Aldehyde dehydrogenase gene ltp and application thereof in construction of 4-hbc high-yield genetically engineered bacteria

Abstract

The present application relates to a kind of high 4-HBC mycobacterium genetic engineering bacteria and its construction method, and its application in the microbial fermentation preparation 22-hydroxy-23, 24-bis-norcholesta-4-en-3-ketone of 4-HBC.The high 4-HBC mycobacterium genetic engineering bacteria is constructed as follows: with mycobacterium as chassis, kshA1, kshA2, kstD1, kstD2, kstD3 gene is knocked out in turn, hsd4A gene is knocked out, ltp3, ltp4 gene is overexpressed, and the high 4-HBC mycobacterium genetic engineering bacteria is obtained.The genetic engineering bacteria provided in the present application can produce 4-HBC, greatly improve the production efficiency of steroidal drugs, help to improve the conversion rate of substrate, reduce production cost, and reaction condition is mild, environment-friendly, suitable for vigorous popularization and application, with higher economic benefit and social benefit.

Description

(I) TECHNICAL FIELD

[0001] The present application relates to an aldehyde condensing enzyme gene ltp and its application in constructing a high-yield 4-HBC genetically engineered bacterium, and a high-yield 4-HBC mycobacterium genetically engineered bacterium, its construction and its application in preparing 22-hydroxy-23, 24-bishomocholesta-4-en-3-one by microbial fermentation. (II) BACKGROUND

[0002] There are more than 300 kinds of steroid drugs approved for listing, which are one of the most important drugs for treating diseases in humans. In terms of the pharmaceutical industry alone, the global demand for steroid substances exceeds 1500 tons per year, and the market for steroid drugs produced in 2015 exceeds 100 billion US dollars, second only to antibiotics. Steroid drugs include adrenocortical hormones, sex hormones, progesterone, salt corticosteroids and non-hormone steroids, which are used in various fields, including medicine, veterinary medicine, aquaculture, agriculture, food industry.

[0003] Chemical synthesis method once had absolute dominance in this field, and diosgenin from Dioscorea plant has a very similar structure to steroid drugs, and can synthesize various steroid drugs by chemical methods. However, the source of raw materials is unstable, and the extraction cost is high, which is difficult to meet the growing market demand. Microbial transformation of phytosterols has become one of the main ways to produce various steroids, which has gradually replaced the traditional chemical synthesis route, and can produce various intermediates for preparing steroid drugs, such as 4-androstene-3, 17-dione (AD), 1, 4-androstadiene-3, 17-dione (ADD), 9-hydroxy-4-androstene-3, 17-dione (9-OH-AD), 22-hydroxy-23, 24-bishomocholesta-4-en-3-one (4-HBC), 22-hydroxy-23, 24-bishomocholesta-1, 4-diene-3-one (1, 4-HBC) and the like.

[0004] With the development of bioinformatics, the mystery of functional genes related to sterol catabolic pathway has been gradually revealed. For example, Figure 1As shown, cholesterol is first converted to 4-cholesten-3-one by chox or 3β-hydroxysteroid oxidase (hsd, 3β-HSD). The side chain degradation process is consistent with the fatty acid oxidation pathway, and sterol C26 monooxygenase (cyp125, cyp142) mainly catalyzes the formation of a terminal carboxyl group in the side chain. After C-27 terminal acylation, the cholesterol side chain is activated by terminal CoA thioester, and then the carboxyl CoA of C-27 enters the β-oxidation reaction. The complete degradation of the side chain can generate AD, ADD and 9-OH-AD, and the side branch metabolic pathway can generate 4-HBC under the action of aldolase and propionyl coenzyme A reductase. Due to the slight difference in the side chain structure between cholesterol and phytosterol, the genes in the metabolic pathway are different, for example, the fadA5 encoded sulfhydrylase cannot complete the conversion of the branched structure substrate with C24, and therefore there are other genes and enzyme elements involved in the reaction.

[0005] Hotse et al. reported that the side chain degradation of sitosterol and brassicasterol was blocked by targeting the inactivation of Rhodococcus rhodochrous ltp3 and ltp4, which showed that Ltp3 and Ltp4 had specific effects in removing the C24 branch, had high similarity with sulfhydrylase, and were involved in the β-oxidation reaction of sterol side chain (such as Figure 2 ) (Hotse M, Wilbrink, Robert, et al. Molecular characterization of ltp3 and ltp4, essential for C24-branched chain sterol-side-chain degradation in Rhodococcus rhodochrous DSM43269. Microbiology, 2012).

[0006] Xu et al. reported a method for producing 4-HBC using genetically engineered Mycobacterium with phytosterol as substrate. Under the condition of 40 g / L substrate concentration and 144 h of conversion, the molar yield of resting cells was less than 50%, and there were AD, 1,4-HBC and other by-products, which made it difficult to apply this technology in industrial production (Xu LQ, Liu YJ, Yao K, et al. Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism, Scientific Repots, 2016, 6:21928). Patent CN 112029701 reported that 3-1 The expression amount of dehydrogenase gene and 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA aldolase gene is reduced, inactivated or knocked out, and the genes encoding acetyl-CoA acetyltransferase / thiolase and DNA binding protein are overexpressed, which improves the yield of the original strain, but in the reaction of adding 20 g / L phytosterol, the molar yield is only 53% (144 h). Li et al. constructed a high-yield AD strain by knocking out the kstd and ksh genes of Mycobacterium HGMS2 strain. It was found that the phytosterol conversion capacity of the knockout mutant HGMS2 Δkstd211+ΔkshB122 of the endogenous kstd and ksh genes increased by 20%, and the molar yield was 51.6% in the reaction of 10 g / L phytosterol (Li, X., Chen, T., Peng, F. et al. Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria. Microbial Cell Factories, 2021, 20(1): 1-15).

[0007] High-yield and specific preparation of 4-HBC is of great significance to the industrial production of steroid drugs. The number of reports on Mycobacterium obtaining 4-HBC by degrading sterols has increased year by year, and the degradation mechanism has been gradually disclosed. Compared with Nocardia and Rhodococcus, which can degrade sterols, the related metabolic genes and enzymes have high conservation, but due to the difference in substrates, the metabolic pathways still have many changes, and these differences contain the potential to improve the production capacity of the strain. (III) SUMMARY

[0008] The purpose of the present application is to provide an aldolase gene ltp and its application in constructing a high-yield 4-HBC genetically engineered strain of Mycobacterium, as well as a high-yield 4-HBC genetically engineered strain of Mycobacterium and its construction and application in the microbial fermentation preparation of 22-hydroxy-23,24-bishomocholesta-4-en-3-one.

[0009] The technical scheme adopted by the present application is:

[0010] An aldolase gene ltp, which is composed of ltp3 and ltp4, characterized in that the nucleotide sequence of the ltp3 gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the ltp4 gene is shown in SEQ ID NO. 3.

[0011] The present application also relates to the application of the aldehyde dehydrogenase gene ltp in constructing a high-yield 4-HBC genetically engineered bacterium.

[0012] A high-yield 4-HBC mycobacterium genetically engineered bacterium is constructed by the following method:

[0013] (1) knocking out kshA1, kshA2, kstD1, kstD2 and kstD3 genes in sequence from a mycobacterium as a chassis bacterium to obtain an AD-producing engineered bacterium Mn-AD;

[0014] (2) further knocking out hsd4A gene from the engineered bacterium Mn-AD as a chassis bacterium to obtain a 4-HBC-producing engineered bacterium Mn-HBC;

[0015] (3) overexpressing ltp3 and ltp4 genes from the engineered bacterium Mn-HBC as a chassis bacterium to obtain the high-yield 4-HBC mycobacterium genetically engineered bacterium.

[0016] Specifically, the ltp3 gene nucleotide sequence is shown in SEQ ID NO. 1, and the ltp4 gene nucleotide sequence is shown in SEQ ID NO. 3.

[0017] The present application also relates to a method for constructing the genetically engineered bacterium, which comprises:

[0018] (1) using the genome of a chassis bacterium mycobacterium as a template, amplifying the upstream and downstream fragments of kshA1, kshA2, kstD1, kstD2, kstD3 and hsd4A genes, respectively, and connecting with the pacI and NotI enzyme-digested linearized pNS plasmid to construct the knockout plasmids pNS-kshA1H, pNS-kshA2H, pNS-kstD1H, pNS-kstD2H, pNS-kstD3H and pNS-hsd4AH; using the genome of a chassis bacterium mycobacterium as a template, amplifying the-ltp3-ltp4 gene, and connecting the amplified fragment with the BamHI and HindIII enzyme-digested linearized pMV261 plasmid to construct the pMV261-ltp3-ltp4 overexpression plasmid;

[0019] (2) using the mycobacterium as a chassis bacterium, using the knockout plasmids pNS-kshA1H, pNS-kshA2H, pNS-kstD1H, pNS-kstD2H and pNS-kstD3H, and using the method of homologous recombination double exchange to knock out kshA1, kshA2, kstD1, kstD2 and kstD3 genes in sequence to obtain the AD-producing engineered bacterium Mn-AD;

[0020] (3) using the engineering bacteria Mn-AD as a chassis bacteria, knocking out the hsd4A gene by using the plasmid pNS-hsd4AH, to obtain the engineering bacteria Mn-HBC producing 4-HBC;

[0021] (4) using the engineering bacteria Mn-HBC as a chassis bacteria, overexpressing the ltp3 and ltp4 genes by using the pMV261-ltp3-ltp4 overexpression plasmid, to obtain the engineering bacteria Mn-HBC pMV261-ltp3,4, i.e. the mycobacterium genetic engineering bacteria producing 4-HBC.

[0022] Preferably, the mycobacterium is Mycobacterium neoaurum ATCC 25795. The present application is also applicable to the same genus of mycobacterium.

[0023] The nucleotide sequence of the ltp3 gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the ltp4 gene is shown in SEQ ID NO. 3.

[0024] The present application also relates to the application of the genetic engineering bacteria in the microbial fermentation preparation of 22-hydroxy-23,24-bishomocholesta-4-en-3-one.

[0025] Specifically, the application is that the genetic engineering bacteria are inoculated into a fermentation medium containing a sterol, and are cultured at 25-40℃ and 100-300rpm for 48-120h, to obtain the 22-hydroxy-23,24-bishomocholesta-4-en-3-one in the fermentation liquor; the sterol is one of the following: cholesterol, stigmasterol, sitosterol, and phytosterol, and is preferably phytosterol.

[0026] The present application has the following beneficial effects: the genetic engineering bacteria strain provided by the present application can produce 4-HBC, greatly improves the production efficiency of steroidal drugs, helps to improve the conversion rate of substrates, reduces the production cost, and has mild reaction conditions, is environment-friendly, is suitable for vigorous promotion and application, and has high economic and social benefits. (Four) Description of Drawings

[0027] Figure 1 It is a metabolic pathway of cholesterol in microorganisms;

[0028] Figure 2 It is a metabolic pathway of phytosterol and cholesterol LTP participation;

[0029] Figure 3 It is a schematic diagram of a knockout plasmid;

[0030] Figure 4 It is an electropherogram of ltp3,4 knockout;

[0031] Figure 5Schematic diagram of overexpression plasmid. (V) DETAILED DESCRIPTION

[0032] The application will be further described in conjunction with specific examples. However, the application is not limited to the following examples:

[0033] Example 1: Construction and transformation screening of knockout plasmid

[0034] (1) The knockout plasmids of kshA1, kshA2, kstD1, kstD2, kstD3, hsd4A and ltp3, ltp4 were constructed according to the following method, respectively.

[0035] PCR amplification was performed with the genome of the starting strain Mycobacterium neoaurum ATCC 25795 as a template. The PCR fragment amplification system: 2 μL of each upstream and downstream primers, 50-100 ng of template, 1 μL of dNTP, 25 μL of 2×Buffer, 1 μL of DNA polymerase, and ddH2O to 50 μL. The PCR fragment amplification program: 95℃ for 5 min, 95℃ for 15 s, (Tm-5℃) for 15 s, 72℃ for 30 s / kb, 72℃ for 10 min, 30 cycles. The primer sequences used are shown in Table 1:

[0036] Table 1: Primers required for constructing knockout plasmids of each gene

[0037]

[0038]

[0039] The amplified upstream and downstream fragments were connected with the pacI and NotI enzyme-digested linearized pNS plasmid by one-step cloning enzyme (such as Figure 3 ), wherein the pNS plasmid is composed of the fragment (hsp60-sacB) inserted into pGOAL19 from the p2NIL vector. The ligation product was transformed into DH5α competent cells, and plated on Kan-resistant LB plates (tryptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L, Kan: 50 μg / ml agar: 2%), and incubated at 37℃ for 16 h, and single colonies were picked, and PCR and sequencing were performed to verify whether the construction was correct, to construct the knockout plasmids pNS-kshA1H, pNS-kshA2H, pNS-kstD1H, pNS-kstD2H, pNS-kstD3H, pNS-hsd4AH and pNS-ltp34H, respectively.

[0040] (2) Transformation and screening

[0041] The obtained knockout plasmid is added to the mycobacterium competent (dissolved on ice) after alkaline treatment, and is placed at 4°C for 20 min; the voltage is set to 2.5 kV, the shock cup aperture is selected to be 2 mm, and the electric shock is performed twice; the electric shock frequency is confirmed to be in the range of 4-5 ms, 600 μL of fresh LB medium is added, the bottom bacteria are fully suspended, and then transferred to a sterile centrifuge tube; incubated at 37°C-180 rpm for 4 h, centrifuged at 5000 rpm for 3 min, the supernatant is discarded, 100 μL of resuspension is performed, and inverted culture is performed at 30°C for 3-5 d.

[0042] The transformants obtained by screening are verified by colony PCR to determine whether single exchange is successful using primers SCO-F: 5'-cgccaagcttcctgctgaacatcaaagg-3' on the sacB gene and primers SCO-R outside the homologous arm downstream of the target gene; correct transformants are verified by electrophoresis and sequencing, transferred to LB liquid medium, and incubated at 37°C-180 rpm for 12 h; 50 μL of the transfer is plated on a sucrose plate (LB without NaCl containing 5% sucrose) to screen double exchange transformants, and inverted culture is performed at 30°C for 3-5 d; the transformants are picked and printed on Kan resistance and non-antibiotic plates, and colony PCR is performed using primers upstream and downstream of the target gene; the target band of the transformants meeting the requirements is verified by electrophoresis, and the transformants growing on the non-antibiotic plate and not growing on the Kan resistance plate are knockout strains, which are verified by sequencing the PCR product.

[0043] Example 2: Construction of AD-producing strain and 4-HBC-producing strain

[0044] Mycobacterium neoaurum ATCC 25795 is used as the starting strain, the knockout plasmid constructed in Example 1 is used, and the kshA1, kshA2, kstD1, kstD2, and kstD3 genes are sequentially knocked out by the method of homologous recombination double exchange; the sequencing of the PCR product is performed, and the fermentation verification is performed using 2 g / L phytosterol as the substrate and LB as the culture medium to obtain the AD-producing engineering strain Mn-AD. Mn-AD is used as the starting strain, the knockout plasmid constructed in Example 1 is used, and the hsd4A gene is further knocked out by the method of homologous recombination double exchange; the sequencing of the PCR product is performed, and the fermentation verification is performed using 2 g / L phytosterol as the substrate and LB as the culture medium to obtain the 4-HBC-producing engineering strain Mn-HBC.

[0045] Example 3: Construction of ltp3 and ltp4 knockout strain

[0046] On the basis of Mn-AD and Mn-HBC strains, the knockout plasmid pNS-ltp34H constructed in Example 1 was used to knock out the ltp3 and ltp4 genes by homologous recombination double exchange method. After single exchange and double exchange screening, the transformants were obtained and verified by colony PCR. The gel electrophoresis (as shown in Figure 4 ) of the product showed that 2000 bp was missing compared with the unknocked out, and the target gene was deleted by about 90%. The sequencing result showed that the knockout was successful, and the Mn-ADAltp3,4 and Mn-HBCAltp3,4 strains were obtained.

[0047] Example 4: Conversion of different substrates

[0048] A loop of bacterial liquid was taken from the glycerol tube, streaked on LB solid medium, and cultured at 30°C for 48h; a single colony was picked and cultured in 5mL LB liquid at 30°C for 36h; 2% was transferred to 90mL M3 medium, and cultured at 30°C for 12h; 10mL of hydroxypropyl cyclodextrin emulsified sterol 100g / L solution was added, and the culture was shaken at 30°C-180rpm; samples were taken at 96h and 120h, 1mL sample was added to 5mL ethyl acetate for extraction, shaken for 30min, and the upper organic phase was taken to EP tube for evaporation, 0.8mL methanol was added for redissolution, and liquid phase detection was performed. The conversion results are shown in Table 2.

[0049] Table 2: Conversion results of ltp3,4 knockout strain in different substrates

[0050]

[0051] Example 5: Construction and transformation of pMV261-ltp3-ltp4 overexpression plasmid

[0052] (1) Construction of pMV261-ltp3-ltp4 overexpression plasmid

[0053] The genome of the starting strain Mycobacterium neoaurum ATCC 25795 was used as the template for PCR amplification. The PCR fragment amplification system: 2μL of each upstream and downstream primer, 50-100ng of template, 1μL of dNTP, 25μL of 2×Buffer, 1μL of DNA polymerase, and ddH2O to 50μL; PCR fragment amplification program: 95°C for 5min, 95°C for 15s, (Tm-5°C) for 15s, 72°C for 30s / kb, 72°C for 10min, 30 cycles.

[0054] The primer sequences used are as follows:

[0055] ltp-F: GGCCAAGACAATTGCGGATCCatgacagatatcgcagtggtgggct,

[0056] ltp-R: CTACGTCGACATCGATAAGCTTtcacctgctcggcttgtccgc;

[0057] The amplified fragment was ligated with the BamHI and HindIII linearized pMV261 plasmid using one-step ligation enzyme (as described in Figure 5 ), and the ligation product was transformed into DH5a competent cells, plated on Kan-resistant LB plates, and incubated at 37°C for 16 h. Single colonies were picked, and PCR and sequencing were performed to verify the absence of errors. The pMV261-ltp3-ltp4 overexpression plasmid was constructed.

[0058] (2) Transformation and screening

[0059] The constructed overexpression plasmid was added to the mycobacterium competent cells (which had been dissolved on ice) and allowed to stand at 4°C for 20 min. The voltage was set to 2.5 kV, the shock cup aperture was set to 2 mm, and the cells were shocked twice. The shock frequency was confirmed to be in the range of 4-5 ms, 600 μL of fresh LB medium was added, the bottom cells were fully suspended, and then transferred to a sterile centrifuge tube. Incubation was performed at 37°C and 180 rpm for 4 h, centrifugation was performed at 5000 rpm for 3 min, the supernatant was discarded, 100 μL of resuspension was performed, and incubation was performed at 30°C for 3-5 d. The transformants were verified by colony PCR, and the correct transformants were verified by electrophoresis and sequencing. The Mn-AD pMV261-ltp3,4 and Mn-HBC pMV261-ltp3,4 strains were obtained.

[0060] Example 6: Transformation reaction of overexpression strains

[0061] A ring of bacterial liquid was taken from a glycerol tube, streaked on LB solid medium, and incubated at 30°C for 48 h. Single colonies were picked and cultured in 5 mL of LB liquid at 30°C for 36 h. 5% was transferred to 50 mL of M3 medium, and incubated at 30°C for 6 h. 10 mL of hydroxypropyl cyclodextrin emulsified sterol 200 g / L solution was added, and incubation was performed at 30°C and 180 rpm. Samples were taken at 120 h, 1 mL of sample was added to 5 mL of ethyl acetate for extraction, and oscillation was performed for 30 min. 100 μL of the upper organic phase was evaporated in an EP tube, 0.8 mL of methanol was added for redissolution, and liquid phase detection was performed. The results are shown in Table 3.

[0062] Table 3: Transformation capacity of overexpression strains

[0063]

[0064]

[0065] Finally, it should be noted that the above is merely to illustrate the technical solutions of the present application, and is not a limitation on the scope of protection of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A genetically engineered mycobacterium that produces high levels of 4-HBCs, constructed using the following method: (1) Mycobacterium ( Mycobacterium neoaurum ATCC 25795 indicates chassis bacteria; remove them sequentially. kshA1、 kshA2, kstD1, kstD2, kstD3 Genes were used to obtain engineered bacteria that produce AD. Mn-AD ; (2) Using engineered bacteria Mn-AD For chassis bacteria, further knockout is needed. hsd4A Genes were used to obtain engineered bacteria that produce 4-HBC. Mn-HBC ; (3) Using engineered bacteria Mn-HBC For chassis bacteria, construct pMV261-ltp3-ltp4 Overexpression plasmid, overexpression LTP3, LTP4 Genes were used to obtain the high-yielding 4-HBC mycobacterial genetically engineered strain; ltp3 The gene nucleotide sequence is shown in SEQ ID NO.

1. ltp4 The gene nucleotide sequence is shown in SEQ ID NO.

3.

2. A method for constructing the high-yielding 4-HBC mycobacterial genetically engineered bacterium of claim 1, the method comprising: (1) Mycobacterium tumefaciens ( ) Mycobacterium neoaurum Using the ATCC 25795 genome as a template, amplification was performed separately. kshA1, kshA2, kstD1, kstD2, kstD3, hsd4A The upstream and downstream fragments of the gene were ligated to the pNS plasmid, which was linearized by digestion with pacI and NotI enzymes, to construct the knockout plasmid pNS- kshA1H、 pNS- kshA2H、 pNS- kstD1H、 pNS- kstD2H、 pNS- kstD3H and pNS- hsd4AH ; with mycobacterium tumefaciens ( Mycobacterium neoaurum Using the ATCC 25795 genome as a template, amplification was performed. ltp3 and ltp4 The gene was amplified and ligated to the pMV261 plasmid, which was linearized by BamHI and HindIII restriction enzymes, to construct the gene. pMV261-ltp3-ltp4 Overexpression plasmid; ltp3 The gene nucleotide sequence is shown in SEQ ID NO.

1. ltp4 The gene nucleotide sequence is shown in SEQ ID NO. 3; (2) Mycobacterium ( Mycobacterium neoaurum ATCC 25795 is a chassis bacterium, and the knockout plasmid pNS- was used. kshA1H、 pNS- kshA2H、 pNS- kstD1H、 pNS- kstD2H and pNS- kstD3H Using the homologous recombination double exchange method, knockouts are sequentially performed. kshA1, kshA2, kstD1, kstD2, kstD3 Genes were used to obtain engineered bacteria that produce AD. Mn-AD ; (3) Using engineered bacteria Mn-AD For the basal bacteria, the knockout plasmid pNS- was used. hsd4AH Knockout hsd4A Genes were used to obtain engineered bacteria that produce 4-HBC. Mn-HBC ; (4) Using engineered bacteria Mn-HBC For chassis bacteria, utilize pMV261-ltp3-ltp4 Overexpression plasmid, overexpression LTP3, LTP4 Genes, to obtain engineered bacteria Mn-HBC pMV261-ltp3,4 That is, the genetically engineered mycobacterium that produces high levels of 4-HBC.

3. The application of the high-4-HBC-producing mycobacterial genetically engineered strain of claim 1 in the microbial fermentation preparation of 22-hydroxy-23,24-bis-cholesterol-4-en-3-one, characterized in that... The application is as follows: the genetically engineered mycobacterium that produces high levels of 4-HBC is inoculated into a fermentation medium containing sterols to obtain the 22-hydroxy-23,24-bischolesterol-4-en-3-one in the fermentation broth; the sterol is one of the following: cholesterol, stigmasterol, or sitosterol.

4. The application as described in claim 3, characterized in that... The application is as follows: the genetically engineered mycobacterium that produces high levels of 4-HBC is inoculated into a fermentation medium containing sterols and cultured at 25-40 °C and 100-300 rpm for 48-120 h with shaking to obtain the 22-hydroxy-23,24-bis-cholest-4-en-3-one from the fermentation broth.

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