency reaches 93.2% in the fermentation process, the androstenedione conversion efficiency is improved by 16.5% compared with that of the MNR of the contemporaneous starting strain, and the highest conversion rate is 2.2 times of that of the starting strain.
2. The invention solves the problems that in the production process of the steroid precursor by a microbiological method, intracellular ATP accumulation of the strain in the later period of fermentation leads to the reduction of sterol conversion rate and consequently leads to the high price of the steroid precursor.
Drawings
FIG. 1 is an electrophoretogram of amplified products of ATPase MNR and ATPase E genes of the present invention; wherein, lane M is a DNA standard marker, lane 1 is an ATPase E gene amplification band, and lane 2 is an ATPase MNR gene amplification band;
FIG. 2 is a colony PCR verification map of the construction process of the vector pMV261-ATPase MNR、pMV261-ATPaseE of the present invention; wherein, lane M is a DNA standard marker, lane 1 is a pMV261-ATPase E gene amplification band, and lane 2 is a pMV261-ATPase MNR gene amplification band;
FIG. 3 is a graph showing ATP changes during the transformation of the strains MNR-ATPase MNR、MNR-ATPaseE.coli and MNR according to the invention;
FIG. 4 is a graph showing changes in viability of the MNR-ATPase MNR、MNR-ATPaseE.coli and MNR-transformed cells of the present invention;
FIG. 5 is a graph showing growth curves during the transformation of the strains MNR-ATPase MNR、MNR-ATPaseE.coli and MNR according to the invention;
FIG. 6 is a diagram showing the production of androstenedione by the strains MNR-ATPase MNR、MNR-ATPaseE.coli and MNR according to the present invention.
Detailed Description
The invention will now be further illustrated by reference to the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.
The various experimental operations involved in the specific embodiments are conventional in the art, and are not specifically noted herein, and may be implemented by those skilled in the art with reference to various general specifications, technical literature or related specifications, manuals, etc. before the filing date of the present invention.
A genetic engineering strain for efficiently converting sterols is characterized in that: the strain is obtained by constructing a gene for expressing an ATPase subunit singly or in combination in a host bacterium, and the metabolic capacity of the strain on ATP is enhanced by enhancing the expression of the gene in the sterol conversion strain, so that the sterol conversion rate is improved.
Preferably, the host bacteria of the genetically engineered strain are bacteria or fungi with sterol conversion capability;
or the steroid precursor is obtained after the genetically engineered strain converts sterols, and comprises hydroxylated derivatives and A-ring degradation products;
or the ATPase subunit is expressed by a vector.
Preferably, the bacterium or fungus having sterol conversion ability is a mycobacterium microorganism or rhodococcus microorganism;
The hydroxylated derivatives and A-ring degradation products include androsta-4-ene-3, 17-dione (androst-4-ene-3, 17-dione, AD), androsta-1, 4-diene-3,17-dione (Androst-1, 4-diene-3,17-dione, ADD), 9 alpha-hydroxyandrosta-4-ene-3, 17-dione (9 alpha-hydroxyandrost-4-ene-3, 17-dione,9 alpha-OH-AD);
the expression vector is pMV261.
Preferably, the Mycobacterium microorganism is selected from the group consisting of Mycobacterium (Mycobacterium sp.) NRRLB-3683, mycobacterium (Mycobacterium sp.) NRRLB-3805, mycobacterium smegmatis (Mycobacterium smegmatism), mycobacterium fortuitum (Mycobacterium fortuitum), mycobacterium fortuitum (Mycobacterium gilvum), mycobacterium neogolden (Mycobacterium neoaurum), mycobacterium phlei (Mycobacterium Phlei), mycobacterium avium (Mycobacterium avium);
The genetically engineered strain expresses an ATPase subunit in the novel Mycobacterium aurum MNR via pMV 261.
Preferably, the Mycobacterium microorganism is Mycobacterium neogold (Mycobacterium sp.) mnrm3Δ KsdD.
Preferably, the ATPase subunits are respectively derived from escherichia coli MG1655 and MNR, and the amino acid sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
or coding genes E.coliATPase-F1, MNRATPASE-F1 of the ATPase subunit, and the nucleotide sequence is SEQ ID NO.3 and SEQ ID NO.4.
The application of the genetically engineered strain in the production of steroid precursors.
A method for improving the production of steroid precursor by enhancing intracellular ATP metabolism includes such steps as preparing genetically engineered strain by single over-expression or combined expression of ATP enzyme subunit genes in host bacteria, and enhancing the expression of said genes in sterol transformed strain to increase ATP metabolism of said strain and increase sterol conversion rate.
A method for producing androstane-4-alkene-3, 17-dione by using the genetic engineering strain comprises the following steps of;
Transferring seed culture solution of the genetically engineered bacteria into a fermentation culture medium according to the inoculum size of 2% -12%, and culturing for 48-168 hours under the conditions of 25-37 ℃ and 140-220 rpm; the molar conversion rate of androstenedione can reach 50% -99%.
Preferably, the seed culture medium comprises the following components: k 2HPO40.5 g/L,MgSO4, 0.5, g/L, ferric ammonium citrate, 0.05g/L, citric acid, 2g/L, ammonium nitrate, 20g/L glycerol, 5g/L glucose, caCO 3, 1, g/L and water as a solvent, wherein the pH value is 7.2;
the composition of the fermentation culture is as follows: (NH 4)2HPO40.1～4g/L,K2HPO40.1～3g/L,MgSO4 -0.1 g/L, ferric ammonium citrate 0.01-0.2 g/L, citric acid 1-5 g/L, reducing sugar 5-50 g/L, sterol 1-50 g/L, water as solvent, pH 6.0-7.5, and sterilizing with high pressure steam.
Specifically, the preparation and detection of the correlation are as follows:
The host Mycobacterium (Mycobacterium sp.) MNR m3Δ KsdD (hereinafter abbreviated as mnr.) used in the present invention is obtained by knocking out the KsdD gene from Mycobacterium (Mycobacterium sp.) MNR M3 (numbered CICC 21097). The construction of a specific strain mnrm3Δ KsdD has been disclosed in Rili Xie,Yanbing Shen,Ning Qin,Yibo Wang,Liqiu Su,Min Wang.Genetic differences in ksdD influence on the ADD/AD ratio of Mycobacterium Neoaurum.Journal of Industrial Microbiology&Biotechnology,2015,42:507-513. wherein the strain mnrm3 has the code TCCC 11028:11028m3 in this article and the same strain as described herein (Mycobacterium sp.) mnrm3, numbered cic 21097.
The invention will be further explained by means of the following embodiments.
Example 1:
the ATPase MNR derived from MNR M3 and the ATPase derived from E.coli MG 1655.
MNR is inoculated to LB culture medium, genomic DNA is extracted, and ATPase MNR gene PCR primer is designed by taking genome as a template:
Matp F：gcggatccagctgcagaattcATGGCAGAGTTGACAATCTCCG
Matp R：acgctagttaactacgtcgacTCACAGCTTGGCGCCGAG
PCR amplification was performed using Matp F and Matp R as primer pairs to obtain the ATPase MNR gene of the ATPase subunit of MNR. The amplified products were detected by nucleic acid electrophoresis (FIG. 1).
E.coli MG1655 is inoculated to LB culture medium, extracting its genome DNA, taking genome as template, designing ATPase E gene PCR primer:
atpE F：gcggatccagctgcagaattcATGCAACTGAATTCCACCGAA
atpE R：acgctagttaactacgtcgacTTAAAGTTTTTTGGCTTTTTCCACA
PCR amplification was performed using atpE F and atpE R as primer pairs to obtain the ATPase E gene, an ATPase subunit of MNR M3. The amplified products were detected by nucleic acid electrophoresis (FIG. 1).
Example 2:
An expression vector for over-expression of ATPase MNR、ATPaseE coding genes is constructed.
And (3) recovering the ATPase MNR、ATPaseE gene PCR product obtained in the last step through a purification kit, performing seamless cloning connection with a pMV261 linearization vector subjected to SalI and BamHI double digestion, constructing an over-expression plasmid pMV261-ATPase MNR、pMV261-ATPaseE, transferring the connection product into E.coli DH5 alpha through a chemical conversion method, and screening positive clones through kanamycin resistance. The positive cloning plasmid is extracted, and the successfully constructed expression vector pMV261-ATPase MNR、pMV261-ATPaseE is obtained after colony PCR and enzyme digestion verification (figure 2) and sequencing.
Example 3:
Construction of ATPase MNR、ATPaseE Gene-enhanced Strain MNR-ATPase MNR、MNR-ATPaseE.coli.
MNR competent preparation: taking a loop of MNR glycerol bacteria by an inoculating loop, culturing for 3 days at 30 ℃ by three dividing lines on an antibiotic-free LB plate, picking single bacterial colonies into an antibiotic-free LB test tube, culturing for 2 days at 30 ℃ at 200r/min, and transferring the single bacterial colonies into a seed culture medium (without calcium carbonate) according to 10% of inoculum size for secondary seed culture; after 36h, 20% (V/V, vol) glycine was added at a concentration of 10% (V/V, vol) and the culture was continued for 24h. Precooling in ice bath, centrifugally collecting thalli at 4 ℃, flushing and centrifuging the suspended thalli by precooled glycerol with the concentration of 10% (V/V, volume concentration) which is 1 time, 3/4 times, 1/2 times and 1/4 times of the volume of the fermentation liquor, finally adding the glycerol suspended thalli with the concentration of 10% (V/V, volume concentration) which is 1/25 times of the volume of the fermentation liquor, and subpackaging for storage;
Electric conversion: 10 mu L of constructed gene overexpression plasmid pMV261-ATPase MNR、pMV261-ATPaseE is taken and added into 100 mu L of competent thalli to be placed for 30min and then transferred into an electric rotating cup for electric shock. The electric shock condition is 2kV/cm,25 mu F,720 omega, and the ice is placed for 5min after 4-6 ms of electric transfer. To the electric rotor, 700. Mu.L of fresh sterilized LB medium was added, mixed well, transferred to a 1.5mL sterilized EP tube, and resuscitated at 30℃for 4 hours at 200 rpm.
Screening and verification: concentrating the resuscitated bacterial liquid, coating the concentrated bacterial liquid on LB solid medium containing 20 mug/mL of kanamycin for culturing for 5 days, picking single colony, extracting plasmid, and sequencing to verify that the sequence is aligned completely or the sequence is mutated unintentionally to obtain MNR M3 delta KsdD-ATPase MNR、MNR M3ΔKsdD-ATPaseE strain which is expressed correctly ATPase MNR、ATPaseE, and is hereinafter referred to as MNR-ATPase MNR、MNR-ATPaseE.coli strain.
Example 4:
MNR-ATPase MNR、MNR-ATPaseE.coli is used for androstenedione production.
Strain activation and seed preparation: respectively transferring the original strain MNR, MNR-ATPase MNR and MNR-ATPase E.coli over-expression strain onto fresh LB culture medium, culturing for 2d at 30 ℃, washing with 20mL of 0.5% (V/V, volume concentration) Tween 80 sterile aqueous solution, sucking the l mL of eluent, adding into 50mL of seed culture medium, and shake culturing for 36h at 30 ℃ and 200rpm to obtain seed solution;
Androstenedione production process: the seed solution obtained in the step 1 is respectively transferred into a fermentation medium according to the inoculation amount of 8 percent, and the seed solution is biologically transformed for 168 hours under the conditions of 30 ℃ and 140 rpm.
The seed culture medium consists of: k 2HPO40.5 g/L,MgSO4, 0.5, g/L, ferric ammonium citrate, 0.05g/L, citric acid, 2g/L, ammonium nitrate, 20g/L glycerol, 5g/L glucose, caCO 3, 1, g/L and the balance water, wherein the pH is 7.2.
The fermentation medium comprises the following components: (NH 4) 2HPO43.5 g/L,K2HPO40.5 g/L,MgSO4 0.4.4 g/L, ferric ammonium citrate 0.05g/L, citric acid 2g/L, glucose 10g/L, phytosterol 5g/L, and water for the rest, and the pH is 6.8-7.2.
Example 5:
ATP content detection during production of the strains MNR-ATPase MNR、MNR-ATPaseE.coli and MNR androstenedione.
The production of androstenedione was carried out according to the method of the previous step using MNR-ATPase MNR、MNR-ATPaseE.coli and MNR, and 1mL of sample was taken every 24h during the production process for intracellular ATP detection of the strain.
Intracellular ATP concentration assay: intracellular ATP concentration was determined by fluorescence. 100. Mu.L of the fermentation broth was added to a black 96-well plate, and 100. Mu. LBacTiterGloTM reagent (Promega, shanghai) was added thereto, and the mixture was shaken at 25℃for 2 minutes at 100r/min, and the fluorescence value was measured in a Lumineancement mode using INFINITE M Pro (Tecan, switzerland). And calculating the ATP concentration corresponding to the fluorescence value by using a standard curve.
Comparison of results:
the results of the detection of intracellular ATP content during fermentation of recombinant strain MNR-ATPase MNR、MNR-ATPaseE.coli and original strain MNR are shown in FIG. 3. The results show that the intracellular ATP content and the ATP growth rate of the recombinant strain are lower than those of the original strain. MNR-ATPase MNR has an intracellular ATP increase rate of k=0.1771, MNR-ATPase E has an intracellular ATP increase rate of k=0.166, and MNR has an intracellular ATP increase rate of k= 0.2304. This result demonstrates that overexpression of ATPase MNR and ATPase E can effectively enhance the metabolic capacity of ATP of the strain, solving the problem of ATP accumulation.
Example 6:
Cell viability detection during production of the MNR-ATPase MNR、MNR-ATPaseE.coli and MNR androstenedione strains.
Production of androstenedione was performed by using MNR-ATPase MNR、MNR-ATPaseE.coli and MNR as in example 4, and 1mL was sampled every 24 hours during production for strain cell viability detection.
Cell viability verification: the bacterial viability detection adopts an improved CCK-8 method, and 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-8) can be reduced into orange-yellow water-soluble formazan by bacterial dehydrogenase in the presence of an electronic carrier, and the generation amount and bacterial activity of the orange-yellow water-soluble formazan are positively correlated. The formazan has a maximum absorption peak at 450nm, and the activity of the thallus can be reflected by detecting the absorption value at 450 nm. After the OD 600 of the fermentation broth in different time periods was adjusted to 1 by using a pH 7.2Tris-HCl buffer, 190. Mu.L of the fermentation broth was added to a 96-well plate, WST-810. Mu.L of the fermentation broth was added to each well, and after incubation at 30 ℃ for 1 hour, the absorbance at 450nm was detected by using an enzyme-labeled instrument (INFINITE M Pro).
Comparison of results:
As shown in FIG. 4, in the middle and late stages of androstenedione production using MNR-ATPase MNR、MNR-ATPaseE.coli and MNR, strain viability tended to decrease due to cytotoxicity resulting from the accumulation of ATP excess. The cell viability decline rate (k= 0.01906) of the recombinant strain MNR-ATPase MNR was lower than MNR (k= 0.02232), the cell viability decline rate was 80% of MNR, the cell viability decline rate (k= 0.02179) of the recombinant strain MNR-ATPase E.coli was lower than MNR (k= 0.02232), the cell viability decline rate was 97% of MNR, and it was demonstrated that enhancing ATP metabolism was beneficial to reducing cytotoxicity resulting from ATP accumulation in excess in the post-production period, maintaining cell viability.
Example 7:
MNR-ATPase MNR、MNR-ATPaseE.coli was compared to the production performance of the original strain MNR androstenedione.
The production of androstenedione was carried out by using MNR-ATPase MNR、MNR-ATPaseE.coli and MNR as in example 4, and 1mL was sampled under aseptic conditions every 24 hours during the reproduction to carry out the detection of the androstenedione production rate.
Sample detection: sampling every 24 hours, taking 1mL of fermentation liquor, adding an equal volume of ethyl acetate, and carrying out ultrasonic treatment for 30min. Centrifuge at 14,000rpm for 10min, aspirate 100. Mu.L of supernatant, dry at room temperature, re-suspend the sample with 80% methanol, sonicate 30min, centrifuge at 14,000rpm for 10min, aspirate supernatant, and perform HPLC analysis.
The parameters of the Agilent 2000 chromatograph are set as follows: c18 (4.6X1250 mm) column, column temperature 30 ℃; mobile phase methanol/water (8:2, V/V), flow rate 1mL/min, detection wavelength 254nm, sample injection amount 10. Mu.L, each sample run for 8min. Androstenedione production and conversion were calculated according to standard curves.
1ML of the sample was sampled by the same method, and the absorbance at a wavelength of 600nm was measured by an ultraviolet spectrophotometer to determine the change in biomass.
Comparison of results:
As shown in FIG. 5, during androstenedione production using MNR-ATPase MNR、MNR-ATPaseE.coli and MNR, the biomass of MNR-ATPase MNR and MNR-ATPase E.coli was slightly lower than that of MNR, indicating that overexpression of ATPase MNR and MNR-ATPase E.coli inhibited mycobacterial growth to some extent, which may be due to enhanced ATP metabolizing capacity at the beginning of transformation.
As shown in FIG. 6, the androstenedione conversion efficiencies of the recombinant strains MNR-ATPase MNR and MNR-ATPase E.coli were progressively higher in the middle and later stages of the transformation than in the original strain MNR. At 120h, the molar conversion of the recombinant strain MNR-ATPase MNR androstenedione was 89.1% and 1.16 times that of the original strain. At 120h, the molar conversion of the recombinant strain MNR-ATPase E.coli androstenedione was 93.2% and 2.2 times that of the original strain.
By combining the analysis of examples 3-6, the overexpression of ATPase MNR and ATPase E genes can effectively enhance the ATP metabolic capacity of the strain, so that the strain is in a favorable Yu Xiong enedione generation state at the later production stage, and the method has obvious enhancement effect on improving the androstenedione production capacity of mycobacterium.
Currently, in article Xiuling Zhou,Yang Zhang,Yanbing Shen,Xiao Zhang,Zehui Zan,Menglei Xia,Jianmei Luo,Min Wang.Efficient repeated batch production of androstenedione using untreated cane molasses by Mycobacterium neoaurum driven by ATP futile cycle.Bioresource Technology,2020,309:123307, ATP empty cycle (PAFC) based on the interconversion of pyruvic acid and phosphoenolpyruvic acid and ATP empty cycle (CAFC) based on the interconversion of oxaloacetic acid and citric acid are used to reduce intracellular ATP content, so as to achieve the purpose of improving androstenedione production efficiency. When the same transformation condition as that of the present invention is adopted, the recombinant strain MNR-C3 has highest androstenedione molar conversion rate of 93.2% in 144 hr, and the transformation time is 24 hr longer than that of the present invention, and the transformation effect is poorer than that of the present invention.
The sequences used in the present invention are as follows:
SEQ ID NO.1 Escherichia coli MG1655 ATPase subunit
MQLNSTEISELIKQRIAQFNVVSEAHNEGTIVSVSDGVIRIHGLADCMQGEMISLPGNRYAIALNLERDSVGAVVMGPYADLAEGMKVKCTGRILEVPVGRGLLGRVVNTLGAPIDGKGPLDHDGFSAVEAIAPGVIERQSVDQPVQTGYKAVDSMIPIGRGQRELIIGDRQTGKTALAIDAIINQRDSGIKCIYVAIGQKASTISNVVRKLEEHGALANTIVVVATASESAALQYLAPYAGCAMGEYFRDRGEDALIIYDDLSKQAVAYRQISLLLRRPPGREAFPGDVFYLHSRLLERAARVNAEYVEAFTKGEVKGKTGSLTALPIIETQAGDVSAFVPTNVISITDGQIFLETNLFNAGIRPAVNPGISVSRVGGAAQTKIMKKLSGGIRTALAQYRELAAFSQFASDLDDATRKQLDHGQKVTELLKQKQYAPMSVAQQSLVLFAAERGYLADVELSKIGSFEAALLAYVDRDHAPLMQEINQTGGYNDEIEGKLKGILDSFKATQSW*MAGAKEIRSKIASVQNTQKITKAMEMVAASKMRKSQDR MAASRPYAETMRKVIGHLAHGNLEYKHPYLEDRDVKRVGYLVVSTDRGLCGGLNINLFKKLLAEMKTWTDKGVQCDLAMIGSKGVSFFNSVGGNVVAQVTGMGDNPSLSELIGPVKVMLQAYDEGRLDKLYIVSNKFINTMSQVPTISQLLPLPASDDDDLKHKSWDYLYEPDPKALLDTLLRRYVESQVYQGVVENLASEQAARMVAMKAATDNGGSLIKELQLVYNKARQASITQELTEIVSGAAAV*MATGKIVQVIGAVVDVEFPQDAVPRVYDALEVQNGNERLVLEVQQQLGGGIV RTIAMGSSDGLRRGLDVKDLEHPIEVPVGKATLGRIMNVLGEPVDMKGEIGEEERWAIHRAAPSYEELSNSQELLETGIKVIDLMCPFAKGGKVGLFGGAGVGKTVNMMELIRNIAIEHSGYSVFAGVGERTREGNDFYHEMTDSNVIDKVSLVYGQMNEPPGNRLRVALTGLTMAEKFRDEGRDVLLFVDNIYRYTLAGTEVSALLGRMPSAVGYQPTLAEEMGVLQERITSTKTGSITSVQAVYVPADDLTDPSPATTFAHLDATVVLSRQIASLGIYPAVDPLDSTSRQLDPLVVGQEHYDTARGVQSILQRYQELKDIIAILGMDELSEEDKLVVARARKIQRFLSQPFFVAEVFTGSPGKYVSLKDTIRGFKGIMEGEYDHLPEQAFYMVGSIEEAVEKAKKL*
SEQ ID NO.2 MNATATInase subunit
MAELTISASDIEGAIEGYVSSFSADTEREEVGTVVDAGDGIAHVEGLPSVMTQELLEFEGGVLGVALNLDEHSVGAVILGEFNKIEEGQQVKRTGEVLSVPVGDAFLGRVVNPLGQPIDGQGDIASDTRRELELQAPSVVQRQGVGEPLQTGIKAIDAMTPIGRGQRQLIIGDRKTGKTAVCVDTILNQRQAWETGDPNQQVRCVYVAIGQKGTTIASVKRALEDGGAMEYTTIVAAPASDPAGFKWLAPYTGSAIGQHWMYDGKHVLIVFDDLSKQADAYRAISLLLRRPPGREAFPGDVFYLHSRLLERCAKLSDELGGGSMTGLPIIETKANDISAFIPTNVISITDGQCFLESDLFNQGVRPAVNVGVSVSRVGGAAQIKAMKEVAGSLRLDLSQYRELEAFAAFASDLDAASKAQLDRGVRLVELLKQPQYSPLAVEDQVVAIFLGTQGHLDSVPAEDVSRFVDELLEHVKASHSDILDGIRETKKLSEEAEQKLVNVINDFKKGFSASDGSSVVVNEADSEALDPEDLEKESVKVRKPAPKKA*MAATLRELRGRIKSASSIKKITKAQELIATSRIAKAQARVDAARPYSTEITNMLTELASASALDHPLLVPRDNPKRAAVLVVSSDRGLCGGYNANVLRRAEELFSLLRDEGKDPVLYVIGRKALGYYNFRQRNVAESWTGFSERPTYEHAKEIADTLVTAFMSGADDDEDGAGADGVLGVDEIHIVSTEFRSMLSQTAVALRVAPMVVEYVGDEEPEDGPRTLFSFEPNAETLFDALLPRYIATRVYAALLEAAASESASRRRAMKSATDNADDLIKALTLAANRERQAQITQEISEIVGGANALADAK*MTAVETKTTTGRVVRITGPVVDVEFPRGAVPGLLNALHAEITFGALAKTLTLEVAQHLGE SLVRCISMQPTDGLVRGQEVTDTGASISVPVGDGVKGHVFNALGDCLDEPGYGKDFEHWSIHRKPPAFADLEPRTEMLETGLKVVDLLTPYVRGGKIALFGGAGVGKTVLIQEMINRIARNFGGTSVFAGVGERTREGNDLWVELADANVLKDTALVFGQMDEPPGTRMRVALSALTMAEFFRDEQGQDVLLFIDNIFRFTQAGSEVSTLLGRMPSAVGYQPTLADEMGELQERITSTRGRSITSMQAVYVPADDYTDPAPATTFAHLDATTELSRAVFSKGIFPAVDPLASSSTILHPSVVGDEHYRVAQEVIRILQRYKDLQDIIAILGIDELSEEDKVLVYRARKIERFLSQNMMAAEQFTGQPGSTVPLKETIEAFDKLAKGEFDHLPEQAFFLIGGLDDLAKKAESLGAKL*
SEQ ID NO.3E.coliATPase-F1
ATGCAACTGAATTCCACCGAAATCAGCGAACTGATCAAGCAGCGCATTGCTCAGTTCAATGTTGTGAGTGAAGCTCACAACGAAGGTACTATTGTTTCTGTAAGTGACGGTGTTATCCGCATTCACGGCCTGGCCGATTGTATGCAGGGTGAAATGATCTCCCTGCCGGGTAACCGTTACGCTATCGCACTGAACCTCGAGCGCGACTCTGTAGGTGCGGTTGTTATGGGTCCGTACGCTGACCTTGCCGAAGGCATGAAAGTTAAGTGTACTGGCCGTATCCTGGAAGTTCCGGTTGGCCGTGGCCTGCTGGGCCGTGTGGTTAACACTCTGGGTGCACCAATCGACGGTAAAGGTCCGCTGGATCACGACGGCTTCTCTGCTGTAGAAGCAATCGCTCCGGGCGTTATCGAACGTCAGTCCGTAGATCAGCCGGTACAGACCGGTTATAAAGCCGTTGACTCCATGATCCCAATCGGTCGTGGTCAGCGTGAATTGATCATCGGTGACCGTCAGACAGGTAAAACCGCACTGGCTATCGATGCCATCATCAACCAGCGCGATTCCGGTATCAAATGTATCTATGTCGCTATCGGCCAGAAAGCGTCCACCATTTCTAACGTGGTACGTAAACTGGAAGAGCACGGCGCACTGGCTAACACCATCGTTGTGGTAGCAACCGCGTCTGAATCCGCTGCACTGCAATACCTGGCACCGTATGCCGGTTGCGCAATGGGCGAATACTTCCGTGACCGCGGTGAAGATGCGCTGATCATTTACGATGACCTGTCTAAACAGGCTGTTGCTTACCGTCAGATCTCCCTGCTGCTCCGTCGTCCGCCAGGACGTGAAGCATTCCCGGGCGACGTTTTCTACCTCCACTCTCGTCTGCTGGAGCGTGCTGCACGTGTTAACGCCGAATACGTTGAAGCCTTCACCAAAGGTGAAGTGAAAGGGAAAACCGGTTCTCTGACCGCACTGCCGATTATCGAAACTCAGGCGGGTGACGTTTCTGCGTTCGTTCCGACCAACGTAATCTCCATTACCGATGGTCAGATCTTCCTGGAAACCAACCTGTTCAACGCCGGTATTCGTCCTGCGGTTAACCCGGGTATTTCCGTATCCCGTGTTGGTGGTGCAGCACAGACCAAGATCATGAAAAAACTGTCCGGTGGTATCCGTACCGCTCTGGCACAGTATCGTGAACTGGCAGCGTTCTCTCAGTTTGCATCCGACCTTGACGATGCAACACGTAAGCAGCTTGACCACGGTCAGAAAGTGACCGAACTGCTGAAACAGAAACAGTATGCGCCGATGTCCGTTGCGCAGCAGTCTCTGGTTCTGTTCGCAGCAGAACGTGGTTACCTGGCGGATGTTGAACTGTCGAAAATTGGCAGCTTCGAAGCCGCTCTGCTGGCTTACGTCGACCGTGATCACGCTCCGTTGATGCAAGAGATCAACCAGACCGGTGGCTACAACGACGAAATCGAAGGCAAGCTGAAAGGCATCCTCGATTCCTTCAAAGCAACCCAATCCTGGTAACGTCTGGCGGCTTGCCTTAGGGCAGGCCGCAAGGCATTGAGGAGAAGCTCATGGCCGGCGCAAAAGAGATACGTAGTAAGATCGCAAGCGTCCAGAACACGCAAAAGATCACTAAAGCGATGGAGATGGTCGCCGCTTCCAAAATGCGTAAATCGCAGGATCGCATGGCGGCCAGCCGTCCTTATGCAGAAACCATGCGCAAAGTGATTGGTCACCTTGCACACGGTAATCTGGAATATAAGCACCCTTACCTGGAAGACCGCGACGTTAAACGCGTGGGCTACCTGGTGGTGTCGACCGACCGTGGTTTGTGCGGTGGTTTGAACATTAACCTGTTCAAAAAACTGCTGGCGGAAATGAAGACCTGGACCGACAAAGGCGTTCAATGCGACCTCGCAATGATCGGCTCGAAAGGCGTGTCGTTCTTCAACTCCGTGGGCGGCAATGTTGTTGCCCAGGTCACCGGCATGGGGGATAACCCTTCCCTGTCCGAACTGATCGGTCCGGTAAAAGTGATGTTGCAGGCCTACGACGAAGGCCGTCTGGACAAGCTTTACATTGTCAGCAACAAATTTATTAACACCATGTCTCAGGTTCCGACCATCAGCCAGCTGCTGCCGTTACCGGCATCAGATGATGATGATCTGAAACATAAATCCTGGGATTACCTGTACGAACCCGATCCGAAGGCGTTGCTGGATACCCTGCTGCGTCGTTATGTCGAATCTCAGGTTTATCAGGGCGTGGTTGAAAACCTGGCCAGCGAGCAGGCCGCCCGTATGGTGGCGATGAAAGCCGCGACCGACAATGGCGGCAGCCTGATTAAAGAGCTGCAGTTGGTATACAACAAAGCTCGTCAGGCCAGCATTACTCAGGAACTCACCGAGATCGTCTCGGGGGCCGCCGCGGTTTAAACAGGTTATTTCGTAGAGGATTTAAGATGGCTACTGGAAAGATTGTCCAGGTAATCGGCGCCGTAGTTGACGTCGAATTCCCTCAGGATGCCGTACCGCGCGTGTACGATGCTCTTGAGGTGCAAAATGGTAATGAGCGTCTGGTGCTGGAAGTTCAGCAGCAGCTCGGCGGCGGTATCGTACGTACCATCGCAATGGGTTCCTCCGACGGTCTGCGTCGCGGTCTGGATGTAAAAGACCTCGAACACCCGATTGAAGTCCCGGTAGGTAAAGCGACTCTGGGCCGTATCATGAACGTACTGGGTGAACCGGTCGACATGAAAGGCGAGATCGGTGAAGAAGAGCGTTGGGCGATTCACCGCGCAGCACCTTCCTACGAAGAGCTGTCAAACTCTCAGGAACTGCTGGAAACCGGTATCAAAGTTATCGACCTGATGTGTCCGTTCGCTAAGGGCGGTAAAGTTGGTCTGTTCGGTGGTGCGGGTGTAGGTAAAACCGTAAACATGATGGAGCTCATTCGTAACATCGCGATCGAGCACTCCGGTTACTCTGTGTTTGCGGGCGTAGGTGAACGTACTCGTGAGGGTAACGACTTCTACCACGAAATGACCGACTCCAACGTTATCGACAAAGTATCCCTGGTGTATGGCCAGATGAACGAGCCGCCGGGAAACCGTCTGCGCGTTGCTCTGACCGGTCTGACCATGGCTGAGAAATTCCGTGACGAAGGTCGTGACGTTCTGCTGTTCGTTGACAACATCTATCGTTACACCCTGGCCGGTACGGAAGTATCCGCACTGCTGGGCCGTATGCCTTCAGCGGTAGGTTATCAGCCGACCCTGGCGGAAGAGATGGGCGTTCTGCAGGAACGTATCACCTCCACCAAAACTGGTTCTATCACCTCCGTACAGGCAGTATACGTACCTGCGGATGACTTGACTGACCCGTCTCCGGCAACCACCTTTGCGCACCTTGACGCAACCGTGGTACTGAGCCGTCAGATCGCGTCTCTGGGTATCTACCCGGCCGTTGACCCGCTGGACTCCACCAGCCGTCAGCTGGACCCGCTGGTGGTTGGTCAGGAACACTACGACACCGCGCGTGGCGTTCAGTCCATCCTGCAACGTTATCAGGAACTGAAAGACATCATCGCCATCCTGGGTATGGATGAACTGTCTGAAGAAGACAAACTGGTGGTAGCGCGTGCTCGTAAGATCCAGCGCTTCCTGTCCCAGCCGTTCTTCGTGGCAGAAGTATTCACCGGTTCTCCGGGTAAATACGTCTCCCTGAAAGACACCATCCGTGGCTTTAAAGGCATCATGGAAGGCGAATACGATCACCTGCCGGAGCAGGCGTTCTACATGGTCGGTTCCATCGAAGAAGCTGTGGAAAAAGCCAAAAAACTTTAA
SEQ ID NO.4 MNRATPase-F1
ATGGCAGAGTTGACAATCTCCGCTTCTGATATCGAAGGTGCCATCGAGGGCTACGTATCCTCGTTTTCCGCCGACACCGAGCGGGAAGAGGTCGGCACCGTCGTCGATGCCGGTGACGGCATCGCCCACGTCGAGGGCCTGCCCTCGGTCATGACCCAGGAATTGCTCGAGTTCGAGGGCGGCGTGCTGGGCGTGGCGCTCAACCTCGACGAGCACAGCGTCGGCGCCGTGATCCTGGGCGAGTTCAACAAGATCGAAGAGGGTCAGCAGGTCAAGCGGACCGGCGAGGTCCTCTCGGTGCCCGTCGGCGACGCCTTCCTCGGCCGCGTGGTCAACCCGCTCGGCCAGCCGATCGACGGCCAGGGCGATATCGCCTCGGACACCCGTCGCGAACTGGAACTTCAGGCCCCCTCGGTGGTTCAGCGCCAGGGTGTCGGCGAGCCGCTGCAGACCGGTATCAAGGCCATCGACGCCATGACCCCGATCGGTCGCGGCCAGCGTCAGCTGATCATCGGCGACCGCAAGACCGGTAAGACCGCGGTCTGCGTCGACACCATCCTGAACCAGCGTCAGGCGTGGGAGACCGGTGACCCGAACCAGCAGGTGCGTTGCGTGTACGTCGCGATCGGCCAGAAGGGCACCACGATCGCCTCGGTCAAGCGTGCGCTGGAAGACGGCGGCGCGATGGAGTACACCACCATCGTCGCGGCCCCGGCGTCCGACCCCGCCGGCTTCAAATGGCTTGCCCCCTACACCGGTTCGGCCATCGGCCAGCACTGGATGTACGACGGCAAGCACGTCCTGATCGTTTTCGACGATCTGTCCAAGCAGGCCGACGCCTACCGCGCCATCTCGCTGCTGCTGCGTCGCCCGCCGGGCCGCGAGGCATTCCCCGGTGACGTGTTCTACCTGCACTCTCGCCTGCTGGAGCGTTGCGCCAAGCTGTCCGATGAGCTCGGTGGTGGTTCGATGACCGGGCTGCCGATCATCGAGACCAAGGCCAACGACATCTCGGCGTTCATCCCGACCAACGTCATCTCGATCACCGACGGTCAGTGCTTCCTGGAGTCCGACCTGTTCAACCAGGGCGTGCGACCGGCCGTCAACGTCGGTGTGTCGGTCTCCCGCGTCGGTGGCGCCGCGCAGATCAAGGCGATGAAAGAGGTTGCGGGTTCGCTGCGTCTGGACCTGTCGCAGTACCGCGAGCTGGAGGCCTTCGCGGCCTTCGCCTCCGATCTGGATGCGGCCTCCAAGGCGCAGCTGGACCGCGGTGTGCGCCTGGTCGAGCTGCTCAAGCAGCCCCAGTACAGCCCGCTGGCCGTCGAGGACCAGGTTGTCGCCATCTTCCTCGGTACTCAGGGCCACCTCGATTCGGTTCCCGCTGAGGACGTTTCGCGCTTCGTCGACGAGCTGCTCGAGCACGTGAAGGCCAGCCACTCCGACATCCTCGACGGCATTCGGGAGACCAAGAAGCTCTCCGAGGAGGCCGAGCAGAAGCTGGTCAACGTCATCAACGACTTCAAGAAGGGCTTCTCGGCGAGCGACGGTAGCTCCGTCGTCGTCAACGAGGCCGACTCCGAAGCTCTGGATCCCGAGGACCTGGAGAAGGAATCGGTCAAGGTCCGTAAGCCTGCTCCCAAGAAGGCCTAGGTAACCAATGGCAGCCACACTGCGCGAGCTACGCGGACGTATCAAATCCGCTTCGTCGATCAAGAAGATCACGAAGGCCCAGGAACTGATCGCCACGTCGCGGATCGCCAAGGCGCAGGCCCGGGTCGACGCGGCCCGGCCCTACAGCACCGAGATCACCAACATGCTCACCGAGCTGGCCAGTGCCAGCGCGCTGGACCACCCGTTGCTCGTGCCGCGGGACAACCCGAAGCGGGCCGCCGTGTTGGTGGTGTCCTCGGATCGCGGTCTGTGCGGTGGGTACAACGCCAACGTGCTGCGTCGCGCCGAAGAACTGTTCTCGCTGCTGCGCGACGAGGGCAAGGATCCGGTGCTCTACGTCATCGGGCGGAAGGCGCTGGGTTACTACAACTTCCGCCAGCGCAATGTCGCCGAGTCCTGGACCGGCTTCTCCGAGCGTCCGACCTATGAGCACGCCAAGGAGATCGCCGACACCCTGGTGACGGCGTTCATGTCGGGCGCCGACGATGACGAGGACGGCGCCGGCGCTGACGGTGTTCTCGGCGTCGACGAGATTCACATCGTGTCGACCGAGTTCCGCTCGATGCTGTCGCAGACCGCGGTGGCACTGCGGGTCGCCCCGATGGTCGTCGAGTACGTGGGGGACGAGGAGCCCGAGGACGGCCCGCGCACGCTGTTCTCCTTCGAACCGAACGCCGAGACGCTGTTCGACGCCCTGCTGCCGCGCTACATCGCCACCCGCGTGTACGCCGCATTGCTGGAGGCGGCTGCCTCGGAGTCGGCCTCGCGCCGGCGCGCCATGAAGTCGGCCACCGACAACGCCGACGATCTGATCAAGGCACTCACGCTGGCGGCCAACCGCGAGCGTCAGGCGCAGATCACCCAGGAAATCAGCGAGATCGTCGGTGGCGCCAACGCGCTGGCCGACGCCAAATAGGCCGACGTAAGAGCGAAAGCGAAGAAGAGATATGACTGCCGTAGAGACCAAGACCACGACGGGTCGCGTTGTCCGCATCACGGGCCCCGTGGTCGACGTCGAGTTCCCGCGTGGCGCCGTGCCCGGACTGCTCAACGCCCTGCACGCCGAGATCACCTTCGGCGCGCTGGCCAAGACCCTGACCCTCGAGGTCGCCCAGCATCTCGGCGAGAGCCTGGTCCGCTGCATCTCCATGCAGCCCACCGACGGCCTGGTCCGTGGCCAGGAAGTCACCGACACCGGTGCGTCGATCTCGGTGCCCGTCGGCGACGGCGTCAAGGGCCATGTGTTCAACGCCCTCGGCGACTGCCTCGATGAGCCGGGCTACGGCAAGGACTTCGAGCACTGGTCCATCCACCGCAAGCCGCCGGCCTTCGCCGACCTGGAGCCCCGCACCGAGATGCTGGAAACCGGTCTGAAGGTCGTCGACCTGCTCACGCCGTACGTGCGTGGTGGAAAGATCGCCCTGTTCGGTGGCGCCGGCGTCGGCAAGACCGTTCTGATCCAGGAGATGATCAACCGCATCGCCCGTAACTTCGGTGGCACCTCGGTGTTCGCCGGCGTGGGGGAGCGCACCCGTGAGGGTAACGACCTGTGGGTCGAGCTCGCGGACGCCAACGTGCTCAAGGACACCGCCTTGGTGTTCGGCCAGATGGACGAGCCGCCGGGCACCCGTATGCGCGTCGCCCTGTCCGCGCTGACCATGGCGGAGTTCTTCCGCGATGAGCAGGGCCAGGACGTGCTGCTGTTCATCGACAACATCTTCCGGTTCACCCAGGCCGGTTCCGAGGTCTCGACCCTGCTGGGTCGTATGCCTTCGGCCGTGGGTTACCAGCCGACGCTGGCCGACGAGATGGGCGAGCTGCAGGAGCGCATCACCTCGACCCGTGGTCGCTCAATCACCTCGATGCAGGCCGTGTACGTGCCCGCCGACGACTACACCGACCCGGCGCCGGCCACCACGTTCGCCCACCTCGATGCCACCACCGAGCTCTCGCGTGCGGTGTTCTCCAAGGGCATCTTCCCCGCGGTGGATCCGCTGGCATCGTCTTCGACGATCCTGCACCCCAGCGTGGTCGGCGACGAGCACTACCGCGTCGCCCAGGAAGTCATCCGGATCCTGCAGCGCTACAAGGATCTCCAGGACATCATCGCCATCCTCGGTATCGATGAGCTGTCCGAAGAGGACAAGGTGCTGGTGTACCGCGCCCGTAAGATCGAGCGCTTCCTGAGCCAGAACATGATGGCGGCCGAGCAGTTCACCGGTCAGCCGGGTTCGACCGTTCCGCTCAAGGAGACCATCGAGGCCTTCGACAAGCTGGCCAAGGGCGAGTTCGATCACCTGCCCGAGCAGGCGTTCTTCCTCATCGGTGGTCTCGACGACCTGGCGAAGAAGGCAGAGTCGCTCGGCGCCAAGCTGTGA Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (10)
1. A genetic engineering strain for efficiently converting sterols is characterized in that: the strain is obtained by constructing a gene expressing an ATPase subunit singly or in combination in a host bacterium.
2. The genetically engineered strain of claim 1, wherein: the host bacteria of the genetic engineering strain are bacteria or fungi with sterol conversion capability;
or the steroid precursor is obtained after the genetically engineered strain converts sterols, and comprises hydroxylated derivatives and A-ring degradation products;
or the ATPase subunit is expressed by a vector.
3. The genetically engineered strain of claim 2, wherein: the bacterium or fungus having sterol conversion ability is a mycobacterium microorganism or rhodococcus microorganism;
The hydroxylated derivatives and A-ring degradation products include androsta-4-ene-3, 17-dione (androst-4-ene-3, 17-dione, AD), androsta-1, 4-diene-3,17-dione (Androst-1, 4-diene-3,17-dione, ADD), 9 alpha-hydroxyandrosta-4-ene-3, 17-dione (9 alpha-hydroxyandrost-4-ene-3, 17-dione,9 alpha-OH-AD);
the expression vector is pMV261.
4. A genetically engineered strain according to claim 3, wherein: the Mycobacterium genus microorganism is selected from the group consisting of Mycobacterium (Mycobacterium sp.) NRRLB-3683, mycobacterium (Mycobacterium sp.) NRRLB-3805, mycobacterium smegmatis (Mycobacterium smegmatism), mycobacterium fortuitum (Mycobacteriumfortuitum), mycobacterium micro Huang Fenzhi (Mycobacterium gilvum), mycobacterium neogolden (Mycobacterium neoaurum), mycobacterium phlei (Mycobacterium Phlei), mycobacterium avium (Mycobacterium avium);
The genetically engineered strain expresses an ATPase subunit in the novel Mycobacterium aurum MNR via pMV 261.
5. The genetically engineered strain of claim 4, wherein: the Mycobacterium microorganism is Mycobacterium neogold (Mycobacterium sp.) mnrm3Δ KsdD.
6. The genetically engineered strain of any one of claims 1 to 5, wherein: the ATPase subunits are respectively derived from escherichia coli MG1655 and MNR, and the amino acid sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2;
or coding genes E.coliATPase-F1, MNRATPASE-F1 of the ATPase subunit, and the nucleotide sequence is SEQ ID NO.3 and SEQ ID NO.4.
7. Use of a genetically engineered strain according to any one of claims 1 to 6 for the production of steroid precursors.
8. A method for enhancing steroid precursor production by enhancing intracellular ATP metabolism, comprising: the method is characterized in that a gene engineering strain is obtained by constructing a gene for expressing ATP enzyme subunits singly or in combination in host bacteria, and the metabolic capacity of the strain to ATP is enhanced by enhancing the expression of the gene in a sterol conversion strain, so that the sterol conversion rate is improved.
9. A method for producing androsta-4-ene-3, 17-dione using the genetically engineered strain of any one of claims 1 to 6, characterized in that: comprises the following steps of;
Transferring seed culture solution of the genetically engineered bacteria into a fermentation culture medium according to the inoculum size of 2% -12%, and culturing for 48-168 hours under the conditions of 25-37 ℃ and 140-220 rpm; the molar conversion rate of androstenedione can reach 50% -99%.
10. The method according to claim 9, characterized in that: the seed culture medium comprises the following components: k 2HPO40.5 g/L,MgSO4, 0.5, g/L, ferric ammonium citrate, 0.05g/L, citric acid, 2g/L, ammonium nitrate, 20g/L glycerol, 5g/L glucose, caCO 3, 1, g/L and water as a solvent, wherein the pH value is 7.2;
the composition of the fermentation culture is as follows: (NH 4)2HPO40.1～4g/L,K2HPO40.1～3g/L,MgSO4 -0.1 g/L, ferric ammonium citrate 0.01-0.2 g/L, citric acid 1-5 g/L, reducing sugar 5-50 g/L, sterol 1-50 g/L, water as solvent, pH 6.0-7.5, and sterilizing with high pressure steam.
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