Mycobacterium gene engineering bacteria with high yield of add and application thereof

Abstract

The present application relates to an aldehyde mutarotase gene salA and its application in constructing a high-ADD-producing genetically engineered mycobacterium, and a high-ADD-producing genetically engineered mycobacterium and its construction and application in preparing 1,4-androstadiene-3,17-dione by microbial fermentation. The high-ADD-producing genetically engineered mycobacterium is obtained by knocking out kshA1, MnOpccR and salA genes in turn using mycobacterium as a chassis. The genetically engineered strain provided by the present application effectively controls the generation of by-products, greatly improves the utilization rate of steroidal substrates and the molar conversion rate of products, simplifies the post-processing process, and the strategy can also be applied to the synthesis of products in the common synthesis path of 1,4-androstadiene-3,17-dione and 9-OH-AD, which is microbial transformation of side chain degradation using phytosterol as a substrate, completely blocks the synthesis of 4-HBC and 1,4-HBC by-products, and reduces the burden of downstream separation while improving the utilization rate of phytosterol substrate. The present application has certain universality, is suitable for vigorous popularization and application, and has high economic and social benefits.

Description

Technical Field

[0001] This invention relates to an aldolase gene. salA The study also explored the application of high-yield ADD genetically engineered bacteria in constructing genetically engineered bacteria, as well as the construction of high-yield ADD mycobacterial genetically engineered bacteria and their application in the microbial fermentation preparation of 1,4-androsadiene-3,17-dione. Background Technology

[0002] There are over 300 approved steroid drugs on the market, making them one of the most important medicines used to treat diseases in humans. In the pharmaceutical industry alone, the global demand for steroid substances exceeds 1,500 tons annually, and the market for steroid drugs produced in 2015 exceeded US$100 billion, second only to antibiotics. Steroid drugs include adrenocortical hormones, sex hormones, progesterone, mineralocorticoids, and non-hormonal steroids, used in various fields, including pharmaceuticals, veterinary medicine, aquaculture, agriculture, and the food industry.

[0003] Chemical synthesis once held absolute dominance in this field. Dioscorea saponins from yam plants have structures very similar to steroid drugs, allowing for the chemical synthesis of various steroid drugs. However, the source of raw materials is unstable, and extraction costs are high, making it difficult to meet the ever-expanding market demand. Microbial transformation of phytosterols has become one of the main methods for producing various steroids, gradually replacing traditional chemical synthesis routes. It can produce various intermediates used in the preparation of steroid drugs, such as 4-androsten-3,17-dione (AD), 1,4-androsadien-3,17-dione (ADD), 9-hydroxy-4-androsten-3,17-dione (9-OH-AD), 22-hydroxy-23,24-dicholest-4-en-3-one (4-HBC), and 22-hydroxy-23,24-dicholest-1,4-dien-3-one (1,4-HBC), etc.

[0004] With the development of bioinformatics, the mysteries of functional genes related to sterol catabolism are gradually being unveiled. Cholesterol is first metabolized by cholesterol oxidase (… chox ChOx) or 3β-hydroxysteroid oxidase ( hsd 3β-HSD) is converted to 4-cholesten-3-one. The side-chain degradation process was confirmed to be consistent with the fatty acid oxidation pathway, involving steroid C26 monooxygenase (C26). cyp125, cyp142 The main catalytic reaction involves the formation of a terminal carboxyl group on the side chain. Following C-27 terminal acylation, the cholesterol side chain is activated by terminal CoA thioesterification, and then the C-27 carboxyl-CoA group enters the β-oxidation reaction. Li et al. demonstrated this by knocking out the HGMS2 strain of Mycobacterium... kstd and ksh Genes were used to construct strains that produce high levels of AD, and it was discovered that knocking out their endogenous genes...kstd and ksh After the gene, its knockout mutant HGMS2 Δkstd211+ΔkshB122 The conversion capacity of phytosterols increased by 20%, and the molar yield of phytosterols in the reaction of 10 g / L phytosterols was 51.6%, but byproducts such as HBC were present, which was not conducive to the separation and extraction of the product. Yao et al. improved the conversion capacity of phytosterols by knocking out the side chain degradation pathway. kstd and ksh Genes were modified and KshA was overexpressed, resulting in efficient accumulation of 9-OH-AD (Metabolic Engineering. 2014 Jul;24:181-91). Recently, with numerous reports in related literature, based on the above modification strategy, blocking key genes in the sterol side-chain degradation metabolic pathway of *Mycobacterium aureum* has become the mainstream synthetic pathway for the production of ADD and 9-OH-AD (e.g., ...). Figure 1 In addition, recent literature reports knockout. hsd4A Genes that facilitate the synthesis of the intermediate product 4-HBC, but this process discover A bifunctional reductase, MnOpccR, can reduce the yield of 4-HBC, but metabolic pathway analysis shows that other metabolic byproducts still exist (Angewandte Chemie, 2021, 60 (10): 5414-5420). Therefore, precisely regulating the side-chain degradation pathways in the microbial transformation of steroid drugs to improve substrate utilization and product molar conversion, reduce byproduct accumulation, and decrease the burden of downstream product separation and purification is currently a key issue in the microbial transformation of steroidal drugs.

[0005] Reports on the production of ADDs by mycobacteria through the degradation of sterols are increasing year by year, and the degradation mechanisms and genetic information are constantly being elucidated. The discovery of key control genes and enzymes, and their modification through genetic engineering, can help reduce byproducts in synthesis, lower energy consumption in the production process, improve substrate utilization, and simplify post-processing procedures. High-yield, specific preparation of ADDs is of paramount importance for the industrial production of steroidal drugs. Summary of the Invention

[0006] The purpose of this invention is to provide an aldolase gene. salA The study also explored the application of high-yield ADD genetically engineered bacteria in constructing genetically engineered bacteria, as well as the construction of high-yield ADD mycobacterial genetically engineered bacteria and their application in the microbial fermentation preparation of 1,4-androsadiene-3,17-dione.

[0007] The technical solution adopted in this invention is:

[0008] an aldolase gene salA Its nucleotide sequence is shown in SEQ ID NO.3.

[0009] This invention also relates to the aldolase gene. salA Application in constructing genetically engineered mycobacterial bacteria that produce high levels of aDD (additive generation). This strategy can also be applied to similar types of *Mycobacterium aureum* (…). Mycobacterium neoaurum This method utilizes phytosterols as substrates and microbial transformation to degrade side chains to synthesize products via common synthetic pathways such as AD and 9-OH-AD. It completely blocks the synthesis of 4-HBC and 1,4-HBC byproducts, thereby improving the utilization rate of the substrate phytosterols while inhibiting the accumulation of byproducts and reducing the burden on downstream separation. It has a certain degree of universality.

[0010] This invention also relates to the use of the genetically engineered bacteria in constructing high-yield... 9-OH-AD Mycobacterium genetically engineered bacteria and high-yield AD Application in genetically engineered mycobacterial bacteria.

[0011] This invention also relates to a genetically engineered mycobacterium that produces high levels of aDD (additive growth factor), which is constructed by the following method:

[0012] (1) Using mycobacteria as the substrate bacteria, knock out kshA1 Genes, to obtain engineered bacteria MnB △k ;

[0013] (2) Using engineered bacteria MnB △k For chassis bacteria, further knockout is needed. MnOpccR Genes, to obtain engineered bacteria MnB △kM ;

[0014] (3) Using engineered bacteria MnB △kM Chassis bacteria, knock out salA Genes, to obtain engineered bacteria MnB △kMS That is, the genetically engineered mycobacterium that produces high levels of ADD; salA The gene nucleotide sequence is shown in SEQ ID NO. 3.

[0015] The present invention also relates to a method for constructing the genetically engineered bacteria, the method comprising:

[0016] (1) Using the genome of Mycobacterium tumefaciens as a template, amplify the following: kshA1, MnOpccR, salA 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- MnOpccRH and pNS- SalH The salA The gene nucleotide sequence is shown in SEQ ID NO. 3;

[0017] (2) Using Mycobacterium as the substrate bacteria, the knockout plasmid pNS- kshA1H Using homologous recombination double exchange method, knockout knockoutkshA1 Genes, to obtain engineered bacteria MnB △k ;

[0018] (3) Using engineered bacteria MnB △k For the basal bacteria, the knockout plasmid pNS- was used. MnOpccRH Knockout MnOpccR Genes, to obtain engineered bacteria MnB △kM ;

[0019] (4) Using engineered bacteria MnB △kM For the basal bacteria, the knockout plasmid pNS- was used. SalH Knockout salA Genes, to obtain engineered bacteria MnB △kMS That is, the genetically engineered mycobacterium that produces high levels of ADD.

[0020] Preferably, the mycobacterium is Mycobacterium neoaurum NRRL B-3683 or Mycobacterium neoaurum ATCC 25795. This invention is also applicable to other mycobacteria of the same genus.

[0021] The present invention also relates to the application of the genetically engineered bacteria in the microbial fermentation preparation of 1,4-androsadiene-3,17-dione.

[0022] Specifically, the application involves inoculating the genetically engineered bacteria into a fermentation medium containing phytosterols and culturing it at 25-40 °C (more preferably 30 °C) with shaking at 100-300 rpm for 48-120 h to obtain the 1,4-androsadiene-3,17-dione in the fermentation broth.

[0023] More preferably, fermentation culture is carried out at 30 °C, and after cell collection, biotransformation of resting cells is performed, wherein the substrate concentration of the phytosterol is 10~30 g / L.

[0024] The beneficial effects of this invention are mainly reflected in the following: the genetically engineered strain provided by this invention effectively controls the generation of by-products, greatly improves the utilization rate of steroid substrates and the molar conversion rate of products, and simplifies the post-processing procedure. This strategy can also be applied to similar types of *Mycobacterium aureum* (…).

[0025] Mycobacterium neoaurum This method utilizes phytosterols as substrates and microbial transformation to degrade side chains, synthesizing products along common synthetic pathways such as AD and 9-OH-AD. It completely blocks the synthesis of 4-HBC and 1,4-HBC byproducts, improving the utilization rate of phytosterol substrates while inhibiting byproduct accumulation and reducing the burden on downstream separation. It has certain universality and is suitable for widespread application, with high economic and social benefits. Attached Figure Description

[0026] Figure 1 Diagram of steroid metabolism;

[0027] Figure 2 This is a schematic diagram of plasmid knockout.

[0028] Figure 3 HPLC analysis of the transformed sample chromatogram. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments:

[0030] Example 1: Construction and Transformation Screening of Knockout Plasmids

[0031] (1) Construct the knockout according to the following method kshA1, MnOpccR, Sal Plasmid.

[0032] Starting strain Mycobacterium neoaurum The genome of NRRL B-3683 was used as a template for PCR amplification. The PCR fragment amplification system consisted of 2 μL each of forward and reverse primers, 50–100 ng of template, 1 μL of dNTPs, 25 μL of 2×Buffer, 1 μL of DNA polymerase, and ddH2O to a final volume of 50 μL. The PCR fragment amplification program was: 95 ℃–5 min, 95 ℃–15 s, (Tm-5 ℃)–15 s, 72 ℃–30 s / kb, 72 ℃–10 min, for 30 cycles. The primer sequences used are shown in Table 1.

[0033] Table 1: Primers required for constructing knockout plasmids for each gene

[0034]

[0035] The amplified upstream and downstream fragments were ligated to the linearized pNS plasmid digested with pacI and NotI using a one-step cloning enzyme (e.g., ...). Figure 2 The pNS plasmid is composed of a fragment (hsp60-sacB) inserted into the pGOAL19 vector from the p2NIL vector. The ligation product was transformed into DH5α competent cells and plated on Kan-resistant LB agar plates (tryptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L, Kan: 50 μg / mL, agar: 2%). The plates were incubated upside down at 37 ℃ for 16 h. Single colonies were picked, and PCR and sequencing were used to verify correct construction. The knockout plasmid pNS- was constructed. kshA1H pNS- MnOpccRH, pNS- Sal.

[0036] (2) Transformation and screening

[0037] The obtained knockout plasmid was treated with alkali and then added to competent mycobacterial cells (dissolved on ice). The cells were incubated at 4 °C for 20 min. The voltage was set to 2.5 kV, the electroporation cup aperture was selected to be 2 mm, and electroporation was performed twice. The electroporation frequency was confirmed to be in the range of 4-5 ms. 600 μL of fresh LB medium was added, and the cells at the bottom were fully resuspended. The cells were then transferred to a sterile centrifuge tube and incubated at 37 °C and 180 rpm for 4 h with shaking. The tube was centrifuged at 5000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 100 μL. The tube was then incubated upside down at 30 °C for 3-5 days.

[0038] The obtained transformants were screened and used sacB Primers SCO-F (5'-cgccaagcttcctgctgaacatcaaagg-3') on the gene and primer SCO-R on the outer side of the downstream homologous arm of the target gene were used to verify the success of single crossover using colony PCR. Transformants that were verified by electrophoresis and sequencing were transferred to LB broth and incubated at 37°C and 180 rpm for 12 h. 50 μL of the transformed strain was transferred to sucrose plates (LB broth contains 5% sucrose and no NaCl) to screen for double crossover transformants, and incubated upside down at 30°C for 3–5 days. Transformants were simultaneously copied onto Kansai resistant and antibiotic-free plates, and colony PCR was performed using the upstream and downstream primers of the target gene. For strains whose target bands met the requirements, those that grew on antibiotic-free plates but did not grow on Kansai resistant plates were identified as knockout strains. The PCR products were then sequenced for verification.

[0039] Example 2: Knockout kshA1 Construction of strains

[0040] exist Mycobacterium neoaurum NRRL B-3683 (MnB3863) Based on the strain, the knockout plasmid pNS- from Example 1 was used. kshA1H Using homologous recombination double exchange, knockout was achieved. kshA1 After screening with single and double crossovers, transformants were obtained and verified by colony PCR. Gel electrophoresis of the products showed a 1070bp deletion compared to the non-knockout result, indicating approximately 90% deletion of the target gene. Sequencing results confirmed successful knockout. MnB3863 △k strains.

[0041] Example 3: Knockout MnOpccR Construction of strains

[0042] exist MnB3863 △k Based on the strain, the knockout plasmid pNS- from Example 1 was used. MnOpccRH Knockout using homologous recombination double exchange method MnOpccRAfter screening with single and double crossovers, transformants were obtained and verified by colony PCR. Gel electrophoresis of the products showed a 1883 bp deletion compared to the non-knockout result, indicating a 94% deletion of the target gene. Sequencing results confirmed successful knockout. MnB3863 △kM strains.

[0043] Example 4: Knockout Sal Construction of strains

[0044] exist MnB3863 △kM Based on the strain, the knockout plasmid pNS- from Example 1 was used. SalH Knockout using homologous recombination double exchange method salA After screening with single and double crossovers, transformants were obtained and verified by colony PCR. Gel electrophoresis of the products showed a 1061 bp deletion compared to the non-knockout result, indicating approximately 88% deletion of the target gene. Sequencing results confirmed successful knockout. MnB3863 △kMS strains.

[0045] Example 5: Growth cells catalyze the conversion of phytosterols into ADD

[0046] One loopful of bacterial culture was taken from a glycerol tube and streaked onto LB solid medium, then incubated at 30 °C for 48 h. Single colonies were picked and placed in 5 mL of LB liquid, then incubated at 30 °C with shaking for 36 h. 5% of the culture was transferred to 100 mL of M3 medium, incubated at 30 °C with shaking for 6 h, and then 6 mL of a 100 g / L or 200 g / L phytosterol solution emulsified with hydroxypropyl cyclodextrin was added. The culture was then incubated at 30 °C and 180 rpm with shaking. Samples were taken every 24 hours. 1 mL of the sample was extracted with 5 mL of ethyl acetate, shaken and mixed for 30 min. 200 μL of the upper organic phase was collected and evaporated in an EP tube, then reconstituted with 0.8 mL of methanol. Liquid chromatography analysis was performed, and the transformation results are shown in Table 2. Figure 3 Compared to strains that were not knocked out MnB Continuous knockout kshA1 , MnOpccR and salA After 144 hours of cell growth reaction, the molar conversion rate of ADD increased by approximately 17% compared to before the modification. It is worth noting that... salA After knockout, liquid chromatography results showed that there was no longer any accumulation of 1,4-HBC, indicating that the synthetic pathway of 1,4-HBC was completely blocked, which solved the difficulty of downstream separation and extraction caused by the accumulation of byproducts and has good prospects for industrial application.

[0047] Table 2: Results of phytosterol conversion catalyzed by cells grown from knockout strains

[0048]

[0049] Example 6: Resting cells catalyze the conversion of phytosterols into ADD

[0050] Take one loopful of bacterial culture from a glycerol tube, streak it onto LB agar, and incubate at 30 °C for 48 h. Pick a single colony and place it in 5 mL of LB liquid, incubate at 30 °C with shaking for 36 h. Transfer 5% of the culture to 100 mL of M3 medium, incubate at 30 °C with shaking for 48 h, centrifuge the culture at 4 °C (5000 rpm, 10 min), and remove the culture medium from the supernatant. Resuspend the centrifuged bacterial cells in phosphate buffer (20 mM, pH 8.0) and wash them, then centrifuge again at 5000 rpm, 10 min to remove the washing buffer. Repeat the washing steps once more. Finally, resuspend the washed bacterial cells in an appropriate amount of phosphate buffer to prepare a stock solution with a bacterial concentration of 200 g / L. 6 mL of a 100 g / L or 200 g / L phytosterol solution emulsified with hydroxypropyl cyclodextrin and 10 mL of a 200 g / L bacterial cell concentration stock solution were added to phosphate buffer (20 mM, pH 8.0) to prepare a 50 mL reaction system. The system was incubated at 30 ℃ with shaking at 180 rpm for 120 h. Samples were taken after 120 h of reaction. The detection method was as follows: 1 mL of sample was extracted with 5 mL of ethyl acetate, shaken and mixed for 30 min, and 200 μL of the upper organic phase was taken and evaporated in an EP tube. 0.8 mL of methanol was added to redissolve the sample, and the C18 strain was analyzed by liquid chromatography. The molar conversion rate of ADD after 120 h of reaction with phytosterol as the substrate was calculated. The results are shown in Table 3. Compared with the non-knockout strain... MnB Knockout kshA1 Subsequently, the degradation ability of ADD was weakened, and knockout... MnOpccR and salA Subsequently, the synthetic pathway of 1,4-HBC was completely blocked, resolving the extraction difficulties caused by 1,4-HBC byproducts. Ultimately, the conversion rate increased by approximately 22% in resting cell catalysis, which is beneficial for improving substrate utilization. Furthermore, increasing the substrate concentration (30 g / L) did not significantly affect the conversion rate, which still reached 67.5%.

[0051] Table 3: Results of phytosterol conversion catalyzed by resting cells of knockout strains

[0052]

[0053] The genetically engineered strain provided by this invention selectively produces products such as ADD and reduces 1,4-HBC byproducts.

[0054] Example 7: Conversion reaction of phytosterols

[0055] Based on the strategy reported in previous literature, using model bacteria Mycobacterium neoaurumStarting with ATCC25795, the synthetic chassis Mn25795 of 9-OH-AD was obtained by knocking out KstD1 and KstD3. △KstD 1,3 Further knockout of KshA1 and KshA2 yielded a synergist that synthesizes AD. Mn25795△KstD 1,3 △KshA 1,2 Further knockout hsd4A To obtain a synthetic chassis of 4-HBC Mn25795△KstD 1,3 △KshA 1,2 △hsd4A Based on these basal bacteria, the method of Example 4 was used to further knock out... salA Genes were used to compare the changes in the molar conversion rate of the target product under different chassis. The specific conversion reaction process is as follows:

[0056] One loopful of bacterial culture was taken from a glycerol tube and streaked onto LB solid medium, then incubated at 30°C for 48 h. Single colonies were picked and placed in 5 mL of LB liquid, then incubated at 30°C with shaking for 36 h. 5% of the culture was transferred to 50 mL of M3 medium, incubated at 30°C with shaking for 6 h, and then 10 mL of a 200 g / L phytosterol solution emulsified with hydroxypropyl cyclodextrin was added. The culture was then incubated at 30°C and 180 rpm with shaking. After 120 hours of reaction, a sample was taken, and 1 mL of the sample was extracted with 5 mL of ethyl acetate. The mixture was shaken and mixed for 30 min. 200 μL of the upper organic phase was collected and evaporated in an EP tube, then reconstituted with 0.8 mL of methanol. The transformation results were analyzed by liquid chromatography and are shown in Table 4. Figure 3 Compared to strains without knockout, the elimination of the aldolase gene... salA Subsequently, the synthetic pathway of 4-HBC was completely blocked, significantly improving the molar conversion rate of AD and 9-OH-AD. This process can effectively improve the utilization rate of the substrate and reduce the accumulation of the byproduct HBC, and has significant industrial application value.

[0057] Table 4: Knockout of *Mycobacterium aureum* strain Mn sal The subsequent transformation results (molar yield of phytosterols)

[0058]

[0059] The above results show that the invention provides... SalA Genetically engineered strains constructed after gene knockout selectively produce products such as AD, ADD, and 9-OHAD, and inhibit the synthesis of byproducts such as 4-HBC.

[0060] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A genetically engineered Mycobacterium neoaurum strain with high yield of ADD, which is obtained by the following method: (1) using mycobacterium Mycobacterium neoaurum NRRL B-3683 as the chassis, knocking out kshA1 the gene, obtaining the engineered bacteria MnB △k ; (2) Taking the engineering bacteria MnB △k as the chassis bacteria, further knocking out MnOpccR genes, obtaining engineering bacteria MnB △kM ; (3) Using engineered bacteria MnB △kM Chassis bacteria, knock out salA Genes, to obtain engineered bacteria MnB △kMS That is, the genetically engineered mycobacterium that produces high levels of ADD; salA The gene nucleotide sequence is shown in SEQ ID NO.

3. 2.A method for constructing the genetically engineered strain of claim 1, the method comprising: (1) Mycobacterium tumefaciens Mycobacterium neoaurum Using the NRRL B-3683 genome as a template, amplification was performed separately. kshA1, MnOpccR, salA 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- MnOpccRH and pNS- SalH The salA The gene nucleotide sequence is shown in SEQ ID NO. 3; (2) using mycobacterium Mycobacterium neoaurum NRRL B-3683 as the chassis, using knock-out plasmid pNS- kshA1H , knocking out the gene by the method of homologous recombination double exchange to obtain the engineering bacteria kshA1 MnB △k ;​ (3) Using engineered bacteria MnB △k For the basal bacteria, the knockout plasmid pNS- was used. MnOpccRH Knockout MnOpccR Genes, to obtain engineered bacteria MnB △kM ; (4) Using engineered bacteria MnB △kM For the basal bacteria, the knockout plasmid pNS- was used. SalH Knockout salA Genes, to obtain engineered bacteria MnB △kMS That is, the genetically engineered mycobacterium that produces high levels of ADD. 3.Use of the genetically engineered strain of claim 1 in microbial fermentation for preparing 1, 4-androstadiene-3, 17-dione.

4. The use according to claim 3, wherein The use is: inoculating the genetically engineered strain into a fermentation medium containing phytosterols, culturing at 25-40 ℃ with 100-300 rpm shaking for 48-120 h, and obtaining the 1, 4-androstadiene-3, 17-dione in the fermentation broth.

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