CN115960931A - Aldolase gene ltp and application thereof in construction of high-yield 4-HBC genetic engineering bacteria - Google Patents

Abstract

The invention relates to a mycobacterium genetic engineering bacterium with high 4-HBC yield, a construction method thereof and application thereof in preparing 22-hydroxy-23, 24-bis-cholesta-4-en-3-one by microbial fermentation. The mycobacterium genetic engineering bacteria with high 4-HBC yield is constructed according to the following method: and (3) knocking out kshA1, kshA2, kstD1, kstD2 and kstD3 genes, knocking out hsd4A genes and overexpressing ltp3 and ltp4 genes by taking the mycobacteria as chassis bacteria in sequence to obtain the mycobacterium genetic engineering bacteria with high 4-HBC yield. The genetic engineering strain provided by the invention can produce 4-HBC, greatly improves the production efficiency of steroid drugs, is beneficial to improving the conversion rate of substrates and reducing the production cost, has mild reaction conditions and environmental friendliness, is suitable for vigorous popularization and application, and has higher economic benefit and social benefit.

Description

Aldolase gene ltp and application thereof in construction of high-yield 4-HBC genetic engineering bacteria

(I) the technical field

The invention relates to an aldolase gene ltp and application thereof in constructing a high-yield 4-HBC genetically engineered bacterium, a high-yield 4-HBC mycobacteria genetically engineered bacterium, construction thereof and application thereof in preparing 22-hydroxy-23, 24-bis-cholesta-4-en-3-one by microbial fermentation.

(II) background of the invention

Steroid drugs are approved for more than 300, and are one of the most important drugs for human use in the treatment of diseases. Worldwide demand for steroid substances is over 1500 tons per year for the pharmaceutical industry alone, with a market for steroid drugs over $ 1000 billion produced 2015, second only to antibiotics. Steroid drugs include adrenocortical hormone, sex hormone, progesterone, mineralocorticoid, and non-hormonal steroids, and are used in various fields including medicine, veterinary drugs, aquaculture, agriculture, and food industry.

The chemical synthesis method has been an absolute principle in the field, diosgenin from dioscorea plants has a structure very similar to that of steroid drugs, and multiple steroid drugs can be synthesized by a chemical mode. But the raw material source is unstable, the extraction cost is high, and the increasingly expanded market demand is difficult to meet. The microbial conversion of phytosterol becomes one of the main ways of producing various steroids currently, and the traditional chemical synthesis routes are gradually replaced, so that various intermediates for preparing steroid drugs can be produced, 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-bis-cholesta-4-ene-3-one (4-HBC), 22-hydroxy-23, 24-bis-cholesta-1, 4-diene-3-one (1, 4-HBC) and the like.

With the development of bioinformatics, the mysteries of functional genes related to sterol catabolic pathways are gradually uncovered. As shown in FIG. 1, cholesterol is first converted to 4-cholesten-3-one by cholesterol oxidase (ChOx) or 3 β -hydroxysteroid oxidase (HSD, 3 β -HSD). The side chain degradation process is shown to be consistent with the fatty acid oxidation pathway, with the steroid C26 monooxygenases (cyp 125, cyp 142) primarily catalyzing the formation of terminal carboxyl groups on the side chains. After acylation at the C-27 terminus, the cholesterol side chain is activated by terminal CoA thioesterification, and then the C-27 carboxylate CoA enters into a beta-oxidation reaction. The side chains are completely degraded to generate AD, ADD and 9-OH-AD, and the side-branch metabolic pathway can generate 4-HBC under the action of aldolase and propionyl-CoA reductase. Since cholesterol is slightly different from phytosterol in side chain structure, resulting in different genes in metabolic pathways, for example, the thiolase encoded by fadA5 cannot complete the conversion of C24 into a substrate with a branched structure, and thus other genes and enzyme elements involved in the reaction exist.

Hotspot et al reported that the degradation of the side chains of sitosterol and rapeseed was hindered by targeted inactivation of the Rhodococcus rhodochrous Ltp3 and Ltp4, and showed that Ltp3 and Ltp4 have specific roles in the removal of the C24 branch, have a high similarity to thiolase, and participate in the β -oxidation of the sterol side chains (see fig. 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).

Xu et al reported that the molar yield of resting cells was less than 50% under the condition of conversion for 144 hours at a substrate concentration of 40g/L using genetically engineered mycobacteria for producing 4-HBC using phytosterols as a substrate, and AD,1,4-HBC, etc. was a by-product, which is difficult to apply in industrial production (Xu LQ, liu YJ, yao K, et al. Underwurling and engineering the production of 23, 24-biosorbornenicorsteroides in sterol metabolism, scientific reports, 2016, 6. Patent CN 112029701 reports 3-sterone-delta in the fungus ¹ The reduced expression, inactivation or knock-out of the-dehydrogenase gene and 17-hydroxy-3-oxo-4-pregnene-20-carboxy-CoA aldolase genes, together with the overexpression of the genes coding for acetyl-CoA acetyltransferase/thiolase and DNA-binding protein, increased the yield of the original strain, but only at a molar yield of 53% (144 h) in the 20g/L phytosterol addition reaction. Li and the like construct a strain with high AD yield by knocking out kstd and ksh genes of a mycobacterium HGMS2 strain. After the endogenous kstd and ksh genes are knocked out, the mutant HGMS2 is knocked out _{Δkstd211+ΔkshB122} The phytosterol conversion capacity of (1) was increased by 20%, and the molar yield was 51.6% in 10g/L phytosterol reaction (Li, X., chen, T., peng, F.et al. Effective conversion of phytosterols inter 4-alkane-3, 17-dione and its C1, 2-dehydrated and 9 α -hydroxylated derivatives by y engineered Mycobacterium Microbiological Cell Factories,2021,20 (1): 1-15).

The high-yield specific preparation of the 4-HBC has extremely important significance for the industrial production of steroid medicaments. The reports on 4-HBC obtained by degrading sterol by mycobacteria are increased year by year, and the degradation mechanism of the mycobacteria is gradually revealed, compared with sterol-degrading bacteria such as Nocardia and Rhodococcus, the conservation degree of related metabolic genes and enzymes is higher, but because of different substrates, the metabolic pathways still have many changes, and the differences contain the potential of improving the productivity of the strains.

Disclosure of the invention

The invention aims to provide an aldolase gene ltp and application thereof in constructing high-yield 4-HBC genetically engineered bacteria, high-yield 4-HBC mycobacteria genetically engineered bacteria, construction thereof and application thereof in preparing 22-hydroxy-23, 24-bis-cholesta-4-en-3-one by microbial fermentation.

The technical scheme adopted by the invention is as follows:

an aldolase gene ltp consisting of ltp3 and ltp4, characterized in that: the nucleotide sequence of the ltp3 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the ltp4 gene is shown as SEQ ID NO. 3.

The invention also relates to application of the aldolase gene ltp in construction of high-yield 4-HBC genetic engineering bacteria. Experiments show that the capability of producing 4-HBC by engineering bacteria through transformation is greatly improved by over-expressing the aldolase gene ltp.

A mycobacterium genetic engineering bacterium with high 4-HBC yield is constructed and obtained by the following method:

(1) Sequentially knocking out kshA1, kshA2, kstD1, kstD2 and kstD3 genes by taking mycobacteria as chassis bacteria to obtain an engineering bacterium Mn-AD for producing AD;

(2) Further knocking out the hsd4A gene by taking the engineering bacteria Mn-AD as chassis bacteria to obtain engineering bacteria Mn-HBC for producing 4-HBC;

(3) And (3) overexpressing ltp3 and ltp4 genes by using the engineering bacterium Mn-HBC as a chassis bacterium to obtain the mycobacterium genetic engineering bacterium with high yield of 4-HBC.

Specifically, the nucleotide sequence of the ltp3 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the ltp4 gene is shown as SEQ ID NO. 3.

The invention also relates to a method for constructing the genetic engineering bacteria, which comprises the following steps:

(1) Taking the genome of mycobacterium of the chassis bacteria as a template, respectively amplifying upstream and downstream fragments of kshA1, kshA2, kstD1, kstD2, kstD3 and hsd4A genes, and connecting the upstream and downstream fragments with a pacI and NotI enzyme digestion linearized pNS plasmid to construct knockout plasmids pNS-kshA1H, pNS-kshA2H, pNS-kstD1H, pNS-kstD2H, pNS-kstD3H and pNS-hsd4AH; amplifying-ltp 3-ltp4 gene by using a mycobacterium genome of Chassis bacteria as a template, connecting the amplified fragment with pMV261 plasmid which is linearized by enzyme digestion of BamHI and HindIII, and constructing to obtain pMV261-ltp3-ltp4 overexpression plasmid;

(2) Taking mycobacteria as chassis bacteria, knocking out plasmids pNS-kshA1H, pNS-kshA2H, pNS-kstD1H, pNS-kstD2H and pNS-kstD3H, and knocking out genes kshA1, kshA2, kstD1, kstD2 and kstD3 in sequence by a homologous recombination double exchange method to obtain an engineering bacterium Mn-AD for producing AD;

(3) Taking the engineering bacterium Mn-AD as a chassis bacterium, knocking out the plasmid pNS-hsd4AH, knocking out the hsd4A gene, and obtaining the engineering bacterium Mn-HBC producing 4-HBC;

(4) Engineering bacteria Mn-HBC are taken as chassis bacteria, a pMV261-ltp3-ltp4 overexpression plasmid is utilized to overexpress ltp3 and ltp4 genes, and the engineering bacteria Mn-HBC pMV261-ltp3,4, namely the high-yield 4-HBC mycobacterium genetic engineering bacteria, are obtained.

Preferably, the Mycobacterium is Mycobacterium neoaurum ATCC 25795. The invention is equally applicable to mycobacteria of the same species.

The nucleotide sequence of the ltp3 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the ltp4 gene is shown as SEQ ID NO. 3.

The invention also relates to application of the genetic engineering bacteria in preparation of 22-hydroxy-23, 24-bis-cholesta-4-en-3-one by microbial fermentation.

Specifically, the application is as follows: inoculating the genetic engineering bacteria to a fermentation medium containing sterol, performing shake culture at the temperature of 25-40 ℃ and the rpm of 100-300 for 48-120 h, and obtaining the 22-hydroxy-23, 24-bis-cholesta-4-ene-3-one in fermentation liquor; the sterol is one of the following: cholesterol, stigmasterol, sitosterol, phytosterol, preferably phytosterol.

The invention has the following beneficial effects: the genetic engineering strain provided by the invention can produce 4-HBC, greatly improves the production efficiency of steroid drugs, is beneficial to improving the conversion rate of substrates and reducing the production cost, has mild reaction conditions and environmental friendliness, is suitable for wide popularization and application, and has higher economic benefit and social benefit.

Description of the drawings

FIG. 1 is a metabolic pathway of cholesterol in a microorganism;

FIG. 2 is a diagram showing the metabolic pathways involved in LTP of phytosterols and cholesterol;

FIG. 3 is a schematic of a knock-out plasmid;

FIG. 4 is an ltp3,4 knockout electropherogram;

FIG. 5 is a schematic representation of an overexpression plasmid.

(V) detailed description of the preferred embodiments

The present invention will be described in further detail with reference to the following examples, but the present invention is not limited to the following examples:

example 1: construction of knock-out plasmids and transformation screening

(1) Plasmids for knock-out of kshA1, kshA2, kstD1, kstD2, kstD3, hsd4A and ltp3, ltp4 were constructed as follows.

PCR amplification was performed using the genome of the original strain Mycobacterium neoaurum ATCC 25795 as a template. PCR fragment amplification System: 2 μ L of each of the upstream and downstream primers, 50-100ng of template, 1 μ L of dNTP, 25 μ L of 2 XBuffer, 1 μ L of DNA polymerase, ddH ₂ O is complemented to 50 mu L; PCR fragment amplification procedure: 95-5min, 95-15 s, (Tm-5 ℃) to 15s, 72-30 s/kb, 72-10min and 30 cycles. Wherein the primer sequences used are as shown in Table 1:

table 1: primers required for construction of knock-out plasmids for each Gene

The amplified upstream and downstream fragments were ligated with pacI and NotI linearized pNS plasmid (see fig. 3) using one-step cloning enzyme, wherein pNS plasmid is a fragment (hsp 60-sacB) of p2NIL vector inserted into pGOAL 19. The ligation products were transformed into DH 5. Alpha. Competent cells, plated on Kan-resistant LB plates (tryptone: 10g/L, yeast extract: 5g/L, sodium chloride: 10g/L, kan: 50. Mu.g/ml agar: 2%), inverted cultured at 37 ℃ for 16H, single colonies were picked, verified by PCR and sequencing for correct construction, and knock-out plasmids pNS-kshA1H, pNS-kshA2H, pNS-kstD1H, pNS-kstD2H, pNS-kstD3H, pNS-hsd4AH and pNS-ltp34H were constructed, respectively.

(2) Transformation and screening

After alkali treatment, the obtained knockout plasmid is added into the mycobacterium infection (dissolved on ice) and is kept stand for 20min at 4 ℃; setting the voltage to be 2.5kV, selecting the aperture of the electric shock cup to be 2mm, and carrying out electric shock for 2 times; confirming that the electric shock frequency is in the range of 4-5ms, adding 600 mu L of fresh LB culture medium, fully suspending the bottom thalli, and transferring to a sterile centrifuge tube; shaking and incubating at 37-180 rpm for 4h, centrifuging at 5000rpm for 3min, discarding supernatant, resuspending at 100 μ L, and performing inverted culture at 30 deg.C for 3-5d.

The resulting transformants were selected and the DNA fragments were isolated using primer SCO-F on sacB gene: 5 'cgccaaagcttcctgctgaactcaaaagg-3' and a primer SCO-R outside the downstream homology arm of the target gene, and whether the single exchange is successful is verified by colony PCR; verifying a correct transformant through electrophoresis and sequencing, transferring the transformant into an LB liquid culture medium, and oscillating at 37-180 rpm for 12 hours; taking 50 mu L of the double-exchange transformant to be coated on a sucrose plate (LB does not contain NaCl and contains 5 percent of sucrose) for screening, and carrying out inverted culture at the temperature of 30 ℃ for 3-5d; selecting transformants, simultaneously photocopying the transformants on Kan resistant and non-resistant plates, and carrying out colony PCR by using upstream and downstream primers of a target gene; and (3) verifying that the target band meets the requirement through electrophoresis, contrasting that the two plates grow in the absence of resistance, and the plate with the Carna resistance does not grow to be the knockout strain, and performing PCR product sequencing verification.

Example 2: construction of AD-producing Strain and 4-HBC-producing Strain

Using Mycobacterium neoaurum ATCC 25795 as a starting strain, adopting the knock-out plasmid constructed in the example 1, sequentially knocking out kshA1, kshA2, kstD1, kstD2 and kstD3 genes by a homologous recombination double-exchange method, sequencing a PCR product, taking 2g/L phytosterol as a substrate and LB as a culture medium for fermentation verification, and obtaining the engineering bacterium Mn-AD for producing AD. And (3) further knocking out the hsd4A gene by using Mn-AD as an original strain and the knock-out plasmid constructed in the embodiment 1 and using a homologous recombination double-exchange method, sequencing a PCR product, and performing fermentation verification by using 2g/L phytosterol as a substrate and LB as a culture medium to obtain the engineering bacterium Mn-HBC for producing the 4-HBC.

Example 3: construction of knockout ltp3 and ltp4 strains

On the basis of Mn-AD and Mn-HBC strains, a knockout plasmid pNS-ltp34H constructed in the embodiment 1 is adopted, a homologous recombination double exchange method is utilized, ltp3 and ltp4 genes are simultaneously knocked out, a transformant is obtained through screening of single exchange and double exchange, colony PCR verification is carried out, gel electrophoresis (shown in figure 4) of a product shows that the transformant is 2000bp less than that of the transformant which is not knocked out, the target gene is about 90% missing, a sequencing result shows that knockout is successful, and Mn-AD delta ltp3,4 and Mn-HBC delta ltp3,4 strains are obtained.

Example 4: transformation studies of different substrates

Taking a ring of bacteria liquid from a glycerol tube, scribing on an LB solid culture medium, and culturing for 48h at 30 ℃; selecting a single clone to be cultured in 5mL of LB liquid with shaking at 30 ℃ for 36h; transferring 2 percent of the mixture into 90mL of M3 culture medium, carrying out shaking culture at 30 ℃ for 12h, adding 10mL of solution with 100g/L of sterol emulsified by hydroxypropyl cyclodextrin, and carrying out shaking culture at 30-180 rpm; and (3) sampling for reaction for 96h and 120h, adding 5mL ethyl acetate into 1mL of sample for extraction, shaking and uniformly mixing for 30min, volatilizing 200 mu L of upper organic phase in an EP tube, adding 0.8mL of methanol for redissolution, and detecting a liquid phase, wherein the conversion result is shown in Table 2.

Table 2: transformation results of ltp3,4 knockout strains in different substrates

Example 5: construction and transformation of pMV261-ltp3-ltp4 overexpression plasmid

(1) Construction of pMV261-ltp3-ltp4 overexpression plasmid

PCR amplification was performed using the genome of the original strain Mycobacterium neoaurum ATCC 25795 as a template. PCR fragment amplification System: 2 μ L of each of the upstream and downstream primers, 50-100ng of template, 1 μ L of dNTP, 25 μ L of 2 XBuffer, 1 μ L of DNA polymerase, ddH ₂ O is complemented to 50 mu L; PCR fragment amplification procedure: 95-5min, 95-15 s, (Tm-5 ℃) to 15s, 72-30 s/kb, 72-10min and 30 cycles.

Wherein the primer sequences used are as follows:

ltp-F：GGCCAAGACAATTGCGGATCCatgacagatatcgcagtggtgggct，

ltp-R：CTACGTCGACATCGATAAGCTTtcacctgctcggcttgtccgc；

the amplified fragment is connected with a pMV261 plasmid which is linearized by digestion with BamHI and HindIII by adopting one-step cloning enzyme (as shown in figure 5), a connection product is transformed into DH5 alpha competent cells, the cells are coated on a Kan resistant LB plate, inverted culture is carried out for 16h at 37 ℃, a single colony is picked, PCR and sequencing verification are carried out without errors, and the pMV261-ltp3-ltp4 overexpression plasmid is constructed.

(2) Transformation and screening

Adding the constructed over-expression plasmid into the susceptible strain of the mycobacterium (dissolved on ice), and standing for 20min at 4 ℃; setting the voltage to be 2.5kV, selecting the aperture of the electric shock cup to be 2mm, and carrying out electric shock for 2 times; confirming that the electric shock frequency is in the range of 4-5ms, adding 600 mu L of fresh LB culture medium, fully suspending the bottom thalli, and transferring to a sterile centrifuge tube; shaking and incubating at 37-180 rpm for 4h, centrifuging at 5000rpm for 3min, discarding supernatant, resuspending at 100 mu L, and performing inverted culture at 30 ℃ for 3-5d. Transformants were verified by colony PCR, correct transformants were verified by electrophoresis and sequencing, and strains Mn-AD pMV261-ltp3,4 and Mn-HBC pMV261-ltp3,4 were obtained.

Example 6: transformation reaction of over-expressed strains

Taking a ring of bacteria liquid from a glycerol tube, scribing on an LB solid culture medium, and culturing for 48h at 30 ℃; selecting a single clone to be cultured in 5mL of LB liquid with shaking at 30 ℃ for 36h; transferring 5% of the mixture into 50mL of M3 culture medium, performing shaking culture at 30 ℃ for 6h, adding 10mL of hydroxypropyl cyclodextrin emulsified sterol 200g/L solution, and performing shaking culture at 30 ℃ and 180 rpm; the reaction is carried out for 120 hours, a sample is taken, 5mL of ethyl acetate is added into 1mL of the sample for extraction, the mixture is shaken and uniformly mixed for 30min, 100 mu L of an upper layer organic phase is taken and volatilized in an EP tube, 0.8mL of methanol is added for redissolution, the liquid phase detection is carried out, and the conversion result is shown in the table 3.

Table 3: transformation ability of over-expressed strains

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Claims (7)

1. An aldolase gene ltp consisting of ltp3 and ltp4, characterized in that: the nucleotide sequence of the ltp3 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the ltp4 gene is shown as SEQ ID NO. 3.

2. The use of the aldolase gene ltp described in claim 1 for constructing a high-yield 4-HBC genetically engineered bacterium.

3. A mycobacterium genetic engineering bacterium with high 4-HBC yield is constructed and obtained by the following method:

(1) Sequentially knocking out kshA1, kshA2, kstD1, kstD2 and kstD3 genes by taking mycobacteria as chassis bacteria to obtain an engineering bacterium Mn-AD for producing AD;

(2) Further knocking out the hsd4A gene by taking the engineering bacteria Mn-AD as chassis bacteria to obtain engineering bacteria Mn-HBC for producing 4-HBC;

(3) The engineering bacterium Mn-HBC is used as a chassis bacterium, and ltp3 and ltp4 genes are overexpressed to obtain the mycobacterium genetic engineering bacterium with high yield of 4-HBC; the nucleotide sequence of the ltp3 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the ltp4 gene is shown as SEQ ID NO. 3.

4. A method for constructing the genetically engineered bacterium of claim 3, the method comprising:

(1) Taking the genome of mycobacterium of the chassis bacteria as a template, respectively amplifying upstream and downstream fragments of kshA1, kshA2, kstD1, kstD2, kstD3 and hsd4A genes, and connecting the upstream and downstream fragments with a pacI and NotI enzyme digestion linearized pNS plasmid to construct knockout plasmids pNS-kshA1H, pNS-kshA2H, pNS-kstD1H, pNS-kstD2H, pNS-kstD3H and pNS-hsd4AH; amplifying-ltp 3-ltp4 gene by using a mycobacterium genome of Chassis bacteria as a template, connecting the amplified fragment with pMV261 plasmid which is linearized by enzyme digestion of BamHI and HindIII, and constructing to obtain pMV261-ltp3-ltp4 overexpression plasmid; the nucleotide sequence of the ltp3 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the ltp4 gene is shown as SEQ ID NO. 3;

(2) Taking mycobacteria as chassis bacteria, knocking out plasmids pNS-kshA1H, pNS-kshA2H, pNS-kstD1H, pNS-kstD2H and pNS-kstD3H, and knocking out genes kshA1, kshA2, kstD1, kstD2 and kstD3 in sequence by a homologous recombination double exchange method to obtain an engineering bacterium Mn-AD for producing AD;

(3) Taking the engineering bacterium Mn-AD as a chassis bacterium, knocking out the plasmid pNS-hsd4AH, knocking out the hsd4A gene, and obtaining the engineering bacterium Mn-HBC producing 4-HBC;

(4) Engineering bacteria Mn-HBC are taken as chassis bacteria, a pMV261-ltp3-ltp4 overexpression plasmid is utilized to overexpress ltp3 and ltp4 genes, and the engineering bacteria Mn-HBC pMV261-ltp3,4, namely the high-yield 4-HBC mycobacterium genetic engineering bacteria, are obtained.

5. The method of claim 4, wherein the Mycobacterium is Mycobacterium neoaurum ATCC 25795.

6. The use of the genetically engineered bacteria of claim 3 in the preparation of 22-hydroxy-23, 24-bis-cholesta-4-en-3-one by microbial fermentation.

7. The use according to claim 6, characterized in that the use is: inoculating the genetic engineering bacteria to a fermentation medium containing sterol, performing shake culture at the temperature of 25-40 ℃ and the rpm of 100-300 for 48-120 h, and obtaining the 22-hydroxy-23, 24-bis-cholesta-4-ene-3-one in fermentation liquor; the sterol is one of the following: cholesterol, stigmasterol, sitosterol, phytosterol.

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