CN111100851A - Alcohol dehydrogenase mutants and their application in the synthesis of chiral biaryl alcohol compounds - Google Patents
Alcohol dehydrogenase mutants and their application in the synthesis of chiral biaryl alcohol compounds Download PDFInfo
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- CN111100851A CN111100851A CN201811252380.2A CN201811252380A CN111100851A CN 111100851 A CN111100851 A CN 111100851A CN 201811252380 A CN201811252380 A CN 201811252380A CN 111100851 A CN111100851 A CN 111100851A
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Abstract
本发明公开了醇脱氢酶突变体及其在手性双芳基醇化合物合成中的应用。本发明的醇脱氢酶突变体是将位于醇脱氢酶TbSADH的催化中心口袋中的氨基酸残基进行突变后得到的蛋白质，具体是将醇脱氢酶TbSADH的氨基酸序列的第39位和/或第42位和/或第84位和/或第85位和/或第86位和/或第104位和/或第110位和/或第294位的氨基酸残基进行突变后得到的蛋白质。本发明应用定向进化技术和方法对醇脱氢酶TbSADH进行了酶改造，获得了对以(4‑氯苯基)吡啶‑2‑甲酮为代表的双芳基酮类有酶活的一系列突变体，并将其应用于光学纯手性双芳基醇化合物的生物催化合成中。The invention discloses an alcohol dehydrogenase mutant and its application in the synthesis of chiral biaryl alcohol compounds. The alcohol dehydrogenase mutant of the present invention is a protein obtained by mutating the amino acid residues located in the catalytic center pocket of the alcohol dehydrogenase TbSADH, specifically the amino acid sequence of the alcohol dehydrogenase TbSADH at the 39th position and/or or the protein obtained by mutating the amino acid residues at positions 42 and/or 84 and/or 85 and/or 86 and/or 104 and/or 110 and/or 294 . In the present invention, the alcohol dehydrogenase TbSADH is enzymatically transformed by using the directional evolution technology and method, and a series of enzymes having enzymatic activity for the biaryl ketones represented by (4-chlorophenyl)pyridine-2-methanone are obtained. mutants and their application in the biocatalytic synthesis of optically homochiral bisaryl alcohol compounds.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to alcohol dehydrogenase mutants obtained by enzyme modification of alcohol dehydrogenase from high-temperature anaerobic bacillus (Thermoanaerobacter crockii) by using directed evolution technology and a method, and application of the mutants as biocatalysts in preparation of optical pure chiral bisaryl alcohol compounds by asymmetric reduction.
Background
The chiral diaryl secondary alcohol as an important building block structure can be used for synthesizing various chiral drugs, and the process for synthesizing the chiral diaryl alcohol from prochiral diaryl ketone through asymmetric reduction reaction has the advantage of high atom economy. Wherein, (S) - (4-chlorophenyl) pyridine-2-methanol is a chiral precursor for synthesizing antihistamines, and can be used for synthesizing antihistamine medicines of carbinoxamine (Barouh et al., J.Med.chem.,1971,14(9):834-836) and bepotastine besylate (Takahashi et al., Clin.exp.Dermatol.,2004,29(5): 526-532).
At present, the chemical asymmetric reduction synthesis method of (S) - (4-chlorphenyl) pyridine-2-methanol mainly comprises the following steps: taking 2-cyanopyridine as an initial substrate, adding 4-chlorobenzene magnesium bromide, concentrated sulfuric acid and a metal catalyst Pd (Phe)3P)4Etc. through three-step synthesis, the ee value can reach 98%, but the N-group on the pyridine ring needs to be protected and deprotected (Corey)&Helal, Tetrahedron Letters,1996,37(32): 5675-. Also used in recent years are Noyori organometallic catalysts such as: ru and Rh complexes and the like can be used for directly hydrogenating and reducing prochiral ketone substrate (4-chlorophenyl) pyridine-2-ketone into chiral alcohol, and the ee value reaches 97-99% (Tao et al, J.Org.chem.,2012,77(1): 612-41616; Yang et al, org.Lett.,2015,17(17): 4144-4147). Although these chemical syntheses can produce products with high ee values, the entire process requires the use of, for example, concentrated sulfuric acid, H2(8-10bar), high pressure and organic metal reagent, etc., which not only can cause harm to the environment, but also has higher labor protection requirement.
The biological asymmetric reduction synthesis method of (S) - (4-chlorphenyl) pyridine-2-methanol mainly comprises the following steps: the corresponding products were synthesized by asymmetric reduction of (4-chlorophenyl) pyridine-2-methanone using calcium alginate-immobilized baker' S yeast or the chiral product was obtained by selective hydrolysis of (4-chlorophenyl) pyridine-2-methanol acetate (Takemoto & Achiwa, chem.Pharm. Bull.,1994,42(4): 802. sub.805; Takemoto et al, Phytochemistry,1996,42(2): 423. sub.426), but the ee value for the (S) - (4-chlorophenyl) pyridine-2-methanol product was only 28%. The authors also reported studies on the synthesis of (S) - (4-chlorophenyl) pyridine-2-methanol from prochiral ketone (4-chlorophenyl) pyridine-2-methanone using immobilized plant cells with ee values of up to 48% (Takemoto et al, Phytochemistry,1996,42(2): 423-. However, these whole cell transformation methods are inefficient, and there is no report on the amino acid sequence of the relevant enzyme. In 2007, Truppo et al reported a study of asymmetric reduction of prochiral ketone to synthesize (S) - (4-chlorophenyl) pyridine-2-methanol using a commercial carbonyl reductase (KRED), but the ee value was only 60%, the enzyme protein sequence was unknown, and the species source was not reported (Truppo et al, org.Lett.,2007,9(2): 335-338). Ni et al screened a Kluyveromyces sp.CCTCCMM2011385 strain by traditional enrichment culture in 2012, which can catalyze (4-chlorophenyl) pyridine-2-methanone to produce (S) - (4-chlorophenyl) pyridine-2-methanol (86.7% ee) (CN 102559520A). However, the wild strain has low content of active enzyme, can only catalyze 2g/L of substrate at most, has low product concentration and high separation cost, and thus cannot meet the practical application. Li Zheng is equal to 2013, a carbonyl reductase PasCR derived from Pichia pastoris GS115 is researched, the carbonyl reductase PasCR can asymmetrically reduce and catalyze diaryl ketone compounds, and the highest conversion rate is only 50% (Li Zheng et al, BioEngineers, 2013, 29: 68-77). In 2016, Zhou et al isolated and purified Kluyveromyces alcohol dehydrogenase (named KPADH), and screened by protein engineering to obtain KPADH mutants M131F, S196Y and S237A, the wild-type alcohol dehydrogenase KPADH and three mutants can catalyze (4-chlorophenyl) pyridine-2-ketone to generate (R) - (4-chlorophenyl) pyridine-2-methanol with the highest conversion rate of 99%, and the ee value of (R) -product is 74.7% -96.1% (Zhou et al, Catal, Sci. Technol.,2016,6(16): 6320-. In 2018, KPADH is modified by Xu and the like through HCSM design, and can catalyze (4-chlorphenyl) pyridine-2-ketone to generate (R) - (4-chlorphenyl) pyridine-2-methanol, wherein the ee percent of the (R) - (4-chlorphenyl) pyridine-2-methanol reaches 99.4% (Xu et al, ACS catalysis, 2018,8, 8330-8345). However, since (S) - (4-chlorophenyl) pyridine-2-methanol is a precursor required for synthesizing a drug intermediate, no good enzyme is available at present for efficiently catalyzing (4-chlorophenyl) pyridine-2-methanone to generate an (S) -product with high stereoselectivity.
The alcohol dehydrogenase TbSADH derived from high temperature anaerobacterium (thermoanaerobacterium brockii) is a super heat-resistant enzyme which is relatively interested in industry, can tolerate the high temperature higher than 86 ℃, and is used for catalyzing and reducing the asymmetric reduction reaction of carbonyl groups with approximate α -and α' substituents on both sides of C ═ O, which is difficult to realize by a chemical method, by the semi-rational directed evolution modified mutant.
Disclosure of Invention
The invention applies directed evolution technology and method to perform enzyme modification on alcohol dehydrogenase TbSADH derived from high-temperature anaerobic bacillus (Thermoanaerobacter braskii), obtains a series of alcohol dehydrogenase TbSADH mutants which have enzyme activity on diaryl ketone substrates represented by (4-chlorphenyl) pyridine-2-ketone, and applies the alcohol dehydrogenase TbSADH mutants to the biocatalytic synthesis of optical homochiral diaryl alcohol compounds.
In a first aspect, the invention features an alcohol dehydrogenase TbSADH mutant.
The alcohol dehydrogenase TbSADH mutant of the present invention is a protein obtained by mutating at least one amino acid residue among amino acid residues located in the catalytic center pocket of alcohol dehydrogenase TbSADH.
The amino acid residues of the catalytic center pocket of the alcohol dehydrogenase TbSADH are the amino acid residues shown in the 39 th, 42 th, 84 th, 85 th, 86 th, 101 th, 104 th, 110 th, 294 th and 295 th positions of the amino acid sequence of the alcohol dehydrogenase TbSADH.
Wherein the amino acid residues at positions 39, 42 and 110 are all located in the large pocket of the catalytic center of alcohol dehydrogenase TbSADH; the amino acid residues at positions 84, 85, 86, 101, 104, 294 and 295 are located in the small pocket of the catalytic center of alcohol dehydrogenase TbSADH. The amino acid residues are key sites influencing the enantioselectivity and the enzyme catalytic activity of the alcohol dehydrogenase TbSADH, and the conversion rate of a substrate can be improved and alcohol products with different chiralities can be selectively obtained by modifying the amino acid residues.
Furthermore, the alcohol dehydrogenase TbSADH mutant is a protein obtained by mutating the amino acid residue shown in the 39 th position and/or the 42 th position and/or the 84 th position and/or the 85 th position and/or the 86 th position and/or the 104 th position and/or the 110 th position and/or the 294 th position of the amino acid sequence of the alcohol dehydrogenase TbSADH.
Further, the alcohol dehydrogenase TbSADH mutant includes at least one of the following mutations: the amino acid sequence of the alcohol dehydrogenase TbSADH is mutated from serine to threonine at the 39 th position, the amino acid sequence of the alcohol dehydrogenase TbSADH is mutated from histidine to threonine or alanine or valine or aspartic acid at the 42 th position, the amino acid sequence of the alcohol dehydrogenase TbSADH is mutated from proline to alanine or valine or serine or threonine or tryptophan at the 84 th position, the amino acid sequence of the alcohol dehydrogenase TbSADH is mutated from alanine to glycine or leucine or valine or serine or cysteine or histidine or aspartic acid at the 85 th position, the amino acid sequence of the alcohol dehydrogenase TbSADH is mutated from isoleucine to serine or proline or cysteine or lysine or valine or leucine or alanine or glutamine or glutamic acid or glycine or threonine or arginine at the 86 th position, the amino acid sequence of the alcohol dehydrogenase TbSADH is mutated from glycine to serine, threonine or arginine at the 104 th position, The 110 th site of the amino acid sequence of the alcohol dehydrogenase TbSADH is mutated from threonine to alanine, and the 294 th site of the amino acid sequence of the alcohol dehydrogenase TbSADH is mutated from leucine to isoleucine, phenylalanine or methionine.
In a specific embodiment of the present invention, the alcohol dehydrogenase TbSADH mutant is any one of the following (1) to (40):
(1) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to alanine and keeping other amino acid sequences unchanged;
(2) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to valine and keeping other amino acid sequences unchanged;
(3) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to serine and keeping other amino acid sequences unchanged;
(4) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to threonine while keeping other amino acid sequences unchanged;
(5) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to tryptophan and keeping other amino acid sequences unchanged;
(6) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to serine and keeping other amino acid sequences unchanged;
(7) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to proline and keeping other amino acid sequences unchanged;
(8) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to cysteine and keeping other amino acid sequences unchanged;
(9) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to lysine and keeping other amino acid sequences unchanged;
(10) protein obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to glutamine and keeping other amino acid sequences unchanged;
(11) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to glutamic acid and keeping other amino acid sequences unchanged;
(12) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to glycine and keeping other amino acid sequences unchanged;
(13) protein obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to threonine while keeping other amino acid sequences unchanged;
(14) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to arginine and keeping other amino acid sequences unchanged;
(15) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to glycine, and mutating the 86 th amino acid from isoleucine to valine, and keeping other amino acid sequences unchanged;
(16) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to glycine, and mutating the 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged;
(17) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to glycine, and mutating the 86 th amino acid from isoleucine to cysteine, and keeping other amino acid sequences unchanged;
(18) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to leucine, and mutating the 86 th amino acid from isoleucine to cysteine, and keeping other amino acid sequences unchanged;
(19) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to valine, and mutating the 86 th amino acid from isoleucine to cysteine, and keeping other amino acid sequences unchanged;
(20) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to serine, and mutating the 86 th amino acid from isoleucine to cysteine, and keeping other amino acid sequences unchanged;
(21) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to serine, and mutating the 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged;
(22) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to leucine, and mutating the 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged;
(23) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to cysteine, and mutating the 86 th amino acid from isoleucine to serine, and keeping other amino acid sequences unchanged;
(24) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to serine, and mutating the 86 th amino acid from isoleucine to serine, and keeping other amino acid sequences unchanged;
(25) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to valine, and mutating the 86 th amino acid from isoleucine to serine, and keeping other amino acid sequences unchanged;
(26) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to histidine, and mutating the 86 th amino acid from isoleucine to serine, and keeping other amino acid sequences unchanged;
(27) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to aspartic acid, and mutating the 86 th amino acid from isoleucine to serine, and keeping other amino acid sequences unchanged;
(28) the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to leucine, mutating the 86 th amino acid from isoleucine to leucine, mutating the 104 th amino acid from glycine to serine, and keeping other amino acid sequences unchanged;
(29) the protein is obtained by mutating the 42 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from histidine to threonine, mutating the 85 th amino acid from alanine to glycine, mutating the 86 th amino acid from isoleucine to alanine, and keeping other amino acid sequences unchanged;
(30) the protein is obtained by mutating the 42 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from histidine to alanine, mutating the 85 th amino acid from alanine to glycine, mutating the 86 th amino acid from isoleucine to alanine, and keeping other amino acid sequences unchanged;
(31) the protein is obtained by mutating the 42 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from histidine to valine, mutating the 85 th amino acid from alanine to glycine, mutating the 86 th amino acid from isoleucine to alanine, and keeping other amino acid sequences unchanged;
(32) the protein is obtained by mutating the 42 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from histidine to aspartic acid, mutating the 85 th amino acid from alanine to glycine, mutating the 86 th amino acid from isoleucine to alanine, and keeping other amino acid sequences unchanged;
(33) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to alanine, and mutating the 110 th amino acid from tryptophan to alanine, and keeping other amino acid sequences unchanged;
(34) the protein is obtained by mutating the 39 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from serine to threonine, and mutating the 86 th amino acid from isoleucine to proline, and keeping other amino acid sequences unchanged;
(35) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to proline, and mutating the 110 th amino acid from tryptophan to alanine, and keeping other amino acid sequences unchanged;
(36) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to proline, and mutating the 294 th amino acid from leucine to isoleucine while keeping other amino acid sequences unchanged;
(37) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to proline, and mutating the 294 th amino acid from leucine to phenylalanine, and keeping other amino acid sequences unchanged;
(38) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to proline, and mutating the 294 th amino acid from leucine to methionine, and keeping other amino acid sequences unchanged;
(39) the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine to proline, mutating the 110 th amino acid from tryptophan to alanine, and mutating the 294 th amino acid from leucine to isoleucine while keeping other amino acid sequences unchanged;
(40) a fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of a protein represented by any one of (1) to (39).
In the protein, the amino acid sequence of the alcohol dehydrogenase TbSADH is shown as SEQ ID No. 2.
The label may be the label shown in the following table.
| Label (R) | Residue of | Sequence of |
| Poly-Arg | 5-6 (typically 5) | RRRRR |
| Poly-His | 2-10 (generally 6) | |
| FLAG | ||
| 8 | DYKDDDDK | |
| Strep- |
8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Nucleic acid molecules encoding the above-described alcohol dehydrogenase TbSADH mutants are also within the scope of the present invention.
The nucleic acid molecule for coding the alcohol dehydrogenase TbSADH mutant is the gene as shown in 1) to 41):
1) mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base G to obtain a DNA molecule;
2) a DNA molecule obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base G, mutating the 251 th position from a base C to a base T, and mutating the 252 th position from a base A to a base T;
3) a DNA molecule obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base A, mutating the 251 th position from a base C to a base G, and mutating the 252 th position from a base A to a base T;
4) mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base A to obtain a DNA molecule;
5) a DNA molecule obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base T, mutating the 251 th position from a base C to a base G, and mutating the 252 th position from a base A to a base G;
6) mutating 257 th site of alcohol dehydrogenase TbSADH wild type gene from base T to base G to obtain DNA molecule;
7) a DNA molecule obtained by mutating the 256 th position of the alcohol dehydrogenase TbSADH wild type gene from a base A to a base C, mutating the 257 th position from a base T to a base C, and mutating the 258 th position from a base T to a base A;
8) the DNA molecule is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild type gene from a base A to a base T and mutating the 257 th site from the base T to a base G;
9) mutating the 257 th site of the alcohol dehydrogenase TbSADH wild type gene from a base T to a base A, and mutating the 258 th site from the base T to the base A to obtain a DNA molecule;
10) a DNA molecule obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild type gene from a base A to a base C, mutating the 257 th site from a base T to a base A, and mutating the 258 th site from a base T to a base A;
11) a DNA molecule obtained by mutating the 256 th position of the alcohol dehydrogenase TbSADH wild type gene from a base A to a base G, mutating the 257 th position from a base T to a base A, and mutating the 258 th position from a base T to a base A;
12) the DNA molecule is obtained by mutating the 256 th position of the alcohol dehydrogenase TbSADH wild type gene from a base A to a base G and mutating the 257 th position from a base T to a base G;
13) mutating the 257 th site of the alcohol dehydrogenase TbSADH wild type gene from a base T to a base C, and mutating the 258 th site from the base T to a base A to obtain a DNA molecule;
14) the DNA molecule is obtained by mutating the 256 th position of the alcohol dehydrogenase TbSADH wild type gene from a base A to a base C and mutating the 257 th position from a base T to a base G;
15) the DNA molecule is obtained by mutating the 254 th site of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base G and mutating the 256 th site from a base A to a base G;
16) the DNA molecule is obtained by mutating the 254 th site of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base G and mutating the 256 th site from a base A to a base C;
17) a DNA molecule obtained by mutating the 254 th site of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base G, mutating the 256 th site from a base A to a base T, and mutating the 257 th site from a base T to a base G;
18) a DNA molecule obtained by mutating the 253 rd position of an alcohol dehydrogenase TbSADH wild-type gene from a base G to a base C, mutating the 254 th position from a base C to a base T, mutating the 256 th position from a base A to a base T, and mutating the 257 th position from a base T to a base G;
19) a DNA molecule obtained by mutating the 254 th base C to the T base, the 256 th base A to the T base and the 257 th base T to the G base of the alcohol dehydrogenase TbSADH wild type gene;
20) a DNA molecule obtained by mutating 253 rd position of alcohol dehydrogenase TbSADH wild type gene from base G to base A, mutating 254 th position from base C to base G, mutating 256 th position from base A to base T, and mutating 257 th position from base T to base G;
21) a DNA molecule obtained by mutating the 253 rd position of the alcohol dehydrogenase TbSADH wild type gene from a base G to a base A, mutating the 254 th position from a base C to a base G and mutating the 256 th position from a base A to a base C;
22) a DNA molecule obtained by mutating the 253 rd position of the alcohol dehydrogenase TbSADH wild type gene from a base G to a base C, mutating the 254 th position from a base C to a base T, and mutating the 256 th position from a base A to a base C;
23) a DNA molecule obtained by mutating the 253 rd position of an alcohol dehydrogenase TbSADH wild-type gene from a base G to a base T, mutating the 254 th position from a base C to a base G, and mutating the 257 th position from a base T to a base G;
24) a DNA molecule obtained by mutating the 253 rd position of an alcohol dehydrogenase TbSADH wild type gene from a base G to a base A, mutating the 254 th position from a base C to a base G, and mutating the 257 th position from a base T to a base G;
25) mutating the 254 th site of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base T, and mutating the 257 th site from the base T to a base G to obtain a DNA molecule;
26) a DNA molecule obtained by mutating the 253 rd position of an alcohol dehydrogenase TbSADH wild type gene from a base G to a base C, mutating the 254 th position from the base G to a base A, and mutating the 257 th position from the base T to the base G;
27) the DNA molecule is obtained by mutating the 254 th site of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base A and mutating the 257 th site from a base T to a base G;
28) a DNA molecule obtained by mutating the 253 rd position of the alcohol dehydrogenase TbSADH wild type gene from a base G to a base C, mutating the 254 th position from a base C to a base T, mutating the 256 th position from a base A to a base C, and mutating the 310 th position from a base G to a base A;
29) a DNA molecule obtained by mutating 124 th site of alcohol dehydrogenase TbSADH wild type gene from base C to base A, mutating 125 th site from base A to base C, mutating 126 th site from base T to base A, mutating 254 th site from base C to base G, mutating 256 th site from base A to base G, mutating 257 th site from base T to base C, and mutating 258 th site from base T to base A;
30) a DNA molecule obtained by mutating 124 th site of alcohol dehydrogenase TbSADH wild type gene from base C to base G, mutating 125 th site from base A to base C, mutating 126 th site from base T to base A, mutating 254 th site from base C to base G, mutating 256 th site from base A to base G, mutating 257 th site from base T to base C, and mutating 258 th site from base T to base A;
31) a DNA molecule obtained by mutating 124 th site of alcohol dehydrogenase TbSADH wild type gene from base C to base G, mutating 125 th site from base A to base T, mutating 254 th site from base C to base G, mutating 256 th site from base A to base G, mutating 257 th site from base T to base C, and mutating 258 th site from base T to base A;
32) a DNA molecule obtained by mutating 124 th site of alcohol dehydrogenase TbSADH wild type gene from base C to base G, mutating 254 th site from base C to base G, mutating 256 th site from base A to base G, mutating 257 th site from base T to base C, and mutating 258 th site from base T to base A;
33) a DNA molecule obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene from a base A to a base G, mutating the 257 th site from a base T to a base C, mutating the 258 th site from a base T to a base A, mutating the 328 th site from a base T to a base G, mutating the 329 th site from a base G to a base C, and mutating the 330 th site from a base G to a base A;
34) a DNA molecule obtained by mutating the 115 th site of the alcohol dehydrogenase TbSADH wild-type gene from a base T to a base A, mutating the 117 th site from a base G to a base A, mutating the 256 th site from a base A to a base C, mutating the 257 th site from a base T to a base C, and mutating the 258 th site from a base T to a base A;
35) a DNA molecule obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene from a base A to a base C, mutating the 257 th site from a base T to a base C, mutating the 258 th site from a base T to a base A, mutating the 328 th site from a base T to a base G, mutating the 329 th site from a base G to a base C, and mutating the 330 th site from a base G to a base A;
36) a DNA molecule obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild type gene from a base A to a base C, mutating the 257 th site from a base T to a base C, mutating the 258 th site from a base T to a base A, mutating the 880 th site from a base C to a base A, and mutating the 882 th site from a base A to a base T;
37) a DNA molecule obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild type gene from a base A to a base C, mutating the 257 th site from a base T to a base C, mutating the 258 th site from a base T to a base A, mutating the 880 th site from a base C to a base T, and mutating the 882 th site from a base A to a base T;
38) a DNA molecule obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild type gene from a base A to a base C, mutating the 257 th site from a base T to a base C, mutating the 258 th site from a base T to a base A, mutating the 880 th site from a base C to a base A, and mutating the 882 th site from a base A to a base G;
39) a DNA molecule obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene from a base A to a base C, mutating the 257 th site from a base T to a base C, mutating the 258 th site from a base T to a base A, mutating the 328 th site from a base T to a base G, mutating the 329 th site from a base G to a base C, mutating the 330 th site from a base G to a base A, mutating the 880 th site from a base C to a base A, and mutating the 882 th site from a base A to a base T;
40) a fusion sequence obtained after connecting a tag coding sequence to the 5 'end and/or the 3' end of the DNA molecule defined in 1) to 39);
41) a DNA molecule having 90% or more identity to the DNA molecule defined in 1) to 40) and encoding the above-mentioned alcohol dehydrogenase TbSADH mutant.
The wild-type gene of the alcohol dehydrogenase TbSADH is shown as SEQ ID No. 1.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
The nucleotide sequence encoding the above-described alcohol dehydrogenase TbSADH mutant of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 90% or more identity with the nucleic acid molecule of the present invention are derived from the nucleic acid molecule of the present invention and are identical to the sequence of the present invention, as long as they encode the above-mentioned alcohol dehydrogenase TbSADH mutant and have the same function, and thus fall within the scope of the present invention.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 90% or more, or 95% or more, or 98% or more, or 99% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No. 2. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
In a second aspect, the invention protects any one of the following biomaterials a1) -a 4):
a1) an expression cassette containing a nucleic acid molecule encoding the above-mentioned alcohol dehydrogenase TbSADH mutant;
a2) a recombinant vector comprising a nucleic acid molecule encoding the above-described alcohol dehydrogenase TbSADH mutant;
a3) a recombinant microorganism containing a nucleic acid molecule encoding the above-mentioned alcohol dehydrogenase TbSADH mutant;
a4) a transgenic cell line comprising a nucleic acid molecule encoding the above-described alcohol dehydrogenase TbSADH mutant.
Further, the expression cassette containing a nucleic acid molecule encoding the alcohol dehydrogenase TbSADH mutant according to a1) is a DNA capable of expressing the alcohol dehydrogenase TbSADH mutant in a host cell, and the DNA may include not only a promoter that initiates transcription of a gene encoding the alcohol dehydrogenase TbSADH mutant, but also a terminator that terminates transcription of a gene encoding the alcohol dehydrogenase TbSADH mutant. Still further, the expression cassette may further comprise an enhancer sequence.
a2) The recombinant vector containing the nucleic acid molecule encoding the alcohol dehydrogenase TbSADH mutant can be a bacterial plasmid (such as an expression vector based on a T7 promoter expressed in bacteria, specifically pET-28a and the like), a bacteriophage, a yeast plasmid (such as a YEp series vector and the like) or a retrovirus packaging plasmid, wherein the bacterial plasmid carries the gene encoding the alcohol dehydrogenase TbSADH mutant.
a3) The recombinant microorganism containing the nucleic acid molecule for coding the alcohol dehydrogenase TbSADH mutant can be yeast, bacteria, algae or fungi, such as agrobacterium and the like, which carry the coding gene of the alcohol dehydrogenase TbSADH mutant.
a4) The transgenic cell line containing the nucleic acid molecule encoding the alcohol dehydrogenase TbSADH mutant does not include propagation material.
In a third aspect, the invention provides a novel use of the above-mentioned alcohol dehydrogenase TbSADH mutant or the above-mentioned nucleic acid molecule or the above-mentioned biomaterial.
The invention protects the application of the alcohol dehydrogenase TbSADH mutant or the nucleic acid molecule or the biological material in any one of the following b1) -b 5):
b1) synthesizing or preparing chiral bisaryl alcohol compounds;
b2) synthesizing or preparing (4-chlorphenyl) pyridine-2-methanol;
b3) synthesizing or preparing (S) - (4-chlorphenyl) pyridine-2-methanol;
b4) catalyzing a bisaryl ketone substrate to produce a bisaryl alcohol compound;
b5) catalyzing (4-chlorphenyl) pyridine-2-ketone to generate (S) - (4-chlorphenyl) pyridine-2-methanol.
The bisaryl ketone substrate can be any one of the following: (2-pyridyl) phenyl ketone, (4-fluorophenyl) pyridine-2-ketone, (4-tolyl) pyridine-2-ketone, (4-methoxyphenyl) pyridine-2-ketone, (2-tolyl) pyridine-2-ketone, (3-chlorophenyl) pyridine-2-ketone, 4-chlorobenzophenone and 4-nitrobenzophenone.
The bisaryl alcohol compound may be any one of: (2-pyridyl) phenylmethanol, (4-fluorophenyl) pyridine-2-methanol, (4-tolyl) pyridine-2-methanol, (4-methoxyphenyl) pyridine-2-methanol, (2-tolyl) pyridine-2-methanol, (3-chlorophenyl) pyridine-2-methanol, 4-chlorodiphenylmethanol, and 4-nitrodiphenylmethanol.
In a fourth aspect, the invention provides a method for synthesizing (S) - (4-chlorophenyl) pyridine-2-methanol.
The synthesis method of (S) - (4-chlorphenyl) pyridine-2-methanol provided by the invention comprises the following steps: the alcohol dehydrogenase TbSADH mutant is used as a biological enzyme catalysis substrate to generate (S) - (4-chlorphenyl) pyridine-2-methanol;
the substrate is (4-chlorphenyl) pyridine-2-ketone.
In the method, the alcohol dehydrogenase TbSADH mutant can perform catalysis in the form of crude enzyme solution, crude enzyme solution freeze-dried powder or whole cells.
Further, the whole cell of the alcohol dehydrogenase TbSADH mutant can be prepared by a method comprising the following steps: expressing the alcohol dehydrogenase TbSADH mutant in a host cell to obtain a recombinant cell, namely the whole cell;
lysing the recombinant cells to obtain the crude enzyme solution;
freeze-drying the crude enzyme solution to obtain the crude enzyme solution freeze-dried powder;
still further, the recombinant cell can be prepared according to a method comprising the following steps: introducing the coding gene of the alcohol dehydrogenase TbSADH mutant into a host cell, and obtaining the recombinant cell expressing the alcohol dehydrogenase TbSADH mutant after induction culture.
Further, the gene encoding the alcohol dehydrogenase TbSADH mutant is introduced into the host cell in the form of a recombinant vector. Wherein, the recombinant vector can be a bacterial plasmid (such as an expression vector based on a T7 promoter expressed in bacteria, specifically pET-28a and the like), a bacteriophage, a yeast plasmid (such as YEp series vectors and the like) or a retrovirus packaging plasmid, wherein the bacterial plasmid carries the coding gene of the alcohol dehydrogenase TbSADH mutant.
In one embodiment of the invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing a small fragment between the NcoI and AvrII cleavage sites of pRSFDuet-1 with the gene encoding the alcohol dehydrogenase TbSADH mutant. The coding gene of the alcohol dehydrogenase TbSADH mutant is the gene of 1) to 41) above.
In the method, the host cell may be a prokaryotic cell or a lower eukaryotic cell.
Further, the prokaryotic cell may specifically be a bacterium; the lower eukaryotic cell may specifically be a yeast cell.
Further, the host cell is specifically Escherichia coli.
In one embodiment of the invention, the host cell is e.coli BL21(DE 3). Accordingly, the induction culture is carried out by adding IPTG to the culture system to a final concentration of 0.05-1.0mmol/L (e.g., 0.1mmol/L) and inducing the culture at 20-30 deg.C (e.g., 20 deg.C) for 10-20 hours (e.g., 18 hours).
In the method, in a reaction in which the substrate is catalyzed by the alcohol dehydrogenase TbSADH mutant as a bio-enzyme to produce (S) - (4-chlorophenyl) pyridine-2-methanol, the reaction system may further contain isopropanol and a coenzyme for the alcohol dehydrogenase TbSADH mutant in addition to the substrate and the alcohol dehydrogenase TbSADH mutant.
Further, the coenzyme may be NADP+Or NAD+。
Further, the reaction of catalyzing the substrate to produce (S) - (4-chlorophenyl) pyridine-2-methanol using the alcohol dehydrogenase TbSADH mutant as a bio-enzyme is carried out in a phosphate buffer at a concentration of 0.001-0.1mol/L (e.g., 0.05mol/L), pH 6-8 (e.g., pH 7.4).
The concentration of the substrate in the reaction system is 1-50mmol/L (such as 5 mmol/L); the concentration of the crude enzyme powder of the alcohol dehydrogenase TbSADH mutant in the reaction system is 1-10g/L (such as 10 g/L); the concentration of the whole alcohol dehydrogenase mutant cells in the reaction system is 50-500g/L (such as 100 g/L); the volume percentage of the isopropanol in the reaction system is 10-30% (such as 10%); the NADP+Or the NAD+The concentration in the reaction system is 0.1-1.0mmol/L (e.g., 1.0 mmol/L).
In the method, in a reaction in which the substrate is catalyzed by the alcohol dehydrogenase TbSADH mutant as a bio-enzyme to produce (S) - (4-chlorophenyl) pyridine-2-methanol, the temperature of the reaction may be 20 to 35 ℃, specifically, may be 30 ℃; the reaction time is based on the completion of the reaction, and may be generally 1 to 24 hours, specifically 24 hours.
In the method, the method also comprises a step of extracting (S) - (4-chlorphenyl) pyridine-2-methanol from the reaction liquid after the reaction is finished according to a conventional method in the field.
The invention applies directed evolution technology and method to carry out enzyme modification on alcohol dehydrogenase TbSADH, obtains a series of mutants which have enzyme activity on diaryl ketones represented by (4-chlorphenyl) pyridine-2-ketone, and the mutants can efficiently and asymmetrically reduce and catalyze (4-chlorphenyl) pyridine-2-ketone to generate (S) - (4-chlorphenyl) pyridine-2-methanol. Experiments prove that: the wild-type alcohol dehydrogenase TbSADH before modification has no activity on a substrate (4-chlorophenyl) pyridine-2-methanone, the modified optimal (S) specific mutant can catalyze the (4-chlorophenyl) pyridine-2-methanone to generate (S) - (4-chlorophenyl) pyridine-2-methanol, the conversion rate is 98%, ee% > 99% (S), and the optimal (R) specific mutant can catalyze the CPMK to generate (R) - (4-chlorophenyl) pyridine-2-methanol, the conversion rate is 63%, ee% 49% (R).
Drawings
FIG. 1 is a SDS-PAGE image of the protein of alcohol dehydrogenase TbSADH at 20 ℃ under 0.1mM IPTG induction conditions. M represents a protein Marker; 1 represents the result of SDS-PAGE of TbSADH whole cell disruption solution; 2 represents the SDS-PAGE result of the supernatant after centrifugation of TbSADH; 3 represents the SDS-PAGE result of the precipitate after TbSADH centrifugation; 4 shows the result of SDS-PAGE of the negative control pRSFDuet-1 whole cell lysate; 5 represents SDS-PAGE of the supernatant after centrifugation of pRSFDuet-1; 6 shows the result of SDS-PAGE of the precipitate after centrifugation of pRSFDuet-1.
FIG. 2 is a diagram showing the catalytic reduction of (4-chlorophenyl) pyridine-2-methanone to (S) - (4-chlorophenyl) pyridine-2-methanol by an alcohol dehydrogenase TbSADH mutant.
FIG. 3 is a HPLC detection result chart of the reaction of catalyzing and reducing (4-chlorophenyl) pyridine-2-ketone to (S) - (4-chlorophenyl) pyridine-2-methanol by the alcohol dehydrogenase TbSADH mutant. A: liquid chromatography results of (4-chlorphenyl) pyridine-2-ketone and racemic (4-chlorphenyl) pyridine-2-methanol standard substances; b: the liquid chromatography result of the reaction liquid of the negative control group; c: experimental group 1 (mutant catalysis) reaction liquid chromatography results; d: experimental group 2 (mutant catalysis) reaction solution liquid chromatography results. The negative control in the figure is the reaction catalyzed by the empty vector plasmid pRSFDuet-1; mutant 1 is TbSADH mutant A85G/I86L; mutant 2 is TbSADH mutant I86P/L294I.
FIG. 4 is a diagram showing the whole-cell reaction process of the TbSADH mutant catalyzing (4-chlorophenyl) pyridine-2-methanone to produce (S) - (4-chlorophenyl) pyridine-2-methanol. The abscissa is the reaction time and the ordinate is the substrate conversion. The mutants tested included A85G/I86A, A85G/I86L and A85V/I86S.
FIG. 5 shows the conversion rate and enantioselectivity results of crude TbSADH mutant enzyme powder catalyzing other diaryl ketone substrates. The mutants tested included A85G/I86L and A85V/I86S.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 alcohol dehydrogenase TbSADH and mutant sequences thereof
The amino acid sequence of the alcohol dehydrogenase TbSADH in this example is shown in SEQ ID No. 2. The alcohol dehydrogenase TbSADH mutant in this example is specifically any one of the following (1) to (39):
(1) alcohol dehydrogenase TbSADH mutant P84A: the protein is obtained by mutating the 84 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from proline (P) to alanine (A) and keeping other amino acid sequences unchanged.
(2) Alcohol dehydrogenase TbSADH mutant P84V: the protein is obtained by mutating the 84 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from proline (P) to valine (V) and keeping other amino acid sequences unchanged.
(3) Alcohol dehydrogenase TbSADH mutant P84S: the protein is obtained by mutating the 84 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from proline (P) to serine (S) and keeping other amino acid sequences unchanged.
(4) Alcohol dehydrogenase TbSADH mutant P84T: the protein is obtained by mutating the 84 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from proline (P) to threonine (T) and keeping other amino acid sequences unchanged.
(5) Alcohol dehydrogenase TbSADH mutant P84W: the protein is obtained by mutating the 84 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from proline (P) to tryptophan (W) and keeping other amino acid sequences unchanged.
(6) Alcohol dehydrogenase TbSADH mutant I86S: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to serine (S) and keeping other amino acid sequences unchanged.
(7) Alcohol dehydrogenase TbSADH mutant I86P: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to proline (P) and keeping other amino acid sequences unchanged.
(8) Alcohol dehydrogenase TbSADH mutant I86C: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to cysteine (C) and keeping other amino acid sequences unchanged.
(9) Alcohol dehydrogenase TbSADH mutant I86K: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to lysine (K) and keeping other amino acid sequences unchanged.
(10) Alcohol dehydrogenase TbSADH mutant I86Q: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to glutamine (Q), and keeping other amino acid sequences unchanged.
(11) Alcohol dehydrogenase TbSADH mutant I86E: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to glutamic acid (E) and keeping other amino acid sequences unchanged.
(12) Alcohol dehydrogenase TbSADH mutant I86G: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to glycine (G) and keeping other amino acid sequences unchanged.
(13) Alcohol dehydrogenase TbSADH mutant I86T: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to threonine (T) and keeping other amino acid sequences unchanged.
(14) Alcohol dehydrogenase TbSADH mutant I86R: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to arginine (R) and keeping other amino acid sequences unchanged.
(15) Alcohol dehydrogenase TbSADH mutant A85G/I86V: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to glycine (G), and mutating the 86 th amino acid from isoleucine (I) to valine (V), and keeping other amino acid sequences unchanged.
(16) Alcohol dehydrogenase TbSADH mutant A85G/I86L: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to glycine (G), and mutating the 86 th amino acid from isoleucine (I) to leucine (L), and keeping other amino acid sequences unchanged.
(17) Alcohol dehydrogenase TbSADH mutant A85G/I86C: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to glycine (G), and mutating the 86 th amino acid from isoleucine (I) to cysteine (C), and keeping other amino acid sequences unchanged.
(18) Alcohol dehydrogenase TbSADH mutant A85L/I86C: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to leucine (L), and mutating the 86 th amino acid from isoleucine (I) to cysteine (C), and keeping other amino acid sequences unchanged.
(19) Alcohol dehydrogenase TbSADH mutant A85V/I86C: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to valine (V), and mutating the 86 th amino acid from isoleucine (I) to cysteine (C), and keeping other amino acid sequences unchanged.
(20) Alcohol dehydrogenase TbSADH mutant A85S/I86C: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to serine (S), and mutating the 86 th amino acid from isoleucine (I) to cysteine (C), and keeping other amino acid sequences unchanged.
(21) Alcohol dehydrogenase TbSADH mutant A85S/I86L: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to serine (S), and mutating the 86 th amino acid from isoleucine (I) to leucine (L), and keeping other amino acid sequences unchanged.
(22) Alcohol dehydrogenase TbSADH mutant A85L/I86L: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to leucine (L), and mutating the 86 th amino acid from isoleucine (I) to leucine (L), and keeping other amino acid sequences unchanged.
(23) Alcohol dehydrogenase TbSADH mutant A85C/I86S: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to cysteine (C), and mutating the 86 th amino acid from isoleucine (I) to serine (S), and keeping other amino acid sequences unchanged.
(24) Alcohol dehydrogenase TbSADH mutant A85S/I86S: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to serine (S), and mutating the 86 th amino acid from isoleucine (I) to serine (S), and keeping other amino acid sequences unchanged.
(25) Alcohol dehydrogenase TbSADH mutant A85V/I86S: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to valine (V), and mutating the 86 th amino acid from isoleucine (I) to serine (S), and keeping other amino acid sequences unchanged.
(26) Alcohol dehydrogenase TbSADH mutant A85H/I86S: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to histidine (H), and mutating the 86 th amino acid from isoleucine (I) to serine (S), and keeping other amino acid sequences unchanged.
(27) Alcohol dehydrogenase TbSADH mutant A85D/I86S: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to aspartic acid (D), and mutating the 86 th amino acid from isoleucine (I) to serine (S), and keeping other amino acid sequences unchanged.
(28) Alcohol dehydrogenase TbSADH mutant A85L/I86L/G104S: the protein is obtained by mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine (A) to leucine (L), mutating the 86 th amino acid from isoleucine (I) to leucine (L), mutating the 104 th amino acid from glycine (G) to serine (S), and keeping other amino acid sequences unchanged.
(29) Alcohol dehydrogenase TbSADH mutant H42T/A85G/I86A: the protein is obtained by mutating the 42 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from histidine (H) to threonine (T), mutating the 85 th amino acid from alanine (A) to glycine (G), and mutating the 86 th amino acid from isoleucine (I) to alanine (A), while keeping the other amino acid sequences unchanged.
(30) Alcohol dehydrogenase TbSADH mutant H42A/A85G/I86A: the protein is obtained by mutating the 42 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from histidine (H) to alanine (A), mutating the 85 th amino acid from alanine (A) to glycine (G), mutating the 86 th amino acid from isoleucine (I) to alanine (A), and keeping other amino acid sequences unchanged.
(31) Alcohol dehydrogenase TbSADH mutant H42V/A85G/I86A: the protein is obtained by mutating the 42 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from histidine (H) to valine (V), mutating the 85 th amino acid from alanine (A) to glycine (G), mutating the 86 th amino acid from isoleucine (I) to alanine (A), and keeping other amino acid sequences unchanged.
(32) Alcohol dehydrogenase TbSADH mutant H42D/A85G/I86A: the protein is obtained by mutating the 42 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from histidine (H) to aspartic acid (D), mutating the 85 th amino acid from alanine (A) to glycine (G), mutating the 86 th amino acid from isoleucine (I) to alanine (A), and keeping other amino acid sequences unchanged.
(33) Alcohol dehydrogenase TbSADH mutant I86A/W110A: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to alanine (A), and mutating the 110 th amino acid from tryptophan (W) to alanine (A), and keeping other amino acid sequences unchanged.
(34) Alcohol dehydrogenase TbSADH mutant S39T/I86P: the protein is obtained by mutating the 39 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from serine (S) to threonine (T), and mutating the 86 th amino acid from isoleucine (I) to proline (P), and keeping other amino acid sequences unchanged.
(35) Alcohol dehydrogenase TbSADH mutant I86P/W110A: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to proline (P), and mutating the 110 th amino acid from tryptophan (W) to alanine (A), and keeping other amino acid sequences unchanged.
(36) Alcohol dehydrogenase TbSADH mutant I86P/L294I: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to proline (P), and mutating the 294 th amino acid from leucine (L) to isoleucine (I), and keeping other amino acid sequences unchanged.
(37) Alcohol dehydrogenase TbSADH mutant I86P/L294F: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to proline (P), and mutating the 294 th amino acid from leucine (L) to phenylalanine (F), and keeping other amino acid sequences unchanged.
(38) Alcohol dehydrogenase TbSADH mutant I86P/L294M: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to proline (P), and mutating the 294 th amino acid from leucine (L) to methionine (M), and keeping other amino acid sequences unchanged.
(39) Alcohol dehydrogenase TbSADH mutant I86P/W110A/L294I: the protein is obtained by mutating the 86 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from isoleucine (I) to proline (P), mutating the 110 th amino acid from tryptophan (W) to alanine (A), and mutating the 294 th amino acid from leucine (L) to isoleucine (I), and keeping other amino acid sequences unchanged.
Example 2 preparation of engineering bacteria of alcohol dehydrogenase TbSADH Gene or its mutant
Preparation of alcohol dehydrogenase TbSADH gene wild type gene engineering strain
1. Optimization of alcohol dehydrogenase TbSADH gene sequence
The method comprises the steps of carrying out codon optimization on an alcohol dehydrogenase TbSADH gene derived from high-temperature anaerobic bacillus (Thermoanaerobacter brockii) by taking escherichia coli as a host cell, carrying out whole-gene synthesis after optimization, wherein the sequence of the optimized alcohol dehydrogenase TbSADH gene is shown as SEQ ID No. 1.
2. Construction of recombinant vector pRSFDuet-1-TbSADH
The DNA fragment shown in SEQ ID No.1 was homologously recombined onto the pRSFDuet-1 plasmid using the primers pRSF-NcoI (ggt ata tct tat taa agt taa aca aaa ttaa tta ttt ctacag) and pRSF-AvrII (taa cct agg ctg ctg cca ccg ctg agc aac). The recombinant plasmid, which was sequenced to show that the small fragment between the restriction sites NcoI and AvrII of the pRSFDuet-1 plasmid was the DNA fragment shown in SEQ ID No.1, was designated pRSFDuet-1-TbSADH.
3. Construction of recombinant bacterium
And (2) electrically transferring the recombinant vector pRSFDuet-1-TbSADH into E.coli BL21(DE3) competent cells, carrying out inverted culture in an LB solid plate containing kanamycin (Kan) resistance for 12-16h, selecting positive transformants, carrying out colony PCR and DNA sequencing verification, and verifying the correct transformants to be alcohol dehydrogenase TbSADH gene wild-type genetic engineering strains.
Preparation of di-alcohol dehydrogenase TbSADH gene mutant engineering strain
Engineering strains of alcohol dehydrogenase TbSADH gene mutants expressing the respective alcohol dehydrogenase TbSADH mutants in example 1 were constructed, respectively. Part of the alcohol dehydrogenase TbSADH mutant is obtained by designing a degenerate primer to construct a mutant library and screening, and the other part of the alcohol dehydrogenase TbSADH mutant is obtained by designing a mutant primer. The designed degenerate primer and mutant primer sequences are shown in tables 1 and 2, respectively.
TABLE 1 primer sequences used for mutant library construction
TABLE 2 mutants and primer sequences used
Note: the mutation sites are underlined.
The specific construction method comprises the following steps:
1. PCR reaction
And (3) taking the recombinant vector pRSFDuet-1-TbSADH constructed in the step one as a template, and respectively carrying out two rounds of PCR reactions by adopting primers corresponding to the mutants. The PCR reaction system and procedure were as follows:
the first round of PCR system is 50 μ L, and comprises the following components: 50ng of template (pRSFDuet-1-TbSADH); PrimeStar DNA polymerase (2.5U/. mu.L) 0.5. mu.L; dNTP (2.5mmol/L) 4. mu.L; 2 XPS Buffer 25 uL; dd H218.5 mu L of O; 1 μ L of the pre-primer (10 μ M); the rear primer (10. mu.M) was 1. mu.L. Specific sequences of primers used for each mutant are shown in tables 1 and 2.
First round PCR procedure: pre-denaturation at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 30 seconds; annealing at 56 deg.C for 15 seconds; extension at 72 ℃ for 40 seconds; final extension 72 ℃ for 10 min. The number of cycles was 32.
The second round of PCR system was 50. mu.L, comprising the following components: template (pR)SFDuet-1-TbSADH)50ng；PrimeStar DNA polymerase(2.5U/μL)0.5μL；dNTP(2.5mmol/L)4μL；2×PS Buffer 25μL；dd H218.5 mu L of O; first round PCR product 2. mu.L.
Second round PCR procedure: pre-denaturation at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 30 seconds; annealing at 60 ℃ for 15 seconds; extension at 72 ℃ for 7 minutes; final extension 72 ℃ for 10 min. The number of cycles was 28.
2. Obtaining and screening of alcohol dehydrogenase TbSADH gene mutant engineering strain
After the step 1 is completed, adding 2 mu L of Dpn I enzyme into each reaction system, digesting for 2 hours at 37 ℃, taking 1 mu L of Dpn I enzyme to electrically transfer into E.coli BL21(DE3) competent cells, putting the E.coli BL21(DE3) competent cells into a 37 ℃ incubator to perform inverted culture for 12-16 hours, when transformants grow out, picking transformants to perform sequencing verification, and obtaining transformants which are alcohol dehydrogenase TbSADH gene mutant engineering strains with correct sequencing verification, wherein the transformants are named as recombinant strain P84A, recombinant strain P84V, recombinant strain P84S, recombinant strain P84T, recombinant strain P84W, recombinant strain I86S, recombinant strain I86P, recombinant strain I86C, recombinant strain I86/C A C/C, recombinant strain I72/C, C A C/C, C I72/C I72, C I72, C I72/C I72, Recombinant bacterium A85L/I86L, recombinant bacterium A85C/I86S, recombinant bacterium A85S/I86S, recombinant bacterium A85V/I86S, recombinant bacterium A85H/I86S, recombinant bacterium A85D/I86S, recombinant bacterium A85L/I86L/G104S, recombinant bacterium H42T/A85T/I86T, recombinant bacterium H42/A85T/I86T, recombinant bacterium H42T/A85T/I86T, recombinant bacterium I86T/W110T, recombinant bacterium I T/L294, recombinant bacterium I86/L T, recombinant bacterium I T/L T/W36294 and recombinant bacterium L T/36294.
The recombinant strain P84A is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84A into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84A is obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base C to a base G.
The recombinant strain P84V is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84V into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84V is obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from base C to base G, mutating the 251 th position from base C to base T, and mutating the 252 th position from base A to base T.
The recombinant strain P84S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84S is obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from base C to base A, mutating the 251 th position from base C to base G, and mutating the 252 th position from base A to base T.
The recombinant strain P84T is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84T into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84T is obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base C to a base A.
The recombinant strain P84W is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84W into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84W is obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from base C to base T, mutating the 251 th position from base C to base G, and mutating the 252 th position from base A to base G.
The recombinant strain I86S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86S is obtained by mutating the 257 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base T to a base G.
The recombinant strain I86P is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86P into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86P is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from the base A to the base C, mutating the 257 th site from the base T to the base C, and mutating the 258 th site from the base T to the base A.
The recombinant strain I86C is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86C into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86C is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from the base A to the base T and mutating the 257 th site from the base T to the base G.
The recombinant strain I86K is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86K into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86K is obtained by mutating the 257 th base of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from T to A and mutating the 258 th base from T to A.
The recombinant strain I86Q is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86Q into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86Q is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from the base A to the base C, mutating the 257 th site from the base T to the base A, and mutating the 258 th site from the base T to the base A.
The recombinant strain I86E is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86E into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86E is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from the base A to the base G, mutating the 257 th site from the base T to the base A, and mutating the 258 th site from the base T to the base A.
The recombinant strain I86G is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86G into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86G is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from the base A to the base G and mutating the 257 th site from the base T to the base G.
The recombinant strain I86T is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86T into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86T is obtained by mutating the 257 th base of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from T to C and mutating the 258 th base from T to A.
The recombinant strain I86R is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86R into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86R is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from the base A to the base C and mutating the 257 th site from the base T to the base G.
The recombinant strain A85G/I86V is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85G/I86V into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85G/I86V is obtained by mutating the 254 th base C to the G base G and the 256 th base A to the G base of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH.
The recombinant strain A85G/I86L is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85G/I86L into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85G/I86L is obtained by mutating the 254 th base C to the G base and the 256 th base A to the C base of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH.
The recombinant strain A85G/I86C is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85G/I86C into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85G/I86C is obtained by mutating the 254 th base C to the G base, the 256 th base A to the T base and the 257 th base T to the G base of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH.
The recombinant strain A85L/I86C is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85L/I86C into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84A is obtained by mutating the 253 rd position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from the base G to the base C, mutating the 254 th position from the base C to the base T, mutating the 256 th position from the base A to the base T, and mutating the 257 th position from the base T to the base G.
The recombinant strain A85V/I86C is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85V/I86C into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85V/I86C is obtained by mutating the 254 th base C to the T base, the 256 th base A to the T base and the 257 th base T to the G base of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH.
The recombinant strain A85S/I86C is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85S/I86C into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85S/I86C is obtained by mutating the 253 rd position of the wild-type gene of the alcohol dehydrogenase TbSADH in pRSFDuet-1-TbSADH from a base G to a base A, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base T, and mutating the 257 th position from a base T to a base G.
The recombinant strain A85S/I86L is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85S/I86L into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85S/I86L is obtained by mutating the 253 rd position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base G to a base A, mutating the 254 th position from a base C to a base G, and mutating the 256 th position from a base A to a base C.
The recombinant strain A85L/I86L is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85L/I86L into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85L/I86L is obtained by mutating the 253 rd position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base G to a base C, mutating the 254 th position from a base C to a base T, and mutating the 256 th position from a base A to a base C.
The recombinant strain A85C/I86S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85C/I86S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85C/I86S is obtained by mutating the 253 rd position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base G to a base T, mutating the 254 th position from a base C to a base G, and mutating the 257 th position from a base T to a base G.
The recombinant strain A85S/I86S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85S/I86S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85S/I86S is obtained by mutating the 253 rd position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base G to a base A, mutating the 254 th position from a base C to a base G, and mutating the 257 th position from a base T to a base G.
The recombinant strain A85V/I86S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85V/I86S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85V/I86S is obtained by mutating the 254 th base C to the 254 th base T and the 257 th base T to the 257 th base G of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH.
The recombinant strain A85H/I86S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85H/I86S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85H/I86S is obtained by mutating the 253 rd position of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base G to a base C, mutating the 254 th position from a base G to a base A, and mutating the 257 th position from a base T to a base G.
The recombinant strain A85D/I86S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85D/I86S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84A is obtained by mutating the 254 th base C to the A base and the 257 th base T to the G base of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH.
The recombinant strain A85L/I86L/G104S is a strain obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant A85L/I86L/G104S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant A85L/I86L/G104S is obtained by mutating the 253 rd position of the wild-type gene of the alcohol dehydrogenase TbSADH in pRSFDuet-1-TbSADH from a base G to a base C, mutating the 254 th position from a base C to a base T, mutating the 256 th position from a base A to a base C, and mutating the 310 th position from a base G to a base A.
The recombinant strain H42T/A85G/I86A is a strain obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant H42T/A85G/I86A into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant H42T/A85G/I86A is obtained by mutating the 124 th site of the wild-type gene of the alcohol dehydrogenase TbSADH in pRSFDuet-1-TbSADH from a base C to a base A, mutating the 125 th site from a base A to a base C, mutating the 126 th site from a base T to a base A, mutating the 254 th site from a base C to a base G, mutating the 256 th site from a base A to a base G, mutating the 257 th site from a base T to a base C, and mutating the 258 th site from a base T to a base A.
The recombinant strain H42A/A85G/I86A is a strain obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant H42A/A85G/I86A into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant H42A/A85G/I86A is obtained by mutating the 124 th site of the wild-type gene of the alcohol dehydrogenase TbSADH in pRSFDuet-1-TbSADH from a base C to a base G, mutating the 125 th site from a base A to a base C, mutating the 126 th site from a base T to a base A, mutating the 254 th site from a base C to a base G, mutating the 256 th site from a base A to a base G, mutating the 257 th site from a base T to a base C, and mutating the 258 th site from a base T to a base A.
The recombinant strain H42V/A85G/I86A is a strain obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant H42V/A85G/I86A into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant H42V/A85G/I86A is obtained by mutating the 124 th site of the wild-type gene of the alcohol dehydrogenase TbSADH in pRSFDuet-1-TbSADH from a base C to a base G, mutating the 125 th site from a base A to a base T, mutating the 254 th site from a base C to a base G, mutating the 256 th site from a base A to a base G, mutating the 257 th site from a base T to a base C, and mutating the 258 th site from a base T to a base A.
The recombinant strain H42D/A85G/I86A is a strain obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant H42D/A85G/I86A into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant H42D/A85G/I86A is obtained by mutating the 124 th site of the wild-type gene of the alcohol dehydrogenase TbSADH in pRSFDuet-1-TbSADH from a base C to a base G, mutating the 254 th site from a base C to a base G, mutating the 256 th site from a base A to a base G, mutating the 257 th site from a base T to a base C, and mutating the 258 th site from a base T to a base A.
The recombinant strain I86A/W110A is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86A/W110A into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86A/W110A is obtained by mutating the 256 th site of the wild-type gene of the alcohol dehydrogenase TbSADH in pRSFDuet-1-TbSADH from the base A to the base G, mutating the 257 th site from the base T to the base C, mutating the 258 th site from the base T to the base A, mutating the 328 th site from the base T to the base G, mutating the 329 rd site from the base G to the base C, and mutating the 330 th site from the base G to the base A.
The recombinant strain S39T/I86P is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant S39T/I86P into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant S39T/I86P is obtained by mutating the 115 th site of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH from a base T to a base A, mutating the 117 th site from a base G to a base A, mutating the 256 th site from a base A to a base C, mutating the 257 th site from a base T to a base C, and mutating the 258 th site from a base T to a base A.
The recombinant strain I86P/W110A is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86P/W110A into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86P/W110A is obtained by mutating the 256 th site of the wild-type gene of the alcohol dehydrogenase TbSADH in pRSFDuet-1-TbSADH from the base A to the base C, mutating the 257 th site from the base T to the base C, mutating the 258 th site from the base T to the base A, mutating the 328 th site from the base T to the base G, mutating the 329 rd site from the base G to the base C, and mutating the 330 th site from the base G to the base A.
The recombinant strain I86P/L294I is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86P/L294I into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86P/L294I is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH from the base A to the base C, mutating the 257 th site from the base T to the base C, mutating the 258 th site from the base T to the base A, mutating the 880 th site from the base C to the base A, and mutating the 882 th site from the base A to the base T.
The recombinant strain I86P/L294F is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86P/L294F into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86P/L294F is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH from the base A to the base C, mutating the 257 th site from the base T to the base C, mutating the 258 th site from the base T to the base A, mutating the 880 th site from the base C to the base T, and mutating the 882 th site from the base A to the base T.
The recombinant strain I86P/L294M is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86P/L294M into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86P/L294M is obtained by mutating the 256 th site of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH from the base A to the base C, mutating the 257 th site from the base T to the base C, mutating the 258 th site from the base T to the base A, mutating the 880 th site from the base C to the base A, and mutating the 882 nd site from the base A to the base G.
The recombinant strain I86P/W110A/L294I is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant I86P/W110A/L294I into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant I86P/W110A/L294I is obtained by mutating the 256 th site of the wild-type gene of the alcohol dehydrogenase TbSADH in pRSFDuet-1-TbSADH from a base A to a base C, mutating the 257 th site from a base T to a base C, mutating the 258 th site from a base T to a base A, mutating the 328 th site from a base T to a base G, mutating the 329 rd site from a base G to a base C, mutating the 330 th site from a base G to a base A, mutating the 880 th site from a base C to a base A, and mutating the 882 th site from a base A to a base T.
Example 3 expression of alcohol dehydrogenase TbSADH Gene or its mutant and preparation of Whole cell, crude enzyme solution and crude enzyme powder
The wild-type gene engineering strain of the alcohol dehydrogenase TbSADH gene prepared in the first step of the embodiment 2 and the 39 engineering strains of the alcohol dehydrogenase TbSADH gene mutants prepared in the second step are induced to express to obtain the wild-type alcohol dehydrogenase TbSADH and each of the alcohol dehydrogenase TbSADH mutants in the embodiment 1. The method comprises the following specific steps:
respectively picking recombinant bacteria E.coli BL21(DE3) transformants containing recombinant plasmids of alcohol dehydrogenase TbSADH genes or mutants thereof to 5mL LB liquid culture medium containing 50 mu g/mL kanamycin, shaking at 37 ℃ and 220rpm overnight for 12-16 hours, respectively inoculating the transformants into TB liquid culture medium containing 50 mu g/mL kanamycin according to the inoculation amount of 1 percent (volume percentage content), and culturing at 37 ℃ until OD is achieved600When the concentration is 0.7, adding IPTG with the final concentration of 0.1mmol/L, inducing expression at 20 ℃ and 220rpm for 18h, centrifuging at 4 ℃ and 4000rpm for 10min, collecting thalli, re-suspending the collected thalli by potassium phosphate buffer (50mM, pH7.4) to obtain alcohol dehydrogenase TbSADH whole cells, and then ultrasonically crushing the thalli cells under the ice bath condition to obtain an ultrasonically crushed sample (namely crude enzyme liquid). After disruption, one portion of the sample was subjected to SDS-PAGE, and the other portion was centrifuged at 12000rpm at 4 ℃ for 10min, and then the supernatant was subjected to freeze-drying to prepare a crude enzyme powder.
The collected protein samples were examined by SDS-PAGE using vertical electrophoresis, the gel concentration of SDS-PAGE was 12%, concentration electrophoresis was performed using 80V followed by separation electrophoresis using 150V. The result shows that the constructed, expressed and purified alcohol dehydrogenase TbSADH and the mutant protein thereof are soluble expression, and the sizes of the TbSADH and the mutant protein thereof are all about 37kDa (shown in figure 1), and are consistent with the expected sizes.
Example 4 alcohol dehydrogenase TbSADH and its mutant crude enzyme powder catalyzing production of (S) - (4-chlorophenyl) pyridine-2-methanone from (4-chlorophenyl) pyridine-2-methanol
The wild-type alcohol dehydrogenase TbSADH and each of the alcohol dehydrogenase TbSADH mutants prepared in example 3 were used to catalyze (4-chlorophenyl) pyridine-2-methanone to produce (S) - (4-chlorophenyl) pyridine-2-methanol, respectively, and the catalytic effect on the asymmetric reduction of prochiral bisarylketone (4-chlorophenyl) pyridine-2-methanone was determined. A schematic diagram of the catalytic reduction of (4-chlorophenyl) pyridine-2-methanone to (S) - (4-chlorophenyl) pyridine-2-methanol is shown in FIG. 2.
The components of the asymmetric reduction reaction system and the concentration thereof in the system are respectively as follows: substrate ((4-chlorphenyl) pyridine-2-ketone) 10mmol/L, recombinant alcohol dehydrogenase TbSADH (alcohol dehydrogenation)Mutant of enzyme TbSADH, crude enzyme powder, prepared in example 3) 10g/L, isopropanol 10% (volume fraction), NADP+1mmol/L, phosphate buffer 50mmol/L, pH 7.4. The asymmetric reduction reaction was carried out at 30 ℃ for 24 hours.
After the reaction was completed, the conversion was calculated and stereoselectivity analysis was performed. The specific method comprises the following steps: and (3) adding 500 mu L of reaction liquid into 500 mu L of ethyl acetate, shaking for 1-2 min, centrifuging at 12000rpm for 2-5 min, taking the supernatant into a centrifuge tube, adding anhydrous sodium sulfate, drying at 4 ℃ overnight, taking the supernatant into the centrifuge tube, adding 500 mu L of chromatographic grade isopropanol when the organic phase is completely naturally volatilized, and carrying out liquid phase analysis on the conversion rate and the ee value. The HPLC detection conditions were as follows: chiralpak AD-H (5 μm, 250 mm. times.4.6 mm) liquid chromatography column, mobile phase n-hexane: isopropanol (90:10, V/V), flow rate of 1mL/min, column temperature of 30 ℃, ultraviolet detection wavelength of 220nm, and sample injection amount of 1 muL.
The HPLC detection result spectrum of the reaction for catalyzing and reducing (4-chlorophenyl) pyridine-2-ketone to generate (S) - (4-chlorophenyl) pyridine-2-methanol by the alcohol dehydrogenase TbSADH mutant is shown in figure 3. Retention time (S) - (4-chlorophenyl) pyridine-2-methanol was 9.8min, and (R) - (4-chlorophenyl) pyridine-2-methanol was 12.2 min. The optical purity of the product was evaluated by the enantiomeric excess (ee): ee ═ aS-AR)/(AS+AR)×100％；AS: peak area values of (S) - (4-chlorophenyl) pyridine-2-methanol obtained by liquid chromatography; a. theR: the peak area value of the obtained (R) - (4-chlorophenyl) pyridine-2-methanol was analyzed by liquid chromatography.
The results are shown in Table 3. As shown in Table 3, the mutant of alcohol dehydrogenase TbSADH can catalyze asymmetric reduction catalysis (4-chlorophenyl) pyridine-2-ketone to generate (S) - (4-chlorophenyl) pyridine-2-methanol, the conversion rate is 0.7-99%, and the stereoselectivity is 20-99%.
TABLE 3 analysis of conversion and stereoselectivity of asymmetric reduction catalysis (4-chlorophenyl) pyridine-2-methanone by alcohol dehydrogenase TbSADH and its mutant
Note: WT stands for wild type TbSADH and "-" for too low an ee value for conversion is not shown.
Example 5 Whole-cell catalysis of alcohol dehydrogenase mutant for production of (S) - (4-chlorophenyl) pyridine-2-methanone
The catalytic effect of (4-chlorophenyl) pyridine-2-methanone on asymmetric reduction of prochiral bisarylketone (4-chlorophenyl) pyridine-2-methanone was determined by whole-cell catalysis of (4-chlorophenyl) pyridine-2-methanone with the alcohol dehydrogenase TbSADH mutants (A85G/I86A, A85G/I86L, and A85V/I86S) with higher conversion rate in Table 3. A schematic diagram of the catalytic reduction of (4-chlorophenyl) pyridine-2-methanone to (S) - (4-chlorophenyl) pyridine-2-methanol is shown in FIG. 1.
The components of the asymmetric reduction reaction system and the concentration thereof in the system are respectively as follows: substrate ((4-chlorophenyl) pyridine-2-one) 50mmol/L, recombinant alcohol dehydrogenase TbSADH (alcohol dehydrogenase TbSADH mutant, whole cell, prepared in example 3) 100g/L, isopropanol 10% (volume fraction), NADP+0.1mmol/L, 50mmol/L phosphate buffer, pH 7.4. The asymmetric reduction reaction condition is 30 ℃, and the reaction is carried out until the substrate is completely converted according to the catalytic rate of different mutants.
After the reaction was completed, the conversion was calculated and stereoselectivity analysis was performed. The specific method comprises the following steps: and (3) adding 500 mu L of reaction liquid into 500 mu L of ethyl acetate, shaking for 1-2 min, centrifuging at 12000rpm for 2-5 min, taking the supernatant into a centrifuge tube, adding anhydrous sodium sulfate, drying at 4 ℃ overnight, taking the supernatant into the centrifuge tube, adding 500 mu L of chromatographic grade isopropanol when the organic phase is completely naturally volatilized, and carrying out liquid phase analysis on the conversion rate and the ee value. The HPLC detection conditions were as follows: chiralpak AD-H (5 μm, 250 mm. times.4.6 mm) liquid chromatography column, mobile phase n-hexane: isopropanol (90:10, V/V), flow rate of 1mL/min, column temperature of 30 ℃, ultraviolet detection wavelength of 220nm, and sample injection amount of 1 muL.
The substrate conversion rate in the reaction process of the TbSADH mutant is shown in FIG. 4, and as can be seen from FIG. 4, the reaction rate of the TbSADH mutant A85G/I86L is fastest, the complete conversion of the substrate (4-chlorophenyl) pyridine-2-ketone (1.09g) can be achieved within 4 hours, while the reaction rates of the other two mutants are slower, and the conversion rate can only reach 92-97% after 40 hours of reaction.
Example 6 catalysis of crude enzyme powder of alcohol dehydrogenase mutant on other bisaryl ketone substrates
Other diaryl ketone substrates are catalyzed by the wild type alcohol dehydrogenase TbSADH and the crude enzyme powder of the alcohol dehydrogenase TbSADH mutant (A85G/I86L, A85V/I86S) prepared in example 3 respectively, so as to widen the substrate spectrum of the mutant. The bisaryl ketone substrates were (2-pyridyl) phenyl ketone (3a), (4-fluorophenyl) pyridine-2-ketone (3b), (4-tolyl) pyridine-2-ketone (3c), (4-methoxyphenyl) pyridine-2-ketone (3d), (2-tolyl) pyridine-2-ketone (3e), (3-chlorophenyl) pyridine-2-ketone (3f), 4-chlorobenzophenone (3g) and 4-nitrobenzophenone (3h), respectively, and the corresponding products were (2-pyridyl) phenyl methanol, (4-fluorophenyl) pyridine-2-methanol, (4-tolyl) pyridine-2-methanol, (4-methoxyphenyl) pyridine-2-methanol, respectively, (2-tolyl) pyridine-2-methanol, (3-chlorophenyl) pyridine-2-methanol, 4-chlorobenzhydrol and 4-nitrobenzyl alcohol.
The components of the asymmetric reduction reaction system and the concentration thereof in the system are respectively as follows: substrate 10mmol/L, recombinant alcohol dehydrogenase TbSADH (alcohol dehydrogenase TbSADH mutant, crude enzyme powder, prepared in example 3) 10g/L, isopropanol 10% (volume fraction), NADP+1mmol/L, phosphate buffer 50mmol/L, pH 7.4. The asymmetric reduction reaction was carried out at 30 ℃ for 24 hours.
After the reaction was completed, the conversion was calculated and stereoselectivity analysis was performed. The specific method comprises the following steps: and (3) adding 500 mu L of reaction liquid into 500 mu L of ethyl acetate, shaking for 1-2 min, centrifuging at 12000rpm for 2-5 min, taking the supernatant into a centrifuge tube, adding anhydrous sodium sulfate, drying at 4 ℃ overnight, taking the supernatant into the centrifuge tube, adding 500 mu L of chromatographic grade isopropanol when the organic phase is completely naturally volatilized, and carrying out liquid phase analysis on the conversion rate and the ee value. The HPLC detection conditions are shown in Table 4 below.
TABLE 4 HPLC detection method for asymmetric reduction catalysis of other bisaryl ketone substrates by alcohol dehydrogenase TbSADH and mutants thereof
aDetection conditions are as follows: the flow rate is 1 mL/min; the temperature is 30 ℃; the detection wavelength is 220 nm;
m1: TbSADH mutant A85G/I86L; m2: TbSADH mutant A85V/I86S.
The results of HPLC measurements are shown in FIG. 5 and Table 5. As shown in FIG. 5 and Table 5, the mutant of alcohol dehydrogenase TbSADH has good catalytic activity on other biaryl ketone substrates, except that the mutant has good catalytic activity and enantioselectivity on the substrate (4-chlorphenyl) pyridine-2-ketone.
TABLE 5 analysis of conversion and stereoselectivity of TbSADH and its mutants by asymmetric reduction catalysis of other bisaryl ketone substrates
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> alcohol dehydrogenase mutant and application thereof in synthesis of chiral diaryl alcohol compound
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<170>PatentIn version 3.5
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ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480
ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540
atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600
ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660
ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720
atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780
accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840
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Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
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Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
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Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
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Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp
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Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ile Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
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Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
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Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
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Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
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Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
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Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
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Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
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Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
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Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
305 310 315 320
Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
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| CN112852770A (en) * | 2020-11-04 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Alcohol dehydrogenase mutant and application thereof in preparing chiral diaryl alcohol compound by efficient asymmetric reduction |
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| CN112852770B (en) * | 2020-11-04 | 2022-01-07 | 中国科学院天津工业生物技术研究所 | Alcohol dehydrogenase mutants and their application in the preparation of chiral biaryl alcohol compounds by efficient asymmetric reduction |
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