3-ketosteroid-delta1-dehydrogenase mutant with improved heat resistance and application of 3-ketosteroid-delta1-dehydrogenase mutant

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a 3-ketosteroid-delta1-dehydrogenase mutant with improved heat resistance and an application of the 3-ketosteroid-delta1-dehydrogenase mutant. The 3-ketosterone-delta1-dehydrogenase AuKsdD gene derived from arthrobacter urate oxydans is subjected to structural analysis and site-directed mutagenesis design, and a mutant with higher catalytic activity and remarkably enhanced heat resistance compared with a wild type AuKsdD is obtained through directed mutation of key sites E244, K267, T301 and A335. The mutant can keep high catalytic efficiency for a long time under a high-temperature condition, and is suitable for industrial synthesis of steroid drug raw materials.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a 3-sterone-Δ1-dehydrogenase mutant with improved heat resistance and its applications. Background Technology

[0002] Steroid drugs occupy an extremely important position in the pharmaceutical field due to their significant anti-inflammatory, anti-allergic, anti-infective, and immunomodulatory effects, and are the second largest class of clinically commonly used drugs after antibiotics. Prednisolone, as a typical example, has a key step in its synthesis process: the dehydrogenation reaction at the C1,2 position of the A ring of the steroid nucleus. This reaction is catalyzed by 3-sterone-Δ1-dehydrogenase (KsdD) and is the core step in achieving efficient biosynthesis of steroid drugs.

[0003] However, natural KsdD generally suffers from poor heat resistance, low specific enzyme activity, and insufficient tolerance to organic solvents, leading to its easy inactivation under high temperature, high substrate concentration, or long reaction conditions, which seriously restricts the industrial-scale green production of steroid drugs. Especially in continuous catalytic and scale-up reaction systems, thermal inactivation of enzymes has become a major bottleneck restricting production efficiency and economy.

[0004] Therefore, molecular modification of KsdD to enhance its heat resistance and catalytic performance has become an urgent need for the efficient, energy-saving, and low-cost biosynthesis of steroid drugs. This invention addresses this problem by using structure-guided protein engineering to perform site-directed mutagenesis on key residues of KsdD, resulting in mutants with both high catalytic activity and excellent heat resistance, providing a new solution for the industrial production of steroid drugs. Summary of the Invention

[0005] This invention aims to address the problems of poor heat resistance and low catalytic efficiency of wild-type 3-sterone-Δ1-dehydrogenase (KsdD) by providing a KsdD mutant and its genetically engineered strain with significant improvements in both heat resistance and enzyme activity, and to realize its efficient application in the C1,2-position dehydrogenation reaction of steroidal compounds.

[0006] To address the aforementioned technical problems, this invention involves structural analysis and site-directed mutagenesis design of the 3-sterone-Δ1-dehydrogenase (AuKsdD) gene derived from Arthrobacter urateux. Through targeted mutations at key sites E244, K267, T301, and A335, several KsdD mutants with superior performance were obtained. Experimental verification shows that these mutants exhibit higher catalytic activity and significantly enhanced heat resistance compared to wild-type AuKsdD, maintaining high catalytic efficiency for extended periods under high-temperature conditions, making them suitable for the industrial synthesis of steroidal drug raw materials.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] One of the technical solutions provided by this invention is a 3-sterone-Δ1-dehydrogenase mutant, which is obtained by at least one of the following mutations based on the wild-type AuKsdD shown in SEQ ID NO:1: glutamic acid (E) at position 244 is mutated to leucine (L), lysine (K) at position 267 is mutated to arginine (R), threonine (T) at position 301 is mutated to glutamic acid (E), and alanine (A) at position 335 is mutated to proline (P). Furthermore, the mutant is the AuKsdD-M4 mutant, which is obtained by simultaneously causing the E244L, K267R, T301E, and A335P mutations on the wild-type AuKsdD shown in SEQ ID NO:1. The amino acid sequence of the AuKsdD-M4 mutant is shown in SEQ ID NO:3.

[0009] This invention also provides the coding gene of the mutant described in one of the technical solutions; Furthermore, the encoding gene is shown in SEQ ID NO:4.

[0010] The second technical solution provided by the present invention is a recombinant vector containing the encoding gene of the 3-sterone-Δ1-dehydrogenase mutant described in the first technical solution; Furthermore, the expression vectors used in the recombinant vector include, but are not limited to, pET series plasmids, such as pET28a plasmid.

[0011] The third technical solution provided by the present invention is a recombinant strain containing the encoding gene of the 3-sterone-Δ1-dehydrogenase mutant described in one of the technical solutions; Furthermore, the host bacteria used in the recombinant strain include, but are not limited to, Escherichia coli, preferably Escherichia coli BL21(DE3).

[0012] The fourth technical solution provided by this invention is the application of the recombinant vector described in the third technical solution or the recombinant bacteria described in the fourth technical solution; particularly its application in the production of the 3-sterone-Δ1-dehydrogenase mutant described in the first technical solution; more particularly its application in the C1,2-position dehydrogenation reaction of steroidal compounds; and even more particularly its application in the preparation of steroidal drugs, such as catalyzing the C1,2-position dehydrogenation reaction of hydrocortisone, androst-4-ene-3,17-dione, or 11α-hydroxy-16,17α-epoxypregnane-1,4-diene-3,20-dione, etc.

[0013] The fifth technical solution provided by this invention is the application of the 3-sterone-Δ1-dehydrogenase mutant described in one of the technical solutions; particularly its application in the C1,2-position dehydrogenation reaction of steroidal compounds; and more particularly its application in the preparation of steroidal drugs, such as catalyzing the C1,2-position dehydrogenation reaction to synthesize prednisolone, androst-1,4-diene-3,17-dione, or 11α-hydroxy-16,17α-epoxypregn-1,4-diene-3,20-dione, etc.

[0014] Beneficial effects: (1) This invention obtained the AuKsdD-M4 mutant by site-directed modification of the amino acid sequence of 3-sterone-Δ1-dehydrogenase (AuKsdD) derived from Arthrobacter uric acid oxidase, with mutation sites E244L, K267R, T301E, and A335P. Compared with wild-type AuKsdD, the AuKsdD-M4 mutant showed a further enhanced activity in catalyzing the synthesis of prednisolone from hydrocortisone, reaching an enzyme activity of 34.5 U / mg, which is 80.6% higher than the specific activity of the wild-type enzyme (19.1 U / mg). Simultaneously, the heat inactivation temperature (T0) of the AuKsdD-M4 mutant was also significantly improved. 50 The half-life of the AuKsdD-M4 mutant was 46.3 °C, which was 10.2 °C higher than that of the wild type, and its half-life at 30 °C reached 19.5 h, which was about 6 times that of the wild type. This indicates that the AuKsdD-M4 mutant maintains excellent catalytic activity and structural stability under high temperature conditions. In the synthesis of prednisolone from hydrocortisone (50 g / L) catalyzed by the AuKsdD-M4 mutant, the conversion rate was increased by 11% (98.2%, reaction time 6 h) compared with that of the wild type AuKsdD (88.6%, reaction time 6 h).

[0015] (2) The AuKsdD-M4 mutant showed good applicability to different steroid substrates: the product formation rate reached 98.5% after 24 h of reaction with androst-4-ene-3,7-dione (120 g / L) in wet cells with 50 g / L, which was 28.3% higher than that of wild-type AuKsdD (76.8%); the product formation rate reached 90.1% after 72 h of reaction with fungal oxide (30 g / L substrate), which was 9.3% higher than that of wild-type AuKsdD (82.4%).

[0016] (3) High-density culture was carried out in a 1.5 L fermenter. The concentration of hydrocortisone was increased to 60 g / L by growing cells and fed in a feed manner. After 60 h of reaction, the prednisolone product was generated at a rate of 98.3%. Attached Figure Description

[0017] Figure 1 This is a schematic diagram showing the locations of the four mutation sites in the three-dimensional protein structure in this invention.

[0018] Figure 2 The half-inactivation temperature curves of the AuKsdD and AuKsdD-M4 mutants in this invention are shown.

[0019] Figure 3 The half-life curves of AuKsdD and AuKsdD-M4 mutants in this invention are shown.

[0020] Figure 4 The curves show the conversion process of AuKsdD and AuKsdD-M4 mutants to prednisolone using 50 g / L hydrocortisone.

[0021] Figure 5 This is the conversion result of AuKsdD and AuKsdD-M4 mutants to prednisolone in a 1.5 L fermenter using 60 g / L hydrocortisone. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this patent clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of this patent and are not intended to limit the scope of the invention.

[0023] The experimental techniques and methods used in this embodiment are conventional unless otherwise specified. For example, experimental methods in the following embodiments without specific conditions are examples of conventional techniques in the field. For parts not specifically annotated herein, those skilled in the art can refer to various commonly used reference books, scientific and technological literature, or related instructions and manuals prior to the filing date of this invention for implementation. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0024] In this invention, the following definitions are used: (1) Nomenclature of amino acids and DNA nucleic acid sequences The IUPAC nomenclature, a recognized system for naming amino acid residues, is used, employing single-letter or three-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0025] (2) Identification of 3-sterone-Δ1-dehydrogenase mutants The mutated amino acid in the 3-sterone-Δ1-dehydrogenase mutant is represented by "original amino acid + position + substituted amino acid". For example, E244L indicates that the 244th amino acid is mutated from glutamic acid (E) to leucine (L), and the position number corresponds to the amino acid sequence number of wild-type AuKsdD in SEQ ID NO. 1.

[0026] In this invention, lowercase italics AuksdD This indicates the encoding gene for the wild-type 3-sterone-Δ1-dehydrogenase AuKsdD from *Arthrobacter uricula-oxatum*, in lowercase italics. AuksdD-M4 The encoding gene for the AuKsdD-M4 mutant is shown in the table below.

[0027]

[0028] The amino acid sequence of the wild-type 3-sterone-Δ1-dehydrogenase AuKsdD described in this invention is shown in SEQ ID NO.1: MTAKSVLSPVKINHSEVDLLVIGSGTGLATALAAKELGLNVAVVEKTKYVGGSTARSGGAFWIPANPVLQRDGAVDSPERGEKYIEAVVAGTSPAERWKAFLEFGDETVRMLERMTQLSFFWAKGYSDYHPEKPGGSAAGRSVE ARPFDLNKLGEQRGRFQPATMAALPMPVTGADYKWLNLIAKAPVKALPRAIKRVIQGIGGLALGRDYGAGGQAIAAGMYAGALEAGIPVWTEAALVDLESSDGVITAAVLEQNGERIAVRAKYGVVLAAGGFDHNMPMRRQYQ SPSLEQDLSLGATGNSGDAIVLAQKLNAQLAGMQQSWWFPAVAPLEAEASPMILLAERSLPGSFMVDSSGRRFINESTDYMSFGQEVLRREASGDPVGEMWLVFDQTYRDSYMFAGQVFPRMALPQSWYAAGIAHKAASIGELA AGMGVPAEALARSLHAFNEAAGTGTDDQFGRGASAYDRYYGDPTQTPNPNLRPLDGKAYYAVKVVLSDLGTCGGILTDGHGQALDTSGNVIEGLYAQGNSAANIFGEVYPGAGATIGQGLVYGTIIATHAAQKAQAHSTAQRSS The amino acid sequence of the AuKsdD-M4 mutant described in this invention is shown in SEQ ID NO.3: MTAKSVLSPVKINHSEVDLLVIGSGTGLATALAAKELGLNVAVVEKTKYVGGSTARSGGAFWIPANPVLQRDGAVDSPERGEKYIEAVVAGTSPAERWKAFLEFGDETVRMLERMTQLSFFWAKGYSDYHPEKPGGSAAGRSVE ARPFDLNKLGEQRGRFQPATMAALPMPVTGADYKWLNLIAKAPVKALPRAIKRVIQGIGGLALGRDYGAGGQAIAAGMYAGALEAGIPVWTEAALVDLLSSDGVITAAVLEQNGERIAVRARYGVVLAAGGFDHNMPMRRQYQ SPSLEQDLSLGAEGNSGDAIVLAQKLGAQLAGMQQSWWFPAVAPLEPEASPMILLAERSLPGSFMVDSSGRRFINESTDYMSFGQEVLRREASGDPVGEMWLVFDQTYRDSYMFAGQVFPRMALPQSWYAAGIAHKAASIGELA AGMGVPAEALARSLHAFNEAAGTGTDDQFGRGASAYDRYYGDPTQTPNPNLRPLDGKAYYAVKVVLSDLGTCGGILTDGHGQALDTSGNVIEGLYAQGNSAANIFGEVYPGAGATIGQGLVYGTIIATHAAQKAQAHSTAQRSS The present invention will be further explained and illustrated below through specific embodiments.

[0029] Example 1: Construction of a mutant of AuKsdD This embodiment aims to obtain a 3-sterone-Δ1-dehydrogenase mutant with high stability and high catalytic efficiency. Based on the wild-type AuKsdD amino acid sequence shown in SEQ ID NO:1, combined with protein structure analysis, this invention screens potential key residues of AuKsdD through systematic sequence consensus analysis, structure prediction, and thermostability hotspot identification. Integrating a combined strategy of computer-aided screening, structural analysis, and experimental verification, the target amino acid sequence SEQ ID NO:3 with high thermostability and high catalytic activity was determined by mutating E244L, K267R, T301E, and A335P. This four-site combination mutant is named the AuKsdD-M4 mutant. The positions of the four mutation sites in the AuKsdD protein structure are shown below. Figure 1 As shown.

[0030] (1) Construction of wild-type recombinant plasmid pET28a-AuKsdD-sGFP Wild-type 3-sterone-Δ1-dehydrogenase gene AuksdD Derived from *Arthrobacter urate-oxidizing bacteria*, its nucleotide sequence is shown in SEQ ID NO:2. The sGFP fusion protein tag encoding gene... sgfp The nucleotide sequence is shown in SEQ ID NO:5. The sGFP tag is only used to improve the soluble expression and correct folding of AuKsdD protein in E. coli, and does not participate in the catalytic reaction process, and has no significant effect on enzyme activity and substrate conversion ability.

[0031] The above-mentioned genes were obtained through artificial synthesis, and seamless cloning primers were designed (as shown in Table 1) for... AuksdD Genes and sgfp The gene was amplified by PCR to obtain the target gene fragment, and the PCR product was purified and recovered.

[0032] pET28a vector was subjected to restriction endonuclease Nco I and Hin Linearization was achieved by double digestion with d III enzymes, and the linearized vector fragment was recovered.

[0033] The three fragments were homologously ligated using a seamless cloning kit to construct the recombinant expression plasmid pET28a-AuKsdD-sGFP. This ligation process introduces the exogenous sequence without relying on additional protective bases or restriction endonuclease recognition sites, thus achieving seamless splicing.

[0034] The ligation product was transformed into E. coli DH5α competent cells, and single colonies were picked for colony PCR and sequencing verification. The plasmid with correct sequencing was the wild-type AuKsdD expression plasmid pET28a-AuKsdD-sGFP.

[0035] Table 1

[0036] (2) Construction of mutant plasmids Using the recombinant plasmid pET28a-AuKsdD-sGFP as a template, site-directed mutagenesis was achieved through seamless cloning.

[0037] Taking the E244L mutation as an example, mutation primers E244L-F and E244L-R were designed. The vector-side primers AuksdD-F and sgfp-R were the same as the amplification primers used when constructing the wild-type recombinant plasmid. Using the wild-type recombinant plasmid pET28a-AuKsdD-sGFP as a template, upstream and downstream DNA fragments containing the mutation site were obtained by PCR amplification. The upstream fragment was obtained by amplification using primers ksdD-F and E244L-R, and the downstream fragment was obtained by amplification using primers E244L-F and sgfp-R. The upstream and downstream fragments had a 15-20 bp homologous overlap sequence at the mutation site. The two PCR fragments were then ligated with the linearized pET28a vector through seamless cloning to construct the single-point mutant plasmid pET28a-AuKsdD(E244L)-sGFP.

[0038] Based on the E244L mutant, the K267R, T301E, and A335P mutations were introduced sequentially using the same method. A multi-site mutant was constructed through a stepwise stacking of mutations, ultimately yielding the recombinant expression plasmid pET28a-AuKsdD-M4-sGFP containing four mutation sites: E244L, K267R, T301E, and A335P. The primer sequences used for each mutation site are shown in Table 2.

[0039] The PCR amplification system was configured according to Table 3, and high-fidelity polymerase was used for amplification. The PCR reaction program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 60 °C annealing for 15 s, 72 °C extension (1 min for every 1 kb), for a total of 35 cycles; final extension at 72 °C for 5 min, and storage at 4 °C.

[0040] Table 2. Primer sequences for site-directed mutagenesis

[0041] Table 3 Site-directed mutagenesis PCR reaction system

[0042] Example 2 Mutant Gene Expression The recombinant plasmids pET28a-AuKsdD-sGFP and pET28a-AuKsdD-M4-sGFP obtained in Example 1 were transformed into competent Escherichia coli BL21(DE3) to obtain recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP and recombinant engineered bacteria BL21 / pET28a-AuKsdD-M4-sGFP, which were used for subsequent induction expression and functional analysis.

[0043] 1. Strain activation and induced expression: (1) Strain resuscitation and primary seed culture: Recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP and BL21 / pET28a-AuKsdD-M4-sGFP, preserved in glycerol at -80 °C, were streaked onto LB solid medium containing kanamycin sulfate for resuscitation and cultured at 37 °C for 16 h. Single colonies were picked and inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin sulfate and cultured with shaking at 37 °C and 200 r / min for 16 h to obtain primary seed culture.

[0044] (2) Secondary seed culture: The primary seed culture was transferred into 50 mL of fresh LB medium (containing 50 μg / mL kanamycin sulfate) at an inoculation rate of 1% (v / v) and cultured for 8 h at 37 °C and 200 r / min to obtain the secondary seed culture.

[0045] (3) Induction of expression and cell collection: The secondary seed culture was transferred to 200 mL of fresh LB medium (containing 50 μg / mL kanamycin sulfate) at an inoculation rate of 1% (v / v), and cultured at 37 °C with shaking until the OD of the cells was reached. 600 When the concentration reached 0.6, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.15 mM was added for induction. Expression was induced for 16 h at 25 ℃ and 200 r / min. After induction, the bacterial cells were collected by centrifugation at 4 ℃ and 8000 r / min for 10 min, and washed twice with 10 mM PBS buffer (pH 7.4) for later use.

[0046] The LB medium consists of: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, prepared with deionized water and sterilized at 121 °C for 20 min before use.

[0047] 2. Preparation of crude enzyme solution: The bacterial cells obtained according to the method described above were washed twice with pre-chilled 50 mM Tris-HCl buffer (pH 8.0) to thoroughly remove any culture medium residue. The bacterial cells were then resuspended in 10 mL of pre-chilled lysis buffer. The lysis buffer contained: 50 mM Tris-HCl, 0.5 M NaCl, 5% (v / v) glycerol, 1 mM benzyl sulfonyl fluoride (PMSF), and deionized water, with the pH adjusted to 8.0.

[0048] After the bacterial cells were fully resuspended, they were disrupted using a high-pressure homogenizer at 4 °C and a pressure of 1200 bar. The disrupted mixture was centrifuged at 4 °C and 6000 r / min for 15 min, and the supernatant was collected. The supernatant obtained was the crude enzyme solution of AuKsdD and AuKsdD-M4 mutants, which was stored on ice for subsequent enzyme activity assays.

[0049] Example 3 Enzyme Activity Test The enzyme activities of AuKsdD and AuKsdD-M4 mutants were determined using the 2,6-dichlorophenolindophenol (DCPIP) colorimetric method. The total reaction volume was 200 μL, and the assay was performed at 25 °C.

[0050] The reaction system consisted of the following components: 50 mM Tris-HCl (pH 7.0); 1.5 mM methyl phenazine sulfate (PMS); 40 μM 2,6-dichlorophenolindophenol (DCPIP); 500 μM hydrocortisone (HC, soluble in dimethyl sulfoxide); 1 μL of crude enzyme solution (with a protein concentration of 300 μg / mL) was added to start each reaction.

[0051] A system without crude enzyme solution was used as a blank control. After the reaction started, the absorbance was continuously monitored at a wavelength of 600 nm. The reaction endpoint was determined when the absorbance at 600 nm no longer showed a significant change. The reaction time was controlled within 10 min. Protein concentration was determined by the BCA method.

[0052] Enzyme activity unit (U) is defined as the amount of enzyme required to reduce 1 μmol DCPIP per minute under the assay conditions. Specific enzyme activity (U / mg) is calculated using the following formula: Specific enzyme activity (U / mg) = (ΔA × Vtotal) / (ε × d × Venzyme × t × c) In the formula: ΔA is the difference between the initial absorbance and the final absorbance; Vtotal is the total reaction volume; ε is the molar absorptivity of DCPIP at 600 nm (18.7 × 10³ L·mol⁻¹). - ¹·cm - ¹); d is the optical path length of the cuvette; Venzyme is the volume of crude enzyme solution added; t is the reaction time; c is the protein concentration of the crude enzyme solution.

[0053] The crude enzyme solutions of AuKsdD and AuKsdD-M4 mutants were subjected to enzyme activity assays. The results are shown in Table 4. The specific enzyme activity of AuKsdD was 19.1 U / mg, and the specific enzyme activity of AuKsdD-M4 mutant was 34.5 U / mg, which was 80.6% higher than that before the mutation.

[0054] Table 4 Results of enzyme activity assay

[0055] Example 4: Heat Resistance and Half-Life Analysis (1) Half-deactivation temperature (T) 50 Assay: Equal amounts of crude enzyme solutions of AuKsdD and AuKsdD-M4 mutants were pre-incubated at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, and 50 ℃ for 15 min, respectively, followed by ice bath for 30 min, and then residual activity was measured at 25 ℃. The enzyme activity without heat treatment was taken as 100%. The half-inactivation temperature (T0) was calculated based on the relationship curve between residual enzyme activity and temperature. 50 ).

[0056] The results are as follows Figure 2 As shown in Table 5, the half-inactivation temperature of the AuKsdD-M4 mutant is 46.3 ℃, and the half-inactivation temperature of AuKsdD is 36.1 ℃.

[0057] (2) Determination of thermal inactivation half-life: Equal amounts of crude enzyme solutions of AuKsdD and AuKsdD-M4 mutants were incubated at 30 °C for different times, and samples were taken at each time point to determine the residual enzyme activity. The time-residual enzyme activity curve was plotted, and the results are as follows: Figure 3 As shown. The time required for the enzyme activity to decrease to 50% of its initial value, assuming an initial activity of 100%, is calculated; this is the heat inactivation half-life (t0). 1 / 2 The results are shown in Table 5. The thermal inactivation half-life of the AuKsdD-M4 mutant was 19.5 h, while that of AuKsdD was 3.3 h.

[0058] Table 5. Half-inactivation temperature (T) 50 Results of thermal inactivation half-life (t1 / 2) determination

[0059] (3) Results and Analysis: Enzymatic property analysis showed that the specific enzyme activity of the AuKsdD-M4 mutant was 34.5 U / mg, which was 80.6% higher than that of AuKsdD. In the thermostability analysis, the half-inactivation temperature (T) of the AuKsdD-M4 mutant was... 50 The heat resistance was 46.3 °C, which is 10.2 °C higher than that of AuKsdD. At 30 °C, the thermal inactivation half-life of the AuKsdD-M4 mutant was 19.5 h, which is about 6 times that of AuKsdD, indicating that the AuKsdD-M4 mutant has significantly improved heat resistance.

[0060] Example 5: Dehydrogenation conversion of hydrocortisone (HC) (1) Preparation of engineered strains: The recombinant engineered strains BL21 / pET28a-AuKsdD-sGFP and BL21 / pET28a-AuKsdD-M4-sGFP were induced to express according to the method in Example 2, and the bacterial cells were collected by centrifugation to construct a whole-cell catalytic system.

[0061] (2) Whole-cell catalytic system: The total reaction volume was 10 mL; the final concentration of HC was 50 g / L; wet cells of recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP or recombinant engineered bacteria BL21 / pET28a-AuKsdD-M4-sGFP were added to make the wet cell concentration in the system 50 g / L (based on wet weight); the final concentration of methanol was 4% (v / v); the final concentration of methyl phenazine sulfate (PMS) was 3 mM; the buffer system was 50 mM Tris-HCl buffer (pH 8.0).

[0062] (3) Reaction conditions: The reaction system was shaken at 30 °C and 200 r / min for 6 h.

[0063] (4) Sample processing and product detection: Take 300 μL of the conversion solution each time, add 600 μL of methanol and 900 μL of ethyl acetate and mix for extraction. Sonicate the mixture for 30 min, then centrifuge at 13800 r / min for 10 min. Take the upper organic phase for high performance liquid chromatography (HPLC) analysis to analyze the conversion of the substrate during the conversion process.

[0064] (5) The specific detection conditions of HPLC were as follows: the chromatographic column was Kromasil 100-5SIL 250 mm × 4.6 mm × 5 μm; the mobile phase was dichloromethane: diethyl ether: methanol (volume ratio 82:12:6, v / v), filtered through a 0.22 μm organic microporous membrane; the flow rate was 1 mL / min; the column temperature was 30°C; the detector was a UV detector with a wavelength of 240 nm; and the injection volume was 10 μL. (6) Results and Analysis: The transformation results are as follows Figure 4 As shown, after 6 h of reaction under the same conditions, the maximum conversion rate of recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP was 88.6%, and the maximum conversion rate of recombinant engineered bacteria BL21 / pET28a-AuKsdD-M4-sGFP was 98.2%. This indicates that the AuKsdD-M4 mutant provided by this invention has significantly improved catalytic efficiency and substrate conversion ability under higher substrate concentration conditions, which is beneficial to improving the conversion efficiency of steroid C1,2-position dehydrogenation reaction.

[0065] Example 6: Effect of Temperature on Catalytic Performance This example analyzes the effect of reaction temperature on the catalytic performance of the AuKsdD-M4 mutant. The whole-cell catalytic system from Example 5 was used, except that the final concentration of hydrocortisone was 80 g / L and the reaction temperatures were set to 30℃, 35℃, and 40℃, respectively, while other conditions remained the same for whole-cell catalytic reactions.

[0066] According to the results in Table 6, at 30 ℃, the reaction was basically completed after 6 h, with a conversion rate of 90.1% for recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP and 93.6% for recombinant engineered bacteria BL21 / pET28a-AuKsdD-M4-sGFP. When the reaction temperature was increased to 35 ℃, the reaction rate increased significantly, and the reaction was completed after 4 h, with a conversion rate of 88.2% for recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP and an increased conversion rate of 95.3% for recombinant engineered bacteria BL21 / pET28a-AuKsdD-M4-sGFP. When the reaction temperature was further increased to 40 °C, the reaction was completed after 4 h. The conversion rate of recombinant engineered bacteria BL21 / pET28a-AuKsdD-M4-sGFP was 87.4%, while the conversion rate of recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP decreased to 57.7%.

[0067] The results showed that as the reaction temperature increased, the reaction rate accelerated, but the enzyme stability decreased, leading to a reduction in conversion rate. Compared to AuKsdD, the AuKsdD-M4 mutant maintained a higher conversion rate at higher temperatures, indicating that the AuKsdD-M4 mutant has better heat resistance and catalytic stability, making it suitable for biotransformation reactions under higher temperature conditions.

[0068] Table 6. Results of conversion at different temperatures

[0069] Example 7 High-density fermentation and cell catalysis Recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP and BL21 / pET28a-AuKsdD-M4-sGFP were used as production strains and were cultured at high density in a 1.5 L duplex parallel fermenter. Cell growth catalysis experiments were also conducted.

[0070] (1) Seed culture: Secondary seed cultures of recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP and BL21 / pET28a-AuKsdD-M4-sGFP were obtained according to the strain activation method in Example 2. The secondary seed cultures were inoculated into TB medium containing antibiotics at an inoculation rate of 2% (v / v) and cultured with shaking at 37 ℃ and 200 r / min until the bacterial cell OD reached the target value. 600 When the concentration reaches 0.6, the fermented seed liquid is obtained.

[0071] The TB culture medium consisted of: 12 g / L tryptone, 24 g / L yeast extract, 12.5 g / L dipotassium hydrogen phosphate (K2HPO4), 2.3 g / L potassium dihydrogen phosphate (KH2PO4), and 12 g / L glucose.

[0072] (2) Fermentation culture: The fermentation seed liquid was inoculated into the fermenter at 3% (v / v), and the fermentation medium was a compound medium. Fermentation conditions: temperature 37 ℃, pH 7.0 (adjusted by ammonia or alkaline solution), dissolved oxygen maintained at 30% (achieved by adjusting the stirring speed and aeration rate), stirring speed 600 r / min, aeration rate 1vvm.

[0073] The fermentation medium consisted of: glucose 25 g / L, peptone 35 g / L, yeast extract 7.5 g / L, ammonium chloride 6.7 g / L, disodium hydrogen phosphate (Na₂HPO₄) 22.3 g / L, potassium dihydrogen phosphate (KH₂PO₄) 8.5 g / L, magnesium sulfate (MgSO₄) 1.25 g / L, and sodium sulfate (Na₂SO₄) 1.8 g / L. Each component of the medium was sterilized separately: the glucose and magnesium sulfate solutions were sterilized at 115 °C for 15 min, and the remaining components were mixed and sterilized at 121 °C for 20 min. After sterilization and cooling to room temperature, the pH of the medium was adjusted to 7.0 using ammonia.

[0074] (3) Fed culture: During fermentation, feeding begins when dissolved oxygen shows an upward trend. The feeding method is intermittent feeding, with the feeding rate controlled at 10 mL / h to maintain dissolved oxygen at 30%.

[0075] The feed solution consisted of: 50 g / L yeast extract, 25 g / L peptone, 400 g / L glucose, and 4 g / L magnesium sulfate (MgSO4). Each component was sterilized separately: the glucose and magnesium sulfate solutions were sterilized at 115 °C for 15 min, while the remaining components were mixed and sterilized at 121 °C for 20 min. After sterilization and cooling, the components were aseptically mixed to obtain the feed solution.

[0076] (4) Induction of expression: When the bacterial cells grow to OD 600When the temperature was 30°C, the culture temperature was rapidly reduced to 30°C, and IPTG, the inducer, was added to the fermentation system to make the final concentration 0.2 mM. The induction time was 16 h, and recombinant engineered bacteria with high expression were obtained.

[0077] (5) Cellular Growth Catalysis: After the induction of expression, cell isolation was not performed, and the cell growth catalysis stage was directly initiated. Feeding was continued via a peristaltic pump at a flow rate of 0.3 mL / min, a temperature of 30 °C, a stirring speed of 600 r / min, and an aeration rate of 1 vvm to maintain dissolved oxygen at 20%. Hydrocortisone (HC) was dissolved in methanol to prepare a high-concentration substrate stock solution (200 g / L), which was continuously added to the fermenter via a peristaltic pump, allowing the substrate to be added dropwise during the reaction to avoid substrate inhibition. The substrate flow rate was controlled at 0.5 mL / min to gradually increase the substrate concentration in the system, ultimately reaching a total substrate concentration of 60 g / L. The total reaction time was 60 h. Samples were taken periodically during the reaction to detect the substrate conversion rate. The sample processing and detection methods were the same as in Example 5.

[0078] (6) Results are as follows Figure 5 As shown, under scale-up fermenter conditions, a biotransformation reaction was carried out using hydrocortisone as the substrate. When the substrate concentration was 60 g / L, the conversion rate of the recombinant engineered strain BL21 / pET28a-AuKsdD-M4-sGFP reached 98.3%, while the conversion rate of the recombinant engineered strain BL21 / pET28a-AuKsdD-sGFP was 91.07%. The results indicate that the AuKsdD-M4 mutant provided by this invention still exhibits high catalytic activity and conversion efficiency in high substrate concentrations and scaled-up reaction systems, demonstrating promising prospects for industrial application.

[0079] Example 8: Dehydrogenation conversion of androst-4-ene-3,17-dione (AD) (1) Preparation of engineered bacteria: The recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP and BL21 / pET28a-AuKsdD-M4-sGFP were induced to express according to the method in Example 2, and the bacterial cells were collected by centrifugation and used to construct a whole-cell catalytic system.

[0080] (2) Whole-cell catalytic system: The total reaction volume was 10 mL; the final concentration of AD was 50 g / L; wet cells of recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP or BL21 / pET28a-AuKsdD-M4-sGFP were added to make the wet cell concentration in the system 50 g / L (based on wet weight); the final concentration of dimethyl sulfoxide was 10% (v / v); the final concentration of PMS was 7 mM; the buffer system was 50 mM Tris-HCl buffer (pH 8.0).

[0081] (3) Reaction conditions: The reaction system was shaken at 30 °C and 200 r / min for 24 h.

[0082] (4) Sample processing and detection: Take 300 μL of the conversion solution each time, perform liquid-liquid extraction with twice the volume of ethyl acetate, combine the organic phases, dry and then use HPLC to analyze the product content.

[0083] (5) The specific detection conditions of HPLC were as follows: chromatographic column: ChromCore 120 C18 (250 mm × 4.6 mm, 5 μm); mobile phase: methanol:water = 80:20 (v / v), filtered through a 0.22 μm organic microporous membrane and degassed by sonication; flow rate: 1 mL / min; column temperature: 30 °C; detector: UV detector, wavelength: 254 nm; injection volume: 10 μL.

[0084] (6) Results and analysis: After 24 h of reaction under the same cell volume and reaction conditions, the substrate conversion rate of recombinant engineered bacteria BL21 / pET28a-AuKsdD-M4-sGFP was 98.5%, which was significantly higher than the 76.8% of recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP.

[0085] When the wet cell concentration of the recombinant engineered strain BL21 / pET28a-AuKsdD-sGFP was further increased to 80 g / L, the conversion rate reached 98.3% under the same reaction time of 24 h.

[0086] In contrast, BL21 / pET28a-AuKsdD-M4-sGFP achieves high conversion rates with lower cell usage, indicating superior catalytic efficiency and reaction economy.

[0087] Example 9: Dehydrogenation conversion of 11α-hydroxy-16,17α-epoxyprogesterone (HEP) (1) Preparation of engineered strains: The recombinant engineered strains BL21 / pET28a-AuKsdD-sGFP and BL21 / pET28a-AuKsdD-M4-sGFP were induced to express according to the method in Example 2, and the bacterial cells were collected by centrifugation and used to construct a whole-cell catalytic system.

[0088] (2) Whole-cell catalytic system: The total reaction volume was 10 mL; the final HEP concentration was 30 g / L; wet cells of recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP or BL21 / pET28a-AuKsdD-M4-sGFP were added to make the wet cell concentration in the system 50 g / L (based on wet weight); the final concentration of dimethyl sulfoxide was 10% (v / v); the final concentration of PMS was 5 mM; the buffer system was 10 mM PBS buffer (pH 7.4).

[0089] (3) Reaction conditions: The reaction system was shaken at 30 °C and 200 r / min for 72 h.

[0090] (4) Sample processing and detection: The sample processing and product detection methods are the same as the HC detection method in Example 5.

[0091] (5) Results and analysis: After 72 h of reaction under the same cell dosage and reaction conditions, the substrate conversion rate of HEP catalyzed by the recombinant engineered bacteria BL21 / pET28a-AuKsdD-M4-sGFP was 90.1%, which was higher than the 82.4% of the recombinant engineered bacteria BL21 / pET28a-AuKsdD-sGFP.

[0092] When the wet cell concentration of the recombinant engineered strain BL21 / pET28a-AuKsdD-sGFP was further increased to 60 g / L, the conversion rate could reach 90.3% under the same reaction time of 72 h.

[0093] In contrast, the recombinant engineered bacterium BL21 / pET28a-AuKsdD-M4-sGFP achieved a high transformation rate under low cell loading conditions, indicating that the AuKsdD-M4 mutant has good catalytic adaptability and transformation ability in different steroid substrate systems.

[0094] This embodiment aims to verify the catalytic conversion ability of the AuKsdD-M4 mutant for different steroid substrates and evaluate its substrate adaptability and industrial application scope. Three steroid substrates—hydrocortisone, androstenedin-4-ene-3,17-dione, and 11α-hydroxy-16,17α-epoxyprogesterone—were selected for the experiment, and the conversion efficiency of the substrates was investigated in different reaction systems.

[0095] These results demonstrate that the AuKsdD-M4 mutant possesses excellent steroidal C1,2-position dehydrogenation catalysis capabilities. Even with significantly reduced catalytic cell usage, this mutant maintains high conversion rates under high substrate loading, exhibiting good reaction economy. Furthermore, the AuKsdD-M4 mutant combines strong substrate tolerance, excellent process scale-up stability, and broad-spectrum catalytic adaptability, effectively reducing production costs, increasing capacity and process efficiency, and demonstrating outstanding potential for industrial application.

[0096] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent.

Claims

1. A 3-sterone-Δ1-dehydrogenase mutant, characterized in that, The mutant was obtained by the following mutations on the wild-type AuKsdD shown in SEQ ID NO:1: glutamic acid at position 244 was mutated to leucine, lysine at position 267 was mutated to arginine, threonine at position 301 was mutated to glutamic acid, and alanine at position 335 was mutated to proline.

2. The 3-sterone-Δ1-dehydrogenase mutant as described in claim 1, characterized in that, The mutant is the AuKsdD-M4 mutant, and its amino acid sequence is shown in SEQ ID NO:

3.

3. The encoding gene of the 3-sterone-Δ1-dehydrogenase mutant of claim 1.

4. The encoding gene as described in claim 3, characterized in that, The encoding gene is shown in SEQ ID NO:

4.

5. A recombinant vector or recombinant strain containing the encoding gene of the 3-sterone-Δ1-dehydrogenase mutant of claim 1.

6. The use of the recombinant vector or recombinant strain of claim 5 in the production of the 3-sterone-Δ1-dehydrogenase mutant of claim 1.

7. The use of the recombinant vector or recombinant strain according to claim 5 in the C1,2-position dehydrogenation reaction of steroidal compounds.

8. The use of the 3-sterone-Δ1-dehydrogenase mutant of claim 1 in the dehydrogenation reaction at the C1,2 position of steroid compounds.

9. The application as described in claim 7 or 8, characterized in that, This includes catalyzing the synthesis of prednisolone from hydrocortisone, catalyzing the synthesis of androst-1,4-diene-3,17-dione from androst-4-ene-3,17-dione, or catalyzing the synthesis of 11α-hydroxy-16,17α-epoxypregnane from 11α-hydroxy-16,17α-epoxypregnane-1,4-diene-3,20-dione.

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