Method for producing 9alpha-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion

Abstract

The present disclosure discloses a method for producing 9α-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion, and belongs to the fields of gene engineering and enzyme engineering. According to the present disclosure, oxidation subunit KshA, reduction subunit KshB and unknown active subunit KshC of 3-ketosteroid-9α-hydroxylase sourcing from Mycobacterium sp. Strain VKM Ac-1817D are successfully expressed in E. coli BL21, and KshC is identified as an oxidation subunit, the enzyme activity of which is far higher than that of KshA. BL21/pET-28a(+)-fdh constructed in the laboratory is used for expressing formate dehydrogenase (FDH), and by using crude enzyme liquid of KSH (KshB+KshC) and FDH engineering bacteria as a biocatalyst and a steroidal compound (AD) as a substrate, optimum reaction temperature is determined as 30° C. and optimum pH is determined as 7.0. In optimum conditions, AD is converted to produce a product 9-OH-AD, and within 20 hours, the output of 9-OH-AD is 4.7 g/L, and the molar conversion rate reaches 96.7%. According to the present disclosure, in production of 9-OH-AD, coupling of a 3-ketosteroid-9α-hydroxylase hydroxylation system and a coenzyme recycling system is realized, and the method has the advantages of being high in efficiency, low in cost, green, environmentally friendly and the like.

Claims

1. A composition comprising an Escherichia coli bacterium comprising genes encoding 3-ketosteroid-9α-hydroxylase reduction subunit KshB and 3-ketosteroid-9α-hydroxylase oxidation subunit KshC.

2. The composition of claim 1, further comprising coenzyme NAD.sup.+, NADH, formate dehydrogenase, or a combination thereof.

3. The composition of claim 1, wherein an amino acid sequence of the 3-ketosteroid-9α-hydroxylase reduction subunit KshB is SEQ ID NO:4.

4. The composition of claim 1, wherein an amino acid sequence of the 3-ketosteroid-9α-hydroxylase oxidation subunit KshC is SEQ ID NO:5.

5. The composition of claim 1, wherein the composition further comprises NADH and formate dehydrogenase.

6. The composition of claim 5, wherein the additive proportion of the 3-ketosteroid-9α-hydroxylase oxidation subunit KshC to the 3-ketosteroid-9α-hydroxylase reduction subunit KshB to the formate dehydrogenase is 1-10:1-10:1-10 according to enzyme activity.

7. The composition of claim 5, wherein NADH is present in an amount of 0.01 to −1 mol/L.

8. A method of preparing a composition, wherein the composition comprises 3-ketosteroid-9α-hydroxylase reduction subunit KshB and 3-ketosteroid-9α-hydroxylase oxidation subunit KshC, and wherein the method comprises: (a) amplifying a nucleotide sequence set forth in SEQ ID NO:2 to obtain a gene encoding 3-ketosteroid-9α-hydroxylase reduction subunit KshB, and cloning the gene encoding 3-ketosteroid-9α-hydroxylase reduction subunit KshB into an Escherichia Coli expression vector pET-28a to obtain a recombinant plasmid pET-28a- kshB; (b) amplifying a nucleotide sequence set forth in SEQ ID NO:3 to obtain a gene encoding 3-ketosteroid-9α-hydroxylase oxidation subunit KshC, and cloning the gene encoding 3-ketosteroid-9α-hydroxylase oxidation subunit KshC to an Escherichia Coli expression vector pET- Duet1(+) to obtain a recombinant plasmid pET-Duet1(+)-kshC; (c) respectively transforming the recombinant plasmids obtained in step (a) and step (b) into Escherichia Coli to obtain a recombinant Escherichia Coli comprising BL21/pET-28a(+)-kshB and BL21/pET-Duet1(+)-kshC; and (d) culturing the recombinant Escherichia Coli obtained in step (3) to obtain the composition.

9. A method for increasing the efficiency of converting androstane-4-alkene-3,17-diketone to produce 9α-hydroxy androstane-4-alkene-3,17-diketone, which comprises adding 3 ketosteroid -9α-hydroxylase reduction subunit KshB and 3-ketosteroid-9α-hydroxylase oxidation subunit KshC as a catalyst to a biotransformation reaction.

10. The method of claim 8, which further comprises: (e) rupturing the recombinant Escherichia Coli cells to obtain a crude enzyme mixture; (f) adding an amount of NADH and androstane-4-alkene-3,17-diketone (AD) to the crude enzyme mixture; (g) incubating the crude enzyme mixture under conditions that support formation of 9α-hydroxy androstane-4-alkene-3,17-diketone from AD; and (h) adding the 9α-hydroxy androstane-4-alkene-3,17-diketone to a medicine.

11. The composition of claim 1, wherein the genes encoding 3-ketosteroid-9α-hydroxylase reduction subunit KshB and 3-ketosteroid-9α-hydroxylase oxidation subunit KshC are Mycobacterium genes.

Description

DETAILED DESCRIPTION

(1) A KSH determination system includes: 105 μM of NADH, 200 μM of substrate AD (dissolved in 100% isopropyl alcohol), 50 m of Tris-HCL (200 μL, pH7.0), and KSH enzyme. Therefore, the activity is defined as the enzyme amount needed for oxidizing a steroid substrate AD in 1 min, and is expressed as enzyme amount needed for oxidizing 1 nmol of NADH in 1 min, and the specific activity unit is nmol min.sup.−1 mg.sup.−1 (U/mg).

(2) An FDH enzyme activity determination method comprises the following steps: preparing 17 mg.Math.mL.sup.−1 of sodium formate solution (pH 7.5) and 3 mg.Math.mL.sup.−1 of NAD.sup.+ solution, taking 1.48 mL of sodium formate solution and putting into a quartz cuvette, sequentially adding 80 μL of NAD.sup.+ solution and 10 μL of crude enzyme liquid, detecting light absorption value change of each minute at 340 nm, wherein enzyme activity defining unit is enzyme amount needed for catalyzing 1 μmol of NAD.sup.+ to be converted into NADH in 1 min.

(3) HPLC analysis: AD and 9-OH-AD both have characteristic absorption peaks at ultraviolet wavelength of 254 nm, and therefore, an HPLC method is adopted to formulate 10 μL, and the flow velocity is 1.0 mL/min.

(4) LB culture medium: peptone 10 g/L, yeast extract 5 g/L, and NaCl 10 g/L (2% agar powder is added to a solid culture medium).

Example 1: 3-Ketosteroid-9α-Hydroxylase Primer Design

(5) According to kshA gene sequence (SEQ ID NO.1) in a whole genome nucleotide sequence of Mycobacterium sp. Strain VKM Ac-1817D in NCBI, PCR primers P1 and P2 (SEQ ID NO.6 and SEQ ID NO.7) of 3-ketosteroid-9α-hydroxylase oxidation subunit KshA are designed.

(6) TABLE-US-00001 P1: 5′-CGGGATCCATGACGACTGAGCACGCCGG-3′ (BamH I) P2: 5′-CCCAAGCTTTCAGCTTGATTGAGCGGTTTC-3′ (Hind III)

(7) According to kshB gene sequence (SEQ ID NO.2) in a whole genome nucleotide sequence of Mycobacterium sp. Strain VKM Ac-1817D in NCBI, PCR primers P3 and P3 (SEQ ID NO.8 and SEQ ID NO.9) of 3-ketosteroid-9α-hydroxylase reduction subunit KshB are designed.

(8) TABLE-US-00002 P3: 5′-CGGGATCCATGACTGATGAACCGTTAGGTAG-3′ (BamH I) P4: 5′-CCCAAGCTTTCACTCGTCGTAGGTCACCTC-3′ (Hind III)

(9) According to kshC gene sequence (SEQ ID NO.3) in a whole genome nucleotide sequence of Mycobacterium sp. Strain VKM Ac-1817D in NCBI, PCR primers P5 and P6 (SEQ ID NO.10 and SEQ ID NO.11) of 3-ketosteroid-9α-hydroxylase KshC are designed.

(10) TABLE-US-00003 P5: 5′-CGGGATCCGATGGCCGGTCTGAACAACGATAG-3′ (BamH I) P6: 5′-CCCAAGCTTTCAGCCGCTGGCCGGGGCGGCC-3′ (Hind III)

Example 2: Clone of 3-Ketosteroid-9α-Hydroxylase Genes kshA, kshB and kshC

(11) By taking synthesized DNA as a template, PCR amplification is performed by using the primers provided above, and the amplification conditions are as follows: pre-denaturation at 94° C., 5 min, one cycle; denaturation at 94° C., 1 min, re-firing at 58° C., 1 min, extension at 72° C., 34 cycles; final extension for 10 min at 72° C. A PCR amplification system: template 1 μL, up-stream and down-stream primers respectively 0.4 μL, dNTP Mix 4 μL, 10×Ex Taq Buffer 5 μL, sterile double distilled water 37 μL, and Ex Taq DNA polymerase 1 μL. A gel extraction kit is adopted to perform purification and recycling on the PCR product, and the concentration of the recycled product is tested by electrophoresis. The recycled product is stored in a 1.5 ml centrifugal tube, and is preserved in a refrigerator at 20° C. for later use. The recycled product is connected with pMD18-T Vector, the connection product converts E. coli JM109, the conversion product is coated on an LB slab with ampicillin resistance, the obtained product is cultured at 37° C. and stays overnight, a bacterial colony is picked to a 10 ml liquid LB culture medium, plasmids are extracted after staying overnight on a shaking table and culturing at 37° C. and are named as pMD18 T-kshA, pMD18 T-kshB and pMD18 T-kshC, and after connection is verified to be successful by enzyme digestion, glycerinum is added until the concentration is 15%˜20% (w/v), and the product is preserved in a refrigerator at −70° C.

Example 3: Construction of Recombinant Expression Vectors pET-28a(+)-kshA, pET-28a(+)-kshB and pET-Duet1(+)-kshC

(12) Plasmids pMD18 T-kshA, pMD18 T-kshB, pMD18 T-kshC and pET-28a (+), pET-Duet1(+) stored in E. coli jM109 are extracted, and double enzyme digestion is performed respectively with BamH I/Hind III, and connection is performed after the product is recycled by using the gel extraction kit, and the connection system is as follows: target gene enzyme-digested product 7 μL, pET-28a (+) or pET-Duet1(+) enzyme-digested product 1 μL, T4DNA ligase buffer 111 L and T4DNA ligase 1μL, and the connection system is connects by staying overnight at 16° C. Connected recombinant plasmids pET-28a(+)-kshA, pET-28a(+)-kshB and pET-Duet1(+)-kshC are converted to competent E. coli jM109, and positive bacterial colony is picked by using LB kanamycin resistant and ampicillin resistant culture medium. Plasmids are extracted after staying overnight on a shaking table and culturing at 37° C. and are named as pET-28a(+)-kshA, pET-28a(+)-kshB and pET-Duet1(+)-kshC, and after it is verified to be correct by enzyme digestion, glycerinum is added until the concentration is 15%˜20% (w/v), and the product is preserved in a refrigerator at −70° C.

Example 4: Conversion of E. coli BL21 with Recombinant Plasmids pET-28a(+)-kshA, pET-28a(+)-kshB and pET-Duet1(+)-kshC

(13) E. coli BL21 competence preparation: (1) picking newly activated single colony on the LB slab, inoculating into 10 mL of LB liquid culture medium for culturing for about 12 h, and then inoculating the bacterial suspension into 50 mL of LB liquid culture medium in the inoculation amount of 1% for culturing for 2-3 h, until OD.sub.600 reaches about 0.5. (2) Putting the bacterial solution into an ice-water bath for 15 min, so that the bacterial solution is rapidly cooled. (3) Packaging the bacterial solution into a plurality of 1.5 mL centrifugal tubes separately, centrifuging for 1 min at 8000 rpm and then abandoning the liquid supernatant, and washing cells for 2-3 times with 1M of CaCl.sub.2 solution. (4) Preserving the cleaned bacterial solution for a long time at −40° C. after being suspended with 80 μL of CaCl.sub.2 and 40 μL of 50% glycerinum.

(14) Conversion: 10 μL of recombinant plasmids pET-28a(+)-kshA, pET-28a(+)-kshB and pET-Duet1(+)-kshC are respectively added into two tubes of E. coli BL21 competent cells, the product is uniformly mixed slightly and then placed on ice for 45 min, then is subjected to water bath heat shock for 90 s at 42° C. and then placed on ice for 5 min, then is added into 800 μL of LB liquid culture medium and then being cultured for 1 h at 37° C., and is centrifuged for 1 min at 8000 rpm, the liquid supernatant is abandoned, the rest bacterial solution is coated to the LB slab with kanamycin resistance and ampicillin resistance, and after being cultured at 370° C., positive bacterial colony is picked, and plasmids are extracted for enzyme digestion verification, so as to obtain recombinant bacteria BL21/pET-28a(+)-kshA, BL21/pET-28a(+)-kshB and BL21/pET-Duet1(+)-kshC.

Example 5: Functional Identification of KshC and Determination of Activity of 3-Ketosteroid-9α-Hydroxylase

(15) Recombinant bacteria BL21/pET-28a(+)-kshA and BL21/pET-28a(+)-kshB constructed in Example 4 as well as original strain BL21/pET-28a(+) are respectively inoculated into 10 ml of LB culture medium containing kanamycin, BL21/pET-Duet1(+)-kshC is inoculated into 10 ml of LB culture medium containing ampicillin, the product stays overnight in shaking culture at 37° C., in the next day, the product is transferred into 50 mL of LB culture medium according to an inoculum size of 1%, after culture is performed for 2 h at 37° C. and when OD.sub.600 is 0.8, IPTG with final concentration of 0.8 mM is added, the product is induced for 9 h at 28° C., then the bacterial solution is centrifuged for 10 min at 10000 r/min at 4° C., a phosphate buffer (Na.sub.2HP0.sub.4—NaH.sub.2P0.sub.4) with pH of 7.5 is used for washing for twice, and finally, 5 ml of phosphate buffer with pH of 7.5 is used for suspension. Crude enzyme liquid is prepared by ultrasonic disruption processing.

(16) A KSH determination system contains 105 μM of NADH, 200 μM of substrate AD (dissolved in 100% isopropanol), 50 m of Tris-HCL (200 μL, pH7.0), and KSH enzyme (including oxidation subunit and reduction subunit). By verification, KshC is the oxidation subunit, and in the condition of ensuring same concentration of induction culture cells, enzyme activity with co-action of KshB crude enzyme liquid+KshC crude enzyme liquid with same additive amount is 30.7 U/mL, which is far higher than the KSH enzyme activity with co-action of KshA and KshB crude enzyme liquid, the former is 5.4 times of the latter, however, KSH enzyme activity is not detected in original bacterium E. coli BL21/pET-28a(+), and thus realizing out of nothing of enzyme activity of 3-ketosteroid-9α-hydroxylase in E. coli BL21.

Example 6: Generation of 9-OH-AD by Conversion of 3-Ketosteroid-9α-Hydroxylase

(17) KshA crude enzyme liquid or KshC crude enzyme liquid and KshB crude enzyme liquid obtained in Example 5 are added into 10 mL of conversion system in a proportion of 1:1.5 of enzyme amount to additive amount: 0.1 M of Tris-Hcl buffer solution, pH of 7.0, 0.32 mMol of NADH, 1 g.Math.L.sup.−1 of substrate AD (firstly dissolved in ethyl acetate) and crude enzyme mixed liquid (KshA+KshB or KshC+KshB), pH is regulated with 20% formic acid and 50% ammonia water, rotation speed is 160 r.Math.min-1, conversion is performed for 20 h at 30° C., and substrate AD and coenzyme NADH need to be supplemented in due time, the result shows that the output of 9-OH-AD in the KshA+KshB crude enzyme liquid conversion system is 0.98 g/L, while the output of 9-OH-AD in the KshC+KshB crude enzyme liquid conversion system is 7.8 g/L. Therefore, oxidation subunit KshC of 3-ketosteroid-9α-hydroxylase has a better effect than KshA.

Example 7: Determination on Activity of E. coli BL21/pET-28a(+)-Fdh Formate Dehydrogenase

(18) Recombinant bacterium E. coli BL21/pET-28a(+)-fdh constructed by the laboratory is inoculated into 10 ml of LB culture medium containing kanamycin, the product is subjected to overnight shaking culture at 37° C., in the next day, the product is transferred into 50 ml of LB culture medium according to an inoculum size of 1%, after culture is performed for 2 h at 37° C. and when OD.sub.600 is 0.8, IPTG with final concentration of 0.8 mM is added, the product is induced for 9 h at 28° C., then the bacterial solution is centrifuged for 10 min at 10000 r/min at 4° C., (Na.sub.2HP0.sub.4—NaH.sub.2P0.sub.4) is used for washing for twice, and finally, 5 ml of phosphate buffer with pH of 7.5 is used for suspension. Crude enzyme liquid is prepared by ultrasonic disruption processing.

(19) 17 mg/mL of sodium formate solution (pH 7.5) and 3 mg/mL of NAD.sup.+ solution are prepared, 1.48 mL of sodium formate solution is taken and put into a quartz cuvette, 80μL of NAD.sup.+ solution and 10μL of crude enzyme liquid are sequentially added, light absorption value change of each minute is detected at 340 nm, and enzyme activity defining unit is enzyme amount needed for catalyzing 1 μmol of NAD.sup.+ to be converted to NADH in 1 min. It is shown by the result that the enzyme activity of recombinant bacterium E. coli BL21/pET-28a(+)-fdh is 0.25 U/m L.

Example 8: Generation of 9-OH-AD by Conversion of AD with 3-ketosteroid-9α-Hydroxylase and Formate Dehydrogenase

(20) 3-ketosteroid-9α-hydroxylase and formate dehydrogenase (FDH) crude enzyme liquid obtained by ultrasonication are added into 25 mL of conversion system in a proportion of 1:0.5 according to enzyme activity additive amount: 0.1 M of Tris-Hcl buffer solution, pH of 7.0, containing any one of 10 g.Math.L.sup.−1 of ammonium formate, 0.25 mmol of NAD.sup.+ or NADH, 10 g. L.sup.−1 of substrate AD (firstly dissolved in ethyl acetate) and crude enzyme mixed liquid (KSH and FDH), pH is regulated with 20% formic acid and 50% ammonia water, rotation speed is 160 r min.sup.−1, conversion is performed for 20 h at 30° C., substrate AD needs to be supplemented in due time, NAD+, NADH or FDH does not need to be supplemented for control experiment, the result is as shown in table 1, and when 3-ketosteroid-9α-hydroxylase is KshC+KshB crude enzyme liquid, and FDH+NADH are added to participate in coenzyme regeneration, the output of 9-OH-AD is 9.7 g/L, and the molar conversion rate is 97%.

(21) To sum up, the obtained output result is as shown in the following table:

(22) TABLE-US-00004 TABLE 1 Synthesis of 9α-Hydroxy Androstane-4-Alkene-3,17-Diketone by Adding of Different Enzyme Liquid and Coenzyme Output of 9α-hydroxy androstane-4-alkene-3,17- Enzyme liquid Type of added coenzyme diketone (g/L) KshA + KshB — 0.3 ± 0.02 KshC + KshB — 2.3 ± 0.05 KshA + KshB NADH 1.2 ± 0.04 KshC + KshB NADH 7.8 ± 0.11 KshA + KshB FDH 0.5 ± 0.03 KshC + KshB FDH 2.9 ± 0.06 KshA + KshB FDH + NAD.sup.+ 0.6 ± 0.02 KshC + KshB FDH + NAD.sup.+ 4.7 ± 0.07 KshA + KshB FDH + NADH 2.6 ± 0.04 KshC + KshB FDH + NADH 9.7 ± 0.15