CN115747238A - Aldolase gene salA and application thereof in construction of high-yield ADD genetic engineering bacteria - Google Patents

Abstract

The invention relates to an aldolase gene salA and application thereof in constructing high-yield ADD genetic engineering bacteria, and high-yield ADD mycobacteria genetic engineering bacteria, construction thereof and application thereof in preparing 1,4-androstadiene-3,17-diketone by microbial fermentation. The high-yield ADD mycobacterium genetic engineering bacteria is obtained by sequentially knocking kshA1, mnOpccR and salA genes out by taking mycobacteria as chassis bacteria. The genetic engineering strain provided by the invention effectively controls the generation of byproducts, greatly improves the utilization rate of steroid substrates and the molar conversion rate of products, simplifies the post-treatment process, can also be applied to the synthesis of products of common synthesis routes such as taking phytosterol as the substrates by the novel mycobacterium aurum of the same type, degrading and synthesizing AD and 9-OH-AD by microbial conversion side chains, completely blocks the synthesis of 4-HBC,1,4-HBC byproducts, improves the utilization rate of the substrate phytosterol, inhibits the accumulation of the byproducts, reduces the burden of downstream separation, has certain universality, is suitable for wide popularization and application, and has higher economic benefit and social benefit.

Description

Aldolase gene salA and application thereof in construction of high-yield ADD genetic engineering bacteria

(I) technical field

The invention relates to an aldolase gene salA and application thereof in constructing high-yield ADD genetic engineering bacteria, and high-yield ADD mycobacteria genetic engineering bacteria, construction thereof and application thereof in preparing 1,4-androstadiene-3,17-diketone by microbial fermentation.

(II) background of the invention

More than 300 steroid drugs have been approved for the market 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 medicine, 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. Microbial conversion of phytosterols is one of the major current ways to produce various types of steroids, and has gradually replaced traditional chemical synthetic routes. Can produce various intermediates for preparing steroid drugs, such as 4-androstene-3,17-diketone (AD), 1,4-androstadiene-3,17-diketone (ADD), 9-hydroxy-4-androstene-3,17-diketone (9-OH-AD), 22-hydroxy-23,24-bis-norcholesta-4-en-3-one (4-HBC), 22-hydroxy-23,24-bis-norcholesta-1,4-diene-3-one (1,4-HBC) and the like.

With the development of bioinformatics, sterol catabolismThe mysteries of pathway-related functional genes are gradually uncovered. 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 of the C-27 terminus, the cholesterol side chain is activated by thioesterification of the terminal CoA, and then the carboxylic acyl CoA of C-27 enters into a β -oxidation reaction. Li and the like construct a high-yield AD strain by knocking off kstd and ksh genes of a mycobacterium HGMS2 strain, and after discovering that endogenous kstd and ksh genes are knocked out, knock-out mutants HGMS2 are knocked out _{Δkstd211+ΔkshB122} The conversion capacity of phytosterol (2) is increased by 20%, and in the reaction of 10g/L of phytosterol, the molar yield is 51.6%, but byproducts such as HBC exist, which are not beneficial to separation and extraction of products. Yao et al achieved efficient accumulation of 9-OH-AD by knocking out the kstd and ksh genes in the side chain degradation pathway and overexpressing KshA (Metabolic engineering.2014Jul; 24. With the recent reports of a large amount of relevant documents, based on the above modification strategies, the blockade of key genes of metabolic pathways of degrading sterol side chains by using new mycobacterium aurum has become the mainstream synthetic pathway for producing ADD, 9-OH-AD (see fig. 1). Furthermore, there has recently been a report of knocking out the hsd4A gene in favor of the synthesis of the intermediate 4-HBC, but this process found that a bifunctional reductase MnOpccR could reduce the production of 4-HBC, but other metabolic byproducts were still present as seen by metabolic pathways (Angewandte Chemie,2021,60 (10): 5414-5420). The key problem of the microbial transformation of the steroid drugs at present is that the side chain degradation path of the microbial transformation process of the sterol is accurately regulated, the utilization rate of the substrate and the molar conversion rate of the product are improved, the accumulation of byproducts is reduced, and the burden of separation and purification of downstream products is reduced.

Reports on ADD obtained by degrading sterol by mycobacteria are increasing year by year, and the degradation mechanism and gene information are being analyzed. The key control gene and enzyme are discovered and are modified by a gene engineering technology, which is beneficial to reducing by-products in synthesis, reducing energy consumption in the production process, improving the utilization rate of a substrate and simplifying the post-treatment procedure. The high-yield specific preparation of ADD has extremely important significance for the industrial production of steroid medicines.

Disclosure of the invention

The invention aims to provide an aldolase gene salA and application thereof in constructing high-yield ADD genetic engineering bacteria, high-yield ADD mycobacteria genetic engineering bacteria, construction thereof and application thereof in preparing 1,4-androstadiene-3,17-diketone by microbial fermentation.

The technical scheme adopted by the invention is as follows:

an aldolase gene salA, the nucleotide sequence of which is shown in SEQ ID NO. 3.

The invention also relates to application of the aldolase gene salA in construction of high-yield ADD mycobacteria genetic engineering bacteria. The strategy can also be applied to the synthesis of products of common synthetic paths such as AD and 9-OH-AD by degrading and synthesizing through microbial conversion side chains by taking phytosterol as a substrate by using the novel Mycobacterium aurum (Mycobacterium neoaurum) of the same type, the synthesis of byproducts of 4-HBC,1,4-HBC is completely blocked, the utilization rate of the substrate phytosterol is improved, the accumulation of the byproducts is inhibited, the burden of downstream separation is reduced, and certain universality is realized.

The invention also relates to application of the genetically engineered bacterium in constructing the genetically engineered bacterium of the mycobacterium with high yield of 9-OH-AD and the genetically engineered bacterium of the mycobacterium with high yield of AD.

The invention also relates to a mycobacterium genetic engineering bacterium with high yield of ADD, which is constructed and obtained by the following method:

(1) Knocking out kshA1 gene by using mycobacteria as chassis bacteria to obtain engineering bacteria MnB delta k;

(2) Taking the engineering bacteria MnB delta k as the chassis bacteria, and further knocking out MnOpccR genes to obtain engineering bacteria MnB delta kM;

(3) Knocking out salA gene by using engineering bacteria MnB delta kM as the chassis bacteria to obtain engineering bacteria MnB delta kMS, namely the high-yield ADD mycobacterium genetic engineering bacteria; the nucleotide sequence of the salA 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) Respectively amplifying upstream and downstream segments of kshA1, mnOpccR and salA genes by taking a mycobacterium genome of chassis bacteria as a template, and connecting the upstream and downstream segments with a pacI and NotI enzyme-digested linearized pNS plasmid to construct knock-out plasmids pNS-kshA1H, pNS-MnOpccRH and pNS-SalH; the nucleotide sequence of the salA gene is shown as SEQ ID NO. 3;

(2) Knocking out kshA1 gene by using mycobacterium as a chassis bacterium and adopting a knock-out plasmid pNS-kshA1H and utilizing a homologous recombination double-exchange method to obtain an engineering bacterium MnB delta k;

(3) Taking engineering bacteria MnB delta k as chassis bacteria, knocking out a plasmid pNS-MnOpccRH and knocking out a MnOpccR gene to obtain engineering bacteria MnB delta kM;

(4) Taking the engineering bacterium MnB delta kM as a chassis bacterium, knocking out a plasmid pNS-SalH, knocking out a salA gene, and obtaining the engineering bacterium MnB delta kMS, namely the high-yield ADD mycobacterium genetic engineering bacterium.

Preferably, the Mycobacterium is Mycobacterium neoaurum NRRL B-3683 or Mycobacterium neoaurum ATCC 25795. The invention is equally applicable to other mycobacteria of the same species.

The invention also relates to application of the genetic engineering bacteria in preparation of 1,4-androstadiene-3,17-diketone by microbial fermentation.

Specifically, the application is as follows: inoculating the genetically engineered bacteria into a fermentation medium containing phytosterol, performing shake culture at 100-300 rpm for 48-120 h at 25-40 ℃ (preferably 30 ℃), and obtaining the 1,4-androstadiene-3,17-diketone from a fermentation liquid.

More preferably, the fermentation culture is carried out at 30 ℃, and after collecting the cells, the biotransformation of resting cells is carried out, wherein the substrate concentration of the phytosterol is 10-30 g/L.

The invention has the following beneficial effects: the genetic engineering strain provided by the invention effectively controls the generation of byproducts, greatly improves the utilization rate of steroid substrates and the molar conversion rate of the products, and simplifies the post-treatment process. The strategy can also be applied to the synthesis of products of common synthetic routes such as taking phytosterol as a substrate by using the novel Mycobacterium aurum (Mycobacterium neoaurum) of the same type to degrade and synthesize AD and 9-OH-AD and the like by converting side chains through microorganisms, the synthesis of byproducts of 4-HBC and 1,4-HBC is completely blocked, the utilization rate of the substrate phytosterol is improved, the accumulation of the byproducts is inhibited while the burden of downstream separation is reduced, and the strategy has certain universality, is suitable for vigorous popularization and application, and has higher economic benefit and social benefit.

(IV) description of the drawings

FIG. 1 is a steroid metabolism diagram;

FIG. 2 is a schematic of a knock-out plasmid;

FIG. 3 is a graph showing the results of HPLC analysis of transformed samples.

(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 and transformation screening of knock-out plasmids

(1) Plasmids kshA1, mnOpccR and Sal were knocked out in the following manner.

PCR amplification was performed using the genome of the original strain Mycobacterium neoaurum NRRL B-3683 as a template. PCR fragment amplification System: 2 mul of each of the upstream and downstream primers, 50-100ng of template, 1 mul of dNTP, 25 mul of 2 xbuffer, 1 mul of DNA polymerase and 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 for constructing knock-out plasmids of each gene

The amplified upstream and downstream fragments were ligated with a pacI and NotI digested linearized pNS plasmid (see FIG. 2) consisting of a p2NIL vector inserted into pGOAL19 (hsp 60-sacB) using a one-step cloning enzyme. 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, PCR and sequencing were performed to verify correct construction, and knock-out plasmids pNS-kshA1H, pNS-MnOpccRH, pNS-Sal 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; incubating at 37 deg.C under shaking at 180rpm 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'-cgccaagcttcctgctgaacatcaaagg-3' and a primer SCO-R outside a downstream homology arm of a target gene, and verifying whether single exchange is successful or not by colony PCR; verifying a correct transformant through electrophoresis and sequencing, transferring the transformant into an LB liquid culture medium, and oscillating at 37 ℃ and 180rpm for 12 hours; taking 50 mu L to be coated on a sucrose plate (LB does not contain NaCl and contains 5 percent of sucrose) to screen double-exchange transformants, and carrying out inverted culture at 30 ℃ for 3-5 d; 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 knock-out kshA1 Strain

The knockout plasmid pNS-kshA1H of example 1 was used on the basis of the Mycobacterium neoaurum NRRL B-3683 (MnB 3863) strain. Knocking out the kshA1 gene by using a homologous recombination double exchange method, and screening single exchange and double exchange to obtain a transformant for colony PCR verification, wherein gel electrophoresis of a product of the transformant is shown to lack 1070bp compared with that of the transformant which is not knocked out, the target gene is deleted by about 90%, and a sequencing result shows that the knockout is successful, so that the MnB3863 delta k strain is obtained.

Example 3: construction of knockout MnOpccR strains

On the basis of the strain MnB3863 delta k, the MnOpccR gene is knocked out by adopting the knock-out plasmid pNS-MnOpccRH in the embodiment 1 by utilizing a homologous recombination double-exchange method, and a transformant is obtained through screening of single exchange and double exchange and is subjected to colony PCR verification, wherein gel electrophoresis of a product of the transformant is 1883bp less than that of the product which is not knocked out, the target gene is deleted by about 94%, and the sequencing result shows that the knock-out is successful, so that the strain MnB3863 delta kM is obtained.

Example 4: construction of knockout Sal Strain

On the basis of the strain MnB3863 delta kM, the salA gene is knocked out by using the knockout plasmid pNS-SalH in the embodiment 1 through a homologous recombination double exchange method, a transformant is obtained through screening of single exchange and double exchange and is subjected to colony PCR verification, gel electrophoresis of a product of the transformant is shown to lack 1061bp compared with that of the product which is not knocked out, the target gene is deleted by about 88%, and the knockout is shown to be successful through a sequencing result, so that the strain MnB3863 delta kMS is obtained.

Example 5: growing cell catalysis phytosterol conversion synthesis ADD

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 100mL of M3 culture medium, carrying out shaking culture at 30 ℃ for 6h, adding 6mL of hydroxypropyl cyclodextrin emulsified phytosterol 100g/L or 200g/L solution, carrying out shaking culture at 30 ℃ and 180 rpm; sampling is carried out at the reaction interval of 24 hours, 5mL ethyl acetate is added into 1mL sample for extraction, oscillation and uniform mixing are carried out for 30min, 200 mu L of upper organic phase is taken and volatilized in an EP tube, 0.8mL methanol is added for redissolution, liquid phase detection is carried out, and the conversion result is shown in table 2 and figure 3. Compared with the strains MnB which are not knocked out, after kshA1, mnOpccR and salA are continuously knocked out, after the growth cell reacts for 144h, the molar conversion rate of ADD is improved by about 17 percent compared with that before modification. It is worth noting that after the salA knockout, the liquid phase result shows that no accumulation of 1,4-HBC exists, and the synthesis path of 1,4-HBC is completely blocked, so that the difficulty of downstream separation and extraction caused by byproduct accumulation is solved, and the method has a good industrial application prospect.

Table 2: knocking out the transformation result of catalyzing phytosterol by growth cells of the strain

Example 6: conversion synthesis of ADD by resting cell catalysis phytosterol

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;5% of the cells were transferred to 100mL of M3 medium, shaking-cultured at 30 ℃ for 48 hours, and the resulting bacterial liquid was centrifuged at 4 ℃ in a large high-speed centrifuge (5000rpm, 10min), and then the medium in the supernatant was removed. The centrifuged cells were then resuspended and washed with phosphate buffer (20mM, pH 8.0), and then centrifuged in a high-speed refrigerated centrifuge (5000 rpm, 10min) to remove the washed buffer. The above washing step was repeated once more. Finally, a proper amount of phosphate buffer solution is used for resuspending the cleaned thalli to prepare mother liquor with the thalli concentration of 200 g/L. Adding 6mL of hydroxypropyl cyclodextrin emulsified phytosterol 100g/L or 200g/L solution and 10mL of mother liquor with thallus concentration of 200g/L into phosphate buffer (20mM, pH8.0), preparing into 50mL of reaction system, and performing shaking culture at 30 ℃ and 180 rpm; the reaction was sampled for 120 h. The detection method comprises the following steps: adding 5mL of ethyl acetate into 1mL of sample for extraction, shaking and uniformly mixing for 30min, taking 200 mu L of an upper organic phase, volatilizing in an EP tube, adding 0.8mL of methanol for redissolution, detecting a liquid phase C18 strain, and calculating the molar conversion rate of ADD after 120h of reaction with phytosterol as a substrate, wherein the results are shown in Table 3. Compared with a strain MnB which is not knocked out, after kshA1 is knocked out, the degradation capability of ADD is weakened, and after MnOpccR and salA are knocked out, the synthetic path of 1,4-HBC is completely blocked, so that the problem of difficulty in extraction caused by a1,4-HBC byproduct is solved. Finally, the conversion rate in the resting cell catalysis is improved by about 22 percent, which is beneficial to improving the utilization rate of the substrate. And after the substrate concentration is increased (30 g/L), the conversion rate is not greatly influenced and still reaches 67.5 percent.

Table 3: knockout strain resting cell catalysis phytosterol transformation result

The genetic engineering strain provided by the invention selectively produces products such as ADD and the like, and 1,4-HBC byproducts are reduced.

Example 7: conversion reaction of phytosterol

According to the previous literature report strategy, starting from the model bacterium Mycobacterium neoaurum ATCC25795, by knocking out KstD1 and KstD3, the synthetic chassis Mn25795 delta KstD of 9-OH-AD is obtained _1,3 Further knocking out KshA1 and KshA2 to obtain combined bacteria Mn25795 delta KstD for synthesizing AD _1,3 △KshA _1,2 Further knocking out hsd4A to obtain a synthetic chassis Mn25795 delta KstD of 4-HBC _1,3 △KshA _1,2 Δ hsd4A. Based on these base plates, the salA gene was further knocked out by the method in example 4, and the molar conversion rate of the target product was compared between the base plates. The specific conversion reaction process is as follows:

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 phytosterol into 50mL of M3 culture medium, performing shaking culture at 30 ℃ for 6h, adding 10mL of hydroxypropyl cyclodextrin emulsified phytosterol solution at 200g/L, and performing shaking culture at 30 ℃ and 180 rpm; after 120 hours of reaction, sampling, adding 5mL of ethyl acetate into 1mL of sample for extraction, shaking and mixing uniformly for 30min, taking 200 mu L of upper organic phase to volatilize in an EP tube, adding 0.8mL of methanol for redissolution, detecting by a liquid phase, and obtaining the conversion result shown in Table 3 and figure 3. Compared with the strains which are not knocked out, after the aldolase gene salA is knocked out, the synthesis path of 4-HBC is completely blocked, the molar conversion rate of AD and 9-OH-AD is obviously improved, the process can effectively improve the utilization rate of the substrate, reduces the accumulation of byproduct HBC, and has obvious industrial application value.

Table 3: transformation results (molar yield of phytosterols) of New M.aureus Mn Strain after knockout of sal

The results show that the genetic engineering strain constructed after the SalA gene is knocked out selectively produces products such as AD, ADD, 9-OHAD and the like, and synthesis of byproducts such as 4-HBC and the like is inhibited.

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 (9)

1. An aldolase gene salA, the nucleotide sequence of which is shown as SEQ ID NO. 3.

2. The application of the aldolase gene salA in the claim 1 in constructing mycobacterium genetic engineering bacteria with high yield of ADD.

3. The use of the aldolase gene salA as defined in claim 1 in constructing a Mycobacterium genetic engineering bacterium with high 9-OH-AD yield.

4. The use of the aldolase gene salA as defined in claim 1 in constructing a Mycobacterium genetic engineering bacterium with high AD yield.

5. A mycobacterium genetic engineering bacterium with high yield of ADD is constructed and obtained by the following method:

(1) Knocking out kshA1 gene by using mycobacteria as chassis bacteria to obtain engineering bacteria MnB delta k;

(2) Taking the engineering bacteria MnB delta k as the chassis bacteria, and further knocking out MnOpccR genes to obtain engineering bacteria MnB delta kM;

(3) Knocking out salA gene by using engineering bacteria MnB delta kM as chassis bacteria to obtain engineering bacteria MnB delta kMS, namely the high-yield ADD mycobacterium genetic engineering bacteria; the nucleotide sequence of the salA gene is shown as SEQ ID NO. 3.

6. A method for constructing the genetically engineered bacterium of claim 5, the method comprising:

(1) Respectively amplifying upstream and downstream segments of kshA1, mnOpccR and salA genes by taking a mycobacterium genome of chassis bacteria as a template, and connecting the upstream and downstream segments with a pacI and NotI enzyme-digested linearized pNS plasmid to construct knock-out plasmids pNS-kshA1H, pNS-MnOpccRH and pNS-SalH; the nucleotide sequence of the salA gene is shown as SEQ ID NO. 3;

(2) Knocking out kshA1 gene by using mycobacterium as a chassis bacterium and adopting a knock-out plasmid pNS-kshA1H and utilizing a homologous recombination double-exchange method to obtain an engineering bacterium MnB delta k;

(3) Taking engineering bacteria MnB delta k as chassis bacteria, knocking out a MnOpccR gene by knocking out a plasmid pNS-MnOpccRH to obtain engineering bacteria MnB delta kM;

(4) Taking the engineering bacterium MnB delta kM as a chassis bacterium, knocking out a plasmid pNS-SalH, knocking out a salA gene, and obtaining the engineering bacterium MnB delta kMS, namely the high-yield ADD mycobacterium genetic engineering bacterium.

7. The method of claim 6, wherein the Mycobacterium is Mycobacterium neoaurum NRRL B-3683 or Mycobacterium neoaurum ATCC 25795.

8. The use of the genetically engineered bacteria of claim 5 in microbial fermentation to prepare 1,4-androstadiene-3,17-dione.

9. The use according to claim 8, characterized in that the use is: inoculating the genetic engineering bacteria to a fermentation culture medium containing phytosterol, performing shake culture at the temperature of 25-40 ℃ and the rpm of 100-300 for 48-120 h, and obtaining the 1,4-androstadiene-3,17-diketone in fermentation liquor.

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