High-throughput screening method of 2-keto-3-deoxy-D-xylonic acid dehydratase mutant library and application of mutant in biosynthesis of alpha-ketoglutaric acid

Abstract

The invention discloses a high-throughput screening method of a 2-keto-3-deoxy-D-xylonic acid dehydratase mutant library and an application of a mutant in biological synthesis of alpha-ketoglutaric acid. The directed evolution modification method comprises the following steps: carrying out fusion expression on a 2-ketone-3-deoxy-D-xylonic acid dehydratase coding gene (xylX), a Rex protein coding gene (rex) and an annularly arranged yellow fluorescent protein coding gene (cpYFP); the method comprises the following steps: firstly, carrying out primary screening on a 2-ketone-3-deoxy-D-xylonic acid dehydratase random mutant library by utilizing a flow cytometry technology, and screening according to a cell fluorescence value to obtain a mutant which possibly has relatively high catalytic activity; then, re-screening the mutants obtained by primary screening through a deep-well plate ultraviolet spectrum method, so as to obtain mutants with obviously improved catalytic activity; and finally, carrying out shake-flask culture on the mutant obtained by secondary screening, and determining the concentration of extracellular alpha-ketoglutaric acid by adopting a high performance liquid chromatography so as to determine the activity of the 2-keto-3-deoxy-D-xylonic acid dehydratase mutant. The high-throughput screening method disclosed by the invention is high in universality, high in sensitivity, simple, effective and high in throughput, and the screened 2-keto-3-deoxy-D-xylonic acid dehydratase mutant can be applied to biosynthesis of alpha-ketoglutaric acid.

Description

Technical Field

[0001] The invention belongs to the field of protein engineering, and specifically relates to a high-throughput screening method for a 2-keto-3-deoxy-D-xylonic acid dehydratase mutant library and application of the mutant in alpha-ketoglutarate biosynthesis. Background Art

[0002] As an important intermediate metabolite in the tricarboxylic acid cycle (TCA cycle), α-ketoglutarate is involved in the synthesis of amino acids, vitamins and organic acids, as well as energy metabolism, and has a wide range of applications in many fields, including food, medicine, chemicals, cosmetics and agriculture. In particular, recent studies have shown that α-ketoglutarate can extend the lifespan of aged mice and reduce their morbidity. Therefore, α-ketoglutarate has great industrial production value. Most of the α-ketoglutarate currently on the market is prepared by chemical synthesis, which uses diethylsuccinic acid and diethyloxalic acid as raw materials to synthesize α-ketoglutarate through condensation and hydrolysis reactions. This process produces a large number of by-products, including cyanide and other toxic substances, heavy metal pollutants and explosive substances, which increase the difficulty of separation, reduce productivity and increase production risks. Therefore, bioconversion processes are a promising alternative that can replace fossil-based raw materials with renewable resources. Biocatalysts, namely enzymes and microorganisms, provide a sustainable way to produce chemicals from lignocellulose-derived sugars.

[0003] Previous studies have found that many microorganisms can synthesize α-ketoglutarate, among which Corynebacterium glutamicum ( Corynebacterium glutamicum ) and Yarrowia lipolytica ( Yarrowia lipoytica ) showed excellent production capacity. Nevertheless, the efficient production of α-ketoglutarate is subject to strict regulation of glucose metabolism, oxaloacetate regeneration reaction, TCA cycle and α-ketoglutarate dehydrogenase complex. The current in vitro enzymatic conversion process only relies on L-glutamate as a substrate.

[0004] The development of biorefining technology driven by the effective use of pretreatment and microbial fermentation technologies has enhanced the interest in utilizing lignocellulose as a sustainable feedstock for biomanufacturing. Among them, lignocellulose is rich in pentoses such as D-xylose and L-arabinose, making it a promising biomanufacturing feedstock. However, their fermentation capacity is limited due to the inefficiency of enzymes and strict metabolic regulation. The Weimberg pathway is a pathway that converts pentoses to xylose-derived α-ketoglutarate without carbon loss, providing a promising route for sustainable chemical production. This non-phosphorylating oxidative metabolic pathway was first described more than 50 years ago, with the notable being Caulobacter crescentus ( Caulobacter crescentus) induces xylose metabolism in the xylXABCD operon. D-xylose is first converted to D-xylolactone by D-xylose dehydrogenase (XDH) and then hydrolyzed to D-xylonic acid ester by D-xylolactonase (XylC). D-xylonic acid is then dehydrated to 2-keto-3-deoxy-D-xylonic acid by D-xylose dehydratase (XylD), which is then acted on by 2-keto-3-deoxy-D-xylonic acid dehydratase (XylX) to generate α-ketoglutarate semialdehyde (KGSA); finally, it is oxidized to α-ketoglutarate by ketoglutarate semialdehyde dehydrogenase (KGSADH). The Weimberg pathway has been widely used in the fermentation process of various microorganisms. However, the relatively low activity of 2-keto-3-deoxy-D-xylonic acid dehydratase in the Weimberg pathway significantly hinders the effective degradation of lignocellulose and becomes a key factor in the advancement of biorefining process. 2-Keto-3-deoxy-D-xylonic acid dehydratase is composed of 384 amino acid residues, catalyzing the conversion of 2-keto-3-deoxy-D-xylonic acid to α-ketoglutaric semialdehyde, and belongs to the fumaryl acetoacetate hydrolase (FAH) family. To date, the protein structure and catalytic mechanism of all identified 2-keto-3-deoxy-D-xylonic acid dehydratases are still not fully understood, which limits the rational modification of the enzyme. Summary of the invention

[0005] The present invention provides a high-throughput screening method for a 2-keto-3-deoxy-D-xylonate dehydratase mutant library and the application of the mutant in α-ketoglutarate biosynthesis. The present invention screens out 2-keto-3-deoxy-D-xylonate dehydratase with high catalytic efficiency through the three steps of flow cytometry primary screening, 96-deep-well plate liquid ultraviolet spectrum secondary screening and high-performance liquid chromatography detection, so as to achieve the purpose of improving sensitivity, and fill the gap in the current high-throughput screening method of 2-keto-3-deoxy-D-xylonate dehydratase mutant library.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The present invention provides a high-throughput screening method for a 2-keto-3-deoxy-D-xylonic acid dehydratase mutant library, comprising the following steps: 1) Preliminary screening: Flow cytometry was used to screen the random mutant library of 2-keto-3-deoxy-D-xylonic acid dehydratase, and mutants with higher catalytic activity were screened based on cell fluorescence values; 2) Rescreening: The mutants obtained from the initial screening were rescreened by deep-well plate UV spectroscopy to obtain mutants with significantly improved catalytic activity; 3) Activity determination: The mutants obtained by repeated screening were cultured in shake flasks, and the extracellular α-ketoglutarate concentration was determined by high performance liquid chromatography to determine the activity of the 2-keto-3-deoxy-D-xylonic acid dehydratase mutant.

[0007] Furthermore, the specific steps of the initial screening are: placing the microorganism containing the gene of the random mutant library of 2-keto-3-deoxy-D-xylonic acid dehydratase in a liquid culture medium, culturing for 20 h under the conditions of 0.4 mmol / L isopropyl-β-D-thiogalactoside (IPTG) and 16 ℃ 200 rpm, collecting the cells by centrifugation at 4 ℃ for 5 min at 6000 rpm, resuspending with Tris-HCl (pH 8.5) buffer, adding xylonic acid dehydratase and ketoglutarate semialdehyde dehydrogenase purified proteins as well as 10 mmol / L D-xylonic acid and 2 mmol / L NAD + The reaction was incubated at 37°C for 30 min, and mutants with higher catalytic activity were screened based on cell fluorescence values.

[0008] Furthermore, the specific steps of the rescreening are: inoculating the cells obtained in the primary screening into a 96-deep-well plate, culturing in liquid, culturing for 20 h under the conditions of 0.4 mmol / L IPTG and 16 °C 200 rpm, collecting the cells by centrifugation at 4 °C 6000 rpm for 5 min, resuspending with Tris-HCl (pH 8.5) buffer, adding xylonate dehydratase and ketoglutarate semialdehyde dehydrogenase purified proteins as well as 10 mmol / L D-xylonic acid and 2 mmol / L NAD + The reaction was incubated at 37 °C for 30 min, and mutants with significantly improved catalytic activity were screened out based on the ultraviolet absorption intensity at 340 nm.

[0009] Furthermore, the specific steps for determining the enzyme activity are as follows: the mutant colonies obtained by repeated screening are cultured in a liquid shake flask under the conditions of 0.4 mmol / L IPTG and 16°C 200 rpm for 20 h, the cells are collected by centrifugation at 6000 rpm for 5 min at 4°C, resuspended in Tris-HCl (pH 8.5) buffer, and purified proteins of xylonate dehydratase and ketoglutarate semialdehyde dehydrogenase as well as 10 mmol / L D-xylonic acid and 2 mmol / L NAD + The reaction was incubated at 37 °C for 30 min, and the extracellular α-ketoglutarate concentration was measured by HPLC. Finally, the mutant with the highest catalytic activity was screened.

[0010] Furthermore, the xylonate dehydratase and ketoglutarate semialdehyde dehydrogenase pure proteins are prepared in the following steps: the coding genes of xylonate dehydratase and ketoglutarate semialdehyde dehydrogenase are respectively inserted into the pET-28a(+) vector, and the obtained strains are respectively cultured in liquid shake flasks, cultured for 20 h under the conditions of 0.5 mmol / L IPTG and 16°C 200 rpm, and the cells are collected by centrifugation at 6000 rpm for 5 min at 4°C, resuspended with Tris-HCl (pH 8.5) buffer, and affinity chromatography purification is performed using Ni-IDA affinity columns (manufacturer TaKaRa, product number 635659) according to the instructions to obtain pure proteins.

[0011] Furthermore, the 2-keto-3-deoxy-D-xylonic acid dehydratase is XylX, and its wild-type amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0012] Further, the 2-keto-3-deoxy-D-xylonic acid dehydratase encoding gene ( xy )、Rex protein encoding gene ( rex ) and a circular yellow fluorescent protein encoding gene ( cpYFP ) fusion expression, construct the recombinant vector pET-28a(+)- rex-cpYFP-xylX . rex-cpYFP The amino acid sequence is SEQ ID No.13, and the nucleotide sequence is SEQ ID No.14.

[0013] Furthermore, according to the method reported in the previous literature (Liang, B., J. Yang, CF Meng, YR Zhang, L. Wang, L. Zhang, J. Liu, ZC Li, S. Cosnier, AH Liu, and JM Yang. 2024. 'Efficient conversion of hemicellulose into high-value productand electric power by enzyme-engineered bacterial consortia', Nature Communication, 15: 8764.), the surface display strain E. coli -XDH, E. coli -XylC, E. coli -XylD, E. coli -KGSADH, E. coli -XylX and E. coli-XylX mutants are mixed to obtain a whole-cell catalyst. The amino acid sequences of XDH, XylC, XylD, and KGSADH are shown in SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, and SEQ ID No.18, and the nucleotide sequences are shown in SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, and SEQ ID No.22. The whole-cell catalytic system achieves controllable proportions, characterized in that: E. coli -XDH: E. coli -XylC, E. coli -XylD: E. coli The ratio of -KGSADH: INP-XylX and its mutants was 3:10:10:10:17. The reaction system was placed in a constant temperature oscillator at 37 °C and 100 rpm for 6 h. The α-ketoglutarate production in the whole cell catalytic system was determined by high performance liquid chromatography, and the yield was calculated based on the concentration of added D-xylose and the concentration of α-ketoglutarate.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention first uses flow cytometry to perform a preliminary screening of a 2-keto-3-deoxy-D-xylonic acid dehydratase mutant library, and reacts the catalytic efficiency according to the fluorescence intensity, but can only perform a range screening on the mutant library; further, the screened mutants are rescreened, and a 96-deep-well plate is used for liquid rescreening, thereby excluding false positive mutants; finally, the activity is determined by shaking flask culture and high performance liquid chromatography detection.

[0015] 2. The screening method of the present invention includes flow cytometry (primary screening), 96-deep-well plate liquid ultraviolet spectroscopy (rescreening) and shake flask culture high performance liquid chromatography detection (activity determination). By organically combining the three methods, high-throughput screening of a dehydratase mutant library, especially a 2-keto-3-deoxy-D-xylonic acid dehydratase mutant library, is achieved. The method of the present invention has strong universality, high sensitivity, simplicity, effectiveness and high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The constructed vector pET-28a(+) -rex-cpYFP-xylX Plasmid map.

[0017] Figure 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis diagram of XylD and KGSADH intracellular protein purification.

[0018] Figure 3Flow cytometry screening results. Induced: the reaction system after adding IPTG to induce the expression of the XylX mutant library, Uninduced: the control system without adding IPTG to induce the XylX mutant library, Induced-sorting: the reaction system after sorting according to fluorescence intensity.

[0019] Figure 4 The characteristic peak diagram of the enzyme reaction products of XylX and its mutants at 340 nm.

[0020] Figure 5 The figure shows the enzymatic activity comparison between XylX mutants and XylX.

[0021] Figure 6 Yields of α-ketoglutarate synthesis for whole-cell catalysts containing wild-type XylX (MC01) and whole-cell catalysts containing XylX mutant 4 (MC02).

[0022] Figure 7 It is the Weimberg xylose metabolic pathway. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

[0024] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased.

[0025] Example 1: Establishment of a flow cytometric screening method for 2-keto-3-deoxy-D-xylonic acid dehydratase (primary screening) 1. pET-28a(+)-rex-cpYFP-xylX Vector construction (1) Using pET-28a(+) (manufacturer Novagen) as a template, in vitro polymerase chain reaction (PCR) was performed with primers pETF / pETR to linearize the pET-28a(+) vector. The PCR amplification system is as follows: Components Dosage (μL) 2 × Phanta Max Master Mix 25 Primer-F (10 μM) 2 Primer-R (10 μM) 2 Template (5 ng) 1 ddH2O 20 The PCR amplification program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 3 min, 30 cycles of amplification, and 72°C final extension for 5 min. The primer sequences are shown in SEQ ID No. 23 and SEQ ID No. 24. The PCR products were subjected to agarose gel electrophoresis to verify the size of the amplified product bands.

[0026] (2) Bacillus subtilis ( Bacillus subtilis) and Caulobacter crescentus ( Caulobacter crescentus ) genome as a template, and in vitro polymerase chain reaction (PCR) was performed with primers rex-cpYFPF / rex-cpYFPR and XylXF / XylXR to amplify the fragment. rex-cpYFP (Zhao Y, Jin J, Hu Q, et al. Genetically Encoded Fluorescent Sensors for Intracellular NADH Detection[J]. Cell Metabolism, 2011, 14(4):555-566.) and XylX, and the PCR amplification system is as follows: Components Dosage (μL) 2 × Phanta Max Master Mix 25 Primer-F (10 μM) 2 Primer-R (10 μM) 2 Template (5 ng) 1 ddH2O 20 The PCR amplification program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 54°C annealing for 15 s, 72°C extension for 1 min 10 s, 30 cycles of amplification, and 72°C final extension for 5 min. The primer sequences are shown in SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, and SEQ ID No. 28. The PCR products were subjected to agarose gel electrophoresis to verify the size of the amplified product bands.

[0027] (3) The PCR product was purified using a gel recovery kit (Vazyme, catalog number DC301-01) according to the instructions.

[0028] (4) The 2-keto-3-deoxy-D-xylonic acid dehydratase encoding gene ( xy )、Rex protein encoding gene ( rex ) and a circular yellow fluorescent protein encoding gene ( cpYFP ) and pET-28a(+) linearized vector for multi-fragment fusion. Please refer to the instruction manual for the steps.

[0029] (4) Seamless cloning product transformation First, take out the competent medium from -80 ℃, place it on an ice box to melt naturally, and handle it with care. Add the digested PCR product to the competent medium in a sterile operating table and place it on ice for 30 min. After that, heat shock it in a 42℃ water bath for 1 min 30 s and place it on ice for 2 min. Add 500 μL LB culture medium to the sterile operating table and culture it in a 37℃ shaker for 1 h. Centrifuge at 6000 rpm for 2 min, aspirate 600 μL supernatant, and leave about 100 μL in the tube. Suspend the suspended bacteria. Spread the bacterial solution on a kanamycin-resistant LB plate and seal it. Invert it in a 37℃ incubator and culture it for 12 h-16 h.

[0030] (5) PCR screening of positive clones, extraction of recombinant plasmid pET-28a (+ )-rex-cpYFP-xylX (like Figure 1 ), and then send the plasmid to Qingke Sequencing Company for sequencing identification.

[0031] 2. Construction of mutant library The constructed recombinant plasmid pET-28a(+) -rex-cpYFP-xylX As a template, in vitro error-prone polymerase chain reaction (error-prone PCR) was performed with primers XylXF / XylXR to amplify fragment XylX. The PCR amplification system is as follows: The PCR amplification program was as follows: 95 °C pre-denaturation for 3 min, 95 °C denaturation for 30 s, 54 °C annealing for 30 s, 72 °C extension for 1 min 10 s, 30 cycles of amplification, and 72 °C final extension for 5 min. The PCR product was subjected to agarose gel electrophoresis to verify the size of the amplified product. Subsequently, seamless cloning, transformation, and verification were performed as described in the previous steps.

[0032] 3. Flow cytometry screening The microorganisms containing the gene of the random mutant library of 2-keto-3-deoxy-D-xylonic acid dehydratase were cultured in liquid culture medium with 0.4 mmol / L IPTG and 16 ℃ 200 rpm for 20 h, and the cells were collected by centrifugation at 6000 rpm for 5 min at 4 ℃, resuspended in Tris-HCl (pH 8.5) buffer, and purified proteins of xylonic acid dehydratase (10 μM) and ketoglutarate semialdehyde dehydrogenase (10 μM) (such as Figure 2 ) and 10 mmol / L D-xylonic acid and 2 mmol / L NAD + The reaction was incubated at 37 °C for 30 min, and then the reaction system was screened using a flow cytometer. The strains with stronger fluorescence intensity were sorted into a 96-well plate. A total of 2,543,355 cells were sorted in 29 min 45 s. The sorting efficiency reached 93%, and the fluorescence value increased from 18.8% to 33%. (like Figure 3 ).

[0033] 1. Initial screening of colonies, 96-well plate activation and expression Transfer 10 μL of the activated bacterial solution to each well of a 96-well plate and incubate it in a 37°C shaker for about 3 h. 600 When the concentration of 1 M IPTG is 0.2, dilute 1 M IPTG 100 times, add 40 μL to each well and place in a 16 ℃ shaker to induce expression for 20 h. After induction, transfer the bacterial solution in each well to a 96-well ELISA plate (250 μL each time, twice in total), centrifuge at 3000 rpm for 5 min using a microplate centrifuge to collect the bacteria, and discard the supernatant. Resuspend the centrifuged bacterial solution with 200 μL Tris-HCl (pH 8.5) buffer, centrifuge at 3000 rpm for 5 min, and discard the supernatant. Finally, resuspend with 100 μL Tris-HCl (pH 8.5) buffer.

[0034] 2. Ultraviolet spectroscopy rescreening The enzymatic activity of XylX and its primary mutants was tested in a coupled assay using XylD and KGSADH as described previously (Liang, B., J. Yang, CF Meng, YR Zhang, L. Wang, L. Zhang, J. Liu, ZC Li, S. Cosnier, AH Liu, and JM Yang. 2024. 'Efficient conversion of hemicellulose into high-value product and electric power by enzyme-engineered bacterial consortia', Nature Communication, 15:8764.). This coupled reaction is accompanied by the production of one molecule of NADH, which can be correlated to the enzymatic activity of XylX by measuring the specific absorbance of NADH at 340 nm. The reaction was added with purified proteins of xylate dehydratase (10 μM) and ketoglutarate semialdehyde dehydrogenase (10 μM) as well as 10 mmol / L D-xylonic acid and 2 mmol / L NAD + , incubate at 37 °C for 30 min, and then screen the reaction system using an ultra-sensitive multifunctional microplate detector (like Figure 4 ), define enzyme activity U as the amount of enzyme required to catalyze 1 μmol D-xylonic acid, and calculate the specific enzyme activity compared with the wild type (such as Figure 5), and the strains with significantly improved catalytic activity were cultured in liquid shake flasks.

[0035] 3. Determination of the activity of mutants by high performance liquid chromatography The mutant strains with significantly improved catalytic activity obtained by repeated screening were cultured in liquid culture medium with 0.4 mmol / L IPTG and 16 ℃ 200 rpm for 20 h, and the cells were collected by centrifugation at 6000 rpm for 5 min at 4 ℃, resuspended in Tris-HCl (pH 8.5) buffer, and purified proteins of xylonate dehydratase (10 μM) and ketoglutarate semialdehyde dehydrogenase (10 μM) as well as 10 mmol / L D-xylonic acid and 2 mmol / L NAD were added. + , incubate the reaction at 37 °C for 30 min, centrifuge the reaction system at 12,000g for 2 min at 4 °C, and remove the supernatant. Then filter the supernatant through a 0.22 μm water filter membrane and put it into a chromatographic bottle. Five mutants with significantly improved catalytic efficiency were identified, among which the catalytic efficiency of mutant 4 amino acid sequence such as SEQ ID No.6 was 2.08 times higher than that of the wild type.

[0036] HPLC detection conditions: Chromatographic column: Aminex HPX87 hydrogen column. Mobile phase: 2.5 mM sulfuric acid. Flow rate: 0.4 mL / min. Column temperature: 55°C. Differential detector temperature: 45°C.

[0037] Example 3: Application of XylX mutant in the production of α-ketoglutarate 1. Whole-cell catalytic strain expression After the in vitro activity determination of XylX and its mutants, the amino acid sequence of the mutant with the highest enzyme activity was determined as SEQ ID No. 6. Starting from the abundant lignocellulosic monosaccharide D-xylose, the de novo biosynthesis of α-ketoglutarate was carried out. To achieve this goal, an in vitro Weimberg pathway was constructed to avoid ATP consumption and intracellular metabolic regulation. Each member of this pathway was displayed on the cell surface of Escherichia coli BL21 (DE3) using the N-terminal region of ice nuclease protein as an anchoring motif, and a whole-cell catalyst was developed, including E. coli -XDH, E. coli -XylC, E. coli -XylD, E. coli -KGSADH, E. coli -XylX and E. coli-XylX, the specific construction steps were reported in previous studies (Liang, B., J. Yang, CF Meng, YR Zhang, L. Wang, L. Zhang, J. Liu, ZC Li, S. Cosnier, AH Liu, and JM Yang. 2024. 'Efficient conversion of hemicellulose into high-value product and electric power by enzyme-engineered bacterial consortia', Nature Communication, 15: 8764.). The α-ketoglutarate synthesis module was constructed by assembling the above-mentioned Escherichia coli immobilized enzyme in vitro multienzyme system. Cell surface display recombinant strain Escherichia coli E. coli -XDH, E. coli -XylD, E. coli -KGSADH was cultured in LB medium containing 50 μg / mL kanamycin. 600 When the value reached 0.5-0.6, the cells were induced with 0.5 mmol / L IPTG at 16 °C for 20 h to express the fusion protein. E. coli -XylX and E. coli The -XylX mutant strain was induced with 0.4 mmol / L IPTG at 16 °C for 20 h to express the fusion protein. E. coli -XylC was induced with 0.5 mmol / L IPTG at 30 °C for 12 h to express the fusion protein.

[0038] 2. Whole-cell catalytic reaction system according to E. coli -XDH: E. coli -XylC: E. coli -XylD: E. coli -KGSADH: E. coli -XylX and its mutants were in a ratio of 3:10:10:10:17, and the whole-cell catalytic reaction system was added as follows: The reaction system was placed in a constant temperature oscillator at 37 °C and 100 rpm for 6 h.

[0039] 3. Determination of α-ketoglutarate yield in whole-cell catalytic reaction system The reaction system of step 2 of Example 3 was centrifuged at 12,000 g for 2 min at 4°C to remove the supernatant. The supernatant was then filtered through a 0.22 μm water filter membrane and placed in a chromatographic bottle.

[0040] HPLC detection conditions: Chromatographic column: Aminex HPX87 hydrogen column. Mobile phase: 2.5 mM sulfuric acid. Flow rate: 0.4 mL / min. Column temperature: 55°C. Differential detector temperature: 45°C.

[0041] like Figure 6 As shown, the detection results showed that the yield of α-ketoglutarate synthesized by the whole cell catalyst (MC01) containing the wild type of XylX was 12.4 mM, and the yield of α-ketoglutarate synthesized by the whole cell catalyst (MC02) containing the XylX mutant 4 was 48.9 mM.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A high-throughput screening method for a 2-keto-3-deoxy-D-xylonic acid dehydratase mutant library, characterized in that: The following steps are involved: 1) Preliminary screening: Flow cytometry was used to screen the random mutant library of 2-keto-3-deoxy-D-xylonic acid dehydratase, and mutants with higher catalytic activity were screened based on cell fluorescence values; 2) Rescreening: The mutants obtained from the initial screening were rescreened by deep-well plate UV spectroscopy to obtain mutants with significantly improved catalytic activity; 3) Activity determination: The mutants obtained by repeated screening were cultured in shake flasks, and the extracellular α-ketoglutarate concentration was determined by high performance liquid chromatography to determine the activity of the 2-keto-3-deoxy-D-xylonic acid dehydratase mutant.

2. The high-throughput screening method according to claim 1, characterized in that The specific steps of the primary screening are: placing a microorganism containing the gene of the random mutant library of 2-keto-3-deoxy-D-xylonic acid dehydratase in a liquid culture medium, culturing for 20 h under the conditions of 0.4 mmol / L isopropyl-β-D-thiogalactoside (IPTG) and 16°C 200 rpm, collecting the cells by centrifugation at 6000 rpm for 5 min at 4°C, resuspending with Tris-HCl (pH 8.5) buffer, adding xylonic acid dehydratase and ketoglutarate semialdehyde dehydrogenase purified proteins as well as 10 mmol / L D-xylonic acid and 2 mmol / L NAD + The reaction was incubated at 37°C for 30 min, and mutants with higher catalytic activity were screened based on cell fluorescence values.

3. The high-throughput screening method according to claim 2, characterized in that: The specific steps of the secondary screening are as follows: the cells obtained from the primary screening are inoculated into a 96-deep-well plate, liquid culture is carried out, 0.4 mmol / L IPTG and 16°C 200 rpm are used for 20 h, the cells are collected by centrifugation at 6000 rpm for 5 min at 4°C, the cells are resuspended in Tris-HCl (pH 8.5) buffer, and the purified proteins of xylonate dehydratase and ketoglutarate semialdehyde dehydrogenase as well as 10 mmol / L D-xylonic acid and 2 mmol / L NAD are added. + The reaction was incubated at 37 °C for 30 min, and mutants with significantly improved catalytic activity were screened out based on the ultraviolet absorption intensity at 340 nm.

4. The high-throughput screening method according to claim 3, characterized in that: The specific steps of determining the activity are as follows: the mutant colonies obtained by repeated screening are cultured in a liquid shake flask, cultured at 0.4 mmol / L IPTG and 16°C 200 rpm for 20 h, centrifuged at 4°C 6000 rpm for 5 min to collect the cells, resuspended in Tris-HCl (pH 8.5) buffer, and xylonate dehydratase and ketoglutarate semialdehyde dehydrogenase purified proteins as well as 10 mmol / L D-xylonic acid and 2 mmol / L NAD + The reaction was incubated at 37 °C for 30 min, and the extracellular α-ketoglutarate concentration was measured by HPLC. Finally, the mutant with the highest catalytic activity was screened.

5. The high-throughput screening method according to claims 2-4, characterized in that: The xylonate dehydratase and ketoglutarate semialdehyde dehydrogenase pure proteins are prepared in the following steps: the coding genes of xylonate dehydratase and ketoglutarate semialdehyde dehydrogenase are respectively inserted into the pET-28a(+) vector, the obtained strains are respectively cultured in liquid shake flasks, cultured at 0.5 mmol / LIPTG and 16°C 200 rpm for 20 h, centrifuged at 6000 rpm for 5 min at 4°C to collect the cells, resuspended in Tris-HCl (pH 8.5) buffer, and affinity chromatography purified using Ni-IDA affinity columns (manufacturer TaKaRa, item number 635659) according to the instructions to obtain pure proteins.

6. A 2-keto-3-deoxy-D-xylonic acid dehydratase mutant obtained by screening by the high-throughput screening method according to any one of claims 1 to 5, characterized in that: The 2-keto-3-deoxy-D-xylonic acid dehydratase mutant has one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID No. 3; (2) the amino acid sequence shown in SEQ ID No. 4; (3) the amino acid sequence shown in SEQ ID No. 5; (4) the amino acid sequence shown in SEQ ID No. 6; (5) The amino acid sequence shown in SEQ ID No.

7.

7. The gene encoding the 2-keto-3-deoxy-D-xylonic acid dehydratase mutant according to claim 6, characterized in that: The coding gene has one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID No. 8; (2) the nucleotide sequence shown in SEQ ID No. 9; (3) the nucleotide sequence shown in SEQ ID No. 10; (4) the nucleotide sequence shown in SEQ ID No. 11; (5) The nucleotide sequence shown in SEQ ID No.

12.

8. An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line comprising a gene encoding the malonate transporter mutant according to claim 7.

9. Use of the mutant according to claim 7 in the biosynthesis of α-ketoglutarate.

10. The mutant according to claim 9 is used for the biosynthesis of α-ketoglutarate, characterized in that: Surface display strains E. coli -XDH, E. coli -XylC, E. coli -XylD, E. coli -KGSADH, E. coli -XylX and E. coli -XylX mutants are mixed to obtain a whole-cell catalyst. The amino acid sequences of XDH, XylC, XylD, and KGSADH are shown in SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, and SEQ ID No.18, and the nucleotide sequences are shown in SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, and SEQ ID No.

22. The whole-cell catalytic system achieves controllable proportions, characterized in that: E. coli -XDH: E. coli -XylC, E. coli -XylD: E. coli The ratio of -KGSADH: INP-XylX and its mutants was 3:10:10:10:

17. The reaction system was placed in a constant temperature oscillator at 37 °C and 100 rpm for 6 h. The α-ketoglutarate production in the whole cell catalytic system was determined by high performance liquid chromatography, and the yield was calculated based on the concentration of added D-xylose and the concentration of α-ketoglutarate.

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