WO2023155474A1 - meso-2,3-丁二醇脱氢酶及其突变体和应用 - Google Patents

meso-2,3-丁二醇脱氢酶及其突变体和应用 Download PDF

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WO2023155474A1
WO2023155474A1 PCT/CN2022/127954 CN2022127954W WO2023155474A1 WO 2023155474 A1 WO2023155474 A1 WO 2023155474A1 CN 2022127954 W CN2022127954 W CN 2022127954W WO 2023155474 A1 WO2023155474 A1 WO 2023155474A1
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meso
mutated
amino acid
butanediol
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于浩然
蒲中机
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浙江大学杭州国际科创中心
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    • C—CHEMISTRY; METALLURGY
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01004—R,R-butanediol dehydrogenase (1.1.1.4)
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    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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  • the invention relates to the technical field of enzyme engineering, in particular to a meso-2, 3-butanediol dehydrogenase and its mutant and application.
  • 2,3-Butanediol is a multifunctional platform chemical used in the manufacture of drugs, cosmetics, food additives, fuels and solvents, among which meso-2,3-butanediol is a precursor of 2-butanol, It is also a preservative and humectant in cosmetics, widely used in biofuel and food industries.
  • 2,3-Butanediol can be synthesized by chemical and biochemical pathways. Biological pathways are more environmentally and economically advantageous due to the use of low-cost renewable carbon sources, reduced greenhouse gas emissions, and selective production of homochiral 2,3-butanediol. Carbon sources in the production process can also use renewable raw materials in agriculture, reducing substrate costs and making biological processes more environmentally friendly.
  • the 2,3-butanediol market is estimated to be worth around USD 220 million by 2027. Therefore, the industrial production of bio-based 2,3-butanediol is expected to be vigorously developed in the coming years.
  • 2,3-butanediol is chemically catalyzed from cracking gases in non-renewable petroleum at 800-900 °C, requiring a lot of energy.
  • a large amount of greenhouse gases are produced, so it is a non-environmentally friendly process.
  • the chemical route is a conventional method for 2,3-butanediol production, it is expensive and complicated, and the 2,3-butanediol produced is a racemic mixture, and its purification is costly.
  • the biological synthesis of 2,3-butanediol only requires mild operating conditions, such as lower temperature and pressure.
  • 2,3-butanediol with high optical purity can be produced from low-cost raw materials and simple reaction conditions.
  • the invention provides a meso-2 , 3-butanediol dehydrogenase and its mutants and applications.
  • the invention provides a meso-2,3-butanediol dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.1.
  • the enzyme has the characteristic of high temperature resistance.
  • the present invention also provides a meso-2,3-butanediol dehydrogenase mutant, which is represented by the 40th, 51st, 72nd, 73rd, and 162nd amino acid sequence shown in SEQ ID NO.1 , No. 192, No. 197, and No. 204 were obtained by single-point mutation or multi-point combination mutation.
  • Aspartic acid at position 202 of the amino acid sequence shown in SEQ ID NO.1 is mutated to cysteine, glutamic acid, glycine, proline or tryptophan;
  • Asparagine at position 197 of the amino acid sequence shown in SEQ ID NO.1 is mutated to glycine, lysine, serine or valine;
  • the 162nd isoleucine of the amino acid sequence shown in SEQ ID NO.1 is mutated to threonine, and the 202nd aspartic acid is mutated to tryptophan;
  • the 162nd isoleucine of the amino acid sequence shown in SEQ ID NO.1 is mutated to threonine, the 192nd histidine is mutated to tryptophan, and the asparagine is mutated to serine.
  • the present invention is derived from Lactococcus lactis meso-2,3-butanediol dehydrogenase (LlBDH, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) AlphaFold 2 Modeling of homotetramer structure, molecular docking with natural substrate acetoin, using accelerated sampling molecular dynamics simulation to determine 8 key amino acid residues in the process of substrate binding/product release, which were subjected to saturation mutation, Meso-2,3-butanediol dehydrogenase mutants were screened by enzyme activity assay and residual enzyme activity after heating in a metal bath at 100°C for 10 minutes. Finally, the sequential iterative mutation of these sites and the combined mutation of the dominant sites were used to obtain highly active and stable meso-2,3-butanediol dehydrogenase mutants.
  • LlBDH Lactococcus lactis meso-2,3-butaned
  • mutants are Q40K, F51M, E72K, K73T, I162T, H192W, N197S, W204Y.
  • Q40K means: the amino acid at position 40 is mutated from glutamine to arginine;
  • F51M means: the amino acid at position 51 is mutated from phenylalanine to methionine;
  • E72K means: the amino acid at position 72 is mutated from glutamine Amino acid is mutated to arginine;
  • K73T means: amino acid at position 73 is mutated from arginine to threonine;
  • I162T means: amino acid at position 162 is mutated from leucine to threonine;
  • H192W means: amino acid at position 192 N197S means that the amino acid at position 197 is mutated from asparagine to serine;
  • W204Y means that the amino acid at position 204 is mutated from tryptophan to tyrosine.
  • meso-2,3-butanediol dehydrogenase variant is obtained by multi-point combination mutation, and the multi-point combination mutation form is as follows:
  • each mutation site and the single-letter abbreviation of the amino acid before and after the mutation are: Q40K, F51M, E72K, K73T, I162T, H192W, N197S, W204Y;
  • glutamate dehydrogenase variant is one of the following multiple point mutations:
  • I162T/H192W means that the 162nd leucine is mutated into threonine, and the 192nd amino acid is replaced by histidine acid mutation to tryptophan.
  • the present invention also provides a coding gene of the above-mentioned meso-2,3-butanediol dehydrogenase or the above-mentioned meso-2,3-butanediol dehydrogenase mutant.
  • the invention also provides a recombinant vector comprising the coding gene. Further, the original expression vector of the recombinant vector is pET28a-SUMO.
  • the invention also provides a genetically engineered bacterium comprising the coding gene. Further, the host cell of the genetic engineering bacteria is E.coli BL21(DE3).
  • the present invention also provides the above-mentioned meso-2,3-butanediol dehydrogenase or the above-mentioned meso-2,3-butanediol dehydrogenase mutant in catalyzing acetoin to generate meso-2, 3-butanediol application.
  • the present invention also provides the application of the above-mentioned genetically engineered bacteria in catalyzing acetoin to produce meso-2,3-butanediol.
  • the present invention has the following beneficial effects:
  • the present invention has discovered a meso-2,3-butanediol dehydrogenase with high temperature resistance, and based on the meso-2,3-butanediol dehydrogenase, by accelerating sampling molecular dynamics Using a chemical simulation method to dynamically describe the product release process, solve the problem of high stability and high activity, and obtain a mutant that can catalyze the preparation of meso-2,3-butanediol, which has very good high temperature resistance. After heat treatment at 100°C for 30 minutes, the residual enzyme activity was 23.9%. The activity of the mutant in catalyzing the formation of meso-2,3-butanediol from acetoin was increased by about 2-5 times.
  • the product meso-2,3-butanediol obtained from the reaction has extremely high optical purity, which provides a broad application prospect for the production of meso-2,3-butanediol through biotransformation.
  • the rational design method used in the present invention can quickly obtain high-stability and high-activity meso-2,3-butanediol dehydrogenase mutants through screening with a small mutant library.
  • Fig. 1 is the electrophoretic analysis of purified meso-2,3-butanediol dehydrogenase LlBDH on SDS-PAGE.
  • the genome extraction kit and DpnI used in the examples of the present invention were purchased from TaKaRa, Treasure Bioengineering (Dalian) Co., Ltd.; the Exnase II seamless cloning kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. ; Plasmid extraction kits and DNA recovery and purification kits were purchased from Axygen Hangzhou Co., Ltd.; E.coli BL21 (DE3), plasmid pET28a-SUMO, etc.
  • acetoin, meso-2,3-butanediol, NAD + and NADH were purchased from Shanghai Macklin Biochemical Technology Co., Ltd.
  • enzyme activity unit (U) Under standard reaction conditions, enzyme activity unit (U) is defined as the amount of enzyme required to consume or produce 1 ⁇ mol NADH per minute.
  • the meso-2,3-butanediol dehydrogenase gene was cloned from Lactococcus lactis, inserted into the pET28a-SUMO plasmid, and then transformed into E.coli BL21(DE3) to obtain pET28a -SUMO-LlBDH expressing strain.
  • Table 1 The primers used for cloning target fragments and linearized vectors
  • the PCR amplified product was purified and recovered with SanPrep Column DNA Gel Recovery Kit for subsequent experiments.
  • the specific operation is as follows:
  • Buffer B2 According to the weight and concentration of the gel block, add Buffer B2 at a ratio of 300-600 ⁇ L per 100 mg of agarose;
  • the recombination reaction system is as follows:
  • Purified meso-2,3-butanediol dehydrogenase uses ULP1 protease to remove SUMO-Tag.
  • ULP1 protease has high specificity and maintains high activity in a wide range of reaction environment systems. To ensure For enzyme activity, digestion was performed at 4°C. Finally, the pure enzyme solution of wild-type meso-2,3-butanediol dehydrogenase is obtained, and the purification results are shown in Figure 1.
  • the molecular weight of the monomeric His6-SUMO-L1BDH protein is 40.61kDa.
  • LlBDH temperature tolerance assay method is as follows:
  • the detection system is: an appropriate amount of enzyme solution (the pure enzyme solution of wild-type meso-2,3-butanediol dehydrogenase obtained in step 2 of this example), 12.5mM meso-2,3-butanediol, 0.56mM NAD + , the total volume is 1000 ⁇ L, and the reaction medium is glycine-NaOH buffer (20 mM, pH 10.0). Enzyme activity without heat treatment was defined as 100%. Finally, it was found that the enzyme still retained 23.9% of its activity after being heated at 100° C. for 30 minutes (Table 1).
  • Gaussian accelerated molecular dynamics is an unconstrained enhanced sampling method. Using LiGaMD to simulate the process of repeated dissociation and binding of small-molecule ligands and enzymes on a nanosecond time scale. Molecular dynamics simulations were performed using Amber20 software. Proteins use the ff19SB force field, and NAD + and NADH cofactors use the force field constructed by Holmberg et al. Add an explicit OPC water molecule, and the distance from the protein to the edge of the box is Electroneutralization was then performed with an ion concentration of 0.15M NaCl. Energy minimization is divided into three stages.
  • the first stage minimizes only the positions of solvent molecules and ions; the second stage minimizes hydrogen atoms; and the third stage minimizes all atoms in the simulated system without constraints.
  • Each stage of minimization consists of 2500 steepest descent steps and 2500 conjugate gradient steps.
  • the system was then heated gently by increasing the temperature from 0 to 300 K with constant volume and using periodic boundary conditions.
  • the force constants are applied to proteins and small molecules, and the temperature is controlled using the Langevin thermostat method. Modeling of long-range electrostatic interactions using the PME approach. Lennard-Jones and electrostatic interactions using cutoff value. Bond lengths involving hydrogen atoms are limited using the SHAKE algorithm.
  • NPT-MD was run for 400 ps with a time step of 2 fs.
  • An initial short conventional molecular dynamics simulation of 3.0 ns was run to calculate GaMD acceleration parameters, followed by a GaMD equilibrium simulation of 60 ns.
  • Three independent 100 ns GaMD production simulations were performed on 10 unbound substrate/product molecules of meso-2,3-butanediol dehydrogenase with random initial atomic velocities. All GaMD simulations are run at "dual-boost" level. One boost potential is applied to the dihedral energy term and the other is applied to the total potential energy term.
  • the mean and standard deviation of the system potential energy were calculated every 300 000 steps (0.6 ns).
  • the upper limit ⁇ 0 of the standard deviation of the boost potential is set to 6.0 kcal/mol, and a frame of trajectory is saved every 1.0 ps for analysis.
  • ⁇ G bind ⁇ E vdw + ⁇ E ele + ⁇ G pol + ⁇ G nopol
  • ⁇ E vdw means non-bonding
  • the van der Waals interactions, ⁇ E vdw for electrostatic interactions, and ⁇ G pol and ⁇ G nopol for polar and nonpolar interactions, respectively, constitute the free energy of solvation.
  • igb is set to 5, and other parameters are default values.
  • a single point saturation mutation was performed on LlBDH.
  • the specific method is as follows:
  • the PCR product was subjected to agarose gel electrophoresis, and after recovery, the plasmid template digestion system was digested with DpnI enzyme: 1 ⁇ L of DpnI enzyme, 45 ⁇ L of PCR product, and 4 ⁇ L of Buffer. Digestion of the template is complete for 1 hour at 37°C.
  • the culture solution was centrifuged at 4000g at 4°C for 15min, the supernatant was discarded, and the bacteria were collected.
  • the collected cells were washed twice with 20 mM Tris-HCl buffer solution of pH 8.0, resuspended in Tris-HCl buffer solution, and ultrasonically disrupted 30 times at 400W power, each time for 3 seconds, with an interval of 7 seconds.
  • the cell disruption solution was centrifuged at 12,000 g at 4°C for 30 min to remove the precipitate, and the obtained supernatant was crude enzyme solution.

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Abstract

本发明公开了一种meso-2,3-丁二醇脱氢酶及其突变体和应用，该meso-2,3-丁二醇脱氢酶的氨基酸序列如SEQ ID NO.1所示。本发明发现了一种具有耐高温特性的meso-2,3-丁二醇脱氢酶，并以该meso-2,3-丁二醇脱氢酶为基础，通过加速采样分子动力学模拟方法，动态描述产物释放过程，解决高稳定性和高活性无法兼顾的问题，获得可以催化制备meso-2,3-丁二醇的突变体，该突变体具有非常好的耐高温特性。在100℃热处理30分钟后，残余酶活为23.9％。突变体催化乙偶姻形成meso-2,3-丁二醇中，活性提高约2-5倍。反应获得的产物meso-2,3-丁二醇具有极高的光学纯度，为生物转化生产meso-2,3-丁二醇提供了广阔的应用前景。

Description

meso-2,3-丁二醇脱氢酶及其突变体和应用 技术领域
本发明涉及酶工程技术领域，尤其涉及一种meso-2,3-丁二醇脱氢酶及其突变体和应用。
背景技术
2,3-丁二醇是一种多功能平台化学品，用于制造药物、化妆品、食品添加剂、燃料和溶剂，其中meso-2,3-丁二醇除了是2-丁醇的前体外，还是化妆品的防腐剂和保湿剂，广泛应用于生物燃料和食品工业。2,3-丁二醇可以通过化学和生化途径合成。由于使用低成本的可再生碳源，减少温室气体排放和选择性生产纯手性的2,3-丁二醇，生物途径更具环境和经济优势。生产过程中的碳源也可以使用农业中的可再生原料，降低底物成本，使生物过程更加环保。预计到2027年，2,3-丁二醇市场将投资约2.2亿美元。因此，生物基2,3-丁二醇的工业生产预计将在未来几年得到大力发展。
传统上，2,3-丁二醇是由800-900℃的不可再生石油中的裂解气体化学催化产生的，需要大量的能量。在裂解过程中，产生大量的温室气体，因此这是一个非环保的过程。虽然化学路线是2,3-丁二醇生产的常规方法，但它是昂贵和复杂的，并且产生的2,3-丁二醇是外消旋混合物，其纯化成本很高。反之，生物途径合成2,3-丁二醇仅需要温和的操作条件，如较低的温度和压力。此外，可以从低成本的原材料和简单的反应条件中生产出高光学纯度的2,3-丁二醇。
然而，微生物发酵法在实验室和工业规模生产2,3-丁二醇的应用经常受到底物和产物抑制。在发酵过程中，酶的活性被抑制一直是提高产量的瓶颈。另一方面野生的meso-2,3-丁二醇脱氢酶无法兼顾高活性和高稳定性。因此，通过基于结构的分子改造双目标协同进化(稳定性和活性)获得meso-2,3-丁二醇脱氢酶，有助于其更好的进行工业应用。
鉴于此，还需要进一步深入研究，通过基于结构的酶设计，来解决NAD(H)特异性的meso-2,3-丁二醇脱氢酶催化生产meso-2,3-丁二醇稳定性和活性无法兼顾的问题。
发明内容
为了解决来源于Lactococcus lactis的原始meso-2,3-丁二醇脱氢酶(氨基酸序列如SEQ ID NO.1所示)稳定性和活性无法兼顾的问题，本发明提供了一种meso-2,3-丁二醇脱氢酶及其突变体和应用。
具体技术方案如下：
本发明提供了一种meso-2,3-丁二醇脱氢酶，其氨基酸序列如SEQ ID NO.1所示。该酶具有耐高温的特性。
本发明还提供了meso-2,3-丁二醇脱氢酶突变体，由SEQ ID NO.1所示氨基酸序列的第40位，第51位，第72位，第73位，第162位，第192位，第197位，第204位进行单点突变或多点组合突变获得。
进一步地，具体突变为以下任意一种：
(1)由SEQ ID NO.1所示氨基酸序列的第40位谷氨酰氨突变为赖氨酸；
(2)由SEQ ID NO.1所示氨基酸序列的第51位苯丙氨酸突变为甲硫氨酸；
(3)由SEQ ID NO.1所示氨基酸序列的第72位谷氨酸突变为赖氨酸；
(4)由SEQ ID NO.1所示氨基酸序列的第73位赖氨酸突变为苏氨酸；
(5)由SEQ ID NO.1所示氨基酸序列的第162位异亮氨酸突变为苏氨酸；
(6)由SEQ ID NO.1所示氨基酸序列的第192位组氨酸突变为甲硫氨酸，精氨酸或酪氨酸；
(7)由SEQ ID NO.1所示氨基酸序列的第202位天冬氨酸突变为半胱氨酸，谷氨酸，甘氨酸，脯氨酸或色氨酸；
(8)由SEQ ID NO.1所示氨基酸序列的第197位天冬酰胺突变为甘氨酸，赖氨酸，丝氨酸或缬氨酸；
(9)由SEQ ID NO.1所示氨基酸序列的第204位色氨酸突变为酪氨酸；
(10)由SEQ ID NO.1所示氨基酸序列的第162位异亮氨酸突变为苏氨酸且第192位组氨酸突变为色氨酸；
(11)由SEQ ID NO.1所示氨基酸序列的第162位异亮氨酸突变为苏氨酸，第202位天冬氨酸突变为色氨酸；
(12)由SEQ ID NO.1所示氨基酸序列的第162位异亮氨酸突变为苏氨酸，第192位组氨酸突变为色氨酸，天冬酰胺突变为丝氨酸。
本发明对来源于Lactococcus lactis的meso-2,3-丁二醇脱氢酶(LlBDH，氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示)进行AlphaFold 2同源四聚体结构建模，用天然底物乙偶姻进行分子对接，使用加速采样分子动力学模拟确定底物结合/产物释放过程中的8个关键氨基酸残基，将其进行饱和突变，通过酶活测定以及金属浴100℃加热10min的残余酶活筛选meso-2,3-丁二醇脱氢酶突变体。最后将这些位点顺序迭代突变以及优势位点组合突变获得高活性和高稳定性的meso-2,3-丁二醇脱氢酶突变体。
更进一步地，所述突变体为Q40K、F51M、E72K、K73T、I162T、H192W、N197S、W204Y。
其中，Q40K表示：第40位的氨基酸由谷氨酰胺突变为精氨酸；F51M表示：第51位的氨基酸由苯丙氨酸突变为甲硫氨酸；E72K表示：第72位的氨基酸由谷氨酸突变为精氨酸；K73T表示：第73位的氨基酸由精氨酸突变为苏氨酸；I162T表示：第162位的氨基酸由亮氨酸突变为苏氨酸；H192W表示：第192位的氨基酸由组氨酸突变为色氨酸；N197S表示：第197位的氨基酸由天冬酰胺突变为丝氨酸；W204Y表示：第204位的氨基酸由色氨酸突变为酪氨酸。
进一步地，所述meso-2,3-丁二醇脱氢酶变体由多点组合突变获得，多点组合突变形式为以下形式：
(1)按照第40位，第51位，第72位，第73位，第162位，第192位，第197位，第204位的排列顺序，依次由相邻的两个或两个以上位点进行顺序迭代突变获得；
其中，各突变位点及其突变前后的氨基酸单字母简写分别为：Q40K、F51M、E72K、K73T、I162T、H192W、N197S、W204Y；
更进一步地，所述谷氨酸脱氢酶变体为以下多点突变中的一种：
I162T/H192W，I162T/D202E，I162T/H192W/N197S,I162T/H192W/N197S/W204Y,I162T/H192W/N197S/W204Y/F51M,I162T/H192W/N197S/W204Y/F51M/Q40K；
上文中的“/”表示“且，”即“/”前后两个位点同时突变；例如：I162T/H192W表示第162位亮氨酸突变为苏氨酸，且第192位的氨基酸由组氨酸突变为色氨酸。
本发明还提供了一种如上所述的meso-2,3-丁二醇脱氢酶或者如上所述的meso-2,3-丁二醇脱氢酶突变体的编码基因。
本发明还提供了一种包含所述编码基因的重组载体。进一步地，重组载体的原始表达载体为pET28a-SUMO。
本发明还提供了一种包含所述编码基因的基因工程菌。进一步地，基因工程菌的宿主细胞为E.coli BL21(DE3)。
本发明还提供了如上所述的meso-2,3-丁二醇脱氢酶或者如上所述的meso-2,3-丁二醇脱氢酶突变体在催化乙偶姻生成meso-2,3-丁二醇中的应用。
本发明还提供了如上所述的基因工程菌在催化乙偶姻生成meso-2,3-丁二醇中的应用。
与现有技术相比，本发明具有以下有益效果：
(1)本发明发现了一种具有耐高温特性的meso-2,3-丁二醇脱氢酶，并以该meso-2,3-丁二醇脱氢酶为基础，通过加速采样分子动力学模拟方法，动态描述产物释放过程，解决高稳定性和高活性无法兼顾的问题，获得可以催化制备meso-2,3-丁二醇的突变体，该突变体具有非常好的耐高温特性。在100℃热处理30分钟后，残余酶活为23.9％。突变体催化乙偶姻形成meso-2,3-丁二醇中，活性提高约2-5倍。反应获得的产物meso-2,3-丁二醇具有极高的光学纯度，为生物转化生产meso-2,3-丁二醇提供了 广阔的应用前景。
(2)本发明所使用的理性设计方法能以较小的突变文库、通过筛选快速获得高稳定性和高活性的meso-2,3-丁二醇脱氢酶突变体。
附图说明
图1为纯化的meso-2,3-丁二醇脱氢酶LlBDH在SDS-PAGE上的电泳分析。
具体实施方式
下面结合具体实施例对本发明作进一步描述，以下列举的仅是本发明的具体实施例，但本发明的保护范围不仅限于此。
本发明中的实验方法如无特别说明均为常规方法，基因克隆操作具体可参见J.萨姆布鲁克等编的《分子克隆实验指南》。
上游基因工程所用试剂：本发明实施例中使用的基因组提取试剂盒、DpnI购自TaKaRa，宝生物工程(大连)有限公司；Exnase II无缝克隆试剂盒购自南京诺唯赞生物科技股份有限公司；质粒提取试剂盒、DNA回收纯化试剂盒购自Axygen杭州有限公司；E.coli BL21(DE3)、质粒pET28a-SUMO等购自Novagen公司；DNA marker、低分子量标准蛋白、琼脂糖电泳试剂购自北京全式金生物技术有限公司；引物合成与序列测序工作由擎科生物工程股份有限公司完成。以上试剂使用方法参考商品说明书。
催化反应所用试剂：乙偶姻、meso-2,3-丁二醇、NAD +和NADH购自上海麦克林生化科技有限公司。
meso-2,3-丁二醇脱氢酶酶活标准检测方法：适量的酶液、12.5mM底物、0.56mM NAD +，总体系为1000μL，反应介质为甘氨酸-NaOH缓冲液(20mM，pH 10.0)。20℃反应5min，样品中生成/消耗的NADH通过酶标仪进行定量分析。
酶活单位(U)的定义：在标准的反应条件下，酶活力单位(U)定义为每分钟消耗或产生1μmol NADH所需要的酶量。
实施例1 meso-2,3-丁二醇脱氢酶的克隆、表达、纯化和温度耐受性测定
一、meso-2,3-丁二醇脱氢酶(简称LlBDH)的克隆
从乳酸乳球菌(Lactococcus lactis)中克隆获得meso-2,3-丁二醇脱氢酶基因，并将该基因插入到pET28a-SUMO质粒中，再转入E.coli BL21(DE3)中得到pET28a-SU MO-LlBDH表达菌株。
具体步骤为：
1、乳酸乳球菌基因组的提取
使用TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0提取Lactococcus lactis基因组，操作如下：
①用1.5mL离心管收集培养好的Lactococcus lactis菌液，12000rpm离心2min，弃上清；
②加入180μL Buffer GL、20μL的Proteinase K(20mg/mL)和10μL的RNase A(10mg/mL)充分振荡混匀，于56℃水浴温育10min，此时溶液应呈透明、澄清状；
③加入200μL的Buffer GB和200μL的100％乙醇，充分吸打混匀；
④将Spin Column安装在Collection Tube上，将处理好的细胞溶解液移至Spin Column中，12000rpm离心2min，弃滤液；
⑤将500μL的Buffer WA加入至Spin Column中，12000rpm离心1min，弃滤液；
⑥将700μL的Buffer WB加入至Spin Column中，12000rpm离心1min，弃滤液(Buffer WB使用前需加入指定体积的100％乙醇，确保沿Spin Column管壁四周加入Buffer WB，这样有助于完全冲洗沾附于管壁上的盐份)；
⑦重复操作步骤⑥；
⑧将Spin Column置于Collection Tube上，12000rpm离心2min；
⑨将Spin Column置于新的1.5mL离心管上，在Spin Column膜的中央处加入35μL 65℃左右温热的灭菌ddH 2O，室温静置10min，12000rpm离心3min洗脱DNA，如需获得更大收量，可将离下液重新加入到Spin Column膜的中央，室温静置10min后，12000rpm离心3min洗脱DNA。
2、PCR反应克隆目的片段及线性化载体
以NCBI中Lactococcus lactis菌株的基因组(NZ_CP059048)为模板进行引物的设计。设计的引物如下：
表1 克隆目的片段及线性化载体所用引物
Figure PCTCN2022127954-appb-000001
引物由北京擎科生物科技有限公司有限公司合成。
PCR反应体系1
Figure PCTCN2022127954-appb-000002
Figure PCTCN2022127954-appb-000003
PCR反应体系2
Figure PCTCN2022127954-appb-000004
PCR反应条件：
Figure PCTCN2022127954-appb-000005
PCR产物的回收：
用SanPrep柱式DNA胶回收试剂盒纯化回收PCR扩增产物，用于后续实验。具体操作如下：
①PCR产物经电泳分离后，在紫外灯下用干净的刀片从琼脂糖凝胶中切割下含目的DNA片段的琼脂糖凝胶块，放入1.5mL离心管中，称重。注意尽可能多地去掉不含目标DNA的琼脂糖，每管琼脂糖凝胶块不要超过400mg，同时尽量减少DNA在紫外灯下曝光时间；
②根据胶块的重量和浓度，按每100mg琼脂糖加300-600μL的比例加入Buffer B2；
③将离心管置于50℃水浴5-10min，间或混匀，直至凝胶完全溶化；
④将溶化好的溶液全部转移入吸附柱，12000rpm离心1min，倒掉收集管中的液体， 将吸附柱放入同一个收集管中；
⑤向吸附柱中加入300μL Buffer B2，12000rpm离心1min，倒掉收集管中的液体，将吸附柱放入同一个收集管中；
⑥向吸附柱中加入500μL Wash Solution，12000rpm离心1min，倒掉收集管中的液体，将吸附柱放入同一个收集管中；
⑦重复步骤⑥一次；
⑧将吸附柱放回收集管中，12000rpm离心2min，清除柱子中残留乙醇；
⑨弃收集管，将吸附柱放入新的1.5mL离心管，在吸附膜中央加入30μL温热的ddH 2O，室温静置10min，12000rpm离心3min，将所得到的DNA溶液置于-20℃保存用于后续实验。
3、目的基因插入线性化的pET28a-SUMO载体
反应结束后，取5μL样品，经1％琼脂糖凝胶电泳检测，使用电泳比较条带亮度的方法对DNA进行定量。克隆载体使用量为0.03pmol，最适插入片段使用量为0.06pmol。
重组反应体系如下：
Figure PCTCN2022127954-appb-000006
①使用移液器轻轻吸打混匀(请勿振荡混匀)，短暂离心将反应液收集至管底。37℃反应30min；降至4℃或立即置于冰上冷却。
②在冰上解冻克隆感受态细胞。
③取10μl重组产物加入到100μl感受态细胞中，轻弹管壁混匀(请勿振荡混匀)，冰上静置30min。
④42℃水浴热激90c后，立即置于冰上冷却2-3min。
⑤加入500μl LB培养基(不添加抗生素)，37℃摇菌1h(转速200rpm)。
⑥5000rpm离心5min，弃掉300μl上清。用剩余培养基将菌体重悬，用无菌涂布棒在含有卡那霉素抗性的平板上轻轻涂匀。
⑦37℃培养箱中倒置培养12-16h。
二、meso-2,3-丁二醇脱氢酶的表达和纯化
醇脱氢酶LlBDH的纯化操作过程为:
(1)含醇脱氢酶LlBDH的pET28a-SUMO-LlBDH表达菌株破碎后，将上清转移到预冷的离心管中并冰浴待用；整个纯化过程的液体流速保持在1mL/min；将预装柱用含3mM咪唑的Tris-HCl缓冲液(20mM，pH＝8.0)以十倍柱体积平衡镍柱。
(2)上清液流过镍柱，此过程含有His标签的目的酶和部分杂蛋白会与镍特异性结合。
(3)用十倍柱体积的含20mM咪唑Tris-HCl缓冲液(20mM，pH＝8.0)溶液清洗镍柱中的蛋白。
(4)用500mM咪唑Tris-HCl缓冲液(20mM，pH＝8.0)洗脱镍柱，将蛋白洗收集。
纯化后的meso-2,3-丁二醇脱氢酶用ULP1蛋白酶去除SUMO-Tag，ULP1蛋白酶具有很高的特异性，且在较宽范围的反应环境体系中保持较高的活力，为保证酶的活性，在4℃下进行酶切。最终得到野生型meso-2,3-丁二醇脱氢酶的纯酶液，纯化的结果如图1所示，单体His6-SUMO-LlBDH蛋白的分子量为40.61kDa蛋白被ULP1蛋白酶切割后，ULP1蛋白酶、His6-SUMO标签(表观分子量13kDa)和未切割的蛋白质留在亲和层析柱中。切割后的LlBDH蛋白的分子量为26.83kDa。SDS-PAGE分析显示蛋白质被成功纯化至95％以上的均一性(图3)。
三、meso-2,3-丁二醇脱氢酶的温度耐受性
LlBDH温度耐受性测定方法如下：
将纯酶和酶活检测体系的各成分孵育在100℃金属浴中2、5、10、20、30、40、50、60min。检测体系为:适量的酶液(本实施例步骤二获得的野生型meso-2,3-丁二醇脱氢酶的纯酶液)、12.5mM meso-2,3-丁二醇、0.56mM NAD +，总体系为1000μL，反应介质为甘氨酸-NaOH缓冲液(20mM，pH 10.0)。未热处理时的酶活定义为100％。最终发现该酶在100℃加热30min仍保留23.9％的活性(表1)。
表1 meso-2,3-丁二醇脱氢酶的100℃耐受性测定
Figure PCTCN2022127954-appb-000007
实施例2 基于产物释放过程的突变位点设计
高斯加速分子动力学(GaMD)是一种无约束增强采样方法。使用LiGaMD以纳秒时间尺度，模拟捕获小分子配体与酶重复解离和结合的过程。分子动力学模拟操作均应用Amber20软件进行。蛋白质使用ff19SB力场，NAD +和NADH辅因子使用Holmberg等人构建的力场。添加显式的OPC水分子，蛋白距盒子边缘为
Figure PCTCN2022127954-appb-000008
然后用0.15M NaCl的离子浓度进行电中和。能量最小化共分为三个阶段。第一阶段仅最小化溶剂分子和离子的位置；第二阶段最小化氢原子；第三阶段不受约束地最小化模拟体系内的所有原子。每一阶段的最小化包括2500步最速下降步骤和2500步共轭梯度步骤。然后，恒定体积和使用周期边界条件，将温度从0增加到300K，对系统进行温和加热。
Figure PCTCN2022127954-appb-000009
的力常数应用在蛋白质和小分子上，并采用Langevin thermostat方法控制温度。使用PME方法对远程静电作用进行建模。Lennard-Jones和静电相互作用采用
Figure PCTCN2022127954-appb-000010
截止值。涉及到氢原子的键长使用SHAKE算法进行限制。随后在恒定压力1atm和温度300K下，运行NPT-MD 400ps，时间步长为2fs。运行3.0ns的初始短的传统分子动力学模拟以计算GaMD加速参数，然后运行60ns的GaMD平衡模拟。10个未结合底物/产物分子的meso-2,3-丁二醇脱氢酶上执行三个独立的100ns GaMD生产模拟，初始原子速度随机。所有GaMD模拟都在“dual-boost”水平下运行。一个升压势应用于二面角能量项，另一个应用于总势能项。对于所有模拟系统，每300 000步(0.6ns)计算一次系统势能的平均值和标准差。对于二面角和总势能项，升压势标准差的上限δ0均设置为6.0kcal/mol，每1.0ps保存一帧轨迹以供分析。使用MM/GBSA方法对0～80ns范围内的每帧进行残基自由能分解，分解过程由四个能量项组成：ΔG bind＝ΔE vdw+ΔE ele+ΔG pol+ΔG nopol，ΔE vdw表示非键范德华相互作用，ΔE vdw表示静电相互作用，ΔG pol和ΔG nopol分别表示极性和非极性相互作用，它们构成了溶剂化自由能。输入文件中igb设置为5，其他参数均为默认值。
目视观察产物释放轨迹，发现H192-D202之间的氢键首先断开，产物从酶催化口袋释放，据此我们确定了H192和D202两个潜在的突变位点。分析产物释放过程中每个氨基酸残基的能量贡献，发现W204、N197、Q40、F51、E72、K73位点；另外根据稳定多聚体界面提高酶活的策略，将I162突变为苏氨酸，在二聚体结合界面引入了新的氢键。
实施例3 单点饱和突变的构建
对LlBDH进行单点饱和突变。具体方法如下：
1、全质粒PCR
以pET28a-SUMO-LlBDH质粒为模版，设计覆盖突变点的上下游引物(表1)进行全质粒PCR:
表2 单点突变库构建所用引物
Figure PCTCN2022127954-appb-000011
PCR扩增体系：
Figure PCTCN2022127954-appb-000012
PCR扩增条件：
1)预变性：95℃5min；
2)变性：98℃10s；退火：60℃15s；延伸：72℃10s；共循环30次；
3)后延伸：72℃10min；
4)4℃保存。
2、模板消化：
将PCR产物进行琼脂糖凝胶电泳，回收后用DpnI酶消化其中的质粒模板消化体系为：DpnI酶1μL，PCR产物45μL，Buffer 4μL。37℃下1小时可完成模板的消化。
3、转化及验证：
消化后产物经核酸琼脂糖凝胶电泳验证后，采用42℃热激法转化大肠杆菌BL21(DE3)感受态细胞。具体过程如下：
(1)将感受态细胞放置在冰上解冻5min；
(2)在无菌环境下将10μL DNA加入100μL感受态细胞中并轻轻混匀，冰上放置30min；
(3)EP管静置于42℃金属浴中热激90s，结束后置于冰上冷却2min；
(4)向EP管中加入500μL LB培养基并用枪头混匀，置于220rpm摇床中37℃孵育60min；
(5)浓缩后取适量体积涂相应的抗性平板，于37℃培养箱中培养12-16h后可出现菌落。每块平板挑出3～4个单菌落进行培养，并测序验证突变是否成功。
4、突变菌株培养及蛋白表达
将测序成功的突变株经平板划线活化后，挑单菌落接种至含50μg/mL卡那霉素的5mL LB液体培养基中，37℃震荡培养12h。按2％的接种量转接至200mL同样含50μg/mL卡那霉素的LB液体培养基中，37℃震荡培养至OD 600达到0.6～0.8左右时，加入IPTG至其终浓度为0.5mM，37℃下诱导培养6h。
培养结束后，将培养液4000g 4℃离心15min，弃上清，收集菌体。将收集到的菌体，用20mM pH 8.0的Tris-HCl缓冲液洗涤两次后，重悬于Tris-HCl缓冲液中，400W功率超声破碎30次，每次超声持续3s，间歇7s。将此细胞破碎液12000g 4℃离心30min去除沉淀，得到的上清为粗酶液。
5、酶活测定
用酶标仪测定上述突变体对底物乙偶姻的酶活，测定结果见表3：
表3 meso-2,3-丁二醇脱氢酶的酶活测定
Figure PCTCN2022127954-appb-000013
Figure PCTCN2022127954-appb-000014
实施例4 基于产物释放路径的顺序迭代突变库的构建与活性测定
基于产物释放路径的顺序，对162，192，202，197共4个氨基酸残基位点进行顺序迭代突变，具体地，设计以下突变体：
1X：I162T
2X-1：I162T/H192W；
2X-2：I162T/D202E；
3X：I162T/H192W/N197S；
具体实验步骤如下：
以pET28a-SUMO-LlBDH-I162T质粒为模版，设计覆盖突变点的上下游引物(表3)，进行全质粒PCR获得突变体2X-1和2X-2，然后以2X-1为模板，以N197S_F和N197S_R为上下游引物进行全质粒PCR获得突变体3X，所用引物见表4，详细PCR操作、化转、菌株培养及蛋白表达步骤与实施例1相同。
表4 多点组合突变构建所用引物
Figure PCTCN2022127954-appb-000015
用酶标仪测定上述突变体对天然底物meso-2,3-丁二醇的酶活，测定结果见表5。
表5 多点组合的meso-2,3-丁二醇脱氢酶的酶活测定
Figure PCTCN2022127954-appb-000016

Claims (8)

  1. meso-2,3-丁二醇脱氢酶，其特征在于，氨基酸序列如SEQ ID NO.1所示。
  2. meso-2,3-丁二醇脱氢酶突变体，其特征在于，由SEQ ID NO.1所示氨基酸序列的第40位，第51位，第72位，第73位，第162位，第192位，第197位，第204位进行单点突变或多点组合突变获得。
  3. 如权利要求2所述的meso-2,3-丁二醇脱氢酶突变体，其特征在于，具体突变为以下任意一种：
    (1)由SEQ ID NO.1所示氨基酸序列的第40位谷氨酰氨突变为赖氨酸；
    (2)由SEQ ID NO.1所示氨基酸序列的第51位苯丙氨酸突变为甲硫氨酸；
    (3)由SEQ ID NO.1所示氨基酸序列的第72位谷氨酸突变为赖氨酸；
    (4)由SEQ ID NO.1所示氨基酸序列的第73位赖氨酸突变为苏氨酸；
    (5)由SEQ ID NO.1所示氨基酸序列的第162位异亮氨酸突变为苏氨酸；
    (6)由SEQ ID NO.1所示氨基酸序列的第192位组氨酸突变为甲硫氨酸，精氨酸或酪氨酸；
    (7)由SEQ ID NO.1所示氨基酸序列的第202位天冬氨酸突变为半胱氨酸，谷氨酸，甘氨酸，脯氨酸或色氨酸；
    (8)由SEQ ID NO.1所示氨基酸序列的第197位天冬酰胺突变为甘氨酸，赖氨酸，丝氨酸或缬氨酸；
    (9)由SEQ ID NO.1所示氨基酸序列的第204位色氨酸突变为酪氨酸；
    (10)由SEQ ID NO.1所示氨基酸序列的第162位异亮氨酸突变为苏氨酸且第192位组氨酸突变为色氨酸；
    (11)由SEQ ID NO.1所示氨基酸序列的第162位异亮氨酸突变为苏氨酸，第202位天冬氨酸突变为色氨酸；
    (12)由SEQ ID NO.1所示氨基酸序列的第162位异亮氨酸突变为苏氨酸，第192位组氨酸突变为色氨酸，天冬酰胺突变为丝氨酸。
  4. 一种如权利要求1所述的meso-2,3-丁二醇脱氢酶或者如权利要求2或3所述的meso-2,3-丁二醇脱氢酶突变体的编码基因。
  5. 一种包含权利要求4所述编码基因的重组载体。
  6. 一种包含权利要求4所述编码基因的基因工程菌。
  7. 如权利要求1所述的meso-2,3-丁二醇脱氢酶或者如权利要求2或3所述的meso-2,3-丁二醇脱氢酶突变体在催化乙偶姻生成meso-2,3-丁二醇中的应用。
  8. 如权利要求6所述的基因工程菌在催化乙偶姻生成meso-2,3-丁二醇中的应用。
PCT/CN2022/127954 2022-02-15 2022-10-27 meso-2,3-丁二醇脱氢酶及其突变体和应用 WO2023155474A1 (zh)

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