Method for producing 1,2-amino alcohol compound by whole cell transformation

Abstract

The present invention discloses a method for producing a 1,2-amino alcohol compound by utilizing whole-cell transformation, and belongs to the technical field of gene engineering and microorganism engineering. According to the present invention, engineered Escherichia coli co-expresses epoxide hydrolase, alcohol dehydrogenase, -transaminase and glutamate dehydrogenase, is capable of realizing whole-cell catalysis of an epoxide in one step to synthesize a 1,2-amino alcohol compound, and meanwhile, can realize regeneration of coenzyme NADP.sup.+ and an amino doner L-Glu; alcohol dehydrogenase expressed by the engineered Escherichia coli is RBS optimized alcohol dehydrogenase, and such RBS optimization can control the expression quantity of alcohol dehydrogenase, so that the catalysis rate of alcohol dehydrogenase and transaminase can achieve an optimum ratio, to eliminate influence caused by a rate-limiting step in a catalyzing course.

Claims

1. An engineered Escherichia coli, wherein the engineered Escherichia coli comprises recombinant plasmid A and recombinant plasmid B; the recombinant plasmid A comprising a target gene A and an expression vector; the recombinant plasmid B comprising a target gene B, a target gene C, and an expression vector; the target gene A being a gene encoding epoxide hydrolase (SpEH) comprising SEQ ID NO: 1; the target gene B being a gene encoding alcohol dehydrogenase (MnADH) comprising SEQ ID NO: 2; the target gene C being a gene encoding w-transaminase (PAK-TA), wherein the amino acid sequence expressed by target gene C comprises SEQ ID NO: 3; and wherein the recombinant plasmid A also comprises a target gene D; the target gene D being a gene encoding glutamate dehydrogenase (GluDH) comprising SEQ ID NO: 5.

2. The engineered Escherichia coli according to claim 1, wherein the alcohol dehydrogenase (MnADH) is optimized by RBS; RBS optimization of alcohol dehydrogenase (MnADH) meaning that an RBS sequence used for regulating alcohol dehydrogenase (MnADH) and located at the upstream of alcohol dehydrogenase (MnADH) on recombinant plasmid B is substituted; the substituted RBS sequence comprising SEQ ID NO: 4.

3. A method for producing a 1,2-amino alcohol compound, wherein the method uses the engineered Escherichia coli described in claim 1.

4. A method for producing a 1,2-amino alcohol compound, the method comprising: providing a catalysis system comprising a substrate selected from epoxyethylbenzene, epoxypropane, epoxybutane, epichlorohydrin or epoxypentane and the engineered Escherichia coli according to claim 1, adding coenzyme NADP+, amino donor L-Glu, and ammonium chloride, and reacting for 10-15 hours.

Description

DETAILED DESCRIPTION

(1) Further descriptions will be made to the present invention below in combination with specific embodiments.

(2) E. coli BL21 competent cell involved in the following embodiments is purchased from Shanghai Sangon Biotech Co., Ltd.

(3) Detection method involved in the following embodiments are as follows:

(4) An SpEH Enzyme Activity Determination Method:

(5) 1 mL of a reaction system includes 150 L of 10 mM epoxyethylbenzene and 50 L of enzyme solution;

(6) enzyme activity definition: enzyme amount for generating phenyl 1,2-glycol by transforming 1 mol of epoxyethylbenzene for 1 min at 37 C. is defined as one enzyme activity unit (U).

(7) A PAK-TA Enzyme Activity Determination Method:

(8) 1 mL of a reaction system includes 150 L of 10 mM epoxyethylbenzene and 50 L of enzyme solution;

(9) enzyme activity definition: enzyme amount for generating 2-amino1-phenethyl alcohol by transforming 1 mol of hydroxyl phenylacetaldehyde for 1 min at 37 C. is defined as one enzyme activity unit (U).

(10) An MnADH Enzyme Activity Determination Method:

(11) 1 mL of a reaction system includes 790 L of 50 mM phosphate buffer solution (pH 8.0), 150 L of 10 mM phenyl 1,2-glycol, 10 L of 1 mol NADP+ and 50 L of enzyme solution;

(12) after reaction is ended, determining activity according to NADPH light absorption value variation of a reaction solution in 340 nm;

(13) enzyme activity definition: enzyme amount needed for generating 1 mol of NADPH in 1 min.

(14) A GluDH Enzyme Activity Determination Method:

(15) 1 mL of a reaction system includes 790 L of 50 mM phosphate buffer solution (pH 8.0), 150 L of 10 mM 2-oxoglutaric acid, 10 L of 1 mol NADPH and 50 L of enzyme solution;

(16) after reaction is ended, determining activity according to NADPH light absorption value variation of a reaction solution in 340 nm;

(17) enzyme activity definition: enzyme amount needed for generating 1 mol of NADPH in 1 min.

(18) An Epoxide Consumption Determination Method:

(19) Chromatographic conditions: a chromatographic column: DinosoilC18 (5 L, 250 nm4.6 nm), a mobile phase: acetonitrile-water (V/V=85:15), column temperature: 30 C., a sample size: 10 L, a flow rate: 1.0 mL/min.

(20) After chromatography is ended, detecting a characteristic absorption peak at 220 nm ultraviolet wavelength, wherein the concentration of a substrate standard sample is 0.5 g/L.

(21) A 1,2-Amino Alcohol Compound Yield Determination Method:

(22) Chromatographic conditions: a chromatographic column: DinosoilC18 (5 L, 250 nm4.6 nm), a mobile phase: acetonitrile-water (V/V=85:15), column temperature: 30 C., a sample size: 10 L, a flow rate: 1.0 mL/min.

(23) After chromatography is ended, detecting a characteristic absorption peak at 220 nm ultraviolet wavelength, wherein the concentration of a product standard sample is 0.5 g/L.

(24) A Determination Method of Intermediate Product Vicinal Diol and 1,2-Amino Alcohol Compound

(25) Chromatographic conditions: a chromatographic column: Sepax Carbomix H-NP (10:8, 7.8 mm*300 mm), a mobile phase: 3 mM perchloric acid solution, column temperature: 50 C., a sample size: 10 L, a flow rate: 1.0 mL/min.

(26) After chromatography is ended, detecting a characteristic absorption peak at 3380 nm ultraviolet wavelength, wherein the concentration of an intermediate product standard sample is 0.5 g/L.

(27) Culture media involved in the following embodiments are as follows:

(28) An LB solid culture medium: 10 g/L of peptone, 5 g/L of yeast extract, 10 g/L of NaCl, 0.2 g/L of agar powder.

(29) An LB liquid culture medium: 10 g/L of peptone, 5 g/L of yeast extract, 10 g/L of NaCl.

Embodiment 1: Construction of Recombinant Plasmid

(30) Specific steps are as follows:

(31) (1) design PCR primers P1 and P2 (SEQ ID NO.6 and SEQ ID NO.7) of epoxide hydrolase SpEH according to a speh gene sequence (SEQ ID NO.1) in a whole genome nucleotide sequence of Sphingomonas sp. HXN-200 in NCBI;

(32) (2) design PCR primers P3 and P4 (SEQ ID NO.8 and SEQ ID NO.9) of alcohol dehydrogenase MnADH according to a mnadh gene sequence (SEQ ID NO.2) in a whole genome nucleotide sequence of Mycobacterium neoaurum in NCBI;

(33) (3) design PCR primers P5 and P6 (SEQ ID NO.10 and SEQ ID NO.11) of -transaminase PAK-TA according to a pak-ta gene sequence (an amino acid sequence being SEQ ID NO.3) in a whole genome nucleotide sequence of Pseudomonas aeruginosa PAK in NCBI;

(34) (4) design PCR primers P7 and P8 (SEQ ID NO.13 and SEQ ID NO.14) of -transaminase Cv-TA according to a cv-ta gene sequence (SEQ ID NO.12) in a whole genome nucleotide sequence of Chromobacterium violaceum in NCBI;

(35) (5) design PCR primers P9 and P10 (SEQ ID NO.16 and SEQ ID NO.17) of transaminase PPTA according to a ppta gene sequence (SEQ ID NO.15) in a whole genome nucleotide sequence of Pseudomonas putida in NCBI;

(36) (6) design PCR primers P11 and P12 (SEQ ID NO.19 and SEQ ID NO.20) of transaminase VFTA according to a vfta gene sequence (an amino acid sequence is as shown in SEQ ID NO.18) in a whole genome nucleotide sequence of Vibrio fluvialis in NCBI;

(37) (7) perform PCR amplification by utilizing the foregoing primers by taking the foregoing genome DNA as a template, amplification conditions being: initial denaturation at 95 C. for 5 min, one cycle; denaturation at 95 C. for 1 min, annealing at 58 C. for 1 min, extension at 72 C. for 1 min 30 s, 30 cycles; final extension at 72 C. for 10 min, and after amplification is ended, purify and recycle PCR products by adopting a gel extraction kit; and

(38) (8) connect recycling products speh and pACYCDuet by PCR after digestion with BamH I and Hind II, connect mnadh, pak-ta and pETDuet by PCR after digestion with BamH I/Hind III and Bgl II/EcoR V respectively, connect mnadh, cv-ta and pETDuet by PCR after digestion with BamH I/Hind III and Bgl II/EcoR V respectively, connect mnadh, ppta and pETDuet by PCR after digestion with BamH I/Hind III and Bgl II/EcoR V respectively, and connect mnadh, vfta and pETDuet by PCR after digestion with amH I/Hind III and Bgl II/EcoR V respectively, to obtain recombinant plasmids pACYCDuet-speh, pETDuet-mnadh-pak-ta, pETDuet-mnadh-cv-ta, pETDuet-mnadh-ppta, pETDuet-mnadh-vfta.

Embodiment 2: Construction of Recombinant Bacteria

(39) Specific steps are as follows:

(40) Put 100 L of E. coli BL21 competent cell into a 1.5 mL centrifugal tube, respectively add 5 L of recombinant plasmids pACYCDuet-speh and pETDuet-mnadh-pak-ta, pACYCDuet-speh and pETDuet-mnadh-cv-ta, pACYCDuet-speh and pETDuet-mnadh-ppta, pACYCDuet-speh and pETDuet-mnadh-vfta needing transformation, blow and suck gently, place on ice for 45 min; perform precise heat shock on the centrifugal tube at 42 C. for 90 s, then place on ice again for 5 min, then add 800 L of LB liquid culture medium, perform shake cultivation for 1-1.5 h at 37 C.; remove most supernatant after centrifugation, block and suck again for suspension, coat residual bacteria solution to an LB dish with ampicillin resistance and chlorampenicol resistance, and extract plasmids for verification after transformants come out, to obtain recombinant plasmids BL21/pACYCDuet-speh&pETDuet-mnadh-pak-ta, BL21/pACYCDuet-speh&pETDuet-mnadh-cv-ta, BL21/pACYCDuet-speh&pETDuet-mnadh-ppta and BL21/pACYCDuet-speh&pETDuet-mnadh-vfta.

Embodiment 3: Verification of Recombinant Bacteria

(41) Specific steps are as follows:

(42) (1) activate recombinant plasmids BL21/pACYCDuet-speh&pETDuet-mnadh-pak-ta, BL21/pACYCDuet-speh&pETDuet-mnadh-cv-ta, BL21/pACYCDuet-speh&pETDuet-mnadh-ppta and BL21/pACYCDuet-speh&pETDuet-mnadh-vfta obtained in embodiment 2 with an LB culture medium and then culture for 12 h in the conditions of 37 C., 160 r/min, to obtain a seed solution;

(43) (2) inoculate the seed solution to 100 mL of LB liquid culture medium at an inoculation amount of 1%, and continue to culture for 2 h until OD.sub.600 is 0.8, to obtain a fermentation broth;

(44) (3) add IPTG with final concentration of 0.8 mM to the fermentation broth, induce for 12 h at 28 C., centrifuge for 10 min in the conditions of 4 C., 8000 r/min and collect thalli;

(45) (4) rinse the thalli with a phosphate buffer solution with pH of 7.5 for two times, then add the thalli into a catalysis system and react for 10 h at 37 C. to obtain a reaction solution, wherein OD.sub.600 of the thalli in the catalysis system is equal to 30, besides thalli, the catalysis system also contains 100 mM of substrate epoxyethylbenzene, 5 mM of L-Glu, 0.02 mM of NADP.sup.+, 0.35 mM of PLP and 275 mM of NH.sub.4Cl (pH 8.0); and

(46) (5) dilute the reaction solution and filter with a 0.22 m filter membrane and then perform HPLC analysis.

(47) An HPLC analysis result shows that: after whole-cell transformation of 100 mM of epoxyethylbenzene with recombinant bacteria BL21/pACYCDuet-speh&pETDuet-mnadh-pak-ta for 10 h, the amount of a product, 2-amino1-phenethyl alcohol, is 22.4 mM, the accumulation amount of an intermediate product, phenyl 1,2-glycol, is 76.5 mM, and another intermediate product, hydroxyl phenylacetaldehyde, is not accumulated;

(48) after whole-cell transformation of 100 mM of epoxyethylbenzene with recombinant bacteria BL21/pACYCDuet-speh&pETDuet-mnadh-cv-ta for 10 h, the amount of a product, 2-amino1-phenethyl alcohol, is 10.2 mM, the accumulation amount of an intermediate product, phenyl 1,2-glycol, is 78.4 mM, and the accumulation amount of another intermediate product, hydroxyl phenylacetaldehyde, is 11.2 mM;

(49) after whole-cell transformation of 100 mM of epoxyethylbenzene with recombinant bacteria BL21/pACYCDuet-speh&pETDuet-mnadh-ppta for 10 h, the amount of a product, 2-amino1-phenethyl alcohol, is 0 mM, the accumulation amount of an intermediate product, phenyl 1,2-glycol, is 82 mM, and the accumulation amount of another intermediate product, hydroxyl phenylacetaldehyde, is 16.5 mM; and

(50) after whole-cell transformation of 100 mM of epoxyethylbenzene with recombinant bacteria BL21/pACYCDuet-speh&pETDuet-mnadh-vfta for 10 h, the amount of a product, 2-amino1-phenethyl alcohol, is 3.5 mM, the accumulation amount of an intermediate product, phenyl 1,2-glycol, is 80.2 mM, and the accumulation amount of another intermediate product, hydroxyl phenylacetaldehyde, is 13.6 mM;

(51) It is known from a result that: when trying to use four kinds of -transaminase from different sources, the yield of -transaminase sourcing from Pseudomonas aeruginosa PAK is the highest, indicating that PAK-TA enzyme activity is higher than the enzyme activity of enzyme from the other three sources, as a result, -transaminase PAK-TA should be selected to construct recombinant bacteria.

(52) Furthermore, it found, by detecting the content of intermediate products, phenyl 1,2-glycol and hydroxyl phenylacetaldehyde, in a catalysis reaction process, that a large amount of intermediate product phenyl 1,2-glycol is accumulated, hydroxyl phenylacetaldehyde is not accumulated, while the action of alcohol dehydrogenase MnADH in a whole catalysis system is to catalyze intermediate product phenyl 1,2-glycol to synthesize hydroxyl phenylacetaldehyde, and the action of -transaminase PAK-TA is to catalyze hydroxyl phenylacetaldehyde to synthesize a final product 2-amino1-phenethyl alcohol; when a crude enzyme solution of alcohol dehydrogenase MnADH is added from the outside, intermediate product phenyl 1,2-glycol may be gradually consumed and finally be totally transformed to be the final product 2-amino1-phenethyl alcohol, in a whole catalysis process, intermediate product hydroxyl phenylacetaldehyde is not accumulated, and the result shows that preparation of a 1,2-amino alcohol compound by transformation of recombinant bacteria BL21/pACYCDuet-speh&pETDuet-mnadh-pak-ta will be limited by relatively low enzyme activity of alcohol dehydrogenase, while the transformation rate of other enzymes is not limited, finally resulting in massive accumulation of intermediate products and relatively low yield of a final product, as a result, it still needs to further improve the enzyme activity of alcohol dehydrogenase in a whole tandem catalysis system.

Embodiment 4: RBS Sequence Optimization of Alcohol Dehydrogenase

(53) Specific steps are as follows:

(54) (1) design PCR upstream primers r1, r2, r3, r4, r5 (sequences being as shown in SEQ ID NO: 25-SEQ ID NO: 29) containing RBS sequences (sequences being as shown in SEQ ID NO: 4, SEQ ID NO: 21-SEQ ID NO: 24) of different intensities according to a T7 promotor on pETDuet plasmid and a gene sequence of MnADH;

(55) (2) constitute primer pairs from upstream primers r1, r2, r3, r4, r5 containing RBS sequences of different intensities and downstream primer r6, r7, r8, r9, r10 (sequences being as shown in SEQ ID NO: 30-SEQ ID NO: 34) by taking recombinant plasmid pETDuet-mnadh-pak-ta as a template, perform PCR to obtain multiple segments of an alcohol dehydrogenase gene containing RBS sequences of different intensities, connect the segments with linearized pETDuet-pak-ta, to obtain co-expression plasmids pETDuet-r1-mnadh-pak-ta, pETDuet-r2-mnadh-pak-ta, pETDuet-r3-mnadh-pak-ta, pETDuet-r4-mnadh-pak-ta, pETDuet-r5-mnadh-pak-ta with different RBS intensities;

(56) (3) transform recombinant plasmids into competent E. coli BL21, and screen correct transformants, to obtain co-expression recombinant bacteria after different RBS optimization of alcohol dehydrogenase;

(57) (4) perform induced expression on constructed recombinant bacteria according to conditions in embodiment 3, then collect and wash, and then re-suspend recombinant Escherichia coli to 10 mL of 50 mM phosphate buffer solution; and

(58) (5) perform ultrasonic disruption on suspended cells, stopping for 3 s after disrupting for 1 s, working for 15 min, put a disruption solution into a centrifugal machine, centrifuge for 25 min at a rate of 10000 r/min at 4 C. to remove precipitates, and measure MnADH enzyme activity in a supernatant.

(59) It is known from a result that: enzyme activity of MnADH in recombinant bacteria pETDuet-r1-mnadh-pak-ta, pETDuet-r2-mnadh-pak-ta, pETDuet-r3-mnadh-pak-ta, pETDuet-r4-mnadh-pak-ta, pETDuet-r5-mnadh-pak-ta is respectively 0.78 U/mL, 0.58 U/mL, 0.52 U/mL, 0.43 U/mL, 0.38 U/mL, which is improved to a certain extent in comparison with original bacterium pETDuet-mnadh-pak-ta without RBS optimization (enzyme activity of MnADH of an original bacterium is 0.32 U/mL).

(60) Therefore, an RBS sequence with sequence of SEQ ID NO: 4 should be selected to optimize alcohol dehydrogenase.

Embodiment 5: Introduction of Glutamate Dehydrogenase

(61) It is found from the foregoing experiments that coenzyme NADP+ and amino donor L-Glu need to be continuously provided for a three-enzyme catalysis system, while introduction of glutamate dehydrogenase may regenerate NADP and L-Glu consumed by a reaction system, and therefore, an attempt may be made to introduction of glutamate dehydrogenase, with specific steps as follows:

(62) (1) design PCR primers P7 and P8 (SEQ ID NO.35 and SEQ ID NO.36) of glutamate dehydrogenase according to gludh gene sequence (SEQ ID NO.5) in a whole genome nucleotide sequence of Escherichia coli Bl21 in NCBI;

(63) (2) perform PCR amplification by utilizing the foregoing primers by taking the foregoing genome DNA as a template, and connect a recycling product and pACYCDuet-speh by PCR after digestion with Nde I and EcoR, to obtain recombinant plasmid pACYCDuet-speh-gludh;

(64) (3) transform recombinant plasmid pACYCDuet-speh-gludh and recombinant plasmid pETDuet-r1-mnadh-pak-ta after RBS sequence optimization into competent E. coli BL21 at the same time, and if verification is correct, obtain recombinant bacterium E. coli BL21 (SGMP) capable of co-expressing four enzymes;

(65) (4) activate obtained recombinant bacterium E. coli BL21 (SGMP) with an LB culture medium and then culture for 12 h in the conditions of 37 C., 160 r/min to obtain a seed solution;

(66) (5) inoculate the seed solution to 100 mL of LB liquid culture medium at an inoculation amount of 1%, and continue to culture for 2 h until OD.sub.600 is 0.8, to obtain a fermentation broth;

(67) (6) add IPTG with final concentration of 0.8 mM to the fermentation broth, induce for 12 h at 28 C., centrifuge for 10 min in the conditions of 4 C., 8000 r/min and collect thalli;

(68) (7) rinse the thalli with a phosphate buffer solution with pH of 7.5 for two times, then add the thalli into a catalysis system and react for 10 h at 37 C. to obtain a reaction solution, wherein OD.sub.600 of the thalli in the catalysis system is equal to 30, besides thalli, the catalysis system also contains 100 mM of substrate epoxyethylbenzene, 5 mM of L-Glu, 0.02 mM of NADP.sup.+, 0.35 mM of PLP and 275 mM of NH.sub.4Cl (pH 8.0); and

(69) (8) dilute the reaction solution and filter with a 0.22 m filter membrane and then perform HPLC analysis.

(70) An HPLC analysis result shows that: after whole-cell transformation of 100 mM of epoxyethylbenzene with recombinant bacteria for 10 h, the amount of a product, 2-amino1-phenethyl alcohol, is 96.5 mM, without accumulation of intermediate products and generation of by-product 2-oxoglutaric acid.

(71) It is known from a result that recombinant bacterium E. coli BL21 (SGMP) may be efficiently transformed to prepare a 1,2-amino alcohol compound, and coenzyme and amino donor do not need to be continuously added in the whole reaction process.

Embodiment 6: Four-enzyme Tandem Co-expression

(72) Specific steps are as follows:

(73) (1) connect the foregoing recycling products speh and gludh with pETDuet-r1-mnadh-pak-ta by PCR respectively after digestion with Kpn I/Xho I and Nde I/EcoR V, to obtain recombinant plasmid pETDuet-r1-mnadh-pak-ta-speh-gludh;

(74) (2) transform pETDuet-r1-mnadh-pak-ta-speh-gludh into competent E. coli BL21, and if verification is correct, obtain recombinant bacterium E. coli BL21/pETDuet-r1-mnadh-pak-ta-speh-gludh with four enzymes expressed on a same plasmid in a tandem mode;

(75) (3) referring to embodiment 5, transform 100 mM of substrate epoxyethylbenzene by using E. coli BL21/pETDuet-r1-mnadh-pak-ta-speh-gludh; and

(76) (4) dilute a reaction solution and filter with a 0.22 m filter membrane and then perform HPLC analysis.

(77) An HPLC analysis result shows that: after whole-cell transformation of 100 mM of epoxyethylbenzene with recombinant bacteria E. coli BL21/pETDuet-r1-mnadh-pak-ta-speh-gludh for 10 h, the amount of a product, 2-amino1-phenethyl alcohol, is 50.6 mM, and intermediate products phenyl 1,2-glycol (24 mM) and hydroxyl phenylacetaldehyde (14.5 mM) are accumulated.

(78) It is known from a result that co-expression of four enzymes on a same plasmid will be limited by unbalance of expression level of each enzyme, and therefore, it is of great importance to select suitable expression plasmids to achieve an optimum adaption relation.

Embodiment 7: Application of Recombinant Bacteria

(79) Specific steps are as follows:

(80) Taking epoxypropane as a substrate: activate recombinant bacterium E. coli BL21 (SGMP) obtained in embodiment 5 with an LB culture medium and then culture for 12 h in the conditions of 37 C., 160 r/min, to obtain a seed solution; inoculate the seed solution to 100 mL of LB liquid culture medium at an inoculation amount of 1%, and continue to culture for 2 h until OD.sub.600 is 0.8, to obtain a fermentation broth; add IPTG with final concentration of 0.8 mM to the fermentation broth, induce for 12 h at 28 C., centrifuge for 10 min in the conditions of 4 C., 8000 r/min and collect thalli; rinse the thalli with a phosphate buffer solution with pH of 7.5 for two times, then add the thalli into a catalysis system and react for 10 h at 37 C. to obtain a reaction solution, wherein OD.sub.600 of the thalli in the catalysis system is equal to 30, besides thalli, the catalysis system also contains 100 mM of substrate epoxypropane, 5 mM of L-Glu, 0.02 mM of NADP.sup.+, 0.35 mM of PLP and 275 mM of NH.sub.4Cl (pH 8.0); and dilute the reaction solution and filter with a 0.22 m filter membrane and then perform HPLC analysis.

(81) Taking epoxybutane as a substrate: activate recombinant bacterium E. coli BL21 (SGMP) obtained in embodiment 5 with an LB culture medium and then culture for 12 h in the conditions of 37 C., 160 r/min, to obtain a seed solution; inoculate the seed solution to 100 mL of LB liquid culture medium at an inoculation amount of 1%, and continue to culture for 2 h until OD.sub.600 is 0.8, to obtain a fermentation broth; add IPTG with final concentration of 0.8 mM to the fermentation broth, induce for 12 h at 28 C., centrifuge for 10 min in the conditions of 4 C., 8000 r/min and collect thalli; rinse the thalli with a phosphate buffer solution with pH of 7.5 for two times, then add the thalli into a catalysis system and react for 10 h at 37 C. to obtain a reaction solution, wherein OD.sub.600 of the thalli in the catalysis system is equal to 30, besides thalli, the catalysis system also contains 100 mM of substrate epoxybutane, 5 mM of L-Glu, 0.02 mM of NADP.sup.+, 0.35 mM of PLP and 275 mM of NH.sub.4Cl (pH 8.0); and dilute the reaction solution and filter with a 0.22 m filter membrane and then perform HPLC analysis.

(82) An HPLC analysis result shows that: after whole-cell transformation of 100 mM of epoxypropane with recombinant bacteria 10 h, the amount of a product, 2-amino1-phenethyl alcohol, is 94.3 mM, after whole-cell transformation of 100 mM of epoxybutane with recombinant bacteria 10 h, the amount of a product, 2-amino1-phenethyl alcohol, is 97.5 mM, and the molar yield of corresponding generated 1,2-amino alcohol compound respectively achieve 94.3% and 97.5%.

(83) Although the present disclosure has been disclosed above through the preferred embodiments, the embodiments are not intended to limit the present disclosure, a person skilled in the art can make various variations and modifications without departing from the spirit and scope of the present invention, therefore, the protection scope of the present invention should be subject to the appended claims.

(84) The present invention discloses a method for producing a 1,2-amino alcohol compound by utilizing whole-cell transformation, and belongs to the technical field of gene engineering and microorganism engineering. According to the present invention, engineered Escherichia coli co-expresses epoxide hydrolase, alcohol dehydrogenase, -transaminase and glutamate dehydrogenase, is capable of realizing whole-cell catalysis of an epoxide in one step to synthesize a 1,2-amino alcohol compound, and meanwhile, can realize regeneration of coenzyme NADP.sup.+ and an amino doner L-Glu; alcohol dehydrogenase expressed by the engineered Escherichia coli is RBS optimized alcohol dehydrogenase, and such RBS optimization can control the expression quantity of alcohol dehydrogenase, so that the catalysis rate of alcohol dehydrogenase and transaminase can achieve an optimum ratio, to eliminate influence caused by a rate-limiting step in a catalyzing course; by utilizing the method disclosed by the present invention, multiple epoxides may be taken as substrates to perform whole-cell transformation to prepare a corresponding 1,2-amino alcohol compound, and therefore, the method has an important industrial application value.