CN117821347A - Method for improving sterol conversion by enhancing side chain degradation capability - Google Patents
Method for improving sterol conversion by enhancing side chain degradation capability Download PDFInfo
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- CN117821347A CN117821347A CN202311510665.2A CN202311510665A CN117821347A CN 117821347 A CN117821347 A CN 117821347A CN 202311510665 A CN202311510665 A CN 202311510665A CN 117821347 A CN117821347 A CN 117821347A
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Abstract
The invention belongs to the technical field of biocatalysis, and particularly relates to a method for improving sterol conversion by enhancing side chain degradation capability. The method is obtained by knocking out a transcription factor FdmR encoding gene in a strain having a steroid drug intermediate production ability and simultaneously overexpressing at least one of isocitrate lyase ICL and succinic dehydrogenase SDH. The invention proves the unique effect of the transcription factor FdmR in the process of producing the steroid medicine intermediate by using sterols for the first time, and provides a new method for improving the production capacity of the steroid medicine intermediate producing strain. The method can also be used for strengthening the side chain degradation capability of other industrial production strains, and has wide application value.
Description
Technical field:
the invention belongs to the technical field of biocatalysis, and particularly relates to a method for improving sterol conversion by enhancing side chain degradation capability.
The background technology is as follows:
steroid drugs are second to antibiotics in the second largest class, and have great market and development prospects. The phytosterol is taken as a raw material, and the producible steroid intermediates mainly comprise two major categories of C19-steroids (AD, ADD, 9-OH-AD) and C22-steroids (20-carboxyl-pregn-4-en-3-one, 4-BNC, 20-hydroxymethyl-pregn-4-en-3-one and 4-BNA). Wherein androstane-4-ene-3, 17-dione (AD) can be used for producing androgen, protein assimilation hormone, spirolactone, etc.; androsta-1, 4-di-ene-3, 17-dione (ADD) produced by dehydrogenation of AD at C1, 2-position can be used to synthesize 19-nor-steroid estrogens such as Estrone (Estrone), norethisterone (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. It follows that AD can synthesize almost all steroid drugs and is therefore extremely popular in the market, with the market scale of steroid intermediates currently being the second to the market for drugs.
Currently, the production process of AD is mainly based on microbial transformation, wherein Mycobacterium microorganisms are the main production strain of steroid drug intermediates. When the mycobacteria microorganism converts sterols such as plant sterols and cholesterol into steroid prodrug, three processes of sterol absorption, preliminary oxidation and sterol side chain degradation are mainly carried out. The sterol conversion to AD is a multiple enzymatic reaction, the most critical step of which is the degradation of the C17 side chain.
The transcription factor FdmR has the capacity of regulating fatty acid degradation, can regulate the expression of a plurality of fatty acid degradation and modification genes, but no related study exists in the prior art between FdmR and sterol side chain degradation.
For the yield increase of AD, the key genes in metabolic pathways are enhanced or weakened mainly by a genetic engineering means so as to realize accumulation of steroid intermediates at different nodes. However, both the original strain and the engineering strain have a longer transformation period (more than or equal to 120 h), and the transformation efficiency of the strain is drastically reduced in the middle and later stages of transformation, which is one of the reasons that the production cost of steroid intermediates is high. NAD in the production of steroid intermediates by sterol side chain degradation + And cofactors such as coenzyme A are converted to NADH, propionyl-CoA and acetyl-CoA. Acetyl coenzyme A is connected with important metabolic pathways such as glycolysis and TCA (ternary content addressable memory) circulation, and is an important center of biosynthesis, and isocitrate cleavage can catalyze isocitrate to form glyoxylic acid and succinic acid to flow into the TCA circulation, so that the glyoxylic acid circulation is enhanced as one branch of the TCA circulation, and the speed of the TCA circulation can be effectively improved. Expression of succinate dehydrogenase can enhance succinic acid metabolism and reduce succinic acid accumulation. In the process of degrading sterol side chains, excessive accumulation of intermediate products has an inhibiting effect on the metabolic process of converting sterols into steroid drug precursors, and is one of reasons for influencing the efficient generation of steroid drug intermediate.
The invention comprises the following steps:
the invention aims to provide a method for improving sterol conversion by enhancing side chain degradation capability, and a genetic engineering bacterium is constructed by using the method, so that the problem that the sterol conversion rate is reduced and the price of a steroid intermediate is high due to lengthy sterol side chain degradation metabolism in the production process of the steroid intermediate by a microbial method is solved. In order to solve the technical problems, the invention adopts the following technical scheme:
one of the technical schemes provided by the invention is a genetically engineered bacterium capable of carrying out sterol transformation, and the strain is obtained by knocking out a transcription factor FdmR coding gene in a starting strain;
further, the genetically engineered bacterium also overexpresses at least one of the isocitrate lyase ICL and the succinate dehydrogenase SDH;
further, the succinic dehydrogenase SDH includes: four subunits, SDHA for the a subunit of succinate dehydrogenase, SDHB for the B subunit of succinate dehydrogenase, SDHC for the C subunit of succinate dehydrogenase and SDHD for the D subunit of succinate dehydrogenase, encoded by sdhA, sdhB, sdhC and SDHD, respectively; over-expression of succinic dehydrogenase SDH is achieved by over-expression of at least one of the four subunits; preferably sdhD;
further, the starting strain is a strain with the capacity of producing a steroid drug intermediate;
the steroid drug intermediates include, but are not limited to, androsta-4-ene-3, 17-dione (AD), 9α -hydroxyandrosta-4-ene-3, 17-dione (9α -hydroxy-ndrost-4-ene-3, 17-dione,9α -OH-AD), androsta-1, 4-diene-3,17-dione (androsta-1, 4-diene-3,17-dione, ADD) and the like;
further, the starting strain is escherichia coli BL21 strain, bacillus subtilis (Bacillus subtilis), mycobacterium (Mycobacterium sp.) NRRLB-3683, mycobacterium (Mycobacterium sp.) NRRLB-3805, mycobacterium smegmatis (Mycobacterium smegmatism), mycobacterium fortuitum (Mycobacterium fortuitum), micro Huang Fenzhi bacillus (Mycobacterium gilvum), mycobacterium neogold (Mycobacterium neoaurum), mycobacterium phlei (Mycobacterium phlei), mycobacterium avium (Mycobacterium avium), and the like;
preferably, the starting strain is Mycobacterium newly grown (Mycobacterium sp.) MNR M3, which is deposited in China center for type culture Collection, accession number CICC 21097;
more preferably, the genetically engineered bacterium capable of sterol transformation is obtained by taking fast-growing new Mycobacterium aurum (Mycobacterium sp.) MNR M3 as an initial strain, knocking out FdmR encoding genes, and overexpressing D subunit encoding genes of isocitrate lyase ICL and succinic dehydrogenase SDH;
further, the coding gene fdmR of the transcription factor FdmR has a nucleotide sequence shown in SEQ ID NO. 1;
further, the coding gene ICL of the isocitrate lyase ICL has a nucleotide sequence shown as SEQ ID NO. 2;
further, the nucleotide sequence of the sdhA is shown as SEQ ID NO. 3;
further, the nucleotide sequence of the sdhB is shown as SEQ ID NO. 4;
further, the nucleotide sequence of the sdhC is shown as SEQ ID NO. 5;
further, the nucleotide sequence of the sdhD is shown as SEQ ID NO. 6;
further, the gene engineering bacteria knocks out the FdmR encoding gene through a knocking-out vector;
further, the genetically engineered bacterium is used for over-expressing at least one coding gene of ICL, SDHA, SDHB, SDHC and SDHD by using a genetically engineered expression vector;
further, the genetic engineering knockout vector is a bacterial knockout vector;
preferably, the bacterial knockout vector is a mycobacterial knockout vector;
further, the genetically engineered expression vector is a bacterial expression vector;
preferably, the bacterial expression vector is a mycobacterial expression vector;
more preferably, the mycobacterial expression vectors are pMV306 and pMV261 mycobacteria-E.coli shuttle expression vectors; the mycobacterium knockout vector contains all or part of the sequences of p2NIL and pGOAL19 plasmids.
The second technical scheme provided by the invention is the application of the genetically engineered bacterium in the production of steroid medicine intermediates, in particular to the application in the production of AD;
further, the application in the production of AD is specifically as follows:
transferring the genetically engineered strain seed culture solution into a fermentation culture medium according to the inoculum size of 2-10%, and culturing for 24-168 hours at the temperature of 25-35 ℃ and the rpm of 50-200; the AD yield can reach 0.3-30 g/L, and the molar conversion rate can reach 50% -100%;
fermentation medium composition: k (K) 2 HPO 4 0.1~3g/L,MgSO 4 0.1-3 g/L, 0.01-0.2 g/L of ferric ammonium citrate, 1-5 g/L of citric acid, 1-10 g/L of diammonium hydrogen phosphate, 5-50 g/L of glucose, 1-50 g/L of phytosterol and the balance of water, wherein the pH value is 6.0-7.5.
The beneficial effects are that:
the invention obtains M3 by independently knocking out fdmR gene in steroid medicine intermediate producing strain MNR M3 ΔfdmR In the fermentation process, the intracellular acetyl coenzyme A content is increased by more than 33% compared with the MNR of the starting strain, the AD conversion rate reaches 85.41% at 96h, and is increased by 14.58% compared with the AD conversion rate of the contemporaneous starting strain M3; meanwhile, after the fdmR gene is knocked out, the cell viability is reduced by 10.41 percent compared with that of the original strain M3 due to the rapid mass accumulation of acetyl-CoA in 96 hours; by at M3 ΔfdmR Obtaining strain M3 by singly and over-expressing icl gene ΔfdmR-icl Enhancing the metabolism of acetyl coenzyme A in the strain, improving the activity of thalli and improving the conversion efficiency of sterols. In the fermentation process, the intracellular acetyl coenzyme A content is increased by more than 21.34 percent compared with the original strain, the cell activity is similar to that of the original strain M3, the highest AD conversion rate reaches 95.25 percent in 120 hours, and the AD conversion rate is increased by 25 percent compared with that of the original strain M3 in the same period; by at M3 ΔfdmR-icl Obtaining strain M3 by expressing sdhD gene in series ΔfdmR-icl-sdhD In the fermentation process, the succinic acid content is reduced by 28.68%, the AD conversion rate can reach 93.34% in 96 hours, the conversion efficiency is obviously improved, the highest AD conversion rate in 120 hours reaches 99.44%, and the AD conversion rate of the strain is improved by 29% compared with that of the strain M3 in the same period. The invention proves the unique effect of the transcription factor FdmR in the process of producing the steroid medicine intermediate by using sterols for the first time, and provides a new method for improving the production capacity of the steroid medicine intermediate producing strain. The method can also be used for strengthening the side chain degradation capability of other industrial production strains, and has wide application value.
As shown in FIG. 8, M3 ΔfdmR 、M3 ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC 、M3 ΔfdmR-icl-sdhD The AD molar conversion rate of each strain is always higher than that of the original strain M3. At 72h, strain M3 ΔfdmR 、M 3ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC And M3 ΔfdmR-icl-sdhD The AD molar conversions of (3) were 72.01%, 75.60%, 66.35%, 74.06%, 64.43% and 83.16%, respectively, 1.38-fold, 1.44-fold, 1.27-fold, 1.41-fold, 1.23-fold and 1.59-fold, respectively, of the original strain M3; at 96h, strain M3 ΔfdmR 、M 3ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC And M3 ΔfdmR-icl-sdhD The AD molar conversion rate of the catalyst is 85.41%, 90.95%, 80.00%, 87.39%, 80.36% and 93.34%, respectively, and the sterol conversion efficiency is obviously improved; at 120h, in particular strain M3 ΔfdmR-icl-sdhD The AD molar conversion of (2) reaches 99.44%.
Description of the drawings:
FIG. 1 is a map of the cleavage verification of the construction of the pYKdel-fdmR knockout plasmid
Wherein lane M is a DNA standard marker and lane pYKdel-fdmR is the result of single cleavage of the pYKdel-fdmR knockout vector by PacI.
FIG. 2 shows the single crossover strain SCO ΔfdmR PCR verification of (C)
Wherein lane M is a DNA standard marker, lane M3 is an amplified band containing the complete fdmR gene, lane SCO ΔfdmR Two long and short amplification bands are arranged at 2840bp and 2240bp after single exchange.
FIG. 3 shows the verification of the genotype of fdmR knockout bacteria
Wherein lane M is a DNA standard marker, lane M3 is an amplified band containing the entire fdmR gene, lane M3 ΔfdmR The amplified band of the fdmR gene with the deletion of 600bp after knockout (the original strain has the amplified band at 2840bp, the knockout strain has the band at 2240bp because of the deletion of fdmR, and the deleted 600bp is the fdmR).
FIG. 4 is a nucleic acid electrophoretogram of the amplified product of icl gene
Wherein lane M is a DNA standard marker and lanes 1-2 are icl gene amplification bands.
FIG. 5 is a map of cleavage verification during construction of pMV261-icl overexpression vector
Wherein lane M is a DNA standard marker and lanes 1-2 are pMV261-icl HindIII single cleavage results.
FIG. 6 is an original strain M3, fdmR knockout strain M3 ΔfdmR And fdmR knockout and icl overexpressing strain M3 ΔfdmR-icl1 Variation of acetyl-CoA content of the strain.
FIG. 7 shows the original strain M3, fdmR knockout strain M3 ΔfdmR Changes in viability of fdmR knockout and icl overexpressing strain m3Δfdmr-icl strain.
FIG. 8 original strain M3, fdmR knockout strain M3 ΔfdmR fdmR knockout icl overexpressing strain M3 ΔfdmR-icl And fdmR knockout, icl and sdh overexpressing strain M3 ΔfdmR-icl1-sdhA 、M3 ΔfdmR-icl1-sdhB 、M3 ΔfdmR-icl1-sdhC 、M3 ΔfdmR-icl1-sdhD AD production profile in seven strains.
The specific embodiment is as follows:
in order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present invention.
The experimental procedure, which is not specified in the following examples, is generally carried out according to conventional conditions, such as those described in the guidelines for molecular cloning experiments (fourth edition) (scientific press 2017).
The invention is further illustrated by the following examples.
EXAMPLE 1 construction of FdmR encoding Gene-deleted engineering bacterium
The mycobacterial gene knockout plasmid was constructed, electrotransformed into mycobacterial competence, and double-resistance screening was performed using hygromycin and kanamycin and simultaneous blue-white screening was performed. Sucrose plates were screened for the correct strains and simultaneously rescreened for kanamycin resistance to obtain positive knock-out clones. And (5) verifying the gene knockout clone by using a PCR method. The method comprises the following specific steps:
preparation of M3 competent cells: inoculating M3 strain (CICC 21097) into LB medium, culturing at 30deg.C to OD 600 About 1.0, transferring the seed culture medium into a seed culture medium according to 10% of inoculation amount for secondary seed culture; after 24h, 2% glycine was added and cultivation continued for 24h. Centrifugally collecting thalli, flushing the suspended thalli with 10% precooled glycerol of which the volume is 1 time, 3/4 times, 1/2 times and 1/4 times of the volume of seed liquid respectively, centrifuging, finally adding 1/25 times of 10% glycerol suspended thalli, and subpackaging for storage;
2. construction of knockout plasmid: the primers of the upper and lower arms of the fdmR gene are designed, and the genome DNA of M3 is used as a template to amplify the upstream and downstream homology arms of the fdmR.
Upper arm primers QC-PDU-F and QC-PDU-R:
QC-PDU-F:taaactaccgcattaaagcttgatatcgcctcgctgtccac;
QC-PDU-R:ccggccgcgatggcatccat;
lower arm primers QC-PDD-F and QC-PDD-R:
QC-PDD-F:atggatgccatcgcggccggggcgggcgaggcgatc;
QC-PDD-R:actatagaatacataggatccgggaagaaccgagagcactga。
the homologous arm genes on the upstream and downstream of the target gene fdmR were amplified by PCR technique and ligated to the BamHI and HindIII double digested Mycobacterium gene knockout plasmid p2NIL, respectively. Then, the above plasmid and pGOAL19 plasmid were digested with PacI, and ligated with T4 ligase to construct a knockout plasmid pYKdel-fdmR, and the digestion was verified (FIG. 1).
3. mu.L of pYKdel-fdmR knockout plasmid was added to 100. Mu. L M3 competent cells and allowed to stand for 30 minutes, followed by transfer to an electric rotating cup for clicking. Electric shock conditions of 2kV/cm,25 mu F and 720 omega are placed on ice for 5min after electric rotation for 3-6 ms, and then the ice is put into fresh sterilized 1.5mL for centrifugation, 500 mu L of fresh sterilized LB culture medium is added, and recovery is carried out for 3-24 hours at 30 ℃ and 200 rpm. Coating on LB solid medium containing hyg 50 mug/ml, kan 20 mug/ml and X-gal 50 mug/ml, culturing for 5-7 days, picking colony with blue spot, utilizing upstream positive directionThe primers (QC-PDU-F) and the downstream reverse primer (QC-PDD-R) were subjected to PCR to verify the correctness (FIG. 2) as a single crossover strain. Coating the verified single-exchange bacteria on a plate containing 2% of sucrose, culturing for 3-7 days, picking white colony to extract genome, and carrying out PCR (figure 3) verification by using an upstream forward primer (QC-PDU-F) and a downstream reverse primer (QC-PDD-R), wherein the fdmR gene deletion strain is named M3 ΔfdmR 。
Example 2 acquisition of ICL encoding Gene, construction of Single Gene overexpression vector
In this example, only the PCR method is used as an example, and the sequence of SEQ ID NO.2 of ICL encoding gene ICL is cloned from M3.
The PCR primers were icl-F and icl-R. PCR amplification was performed using the genomic DNA of M3 as a template to obtain the icl gene of M3 (shown in SEQ ID NO. 2). Amplified products were sequenced after detection by nucleic acid electrophoresis (FIG. 4, 1284 bp). Sequencing results show that the obtained nucleotide fragment is the icl gene.
Constructing a genetic engineering expression vector for over-expression of icl genes by using a pMV261 plasmid, wherein the process comprises the following steps: and (3) recovering the obtained icl gene gel, mixing the obtained icl gene gel with pMV261 plasmid recovered by EcoRI and HindIII double enzyme digestion gel, connecting the obtained icl gene gel by using a Minerva Super Fusion Cloning Kit seamless cloning kit, transferring the connected product into Escherichia coli DH alpha by a chemical conversion method, and screening by kanamycin to obtain positive clones. The plasmid in the extracted positive clone is verified by HindIII single enzyme digestion (FIG. 5, pMV261-icl plasmid has a band at 5771bp after being cut by HindIII single enzyme digestion) and sequenced, thus obtaining the expression vector pMV261-icl for the self over-expression of icl gene successfully constructed.
Table 1ICL, SDHA, SDHB, SDHC and primers for obtaining SDHD coding genes
Example 3SDHA, SDHB, SDHC and SDHD coding Gene acquisition, construction of tandem overexpression vectors
Using the genomic DNA of M3 as a template, amplification products containing SDHA, SDHB, SDHC and SDHD encoding genes were obtained by amplification using primers sdhA-F/R, sdhB-F/R, sdhC-F/R, sdhD-F/R in Table 1, respectively. And (3) respectively carrying out gel recovery on each amplification product, and then carrying out seamless cloning connection on the amplification products and the pMV261-icl enzyme digestion products recovered by HindIII and HpaI double enzyme digestion gel to construct corresponding plasmids. The genes contained therein were designated pMV261-icl-sdhA, pMV261-icl-sdhB, pMV261-icl-sdhC, and pMV261-icl-sdhD, respectively.
EXAMPLE 4 construction of FdmR Gene-deleted ICL, SDHA, SDHB, SDHC and SDHD-encoding Gene-enhanced Strain
M3 ΔfdmR Competent cell preparation: m3 ΔfdmR Inoculating the strain into LB culture medium, culturing at 30deg.C to 0D 600 About 1.0, transferring the seed culture medium into a seed culture medium according to 10% of inoculation amount for secondary seed culture; after 24h, 2% glycine was added and cultivation continued for 24h. Centrifugally collecting thalli, flushing the suspended thalli with 10% precooled glycerol of which the volume is 1 time, 3/4 times, 1/2 times and 1/4 times of the volume of the fermentation liquid respectively, centrifuging, finally adding 1/25 times of 10% glycerol suspended thalli, and subpackaging for storage;
the fdmR gene-deleted icl gene-enhanced strain was added to 100. Mu. L M3 by taking 10. Mu.L of the pMV261-icl gene-engineering expression vector obtained in example 2 ΔfdmR And placing the competent thalli in the cup for 30 minutes, and then transferring the competent thalli into an electric rotating cup for clicking. The conditions for electric shock were the same as in example 1, and after 5min on post-ice, the mixture was centrifuged in fresh sterilized 1.5mL and 500. Mu.L of fresh sterilized LB medium was added thereto for resuscitation at 30℃and 200rpm for 3 to 6 hours.
Recombinant screening and verification: the resuscitated culture is coated on an LB culture medium plate containing kanamycin (50 mg/L), and is subjected to stationary culture for 4-7 d at 30 ℃, single colony is picked up to the LB culture medium for 2-3 d, and then plasmid extraction is verified by sequencing. Verification of correct Positive transformants were designated as recombinant M3 ΔfdmR-icl 。
In recombinant bacterium M3 ΔfdmR Based on the above, the overexpression vectors pMV261-icl-sdhA, pMV261-icl-sdhB, pMV261-icl-sdhC, pMV261-icl-sdhD were transformed into M3, respectively, in the same manner as described above ΔfdmR In the sense, one of icl, SDHA, SDHB, SDHC and SDHD coding genes is over-expressed, and the obtained positive transformants are named respectivelyIs M3 ΔfdmR-icl-sdhA ,M3 ΔfdmR-icl-sdhB ,M3 ΔfdmR-icl-sdhC ,M3 ΔfdmR-icl-sdhD 。
Example 5 M3, M3 ΔfdmR 、M3 ΔfdmR-icl Comparison of intracellular acetyl-CoA content during side chain degradation
1. Strain activation and seed preparation
Strains M3 and M3 ΔfdmR 、M3 ΔfdmR-icl Respectively transferring to fresh LB culture medium, culturing at 30deg.C for 3d, washing with 20mL of 0.5% Tween 80 sterile water solution, absorbing l mL of eluent, adding into 50mL of seed culture medium, and shake culturing at 30deg.C and 200rpm for 36h to obtain seed solution;
seed medium composition: k (K) 2 HPO 4 0.5g/L,MgSO 4 0.5g/L, ferric ammonium citrate 0.05g/L, citric acid 2g/L, ammonium nitrate 2g/L, glycerin 20g/L, glucose 5g/L, caCO 3 1g/L, the balance being water, pH 7.2.
AD production Process
Transferring the seed culture solution obtained in the step 1 into 250mL baffle bottles filled with fermentation culture medium respectively according to the inoculum size of 5%, and carrying out shake cultivation for 120h at 30 ℃ and 150 rpm;
the fermentation medium consists of K 2 HPO 4 0.5g/L,MgSO 4 0.5g/L, 0.05g/L ferric ammonium citrate, 2g/L citric acid, 3.5g/L diammonium phosphate, 10g/L glucose, 38g/L hydroxypropyl beta-cyclodextrin, 5g/L phytosterol, and the balance of water, wherein the pH is 7.2. Samples were taken every 24 hours.
3. acetyl-CoA content assay
Taking fermentation liquor at fixed time, centrifugally collecting 0.1g of thalli, quickly freezing and crushing, deproteinizing a sample through PCA precipitation, and then detecting acetyl coenzyme A. acetyl-CoA assay kits were purchased from merck life sciences and used for determination of intracellular acetyl-CoA content according to the reagent ratios of Table 2.
First, sample preparation and background removal: 50. Mu.L of standard was taken in the standard well and 50. Mu.L of sample was added to the sample well and sample background well, respectively. To correct for background generated by free coa and succinyl-coa, 10 μl of acetyl coa quencher was added to standard wells, sample wells and sample background wells. Incubate for 5 minutes at room temperature. Then 2. Mu.L of quenching remover was added, mixed well and incubated for another 5 minutes.
Next, the reaction mixture was prepared according to the scheme in table 2. 50. Mu.L of standard well and sample well reaction mixture was added to each standard well and sample well, 50. Mu.L of background reaction mixture was added to each background sample well, and the mixture was mixed well with a horizontal shaker or a pipette, and incubated at 37℃for 10 minutes in the absence of light. Finally, the fluorescence intensity was measured using an enzyme-labeled instrument, with a maximum excitation wavelength of 535nm and an emission wavelength of 587nm.
Table 2 reaction mixture (reagents from acetyl CoA assay kit)
Composition of the components | Standard and sample reaction mixtures | Sample background reaction mixture |
acetyl-CoA assay buffer | 40μL | 41μL |
Acetyl-coa substrate mixtures | 2μL | 2μL |
Invertase enzyme | 1μL | - |
Acetyl-coa enzyme mixtures | 5μL | 5μL |
Fluorescent probe | 2μL | 2μL |
4. Comparison of results
FIG. 6 shows M3, M3 ΔfdmR 、M3 ΔfdmR-icl1 Variation of intracellular acetyl-CoA content of each strain. The combined application of the knockout of the fdmR and the overexpression of the icl can increase the intracellular acetyl-CoA level, wherein the intracellular acetyl-CoA content is increased by more than 21%, the knockout effect of the fdmR is most obvious, the intracellular acetyl-CoA content can be increased by 97% at 48 hours, but excessive accumulation can inhibit the cell growth, and the combined application of the knockout of the fdmR and the overexpression of the icl effectively relieves the accumulation of the acetyl-CoA in the cell.
Example 6 M3, M3 ΔfdmR 、M3 ΔfdmR-icl Cell viability level in AD production
1. Strain viability detection
The use of M3, M3 was carried out as in example 5 ΔfdmR 、M3 ΔfdmR-icl The strain was subjected to AD production, and 1mL was sampled under aseptic conditions for strain viability detection.
The strain activity detection adopts an improved CCK-8 method, and the detection method comprises the following steps: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-8) can be reduced to orange-yellow water-soluble formalzan by bacterial dehydrogenase in the presence of an electron carrier, and the yield and the activity of the strain are positively correlated. Formazan has a maximum absorption peak at 450nm, and the activity of the cells can be reflected by detecting the absorbance at 450nm (A450). OD of the samples was performed with pH 7.2Tris-HCl buffer 600 After the value is adjusted to 1, 190 mu L of the mixture is added into a 96-well plate, WST-810 mu L is added into each well respectively, and the mixture is incubated for 1 hour at 30 DEG CThe absorbance at 450nm was measured with Infinite M200 Pro.
2. Comparison of results
M3、M3 ΔfdmR 、M3 ΔfdmR-icl As shown in FIG. 7, the change of the bacterial viability is shown in the trend of rising and then falling of the viability of all bacteria in the whole production process, and the bacterial strain M3 ΔfdmR-icl Always higher than M3, M3 ΔfdmR . In particular at 96h, M3 ΔfdmR The strain viability of (2) is 10.41% lower than that of M3, and M3 ΔfdmR-icl The activity of the bacterial cells is similar to that of M3. Therefore, the combined application of the knockout of the fdmR and the overexpression of icl improves the strain activity, improves the bacterial activity of the single knockout fdmR strain, and also verifies that the accumulation of the intracellular acetyl-CoA content can inhibit the cell activity.
Example 7 M3, M3 ΔfdmR 、M3 ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC 、M3 ΔfdmR-icl-sdhD Succinic acid content variation in AD production
1. Succinic acid content detection
Using M3, M3 as in example 5 ΔfdmR 、M3 ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC 、M3 ΔfdmR-icl-sdhD The strain was subjected to AD production, 1mL was sampled under aseptic conditions during the production, and the supernatant was collected by centrifugation at 12000rpm for 5 minutes at 4℃for detection of succinic acid content.
The succinic acid content is detected by adopting a high-efficiency liquid phase method: chromatographic column Aminex HPX-87H 300X7.8 (mM), ultraviolet detector wavelength 215nm, mobile phase 5mM H 2 SO 4 The temperature was measured at 30℃and at a flow rate of 0.6 mL/min.
2. Comparison of results
M3、M3 ΔfdmR 、M3 ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC 、M3 ΔfdmR-icl-sdhD As shown in Table 3, the succinic acid content was changed, and the combination of the knockout of fdmR and the overexpression of icl was usedCan increase succinic acid level; the combined use of knockout of fdmR and overexpression of icl can reduce the succinic acid content by serially expressing sdh, wherein the strain serially expressing sdhA can directly contact succinic acid due to subunit A and subunit B, and the succinic acid is completely metabolized to reduce the succinic acid content to 0g/L in the later period of fermentation.
TABLE 3 M3, M3 ΔfdmR 、M3 ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC 、M3 ΔfdmR-icl-sdhD Succinic acid content (g/L) in AD production
M3 | M3 ΔfdmR | M3 ΔfdmR-icl | M3 ΔfdmR-icl-sdhA | M3 ΔfdmR-icl-sdhB | M3 ΔfdmR-icl-sdhC | M3 ΔfdmR-icl-sdhD | |
72h | 0.345 | 0.367 | 0.421 | 0.177 | 0.288 | 0.152 | 0.350 |
96h | 0.227 | 0.325 | 0.380 | 0.065 | 0.230 | 0.134 | 0.282 |
120h | 0.265 | 0.288 | 0.351 | 0 | 0.106 | 0.120 | 0.189 |
144h | 0.247 | 0.233 | 0.335 | 0 | 0 | 0.086 | 0.168 |
Example 8 M3, M3 ΔfdmR 、M3 ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC 、M3 ΔfdmR-icl-sdhD Conversion Capacity comparison in AD production
Using M3, M3 as in example 5 ΔfdmR 、M3 ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC 、M3 ΔfdmR-icl-sdhD The strain was subjected to AD production, and 0.8mL was sampled under aseptic conditions during the production.
Detection of AD production: the sample was extracted with an equal volume of ethyl acetate by ultrasonic extraction, centrifuged at 10000rpm for 10min, and 0.1mL of the supernatant was taken in a 1.5mL tube, and after natural air drying, the mobile phase was added for dissolution, and after passing through a 0.22 μm membrane, HPLC analysis was performed. Chromatographic conditions: c18 column, mobile phase methanol/water (4:1) flow rate of 1mL/min, column temperature of 30deg.C, and detection wavelength of 254nm. The molar conversion was calculated as: molar yield% = (cp×ms)/(cs×mp) ×100%, where Cp is the product concentration (g/L), cs is the substrate concentration (g/L), mp is the product molar mass, and Ms is the substrate molar mass (both measured and calculated using this method in the present invention).
3. Comparison of results
As shown in FIG. 8, M3 ΔfdmR 、M3 ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC 、M3 ΔfdmR-icl-sdhD The AD molar conversion rate of each strain is always higher than that of the original strain M3. At 72h, strain M3 ΔfdmR 、M 3ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC And M3 ΔfdmR-icl-sdhD The AD molar conversions of (3) were 72.01%, 75.60%, 66.35%, 74.06%, 64.43% and 83.16%, respectively, 1.38-fold, 1.44-fold, 1.27-fold, 1.41-fold, 1.23-fold and 1.59-fold, respectively, of the original strain M3; at 96h, strain M3 ΔfdmR 、M 3ΔfdmR-icl 、M3 ΔfdmR-icl-sdhA 、M3 ΔfdmR-icl-sdhB 、M3 ΔfdmR-icl-sdhC And M3 ΔfdmR-icl-sdhD The AD molar conversion rate of the catalyst is 85.41%, 90.95%, 80.00%, 87.39%, 80.36% and 93.34%, respectively, and the sterol conversion efficiency is obviously improved; at 120h, in particular strain M3 ΔfdmR-icl-sdhD The AD molar conversion of (3) reaches99.44%. Therefore, the knockout of the negative control factor fdmR and the overexpression of the icl and sdhD genes are beneficial to the improvement of the AD conversion rate, and from the result of 120h, the different subunits of the succinate dehydrogenase SDH do not all have positive synergistic effect with the knockout of the negative control factor fdmR and the overexpression of the icl, and the knockout of the negative control factor fdmR and the overexpression of the icl and sdhD genes have unexpected technical effects.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Accordingly, the protection scope of this patent shall be subject to the appended claims.
Claims (10)
1. The genetically engineered bacterium capable of carrying out sterol transformation is characterized in that the strain is obtained by knocking out a transcription factor FdmR coding gene in a starting strain;
the starting strain is a strain with the capacity of producing steroid drug intermediates.
2. The genetically engineered bacterium capable of sterol conversion according to claim 1, wherein the genetically engineered bacterium further overexpresses at least one of isocitrate lyase ICL and succinic dehydrogenase SDH.
3. A genetically engineered bacterium capable of sterol conversion according to claim 2, wherein said succinate dehydrogenase SDH comprises: four subunits, SDHA for the a subunit of succinate dehydrogenase, SDHB for the B subunit of succinate dehydrogenase, SDHC for the C subunit of succinate dehydrogenase and SDHD for the D subunit of succinate dehydrogenase, encoded by sdhA, sdhB, sdhC and SDHD, respectively; overexpression of succinic dehydrogenase SDH is achieved by overexpressing at least one of the four subunits described above.
4. The genetically engineered bacterium capable of sterol transformation of claim 1, wherein the starting strain is Mycobacterium newly rapid growth (Mycobacterium sp.) MNR M3, accession number cic 21097.
5. The genetically engineered bacterium capable of sterol transformation according to claim 1, wherein the genetically engineered bacterium is obtained by taking fast-growing Mycobacterium new gold (Mycobacterium sp.) MNR M3 as an initial strain, knocking out FdmR encoding genes, and overexpressing D subunit encoding genes of isocitrate lyase ICL and succinic dehydrogenase SDH.
6. The genetically engineered bacterium capable of carrying out sterol transformation according to claim 1, wherein the coding gene FdmR of the transcription factor FdmR has a nucleotide sequence shown in SEQ ID NO. 1;
the nucleotide sequence of the coding gene ICL of the isocitrate lyase ICL is shown as SEQ ID NO. 2;
the nucleotide sequence of the sdhA is shown as SEQ ID NO. 3;
the nucleotide sequence of the sdhB is shown as SEQ ID NO. 4;
the nucleotide sequence of the sdhC is shown as SEQ ID NO. 5;
the nucleotide sequence of the sdhD is shown as SEQ ID NO. 6.
7. Use of the genetically engineered bacterium of any one of claims 1-6 in steroid transformation.
8. The use according to claim 7, in the manufacture of AD.
9. Use according to claim 8, wherein the method of producing AD is as follows:
the seed culture solution of the genetic engineering strain is transferred into a fermentation culture medium according to the inoculation amount of 2-10 percent, and is cultured for 24-168 hours under the conditions of 25-35 ℃ and 50-200 rpm.
10. The use according to claim 9, wherein the fermentation medium consists of: k (K) 2 HPO 4 0.1~3g/L,MgSO 4 0.1-3 g/L, 0.01-0.2 g/L of ferric ammonium citrate, 1-5 g/L of citric acid, 1-10 g/L of diammonium hydrogen phosphate, 5-50 g/L of glucose, 1-50 g/L of phytosterol, and the balance of water, wherein the pH value is 6.0-7.5.
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