PQQ dependent alcohol dehydrogenase mutant and application thereof

Abstract

The invention discloses a PQQ dependent alcohol dehydrogenase mutant and an application thereof. According to the PQQ dependent alcohol dehydrogenase mutant, an amino acid sequence as shown in SEQ ID NO.1 is taken as a benchmark, and the alcohol dehydrogenase mutant comprises one or more of the following mutations: F412S, W554S, G556S and A557N. The invention also constructs an engineering bacterium for producing FDCA, which can efficiently catalyze HMF to generate FDCA, and provides a new way for synthesizing FDCA by a biological method.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a PQQ-dependent alcohol dehydrogenase mutant and an application thereof. Background Art

[0002] Biomass has become the most attractive feedstock for the biochemical industry. Among bio-based chemicals, furan compounds are the most promising for synthetic chemistry. Specifically, 5-hydroxymethylfurfural (HMF) is considered to be the key to the transition from a fossil economy to a sustainable economy. HMF, known as the "sleeping giant", is a versatile intermediate derived from the dehydration of abundant C6 carbohydrates, which can be converted into high value-added chemicals through oxidation, hydrogenation, polymerization and ring-opening reactions of its furan ring, aldehyde and alcohol groups. For example, the oxidation products of HMF include 2,5-diformylfuran (FFF), 5-hydroxymethyl-2-furancarboxylic acid (HMFA), 5-formyl-2-furancarboxylic acid (FFA) and 2,5-furandicarboxylic acid (FDCA), which can be widely used in fine chemicals, pharmaceuticals, chiral catalysis, molecular recognition and polymers. In particular, FDCA, listed by the U.S. Department of Energy as one of the top 12 most promising platform chemicals from biomass, can be used as a “green” alternative to terephthalic acid in the production of polyethylene terephthalate (PET), the most widely used plastic.

[0003] Catalytic synthesis of FDCA from HMF has aroused great interest among researchers. Currently, FDCA is mainly synthesized by chemical oxidation of HMF. However, traditional chemical catalytic methods usually require harsh reaction conditions, such as high temperature and pressure, organic solvents, toxic solvents, metal salts, etc., which are not only complicated to operate but also pollute the environment. In addition, chemical catalysis often shows poor selectivity in oxidation reactions. Biotransformation can overcome the above disadvantages because it is usually carried out under mild conditions, is simple to operate, environmentally friendly, and has excellent selectivity. At present, biotransformation for the production of FDCA mainly involves enzymatic conversion and whole-cell catalysis. It is very challenging to produce FDCA from HMF using a single enzyme because most enzymes are limited to alcohol or aldehyde oxidation, while a complete oxidation of HMF to FDCA requires an enzyme that can accept both groups at the same time. So far, only two enzymes, 5-hydroxymethylfurfural oxidase (HMFO) and aryl alcohol oxidase (AAO), have been reported to be able to perform continuous three-step oxidation to convert HMF to FDCA. However, the activities of these two enzymes are relatively low, and even with the use of evolved mutants, only low concentrations of substrate can be converted. The conversion efficiency of whole-cell catalysis also depends on the activity of specific enzymes within the cell.

[0004] The inventors have found in previous studies that rare earth-dependent alcohol dehydrogenase PedH from Pseudomonas putida can continuously oxidize HMF to FDCA in multiple steps, but its activity is low and there is still a long way to go before industrialization. Therefore, how to molecularly modify PedH, improve its activity in catalyzing HMF to produce FDCA, and use Escherichia coli expressing rare earth-dependent alcohol dehydrogenase PedH to efficiently synthesize FDCA are still urgent problems to be solved in the field. Summary of the invention

[0005] The invention provides a PQQ-dependent alcohol dehydrogenase mutant and application thereof, which solves the technical problem of how to improve the activity of alcohol dehydrogenase PedH from Pseudomonas putida through genetic modification and increase the yield of enzyme-catalyzed synthesis of 2,5-furandicarboxylic acid.

[0006] The present invention reports that PedH can continuously oxidize HMF to FDCA in multiple steps. In order to increase the content of FDCA, the heat-resistant variant 4M, i.e., the parent 4ZM (SEQ ID NO.1), was mutated, and an automated high-throughput screening method was developed to improve the screening efficiency of mutants. At the same time, potential mutation sites were determined based on binding free energy calculations and the deep learning model MutCompute. The mutation targets were determined by high-throughput screening, and single mutants or multiple mutants with significantly improved FDCA yields were obtained using an iterative saturation mutation strategy, including 4M-F412S, 4M-W554S, 4M-A557N, 5M, 6M, 7M and 8M.

[0007] The specific technical solutions are as follows:

[0008] The present invention provides a PQQ-dependent alcohol dehydrogenase mutant. Based on the amino acid sequence shown in SEQ ID NO.1, the alcohol dehydrogenase mutant comprises one or more of the following mutations: F412S, W554S, G556S and A557N.

[0009] The PQQ-dependent alcohol dehydrogenase mutant of the present invention, when catalyzing the substrate HMF, has an FDCA yield that is 3-349 times higher than that of the amino acid sequence shown in SEQ ID NO.1 (i.e., the parent 4M mutant), and an HMF conversion rate that is 3.6-20.9 times higher.

[0010] In certain embodiments, the amino acid sequence shown in SEQ ID NO.1 is an alcohol dehydrogenase from Pseudomonas putida.

[0011] MTRSPRRPLFAVSLVLSAMLLAGAAHAAVSNEEILQDPKNPQQIVTNGLGVQGQRYSPLDLLNVNNVKELRPVWAFSFGGEKQRGQQAQPLIKDGVMYLTGSYSRVFAVDARTGKKLWQYDARLPDDIRPCCDVINRGVALYGNLVFFGTLDAKLVALNKDTGKVVWSKKVADHKEGYSITAAPMIVNGKLITGVAGGEFGVVGKIQAYNPENGELLWMRPTVEGHMGYVYKDGKAIENGITGGEAGKTWPGDLWKTGGAAPWLGGYYDPETNLILFGTGNPAPWNSHLRPGDNLYSSSRLALNPDDGTIKWHFQSTPHDGWDFDGVNELISFNYKDGGKEVKAAATADRNGFFYVLDRTNGKFIRGFPFVDKITWATGLDKDGRPIYNDASRPGAPGSEGKGSSVFVAPAALGAKNWMPMAYNKDTGLFYVPSNEWGMDIWNEPIAYKKGAAFLGAGFTIKPLNEDYIGVLRAIDPVSGKEVWRHKNYAPLWGGVLTTKGNLVFTGTPEGFLQAFNAKTGDKVWEFQTGSGVLGSPVTWEMDGEQYVSVVSGSGSNVPLWGGEVAKRVKDFNQGGMLWTFKLPKQLQQTASVKP(SEQ ID NO.1)

[0012] The base sequence encoding the amino acid sequence shown in SEQ ID NO.1 contains the sequence shown in SEQ ID NO.2.

[0013]

[0014] In certain embodiments, the alcohol dehydrogenase mutant comprises F412S. The FDCA production of this single point mutant is increased by 4 times compared to the parent 4M mutant.

[0015] In certain embodiments, the alcohol dehydrogenase mutant comprises W554S. The FDCA production of this single point mutant is increased by 3 times compared to the parent 4M mutant.

[0016] In certain embodiments, the alcohol dehydrogenase mutant comprises A557N. The FDCA production of this single point mutant is increased by 3 times compared to the parent 4M mutant.

[0017] In certain embodiments, the alcohol dehydrogenase mutant includes G556S, which is the 5M mutant in the embodiment; compared with the parent 4M mutant, the FDCA production of this single point mutant is increased by 159 times.

[0018] In certain embodiments, the alcohol dehydrogenase mutants include F412S and G556S, which is the 6M mutant in the embodiment; compared with the parent 4M mutant, the FDCA production of the 6M mutant is increased by 233 times.

[0019] In certain embodiments, the alcohol dehydrogenase mutants include F412S, W554S and G556S, i.e., the 7M mutant in the embodiment; compared with the parent 4M mutant, the FDCA production of the 7M mutant is increased by 348 times.

[0020] In certain embodiments, the alcohol dehydrogenase mutant includes F412S, W554S, G556S and A557N, i.e., the 8M mutant in the embodiment; compared with the parent 4M mutant, the FDCA production of the 8M mutant is increased by 349 times.

[0021] The present invention also provides an isolated polynucleotide encoding the alcohol dehydrogenase mutant as described above.

[0022] The present invention also provides a recombinant vector comprising the polynucleotide as described above. The original expression vector of the recombinant vector of the present invention can be pET-30a, pET-21a, pET-22b, pET-28a, pETDuet-1, pACYCDuet-1, pCDFDuet-1 and RSFDuet-1, but is not limited to these vectors. Preferably, it is pET-28a.

[0023] The present invention also provides a recombinant microorganism or cell, comprising the above-mentioned recombinant vector or gene in which the above-mentioned polynucleotide is integrated. The host cell of the recombinant microorganism or cell used in the present invention is Escherichia coli, preferably E. coli BL21 (DE3).

[0024] The present invention also provides the use of the alcohol dehydrogenase mutant as described above, or the polynucleotide as described above, or the recombinant vector as described above, or the recombinant microorganism or cell as described above in at least one of the following:

[0025] 1) Use in regulating the production of 2,5-furandicarboxylic acid;

[0026] 2) Use in the preparation of 2,5-furandicarboxylic acid;

[0027] 3) Use in constructing genetically engineered bacteria that produce 2,5-furandicarboxylic acid.

[0028] The present invention also provides a method for preparing 2,5-furandicarboxylic acid, using the alcohol dehydrogenase mutant described above, the recombinant vector described above, or the recombinant microorganism or cell described above to catalyze a substrate reaction to obtain 2,5-furandicarboxylic acid (FDCA).

[0029] In certain embodiments, the substrate is selected from 5-hydroxymethylfurfural.

[0030] In certain embodiments, the reaction system further comprises one or more of a lanthanide metal ion, an artificial electron acceptor, and pyrroloquinoline quinone.

[0031] In certain embodiments, the lanthanide metal ion is selected from Pr 3+ ,La 3+ 、Ce 3+ 、Nd 3+ 、Sm 3+ 、Eu 3+ , Gd 3+ and Tb 3+ One or more of .

[0032] In certain specific embodiments, the lanthanide metal ion is selected from Nd 3+ The present application found that when the lanthanide metal ion is selected from Nd 3+ The conversion rate of HMF was the highest when

[0033] In some embodiments, the artificial electron acceptor is WB (Worster Blue), which can be purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0034] In certain embodiments, the reaction temperature is 15-40°C, or 15-25°C, or 20-35°C, or 30-40°C. Preferably, it is 30°C.

[0035] In certain embodiments, the reaction time is 1-72 hours, or 1-30 hours, or 20-60 hours, or 50-72 hours, and preferably 24 hours.

[0036] In certain embodiments, the pH value of the reaction is 7.0 to 10.5, or 7.0 to 8.0, or 7.5 to 9.0, or 8.5 to 10.5. Preferably, it is 8. Specifically, it is a Tris-HCl buffer with a pH of 8.0.

[0037] In certain embodiments, the recombinant microorganism or cell includes whole cells or cell lysate. The whole cells are obtained by centrifuging the bacterial solution at 4,000×g and 4°C for 15 min after the recombinant microorganism is cultured, discarding the supernatant, and collecting the bacterial bodies, which are whole cells; the cell lysate is obtained by ultrasonically disrupting the whole cells.

[0038] In some specific embodiments, based on the total volume of the reaction system, the concentration of the artificial electron acceptor is 0.5-50 mM, 0.5-3 mM, 2-20 mM, 15-35 mM, 30-50 mM, preferably 1 mM.

[0039] In some specific embodiments, based on the total volume of the reaction system, the concentration of pyrroloquinoline quinone (PPQ) is 2-250 μM, 2-50 μM, 30-100 μM, 80-150 μM, or 14-250 μM. Preferably, it is 5 μM.

[0040] In some specific embodiments, based on the total volume of the reaction system, the concentration of the lanthanide metal ion is 2-250 μM, 2-50 μM, 30-100 μM, 80-150 μM, or 14-250 μM. Preferably, it is 5 μM.

[0041] In some specific embodiments, based on the total volume of the reaction system, the concentration of the substrate is 2-200 mM, 2-80 mM, 60-160 mM, or 120-200 mM, preferably 40 or 30 mM.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The present invention constructs a PQQ-dependent alcohol dehydrogenase mutant, which, when catalyzing the substrate HMF, increases the FDCA yield by 3-349 times compared with the parent 4M mutant, and increases the conversion rate of the substrate HMF by 3.6-20.9 times, among which the FDCA yield of the 8M mutant is the highest at 3.49 mM; it is further found that the specific enzyme activity of the 8M mutant on the substrates HMF, FFF, and FFA is 29.5, 23.1, and 20.4 times higher than that of the parent 4M, respectively; and it also has activity in catalyzing the substrate HMFA, while the parent 4M mutant has no activity on HMFA.

[0044] (2) The present invention constructs an engineered bacterium that produces FDCA, which can efficiently catalyze HMF to produce FDCA, providing a new way for the biosynthesis of FDCA, thereby avoiding the problem of inconsistent reaction conditions of different enzymes during multi-enzyme catalysis.

[0045] (3) The present invention further provides a method for preparing FDCA. By optimizing the reaction conditions, the yield of FDCA is increased from 6.98% to 96.4%, an increase of 13.8 times, and the conversion rate of substrate HMF reaches 100%.

[0046] (4) The yield of FDCA synthesized by the PQQ-dependent alcohol dehydrogenase mutant of the present invention is 349 times higher than that of the 4M parent, which provides a new way for the biological synthesis of FDCA. In addition, it is of great significance to improve the problems of resource shortage and environmental pollution and promote the green biomanufacturing of FDCA. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a graph showing the change in yield of FDCA produced by continuous oxidation of HMF by PedH in Example 1 over time.

[0048] Figure 2 For the substrate surrounding in Example 2 Binding free energy change diagram for single point mutations of some amino acids in the range.

[0049] Figure 3 is the relative activity of the top 20 mutants predicted by MutCompute in Example 2.

[0050] Figure 4 This is the whole-cell catalytic diagram of the forward mutant after high-throughput screening in Example 3.

[0051] Figure 5 This is the evolutionary path of iterative saturation mutation in Example 4.

[0052] Figure 6 This is the whole-cell catalytic diagram of the forward mutant in Example 4.

[0053] Figure 7The concentration of the artificial electron acceptor WB in the preparation method of 2,5-furandicarboxylic acid in Example 7 is optimized.

[0054] Figure 8 The method for preparing 2,5-furandicarboxylic acid in Example 7 optimizes the types of lanthanide elements.

[0055] Fig. 9 The optimal pH in the preparation method of 2,5-furandicarboxylic acid in Example 7 was explored.

[0056] Fig.10 The most suitable PQQ, Nd in the preparation method of 2,5-furandicarboxylic acid in Example 7 3+ Concentration exploration.

[0057] Fig.11 The optimal temperature in the preparation method of 2,5-furandicarboxylic acid in Example 7 was explored.

[0058] Fig.12 The cell concentration, substrate concentration and biocatalyst form in the preparation method of 2,5-furandicarboxylic acid in Example 7 were optimized. DETAILED DESCRIPTION

[0059] The present invention will be further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

[0060] Reagents used in upstream genetic engineering: E. coli BL21 (DE3) used in the examples of the present invention was purchased from Novagen; plasmid pET28a and the like were purchased from Novagen; gene synthesis, primer synthesis and sequence sequencing of PedH were completed by Qingke Biotechnology Co., Ltd.

[0061] Reagents used in the catalytic reaction: Tris (trishydroxymethylaminomethane) and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; pyrroloquinoline quinone (PQQ) was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; praseodymium chloride and 5-hydroxymethylfurfural (HMF) were purchased from Aladdin Reagent (Shanghai) Co., Ltd.; DCPIP (2,6-dichloroindophenol) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; PES (phenazine ethyl sulfate) was purchased from Shanghai Eon Chemical Technology Co., Ltd.; WB (Worcester Blue) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0062] Definition of enzyme activity unit (U): the amount of enzyme required to consume 1 μM DCPIP in the reaction system per minute.

[0063] Example 1

[0064] Continuous multi-step oxidation of HMF to FDCA by rare earth-dependent alcohol dehydrogenase (PedH)

[0065] The reaction was carried out at 100 mM Tris-HCl buffer (pH 8.0), substrate HMF 0.25 mM, PedH 1 μM, and 30°C for 24 hours. The compounds in the reaction system were detected at 0 h, 4 h, 8 h, 12 h, and 24 h. The results are shown in Figure 1 Wherein, PedH refers to the 4M mutant, the amino acid sequence of the 4M mutant is shown in SEQ ID NO.1, and the base sequence is shown in SEQ ID NO.2.

[0066] Detection of compounds in the reaction system: The compounds were quantitatively analyzed by high performance liquid chromatography (HPLC). The chromatograph was an Agilent 1260 series instrument equipped with an Aminex HPX-87H column (300×7.8mm, 9μm). Sulfuric acid (5mM) was used as the mobile phase with a flow rate of 0.8mL / min. The column temperature was maintained at 60°C. A UV detector was used to inject 5μL of sample at a fixed wavelength of 264nm for quantitative analysis of the compounds. The retention times of 5-hydroxymethylfurfural (HMF), 2,5-diformylfuran (FFF), 5-hydroxymethyl-2-furancarboxylic acid (HMFA), 5-formyl-2-furancarboxylic acid (FFA) and 2,5-furandicarboxylic acid (FDCA) were 21.02min, 26.83min, 15.83min, 16.83min and 11.13min, respectively. The content (Concentration) results of each compound are shown in Figure 1 .

[0067] from Figure 1 It can be seen that after 24 hours, the reaction system contained 37.3 μM FFF (2,5-diformylfuran), 9.4 μM HMFA (5-hydroxymethyl-2-furancarboxylic acid), 14.1 μM FFA (5-formyl-2-furancarboxylic acid) and 1.1 μM FDCA (2,5-furandicarboxylic acid). This indicates that PedH can oxidize HMF to FDCA in multiple steps with a single enzyme, although the yield is very low.

[0068] Example 2

[0069] In this Example 2, potential mutation sites of wild-type alcohol dehydrogenase (PedH) were selected by combining free energy calculation and deep learning model MutCompute, etc. The wild-type alcohol dehydrogenase is derived from Pseudomonas putida KT2440, and the specific sequence is shown in CN 116376855 A.

[0070] 2.1. Molecular docking of HMF and wild-type PedH was performed using AutoDock software. Virtual saturation mutations were performed on amino acids within the range and the binding free energy changes of all individual variants were calculated. The results of the changes in binding free energy (Mutation Energy) of single-point mutations of some amino acids in the range are shown in Figure 2 .

[0071] from Figure 2 It can be seen that 20 residues with significant changes in mutation energy were identified, namely G197, G198, G259, A261, W263, G280, W285, R350, F412, L413, G414, N417, W418, W437, L455, I461, W493, G556, A557 and W561.

[0072] 2.2. The high-performance mutants of wild-type PedH were predicted using the deep learning-based MutCompute online design tool (https: / / mutcompute.com / ). The top 20 single-point mutants in the predicted ranking were selected for pure enzyme activity characterization.

[0073] The first 20 single-point mutants obtained were transformed into E. coli BL21 (DE3) competent cells using the transformation method in step 3.2 of Example 3, and then whole-cell catalysis was performed to transform HMF. The reaction system and enzyme activity assay were the same as in Example 6. At the same time, Ethanol (ethanol) was used as a control. It is generally recognized that the activity of wild-type PedH to ethanol is relatively high. Using it as a control is conducive to convenient and intuitive discovery of mutation sites that affect activity.

[0074] The relative activity (%) results of the top 20 mutants are shown in Figure 3 The relative activity refers to the activity of the wild-type PedH on HMF or Ethanol (ethanol) as 100%, and the activities of other mutants on HMF or Ethanol are calculated relative to it.

[0075] from Figure 3 It can be seen that the 11 sites that have a greater impact on the activity are 135V, W554F, L254M, A75S, S30T, S316T, Q528D, Q586E, N136H, N144D and R300Q. These 11 sites were selected for subsequent saturation mutagenesis.

[0076] Example 3

[0077] High-throughput screening of rare earth-dependent alcohol dehydrogenase mutants (PedH).

[0078] In this Example 3, site-directed saturation mutagenesis was performed on a total of 31 mutation sites obtained in Example 2, and high-throughput screening was performed to improve the efficiency of mutant screening.

[0079] The specific method is as follows:

[0080] 3.1. Whole plasmid PCR

[0081] Using pET28a-PedH-4M plasmid as template, upstream and downstream primers covering the mutation site were designed (Table 1) for whole plasmid PCR.

[0082] Table 1 Primers used for site-directed mutagenesis construction

[0083]

[0084]

[0085] PCR amplification system:

[0086]

[0087]

[0088] PCR amplification conditions:

[0089] 1) Pre-denaturation: 98℃ for 3min;

[0090] 2) Denaturation: 98°C for 10 s; annealing: 60°C for 15 s; extension: 72°C for 1 min; 33 cycles in total;

[0091] 3) Post-extension: 72°C for 5 min;

[0092] 4) Store at 4℃.

[0093] 3.2 Conversion and Verification

[0094] The PCR product of step 3.1 was directly transformed into E. coli BL21 (DE3) competent cells using the heat shock method. After incubation for 12 hours, single clones were picked into 96-well deep-well plates with 800 μL LB medium per well and cultured at 37°C for 8 hours. 50 μL of bacterial solution was transferred to 750 μL LB medium containing 10 g / L lactose and cultured at 30°C for 12 hours. The culture was then centrifuged to remove the supernatant. Fresh reaction solution (500 μL) was added to each well, the cells were resuspended and incubated in the dark for 24 hours. The reaction solution was then centrifuged, 200 μL of supernatant was transferred to a 96-well ELISA plate, and the OD was measured using an ELISA reader. 610 . Select OD610 The mutants with lower values ​​than the 4M mutant were sequenced and verified, and 47 single-point mutants were identified, which showed improved enzyme activities compared with the 4M mutant.

[0095] The 47 single-point mutants obtained in the initial screening were subjected to whole-cell catalysis to convert 50mM HMF. After 24 hours of reaction, the HMF conversion rate and the generation of various intermediates of all variants were detected. That is, the whole cells of the 47 mutants were taken and added to 10mL of a reaction solution containing 100mM Tris-HCl (pH 8), 20μM PQQ, 20μM PrCl3, 2mM WB, and 50mM HMF, and the final cell concentration was OD 600 =10. The reaction was carried out at 30°C and 220 rpm for 24 h. The reaction was then terminated by centrifugation at 12,000 g for 10 min. The supernatant was diluted to an appropriate ratio and filtered to obtain the sample to be tested. The content of each compound in the system after 24 h of reaction was detected by the same high performance liquid chromatography (HPLC) as in Example 1. The results are as follows: Figure 4 shown.

[0096] from Figure 4 It can be seen that the mutations with higher total production of FFF, HMFA, FFA, and FDCA are 4M-R300G, 4M-F412S, 4M-W554S, 4M-G556S, and 4M-W561G.

[0097] Example 4

[0098] Iterative Saturation Mutagenesis of Rare Earth-Dependent Alcohol Dehydrogenase

[0099] In this Example 4, amino acids R300, F412, W554, G556, and W561 with higher FDCA yields in Example 3 were selected for perfect iterative saturation mutagenesis, i.e., all mutations at one amino acid site were constructed by site-directed mutagenesis, rather than saturation mutagenesis by NNK. The mutant with the highest FDCA yield in each round was selected as the template for the next round, and site-directed saturation mutagenesis at other sites was superimposed.

[0100] The most active mutant 5M (4M-G556S) was obtained in the first round, the most active mutant 6M (4M-G556S-F412S) was obtained in the second round, and the most active mutant 7M (4M-G556S-F412S-W554S) was obtained in the third round. In the fourth round of evolution, no further increase in FDCA production was observed, so the three new sites (G198, A261, and A557) identified in the initial screening were included in the iterative saturation process, and finally mutant 8M (4M-G556S-F412S-W554S-A557N) was obtained. The new sites G198 and A261 were abandoned due to poor effects. The evolutionary path is as follows Figure 5 shown.

[0101] The obtained 7 mutants were transformed into E. coli BL21 (DE3) competent cells by the transformation method of step 3.2 in Example 3, and then whole-cell catalysis was performed to transform 50 mM HMF. After 24 hours of reaction, the HMF conversion rate and FDCA production of all mutants were detected. The results are shown in FIG. Figure 6 Specifically, whole cells of seven mutants were taken and added into 10 mL of reaction solution containing 100 mM Tris-HCl (pH 8), 20 μM PQQ, 20 μM PrCl3, 2 mM WB, and 50 mM HMF. The final cell concentration was OD 600 =10. The reaction was carried out at 30°C and 220rpm for 24h. The reaction was then terminated by centrifugation at 12,000g for 10 minutes, the supernatant was diluted to an appropriate ratio, and filtered to obtain the sample to be tested. The content of each compound in the system after the reaction was completed was detected by the same high performance liquid chromatography (HPLC) as in Example 1. The conversion rate of HMF was calculated by subtracting the content of HMF in the reaction system after the reaction was completed from the content of HMF in the reaction system before the reaction, divided by the content of HMF in the reaction system before the reaction. The results are shown in Tables 2 and Figure 6 .

[0102] Table 2

[0103]

[0104] From Table 2 and Figure 6It can be seen that compared with 4M, the FDCA production of 4M-F412S, 4M-W554S, 4M-A557N, 5M (4M-G556S), 6M (4M-G556S-F412S), 7M (4M-G556S-F412S-W554S) and 8M (4M-G556S-F412S-W554S-A557N) increased by 4 times, 3 times, 3 times, 159 times, 233 times, 348 times and 349 times, respectively, among which the production of 8M mutant was the highest at 3.49 mM; compared with 4M, The substrate HMF conversion rates of 4M-F412S, 4M-W554S, 4M-A557N, 5M (4M-G556S), 6M (4M-G556S-F412S), 7M (4M-G556S-F412S-W554S) and 8M (4M-G556S-F412S-W554S) were increased by 11.1 times, 7.7 times, 3.6 times, 16.9 times, 19.0 times, 19.2 times and 20.9 times, respectively, among which the substrate HMF conversion rate of 8M was the highest at 60.5%. The FDCA yield of mutant 8M was 6.98%.

[0105] Example 5

[0106] Strain culture, protein expression and purification.

[0107] The recombinant bacteria in Example 4 (the original rare earth-dependent alcohol dehydrogenase recombinant bacteria, i.e., 4M strain and the rare earth-dependent alcohol dehydrogenase mutant strain, i.e., 8M strain) were picked up and placed in 5 mL LB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37° C. for 12 h with shaking. The inoculum was transferred to 50 mL LB liquid medium containing 50 μg / mL kanamycin and 10 g / L α-lactose at a 2% inoculum size and cultured at 30° C. for 12 h with shaking.

[0108] After the culture is completed, the bacterial solution is centrifuged at 4,000×g for 15 minutes at 4°C, the supernatant is discarded, and the bacterial cells are collected. After washing the bacterial cells with 100mM Tris-HCl buffer at pH 8.0, they are resuspended in Tris-HCl buffer and placed in an ice-water bath for ultrasonic cell disruption until clarified. The cell disruption solution is centrifuged at 8,000×g at 4°C for 20 minutes, and the supernatant is subjected to affinity chromatography using a nickel column. The impurities are washed with a washing buffer (100mM Tris-HCl buffer, 150mM NaCl, 50mM imidazole, pH 8.0), and the target protein is eluted with an elution buffer (100mMTris-HCl buffer, 150mM NaCl, 250mM imidazole, pH 8.0). The separated target protein is placed in an ultrafiltration centrifuge tube for sufficient desalting and concentration to obtain a pure target protein, that is, the purified 4M mutant protein or the purified 8M mutant protein.

[0109] Example 6

[0110] In this Example 6, the enzyme activity of rare earth-dependent alcohol dehydrogenase was determined.

[0111] The amount of DCPIP consumed in the reaction system was quantitatively analyzed using an ELISA instrument, and the specific enzyme activities of the original rare earth-dependent alcohol dehydrogenase (4M) and the rare earth-dependent alcohol dehydrogenase mutant (8M) on four substrates HMF, FFF, HMFA, and FFA were obtained by calculation.

[0112] Based on the total volume of the reaction system, each reaction system contains 0.5 μM of the purified 4M mutant protein or the purified 8M mutant protein obtained in Example 5, as well as 1.5 μM PQQ, 1.5 μM PrCl3, and 15 mM substrate; the total reaction system is 200 μL. The reaction medium is Tris-HCl buffer (100 mM, pH 8.0), which contains 1 mM of artificial electron acceptor PES (phenazine ethyl sulfate) and 150 μM DCPIP (2,6-dichloroindophenol). The other reaction systems except the substrate are placed in an ELISA instrument and incubated at 30°C until the background reaction subsides, and then the substrate is added. By measuring the OD within the first minute after adding the substrate 600 The enzyme activity was determined by the change in the enzyme concentration, and the specific enzyme activity was calculated based on the ratio of the enzyme activity to the enzyme concentration.

[0113] The results showed that the specific enzyme activity of mutant 8M on the four substrates was significantly improved compared with the parent 4M. The activities of mutant 8M on HMF, FFF, and FFA were 0.826U / mg, 1.226U / mg, and 0.35U / mg, respectively, which were 29.5, 23.1, and 20.4 times higher than those of the parent 4M.

[0114] The parent 4M had no activity against HMFA, while the mutant 8M had a specific enzyme activity of 0.069 U / mg against HMFA.

[0115] Example 7

[0116] Whole-cell catalytic screening

[0117] The whole cell biocatalytic reaction was carried out in a 50 mL centrifuge tube at 30 °C and 220 rpm with 10 mL of reaction solution. Based on the total volume of the reaction solution, the reaction solution contained 100 mM Tris-HCl (pH 8), 20 μM PQQ, 20 μM PrCl3, 2 mM WB, 50 mM HMF and whole cells (OD 600=10). After the catalytic reaction is completed, the reaction is terminated by centrifugation at 12,000 g for 10 minutes, the supernatant is diluted to an appropriate ratio, and filtered to obtain the sample to be tested. Among them, the whole cell is the whole cell obtained by centrifuging the bacterial solution at 4,000 × g and 4°C for 15 minutes after the recombinant bacteria culture in Example 5 is completed, and the supernatant is discarded.

[0118] 7.1 Investigation of artificial electron acceptor WB concentration and lanthanide element types

[0119] 1) The optimal concentration of the artificial electron acceptor WB was investigated, and the concentration range was set to 0.5, 1, 2, 4 and 8 mM. After 24 hours of catalytic reaction, the content of each compound was detected (Concentration). The results are shown in Figure 7 The detection methods of each compound are the same as those in Example 1.

[0120] from Figure 7 It can be seen that when the WB concentration was 1 mM, the HMF conversion rate and FDCA yield were the highest, which were 64.4% and 5.2%, respectively.

[0121] 2) Since the activity of rare earth-dependent alcohol dehydrogenase is affected by the type of rare earth element, we further evaluated the addition of various lanthanide elements (except Pr 3+ In addition, it also includes La 3+ 、Ce 3 + 、Nd 3+ 、Sm 3+ 、Eu 3+ , Gd 3+ and Tb 3+ ), that is, LaCl3, CeCl3, NdCl3, SmCl3, EuCl3, GdCl3 and TbCl3 were used to replace PrCl3. After 24 hours of catalytic reaction, the content of each compound was detected. The results are shown in Figure 8 The detection methods of each compound are the same as those in Example 1.

[0122] from Figure 8 It can be seen that it was found that 3+ With the addition of Nd, the conversion rate of HMF is as high as 66.6%. 3+ Conduct follow-up experiments.

[0123] 7.2 pH investigation

[0124] On the basis of selecting Nd as the lanthanide element at 7.1, the pH value was further optimized within the range of 7.0 to 10.5.

[0125] Since FDCA is an acid, the pH decreases as the reaction proceeds. The 100mM Tris-HCl buffer used initially is not enough to maintain the stability of the pH during the reaction. Therefore, the buffer concentration was increased to 400mM to provide sufficient buffering capacity. When the pH is 7.0-8.0, HEPES buffer is used; when the pH is 8.0-9.0, Tris-HCl buffer is used; when the pH is 9.0-10.5, Gly-NaOH buffer is used. After 24 hours of catalytic reaction, the content of each compound is detected. The results are shown in Fig. 9 The detection methods of each compound are the same as those in Example 1.

[0126] from Fig. 9 It can be seen that in the Tris-HCl buffer with a pH of 8.0, the conversion rate of HMF reached 89.7%, at which time the yields of FDCA and FFA were the highest. Therefore, the Tris-HCl buffer with a pH of 8.0 was selected for subsequent experiments.

[0127] 7.3, PQQ, Nd 3+ Concentration and temperature investigation

[0128] 1) PQQ and Nd were studied 3+ Effect of concentration on catalytic reaction. Set the concentration range to 2-50 μM. After 24 hours of catalytic reaction, detect the content of each compound (Concentration). The results are shown in Fig.10 The detection methods of each compound are the same as those in Example 1. The columns from left to right represent 2 μM PQQ and 2 μM Nd 3+ (first column), 5 μM PQQ and 5 μM Nd 3+ (second column), 10 μM PQQ and 10 μM Nd 3+ (third column), 20 μM PQQ and 20 μM Nd 3+ (the fourth column), 30 μM PQQ and 30 μM Nd 3+ (fifth column), 40 μM PQQ and 40 μM Nd 3+ (the sixth column) and 50 μM PQQ and 50 μM Nd 3+ (Seventh Pillar).

[0129] from Fig.10 It can be seen that PQQ and Nd 3+ When the concentration was 5 μM, the conversion rate of HMF reached 89.6%, which was lower than that of 20 μM PQQ and 20 μM Nd 3+ It decreased slightly, but the yields of FFA and FDCA were the highest, so this concentration was selected for subsequent experiments.

[0130] 2) Then, after determining PQQ and Nd 3+ The concentration was 5 μM, and the effect of temperature in the range of 20-40°C was investigated. The temperature was set at 20°C, 25°C, 30°C, 35°C and 40°C, respectively. After 24 hours of catalytic reaction, the content of each compound was detected. The results are shown in Fig.11 The detection methods of each compound are the same as those in Example 1.

[0131] from Fig.11 It can be seen that when the reaction temperature is 30°C, the conversion rate of HMF reaches 89.6%.

[0132] 7.4. Optimization of cell concentration, substrate concentration and biocatalyst form

[0133] The cell concentration, substrate concentration and form of the biocatalyst were adjusted to further increase the yield of FDCA. After 24 hours of catalytic reaction, the content of each compound was detected. The results are shown in Fig.12 1×, 2×, and 5× refer to OD 600 The whole cell catalysts were 10, 20, and 50, and the HMF concentration was 50 mM; 5×L refers to the amount of HMF equivalent to OD 600 =50 cell lysate, at this time the HMF concentration is 50mM; 5×L -40 , 5×L -30 Refers to the equivalent of OD 600 =50 cell lysate, at which the HMF concentration was adjusted to 40 mM and 30 mM respectively. The cell lysate used was obtained by disrupting equal amounts of cells using an ultrasonic cell disruptor. The detection method of each compound was the same as in Example 1.

[0134] from Fig.12 It can be seen that when whole-cell catalysis is used, after 24 hours of reaction, the cell OD 600 As the final concentration OD increased from 10 to 50, the FDCA yield increased from 5.4% to 30.3%. 600 =50 cells were lysed by ultrasonic and then catalyzed, and the FDCA yield reached 63.7% in 24 hours and further increased to 90.4% in 48 hours.

[0135] from Fig.12 It can be seen that when the HMF concentration dropped to 40 mM and 30 mM, the FDCA yields after 48 h reached 96.4% and 96.1%, respectively, and the by-product detected in the reaction system was only a small amount of HMFA.

[0136] By optimizing the artificial electron acceptor concentration, rare earth ion type, reaction pH, cofactor PQQ and rare earth ion concentration, reaction temperature, cell concentration, substrate concentration, biocatalyst type and other conditions, when the reaction system includes the following: 40mM HMF, 1mM WB, 5μM NdCl3, Tris-HCl buffer with pH 8.0, 5μM PQQ, 30℃, concentration OD 600 =50 cells ultrasonically lysed, the highest yield of FDCA was 96.4%, the FDCA yield was 38.6mM, and the substrate HMF conversion rate reached 100%; compared with the 8M mutant in Example 4, the yield of FDCA increased from 6.98% to 96.4% after optimizing the reaction system, an increase of 13.8 times. Therefore, the PQQ-dependent alcohol dehydrogenase mutant of the present invention and its application are of great significance to promoting the green biomanufacturing of FDCA.

Claims

1. A PQQ-dependent alcohol dehydrogenase mutant, characterized in that: Based on the amino acid sequence shown in SEQ ID NO.1, the alcohol dehydrogenase mutant includes one or more of the following mutations: F412S, W554S, G556S and A557N.

2. The alcohol dehydrogenase mutant according to claim 1, characterized in that The alcohol dehydrogenase mutants include F412S and G556S; and / or, the alcohol dehydrogenase mutants include F412S, W554S and G556S; And / or, the alcohol dehydrogenase mutant comprises F412S, W554S, G556S and A557N.

3. An isolated polynucleotide, characterized in that Encoding the alcohol dehydrogenase mutant according to claim 1 or 2.

4. A recombinant vector, characterized in that: Comprising the polynucleotide as claimed in claim 3.

5. A recombinant microorganism or cell, characterized in that The polynucleotide according to claim 3 is integrated into the recombinant vector or gene according to claim 4.

6. Use of the alcohol dehydrogenase mutant according to claim 1 or 2, or the polynucleotide according to claim 3, or the recombinant vector according to claim 4, or the recombinant microorganism or cell according to claim 5 in at least one of the following: 1) Use in regulating the production of 2,5-furandicarboxylic acid; 2) Use in the preparation of 2,5-furandicarboxylic acid; 3) Use in constructing genetically engineered bacteria that produce 2,5-furandicarboxylic acid.

7. A method for preparing 2,5-furandicarboxylic acid, characterized in that: The alcohol dehydrogenase mutant according to claim 1 or 2, the recombinant vector according to claim 4, or the recombinant microorganism or cell according to claim 5 is used to catalyze a substrate reaction to obtain 2,5-furandicarboxylic acid.

8. The preparation method according to claim 7, characterized in that: The substrate is selected from 5-hydroxymethylfurfural; and / or, the reaction system further comprises one or more of lanthanide metal ions, artificial electron acceptors and pyrroloquinoline quinone; And / or, the reaction temperature is 15-40°C; And / or, the reaction time is 1-72h; And / or, the recombinant microorganism or cell comprises whole cells or cell lysate.

9. The preparation method according to claim 8, characterized in that: The concentration of the artificial electron acceptor is 0.5-50 mM; and / or, the concentration of pyrroloquinoline quinone is 2-250 μM; And / or, the concentration of the lanthanide metal ions is 2-250 μM.

10. The preparation method according to claim 8, characterized in that: The artificial electron acceptor is selected from Worcester Blue.

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