CN111808900A - Method for reducing NAD analogue by formic acid - Google Patents

Abstract

The invention discloses a method for reducing NAD analogue by formic acid and application thereof. In the method, the reducing agent is a formic acid compound, the catalyst is formic acid dehydrogenase which can utilize the formic acid compound, and the NAD analogue is converted into the reduction state of the formic acid compound while the formic acid dehydrogenase oxidizes the formic acid compound. The method can be used for producing the reduced NAD analogue or the deuterated reduced analogue and can also provide reducing power for the enzymatic reaction consuming the reduced NAD analogue, and the reduced NAD analogue can be used as a coenzyme for the enzymatic reduction reactions of malic enzyme ME-L310R/Q401C, D-lactate dehydrogenase DLDH-V152R, saccharomyces cerevisiae alcohol dehydrogenase and the like, thereby being beneficial to the wide application of the NAD analogue. The NAD analogue reduction method can regenerate a reduced NAD analogue to be used for preparing malic acid or lactic acid under mild conditions; can also be used as a redox force to regulate the metabolic strength of malic acid or lactic acid in the microorganism.

Description

Method for reducing NAD analogue by formic acid

Technical Field

The invention belongs to the technical field of biology, and relates to an enzyme catalytic reduction method of coenzyme Nicotinamide Adenine Dinucleotide (NAD) analogue and application thereof, in particular to an enzyme catalytic reduction method for converting the NAD analogue into a reduction state thereof under enzyme catalysis by using a formic acid compound as a reducing agent, and the NAD analogue can be used as coenzyme by other enzymes to be applied to reduction reaction.

Background

Nicotinamide Adenine Dinucleotide (NAD) and its reduced NADH are important coenzymes in life processes, and participate in redox metabolism and other important biochemical processes in life bodies. These coenzymes can be used for producing chiral chemicals and for preparing isotopic labels. Since many oxidoreductases use NADH or NADPH as a coenzyme, any manipulation to change the NAD concentration and its redox state can have a global effect on the cell, making it difficult to control a particular oxidoreductase in the biological system at the coenzyme level. As NADH can be consumed by various pathways in a metabolic network, the utilization efficiency of the target pathway on reducing power is influenced. When NAD analogues are used to deliver reducing power, the analogues can only be recognized by mutant oxidoreductases, and thus the regulation of the target redox process at the coenzyme level is of great significance for biocatalysis and synthetic biology research (Ji DB, et al creation of biological redox systems dependent on an anionic amino derivative. J Am Chem Soc,2011,133, 20857-mentioned 20862; WangL, et al synthetic factor-linked catalytic reagents for selected energetic enerytranducer ACS, 2017, 1977-1983).

Nicotinamide Cytosine Dinucleotide (NCD) is a reported analogue of NAD with good biocompatibility (JiDB, et al, creation of biological redox systems pending on nicotinic amino flucytosoline di-glucoside. J Am Chem Soc,2011,133, 20857-20862; Ji DB, et al, Synthesis of NAD analogues to novel op biological redox system Sci Chinacchem, 2013,56, 296-300). Meanwhile, some enzymes that recognize NCD, such as D-lactate dehydrogenase (DLDH, Gnebank CAA47255) V152R mutant, malic enzyme (ME, Genbank P26616) L310R/Q401C mutant, have also been reported.

By using NAD analogues and enzymes for recognizing the NAD analogues, a more economical and effective specific biocatalysis system (Jiidenbin and the like, an artificial oxidase system is used for catalyzing L-malic acid oxidative decarboxylation reaction, catalytic journal, 2012,33,530 and 535) can be constructed, and the control of a complex biocatalysis conversion system at the coenzyme level is realized. Currently, regulation of intracellular metabolic reactions using NAD analogs has been achieved, and specific biocatalytic regulation is achieved by transporting NCD into the cell, where DLDH-V152R can reduce pyruvate to lactate using NCD (Wang L, et al synthetic cofactor-linked metabolic catalysis for selective energy transfer. acs cat, 2017,7, 1977-.

Like the use of other redox coenzymes, NAD analogs also require regeneration cycles. The coenzyme regeneration methods mainly include an enzymatic method, an electrochemical method, a chemical method and a photochemical method. The enzyme method has the advantages of high selectivity, compatibility with synthetase, high conversion number and the like. At present, glucose dehydrogenase, formate dehydrogenase or phosphite dehydrogenase is often used for regenerating the reduced coenzyme NADH. Wherein the formate dehydrogenase can utilize a formate compound to catalyze the oxidation of the formate compound to carbon dioxide and the reduction of NAD to NADH. The reaction is irreversible under normal conditions, and the reaction yield reaches 99-100%. Furthermore, since formate compounds are relatively inexpensive and the generated carbon dioxide is easily separated from the reaction system to facilitate product purification, the use of formate dehydrogenase to regenerate NADH has significant advantages (Tishkov VI, et al. protein engineering of Formate hydrogene. Biomol Eng,2006,23, 89-110). Although the regeneration cycle of the NAD analogue has important significance in the fields of biological catalysis, synthetic biology and the like, few documents exist for efficiently reducing the NAD analogue by modifying the structure of enzyme at present, and no document reports how to modify formate dehydrogenase to efficiently reduce the NAD analogue. The reported NAD analog reduction method (Zhao Zongbao et al, a reduction method of NAD analog, Chinese patent 201010524767.6) utilizes phosphite dehydrogenase to reduce NAD analog to NADH with phosphorous acid as a substrate, and at the same time, generates phosphoric acid as a byproduct. Compared with the reaction of catalyzing a phosphorous acid compound to generate a product phosphoric acid compound by using the phosphite dehydrogenase, the carbon dioxide generated in the reaction of catalyzing the formic acid compound by using the formate dehydrogenase is easy to separate from a reaction system, so that the reaction is more completely carried out. Furthermore, the inert nature of the product carbon dioxide does not inhibit or promote the enzymatic reaction, does not interfere with the catalytic activity of formate dehydrogenase and other catalytic reactions coupled thereto, and does not affect the isolation and purification of other substances in the coupling reaction, thus having a distinct advantage (Jiang W, ethyl. identification of catalysis, substrate, and enzyme binding sites and catalytic reaction of formaldehyde from microbial Biotechnol,2016,100, 8425-8437).

Based on the background and the advantages, the directed evolution method is utilized to obtain the mutant with the further improved NAD analogue reduction efficiency on the basis of the existing method for reducing the NAD analogue by formic acid.

Disclosure of Invention

The invention relates to an enzyme catalytic reduction method of coenzyme NAD analogue, in particular to a method for converting NAD analogue into a corresponding reduction state by taking a formic acid compound as a reducing agent and taking an enzyme capable of utilizing the formic acid compound as a catalyst. Reduced NAD analogs can be used as coenzymes for other oxidoreductases for reduction reactions. Therefore, the method can be applied to the fields of biological catalysis and biological conversion and has important value.

The invention relates to a method for reducing NAD analogues, which is characterized in that: the method is characterized in that one or more than two of formic acid, formate, deuteroformic acid and deuteroformate are combined at any ratio to be used as a reducing agent, an enzyme of a formic acid compound can be used as a catalyst, the reaction temperature is 10-40 ℃, the final concentration of the enzyme is 4-500 mu g/mL, the final concentration of an NAD analogue is 0.01-100 mM, and the final concentration of the formic acid compound is 0.4-25 mM in a buffer solution with the pH of 3-9, and the buffer system comprises but is not limited to one or more than two of phosphate buffer solution, Tris-HCl buffer solution, HEPES buffer solution, MES buffer solution, PIPES buffer solution and acetic acid-sodium acetate buffer system.

The NAD analogue related to the invention is Nicotinamide Cytosine Dinucleotide (NCD), Nicotinamide Thymine Dinucleotide (NTD) and Nicotinamide Uracil Dinucleotide (NUD), and the chemical structure of the NAD analogue is as follows:

the NAD analogs of the present invention were prepared by the literature reference method (Ji DB, et al. Synthesis of NADalaologs to Develop biolistichonal redox system. Sci China Chem,2013,56, 296-300).

The formate dehydrogenase used in the invention is an active protein which takes a formate compound as a reducing agent and catalyzes NAD analogue to be in a corresponding reduction state. These enzymes are mutants derived from formate dehydrogenase of Pseudomonas sp.101 (NCBI protein database numbering P33160.3, containing complete amino acid sequence from 1 to 401 site) (mutation sites are represented by amino acid numbers and amino acid names before and after mutation, such as V198I representing that amino acid at position 198 is mutated from V to I, and the rest sites are similar), including mutant E I (V198I/C256I/P260I/E261I/S381I), mutant A I (C256I/E261/S381I), mutant 3C I (A199I/E261/S381I), mutant 2A I (A199I/E261I/S381I), mutant 4C I (V198I/C256I/P260/E261/S261/381I/S381I/381I), mutant C I/P260/S261/I/C I/P I/C I/P/I/C/I/P I/P/I/P60/I/P/I/P72/36 E261P/S381N/S383F). V198I represents that the 198 th amino acid is mutated from V to I, C256I represents that the 256 th amino acid is mutated from C to I, and the like. Expression and purification of these enzymes was performed according to literature methods for expressing other oxidoreductases in E.coli (Ji DB, et al., creation of biochemical redox system dependent on an amino reductase. J Am Chem Soc,2011,133, 20857-20862).

The NAD analogs of the present invention contain a nicotinamide mononucleotide unit, the reduced state of which is 1, 4-dihydronicotinamide mononucleotide. Therefore, the reduced product has strong absorption in the ultraviolet spectral region near 340nm and molar extinction coefficient₃₄₀About 6220M^-1·cm^-1(Ji DB, et al. creation of bioorganic redox system for determining on a nicotinamide flucytoside. J Am Chem Soc.2011,133, 20857-20862). The present invention utilizes this property to analyze the NAD analog reduction process. The conditions for quantifying the NAD analogue and its reduced product by liquid chromatography are as follows: the liquid chromatograph was Agilent 1100, the analytical column was Zorbax 150 mM. times.3.0 mM (3.5 μm), the mobile phase was 5mM tetrabutylammonium sulfate, and the flow rate was 0.5 mL/min. Each sample was tested for 20 min. The detection wavelengths are 260nm (the cofactor and the reduced coenzyme thereof have stronger light absorption at 260 nm) and 340nm (the reduced coenzyme has stronger light absorption at 340 nm).

The prepared reduced product of the NAD analogue can be used as coenzyme by other enzymes and applied to reduction reaction. Thus, the present invention can be viewed as a technique for regenerating a cyclic NAD analog. By the technique of the present invention, the reducing power of a formate compound is transferred and stored in a reduced NAD analog to facilitate selective reduction of other substrates.

When the formate dehydrogenase is used for reducing NAD analogues to provide reduced coenzymes for ME-L310R/Q401C, DLDH-V152R, saccharomyces cerevisiae alcohol dehydrogenase, hydroxyl butanone dehydrogenase or D-xylose dehydrogenase, a buffer system with the pH of 3-9 is adopted, and the reaction temperature is 10-40 ℃.

The enzyme capable of utilizing a formate compound is expressed in the cells of the microorganism, while the NAD analog and the formate compound enter the cells, and the reduction reaction is carried out in the cells.

The microbial cells expressing formate dehydrogenase and used for intracellular reduction of NAD analogue include but are not limited to prokaryotic microorganisms such as Escherichia coli, lactococcus lactis and the like or eukaryotic microorganisms such as Saccharomyces cerevisiae or Trichoderma reesei or Rhodotorula toruloides and the like.

The invention has the advantages and beneficial effects that: the formic acid compound as the reducing agent is low in price, and the oxidation product carbon dioxide is easy to separate from the reaction system; the enzyme catalytic reduction condition is mild, the reaction efficiency is high, and the reaction is irreversible under normal conditions; the product has high selectivity, and can selectively transfer the reducing power of the formic acid compound to a target metabolic reaction when being applied to an endosomal system. The formate dehydrogenase in the invention has higher activity to NAD analogue, and is beneficial to the regeneration of reduced NAD analogue. In addition, by using deuterated formic acid or deuterated formate, the reduced state of the deuterated NAD analog can be obtained for preparing high-purity deuterium-substituted biocatalytic products.

Drawings

FIG. 1 is a crystal structure diagram of 3A3-NCD complex;

FIG. 2 is a crystal structure diagram of A2-NCD complex.

Detailed Description

The following examples will assist one of ordinary skill in the art in further understanding the invention, but are not intended to limit the invention in any way.

Comparative example 1: reaction of formic acid with NAD analogs in the absence of enzymes

NTD reference methods (Ji DB, et al. Synthesis of NAD analogues to Developbiolistic redox system. Sci China Chem,2013,56, 296-300). A20 mM solution was prepared with water and was used.

1mM NTD substrate and 4mM formic acid were dissolved in 1mL of 50mM Tris-HCl buffer, pH7.5, mixed, reacted at 30 ℃ for 2 hours, and 20. mu.L of the mixture was analyzed.

The NTD substrate and its reduced product were detected by HPLC. The liquid chromatograph was Agilent 1100, the analytical column was Zorbax 150 mM. times.3.0 mM (3.5 μm), the mobile phase was 5mM tetrabutylammonium sulfate, and the flow rate was 0.5 mL/min. Each sample was tested for 20 min. The detection wavelength is 260nm (strong absorption of cofactor and its reduced state at 260 nm) and 340nm (strong light absorption of reduced coenzyme at 340 nm).

Analysis revealed that all the reacted samples had no characteristic peak at 340nm, and only the characteristic peak at 260nm was detected, which was identical to the NTD retention time. Indicating that formic acid cannot directly reduce NTD without enzyme.

Comparative example 2: reaction of formic acid with NAD analogs under enzyme-inactivating conditions

A formate dehydrogenase derived from Pseudomonas sp.101 (UniProtKB/Swiss-Prot P33160.3) mutant E4(V198I/C256I/P260S/E261P/S381N) was heated in a water bath at 100 ℃ for 60min for use. Detection of NAD as a substrate indicated that the sample lost the activity of catalytically reducing NAD to NADH was performed as described in the literature (Alekseeva1 AA, et al, the role of Ala198 in the stability and coenzymes specificity of bacterial formulations ACTA NATURAE,2015,7, 60-69).

The NCD was reacted as follows: 1mM NCD, 4mM formic acid and 40. mu.g of the inactivated formate dehydrogenase mutant E4(V198I/C256I/P260S/E261P/S381N) were dissolved in 1mL of 50mM Tris-HCl buffer solution (pH 7.5) and mixed, reacted at 30 ℃ for 2 hours, and 20. mu.L of the mixture was analyzed.

According to the analysis of the method of comparative example 1, the reacted sample has no characteristic peak at 340nm, and only the characteristic peak with the same retention time as that of the NAD analogue is detected at 260 nm. Indicating that the heat-inactivated enzyme is unable to catalyze formic acid reduction of NCD.

Example 1: using formic acid as reducing agent, catalytic reduction NAD analogue by formic acid dehydrogenase

The NAD analogue and formate dehydrogenase mutant E (V198/C256/P260/E261/S381), mutant A (C256/E261/S381), mutant 3C (A199/E261/S381), mutant 2A (A199/E261/S381), mutant 4C (V198/C256/P260/E261/S381/H224), mutant 4C (T197/C256/H260/E261/S381), mutant 4B (T197/C256/H260/E261/S381), mutant 4A (T197/C256/H260/E261/S381) and 3A (V198/C256/P260/E261/S381/S383) are subjected to NAD analogue-formate dehydrogenase combination one by one, and the reaction is carried out according to the following method: 1mM NAD analogue, 4mM formic acid and 40. mu.g formate dehydrogenase were dissolved in 1mL HEPES buffer solution of 50mM concentration, pH7.5, mixed well, reacted at 30 ℃ for 20min, and 20. mu.L thereof was analyzed.

According to the analysis method of comparative example 1, the samples have characteristic absorption peaks at 340nm, but the absorption peak intensities obtained by different combinations are obviously different, which indicates that formate dehydrogenase can catalyze formate to reduce NAD analogues. Molar extinction coefficient of reduced products due to NAD analogs₃₄₀About 6220M^-1·cm^-1The curve was plotted using NADH standards in the same manner as NADH to obtain quantitative results (Table 1). The activity of each mutant was better overall for NCD than for NTD and NUD. The NCD reduction efficiency of the mutants A2 and 3A3 is 98% and 97%, respectively, which is improved compared with the catalytic efficiency of the mutants pseFDH-H223S/L257R, cboFDH-G171Y and cboFDH-I170T/A229S obtained in the previous patent (96%, 44% and 82%, respectively, Chinese patent application No. 201711230338.6). The mutant E has 90% of NUD reduction efficiency, which is higher than the activity of the mutant obtained in the previous patent on NUD (the highest conversion rate of the mutant cboFDH-I170T/A229S on NUD is 89%, Chinese patent application No. 201711230338.6). The mutant 4C4 has an NTD reduction efficiency of 81% which is higher than that of the mutant obtained in the previous patent (cboFDH-I170T/A229S has the highest NTD conversion rate of 69%, and the patent application number is 201711230338.6). furthermore, the mutant of formate dehydrogenase related to the invention has catalytic efficiency on NTD and NUDOverall higher than existing mutants (see above patent, patent application No. 201711230338.6).

Reducing NCD with non-enzymatic system to obtain 4mM Na₂S₂O₄1mM NCD and 4mM NaHCO₃Mixing, reacting at 30 deg.C for 20min, and analyzing 20 μ L. According to the analysis method of comparative example 1, the sample showed a characteristic absorption peak at 340nm, and the quantitative result showed that 0.75mM NCDH was produced. Compared with the table 1, the NCDH yield is lower than that of formate dehydrogenase mutant A2(C256I/E261P/S381I), mutant 3C4(A199C/E261P/S381N), 4B4(T197C/C256I/H260S/E261P/S381N) and 3A3(V198I/C256I/P260S/E261P/S381N/S383F) when NCD is reduced by the chemical method.

The result of example 1 shows that the formate dehydrogenase mutant can effectively catalyze formate to reduce the NAD analogue related to the invention, and prepare a corresponding reduction product, and the reduction level is equivalent to or higher than that of the NAD analogue reduced by a chemical method. The results of the example 1, the comparative example 1 and the comparative example 2 are combined to show that the formate dehydrogenase mutant has the characteristics of high catalytic efficiency, mild reaction and single reaction product when the formate is used as a reducing agent to reduce the NAD analogue. And compared with a chemical method, the method has no pollution problem in large-scale application.

TABLE 1 results of experiments in which formate dehydrogenase mutants catalyze the reduction of NAD analogs by formate

Example 2: preparation of reduced NAD analogs

The reaction system of example 1 was scaled up and used to prepare reduced NCD. 20mM NCD, 25mM sodium formate and 5mg formate dehydrogenase A2(C256I/E261P/S381I) were dissolved in 10mL of 50mM sodium phosphate buffer solution at pH 5.7, and the mixture was mixed and reacted at 30 ℃ for 80 min. Directly freeze-drying after the reaction is finished, concentrating to total volume of about 4mL, separating with formic acid type anion exchange resin column, collecting product by tracking at ultraviolet wavelength of 340nm, and freeze-drying to obtain white powder 11.6mg with yield of about 90%.

Subjecting the white powder sample to high resolutionMass spectrometry to determine the exact molecular weight (M + H)⁺640.1118, theoretical molecular weight (C) of NCDH₂₀H₂₇HN₅O₁₅P₂ ^-640.1125), indicating that the product NCDH in reduced form was obtained.

The NAD analogue was produced according to the method of example 2 using the same amounts of NCD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R to give NCDH in reduced state with a yield of about 89%. The results show that both formate dehydrogenase A2(C256I/E261P/S381I) and phosphite dehydrogenase rsPDH-I151R can catalyze the corresponding substrates to produce NAD analogues with approximate yields.

Example 3: uses sodium formate as reducing agent, formate dehydrogenase catalytic reduction NAD analogue

0.1mM NCD, 0.4mM sodium formate and 4. mu.g of formate dehydrogenase 3C4(A199C/E261P/S381N) were dissolved in 1mL of PIPES buffer solution of 50mM concentration and pH 8.0, mixed, reacted at 40 ℃ for 3min, and 20. mu.L of the mixture was analyzed.

The sample was analyzed by the method of comparative example 1 and found to have a characteristic absorption peak at 340 nm. The concentration of the generated NCDH reaches 78 μ M, namely the yield reaches 78%.

The results of examples 1 and 3 show that, in the reaction for catalytic reduction of NCD by formate dehydrogenase, NCD can be reduced by using sodium formate or formic acid as a reducing agent.

NAD analogues were produced using the same amounts of NCD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R as in example 3, giving a concentration of NCDH of 73. mu.M, i.e.a yield of 73%. The results show that both formate dehydrogenase 3C4(A199C/E261P/S381N) and phosphite dehydrogenase rsPDH-I151R can catalyze the corresponding substrates to produce NAD analogues, and the reaction yield catalyzed by formate dehydrogenase 3C4(A199C/E261P/S381N) is higher than that catalyzed by phosphite dehydrogenase rsPDH-I151R.

Example 4: catalytic reduction of NAD analogue by formic acid dehydrogenase using deuterated formic acid as reducing agent

Formic acid was dissolved in D.sup.FEBS J,2005,272,3816-3827 according to the literature method (Wood R, ethyl. mechanical agitation of a high chlorinated phosphorus hydrolysis and its application for NADPHregistration)₂In the presence of oxygen in the atmosphere of O,freeze drying, repeating for 4 times to obtain deuterated formic acid for use.

1mM NCD, 4mM deuterated formic acid and 40. mu.g formate dehydrogenase 3A3(V198I/C256I/P260S/E261P/S381N/S383F) were dissolved in 1mL MES buffer solution with a concentration of 50mM and a pH of 5.0, mixed, reacted at 10 ℃ for 120min, and 20. mu.L was taken for analysis.

According to the analysis method of comparative example 1, the sample shows a characteristic absorption peak at 340 nm. The product NCDH concentration reached 0.95mM, i.e., the yield was 95%.

Subjecting the sample to high resolution mass spectrometry to determine the precise molecular weight (M-H)^-641.1118, and NCD²Theoretical molecular weight of H (C)₂₀H₂₇ ²HN₅O₁₅P₂ ^-641.1125), indicating that a deuterated NCD reduced product is obtained.

The results of example 4 show that formate dehydrogenase can catalyze the reduction of NCD to the corresponding deuterated reduced product using deuterated formic acid as the reducing agent.

The deuterated NAD analogue was produced according to example 4 using the same amounts of NCD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R/E213C, the product NCDH concentration reaching 0.88mM, i.e.a yield of 88%. The results show that formate dehydrogenase 3A3(V198I/C256I/P260S/E261P/S381N/S383F) and rsPDH-I151R/E213C can catalyze corresponding substrates to produce NAD analogues, and the reaction yield of formate dehydrogenase 3A3(V198I/C256I/P260S/E261P/S381N/S383F) is higher than that of phosphite dehydrogenase rsPDH-I151R/E213C.

Example 5: preparation of malic acid by catalytic reduction and carboxylation of pyruvate by formate dehydrogenase, malic enzyme ME-L310R/Q401C and NAD analogue system

Malic enzyme ME-L310R/Q401C was purified for use by the method described in the reference (Ji DB, et al., creation of bioorganic redox system dependent on a nicotinic amide flucytoside. J Am Chem Soc,2011,133, 20857-20862). ME-L310R/Q401C prefers the analog NCDH but has low activity towards NADH, requiring NCDH as a cofactor.

The malic enzyme ME-L310R/Q401C can catalyze the reaction: pyruvic acid + CO₂+ NCDH → malic acid + NCD. The reaction catalyzed by formate dehydrogenase is: formic acid+NCD→CO₂+ NCDH. The two reactions were combined and the net reaction was: formate + pyruvate → malate. Therefore, the system consisting of formate dehydrogenase and malic enzyme can reduce and carboxylate pyruvic acid to malic acid by using formate as a reducing agent. In the system, the NAD analogue is regenerated and recycled, and the reaction does not have any by-product and has certain potential. A representative experimental procedure is as follows:

with 50mM Tris-HCl buffer system, pH 5.0, a 100. mu.L reaction system consisted of: 4.0mM formic acid, 50mM pyruvic acid, 0.01mM NCD, 1.0mM MnCl₂10mM sodium bicarbonate, 0.05mg/mL3C4(A199C/E261P/S381N) and 0.06mg/mL ME-L310R/Q401C. After 120min at 10 ℃, 900. mu.L of acetonitrile-methanol-water mixture (acetonitrile: methanol: water: 4:1) was added to terminate the reaction.

The content of malic acid, pyruvic acid and formic acid in the reaction solution is analyzed and determined by an ICS-2500 ion chromatography system of Daian, USA under an ED50 pulse electrochemical detection mode. IonPac AG11-HC anion exchange column (200 mm. times.4 mm) was used, and IonPac AS11-HC anion exchange column was used, and the column was protected (50 mm. times.4 mm). Analysis conditions were as follows: the mobile phase is 24mM NaOH, the flow rate is 1mL/min, and the column temperature is as follows: the sample size was 25. mu.L at 30 ℃. As a result of the examination, the reaction solution contained 0.1mM formic acid, 46.1mM pyruvic acid and 3.6mM malic acid.

In carrying out the above reaction, 4 additional control experiments were set up, each lacking one of formic acid, NCD, 3C4(A199C/E261P/S381N) or ME-L310R/Q401C, and analysis revealed that these reactions did not produce malic acid. According to the stoichiometric relationship of the reaction, the NCD is recycled 360 times.

In the above reaction, 1 set of experiments was also set, and the reaction solution was reduced in pyruvic acid concentration to 46.3mM and generated 3.5mM malic acid, which indicates that the system can use deuterated formic acid as the reducing agent to achieve the efficiency equivalent to formic acid as the reducing agent, with deuterated formic acid replacing formic acid, and other components and conditions being the same.

Malic acid was prepared by catalytic reductive carboxylation of pyruvate using the phosphite dehydrogenase rsPDH-I151R/E213C, malic enzyme ME-L310R/Q401C and NAD analog system as described in example 5. Using the same reaction system and analysis method, the results of the examination revealed that the reaction liquid contained 0.2mM of phosphorous acid, 47.1mM of pyruvic acid and 2.6mM of malic acid. The catalytic activity of the system of formate dehydrogenase, malic enzyme ME-L310R/Q401C and NAD analogue in preparing malic acid by reduction and carboxylation of pyruvic acid is higher than that of the system of phosphorous dehydrogenase rsPDH-I151R/E213C, malic enzyme ME-L310R/Q401C and NAD analogue.

Example 6: preparation of lactic acid by catalyzing reduction of pyruvate with formate dehydrogenase, D-lactate dehydrogenase DLDH-V152R and NAD analog system

D-lactate dehydrogenase DLDH-V152R was purified for use according to the reference (Ji DB, et al., creation of bioorganic redox system dependent on a microbial enzyme hydrolysate. J Am Chem Soc,2011,133, 20857-20862). D-lactate dehydrogenase DLDH-V152R prefers the NAD analog NUD, requiring a reduced analog as a cofactor.

The reaction catalyzed by lactate dehydrogenase is: pyruvate + NUDH → D-lactate + NUD. The reaction catalyzed by formate dehydrogenase is: formic acid + NUD → CO₂+ NUDH. The two reactions were combined and the total reaction was: formic acid + pyruvic acid → D-lactic acid + CO₂. Therefore, the system comprising formate dehydrogenase and D-lactate dehydrogenase can reduce pyruvic acid to D-lactate using formate as a reducing agent. In the system, the NAD analogue is recycled, and has certain application potential. A representative experimental procedure is as follows:

using a HEPES buffer system at 50mM, pH 8.0, a 100. mu.L reaction system consisted of: 4.0mM formic acid, 4.0mM pyruvic acid, 0.1mM NUD, 0.05mg/mL E4(V198I/C256I/P260S/E261P/S381N) and 0.06 mg/mLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDH-V152R. After 10min at 40 ℃, the reaction was terminated by adding 900 μ L of a mixture of acetonitrile and methanol (acetonitrile: methanol: water: 4: 1).

As a result of analysis by the ion chromatography system in accordance with the method in example 5, the reaction mixture contained 0.5mM formic acid, 3.3mM D-lactic acid and 0.6mM pyruvic acid.

Experimental results show that the system utilizes formic acid as a reducing agent to reduce pyruvic acid to lactic acid nearly quantitatively, and high raw material utilization efficiency is achieved. From the stoichiometric relationship of the reaction, NUD regeneration was recycled 33 times.

Lactate was prepared by the method of example 6 using formate dehydrogenase cboFDH-I170T/A229S (see earlier patent application No. 201711230338.6), D-lactate dehydrogenase DLDH-V152R and NAD analog system to catalyze the reduction of pyruvate. Using the same reaction system and analysis method, the results of the tests showed that the reaction mixture contained 0.6mM A, 3.0mM D-lactic acid, and 0.8mM pyruvic acid. The catalytic activity of the formate dehydrogenase mutant E4, the D-lactate dehydrogenase DLDH-V152R and the NAD analogue system for preparing lactic acid by reduction of pyruvate is higher than that of the formate dehydrogenase mutant cboFDH-I170T/A229S, the D-lactate dehydrogenase DLDH-V152R and the NAD analogue system.

Example 7: preparation of lactic acid by catalyzing reduction of pyruvate with formate dehydrogenase, D-lactate dehydrogenase DLDH-V152R and NAD analog system

According to the same reaction conditions and analysis methods as those of example 6 in which the buffer system of the reaction was changed to Tris-HCl buffer solution at pH 9.0, the reaction mixture after the termination of the reaction was found to contain 0.8mM formic acid, 2.9mM D-lactic acid and 0.9mM pyruvic acid. It was demonstrated that a system containing E4(V198I/C256I/P260S/E261P/S381N) can reduce pyruvic acid to lactic acid in a nearly quantitative manner using formic acid as a reducing agent under a pH of 9.0, and that a high raw material utilization efficiency can be obtained. But the conversion efficiency was slightly lower than for the reaction system at pH 8.0.

Example 8: preparation of lactic acid by catalyzing reduction of pyruvate with formate dehydrogenase, D-lactate dehydrogenase DLDH-V152R and NAD analog system

According to the same reaction conditions and analysis methods as those of example 6, the buffer system of the reaction was changed to acetic acid-sodium acetate buffer solution having a pH of 4.0, and it was found that the reaction mixture after the termination of the reaction contained 2.0mM formic acid, 1.7mM D-lactic acid, and 2.2mM pyruvic acid. It was demonstrated that, in a system containing E4(V198I/C256I/P260S/E261P/S381N) at pH 4.0, pyruvic acid can be reduced to lactic acid in a near quantitative manner using formic acid as a reducing agent, and the raw material utilization efficiency is correspondingly high. But the conversion efficiency was lower than in the reaction systems of pH 8.0 and pH 9.0.

Example 9: intracellular reduction NAD analogue mediated by formate dehydrogenase and application thereof

The formate dehydrogenase of the NCD, the NCD-preferred oxidoreductase and the NAD transporter can be expressed simultaneously in the host, forming an NCD-dependent biocatalytic system. This biocatalytic system is initiated when formate compounds and NCD in the culture medium enter the host cell. Thus, using the technology of formate dehydrogenase mediated intracellular reduction of NCD, extracellular reducing power can be selectively delivered to intracellular target redox reactions. The construction of an engineered strain for malic acid production will be described below by way of example, using the transformed Escherichia coli XZ654(Zhang X, et al. L-malt production by Escherichia coli. appl Environ Microbiol,2011,77, 427-434).

The NAD transporter AtNDT2(Accession NO. NC-003070) has a broader substrate spectrum (PalmieriF, et al. molecular identification and functional characterization of arabidopsis thaliana restriction and chloroplastic NAD carrier proteins. JBiol Chem,2009,284,31249-31259), and can transport NCD. The gene of AtNDT2 expressing transporter was encoded by gapAP1 promoter (Charpentier B, et al, the Escherichia coli gapA gene istranscribed by polymerase holoenzyme E. sigma.)⁷⁰and by the RNA polymerase Eσ³²JBacteriol,1994,176,830-839) controls expression. The gene coding 3A3(V198I/C256I/P260S/E261P/S381N/S383F) and the gene coding ME-L310R/Q401C are controlled by an isopropyl thiogalactose (IPTG) -induced lac promoter, and the three expression cassettes are cloned to the same plasmid through a LacZ gene replacing pUC18 to obtain an engineering plasmid.

The engineering plasmid is introduced into E.coli XZ654 to obtain an engineering strain E.coli GXJ 01. Inducing engineering strain E.coli GXJ 01 in LB culture medium to express the above three functional proteins, adding 100 μ g/mL ampicillin and 1mM IPTG into the culture medium, culturing in a shaker at 25 deg.C and 200rpm for 48h to obtain the final product with OD_600nmThe cells were collected by centrifugation at 2000 Xg for 6min at 4.5.

The suspended cells were washed with MOPS medium at pH7.5, and the cell density OD was determined_600nmAdjusted to 9. Adding 10mM sodium bicarbonate, 10mM pyruvic acid, 5mM formic acid, and 0.1mM NCD into the above engineering bacteria suspension, respectively, 200 deg.C at 16 deg.C, 30 deg.C, and 42 deg.CAnaerobic reaction is carried out for 4h in a shaking table at rpm, 100 mu L of acetonitrile methanol water mixed solution is added with 900 mu L of acetonitrile methanol (methanol: water is 4:4:1) to terminate the reaction.

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 1.8mM formic acid, 2.8mM malic acid, and 7.1mM pyruvic acid at 16 ℃. The reaction solution contained 0.2mM formic acid, 4.3mM malic acid, and 5.4mM pyruvic acid at 30 ℃. The reaction solution at 42 ℃ contained 0.5mM formic acid, 3.8mM malic acid, and 5.6mM pyruvic acid.

In the control experiments with and without addition of formic acid and NCD, malic acid concentrations were 2.2mM, 1.9mM and 1.9mM, respectively.

The experimental result shows that in the whole-cell catalytic process, formate dehydrogenase 3A3(V198I/C256I/P260S/E261P/S381N/S383F) provides NCDH for ME-L310R/Q401C by oxidizing formate, and catalyzes reduction and carboxylation of pyruvate to malic acid, so that the yield of the malic acid is increased from 1.9mM to 4.3 mM. The malic acid yield did not increase significantly when formic acid alone was added, and malic acid did not increase when NCD alone was added.

Example 9 demonstrates that intracellular formate dehydrogenase can provide reduced NAD analogs via oxidation of formate in whole cell catalytic processes, which is used as a coenzyme in the reduction reaction by ME-L310R/Q401C as a means of regulating the metabolic strength of malate in microorganisms by providing redox.

According to the method of example 7, the gene expressing rsPDH-I151R/E213C and the gene of ME-L310R/Q410C were controlled by an Isopropylthiogalactose (IPTG) -induced lac promoter to construct corresponding engineered strains, and the contents of the respective components were determined by the same experiment and analysis method. The results showed that the reaction mixture at 16 ℃ contained 1.8mM of phosphorous acid, 2.8mM of malic acid, and 7.1mM of pyruvic acid. The reaction solution contained 0.1mM of phosphorous acid, 4.2mM of malic acid, and 5.6mM of pyruvic acid at 30 ℃. The reaction solution at 42 ℃ contained 0.5mM phosphorous acid, 3.8mM malic acid, and 5.6mM pyruvic acid. The results show that compared with the similar catalytic system in which phosphite dehydrogenase rsPDH-I151R/E213C participates, the catalytic system in which formate dehydrogenase 3A3(V198I/C256I/P260S/E261P/S381N/S383F) participates has slightly higher catalytic efficiency at 30 ℃ and equivalent catalytic efficiency at 16 ℃ and 42 ℃.

Example 10: intracellular reduction NAD analogue mediated by formate dehydrogenase and application thereof

The NCD formate dehydrogenase identified, the NCD-preferred oxidoreductase and the NCD transporter can be expressed simultaneously in the host, forming an NCD-dependent biocatalytic system. The construction of an engineered strain for producing lactic acid is described below by way of example by engineering Escherichia coli XZ654(Zhang X, et al. L-lactate production by means of Escherichia coli. appl Environ Microbiol,2011,77, 427-434).

NAD transporter NTT4(Haferkamp I, et al. A. candidate NAD⁺transporter in intracellular bacterial system related to Chlamydiae, Nature,2004,432, 622-. The three genes expressing the transporter NTT4 are expressed under the control of gap P1 promoter. The gene coding for 4C1(T197A/C256I/H260S/E261P/S381N) and the gene coding for DLDH-V152R are controlled by the isopropyl thiogalactoside (IPTG) induced lac promoter, and the three expression cassettes are cloned on the same plasmid by replacing the LacZ gene of pUC18 to obtain an engineered plasmid.

The engineering plasmid is introduced into E.coli XZ654 to obtain an engineering strain E.coli GXJ 02. Inducing engineering bacteria E.coli GXJ 02 in LB culture medium to express the three functional proteins, adding 100. mu.g/mL ampicillin and 1mM IPTG into the culture medium, and culturing in a shaker at 200rpm at 25 ℃ for 48h to OD_600nmThe cells were collected by centrifugation at 2000 Xg for 6min at 4.5.

The resuspended cells were washed with M9 medium at pH 8.0 and the density OD was determined_600nmAdjusted to 9. 10mM pyruvic acid, 5mM formic acid and 0.1mM NCD are added into the engineering bacteria suspension, anaerobic reaction is carried out for 3h in a shaking table with 200rpm at the temperature of 30 ℃, and 100 mu L of acetonitrile-methanol mixed solution (acetonitrile: methanol: water is 4:4:1) is added to terminate the reaction.

As a result of analysis by the ion chromatography system in accordance with the method of example 5, the reaction solution contained 1mM formic acid, 4.9mM lactic acid and 4.7mM pyruvic acid at 30 ℃.

In the control experiments with and without addition of formic acid and NCD, the lactic acid concentrations were 1.0mM, 0.9mM and 0.6mM, respectively.

The experimental results show that formate dehydrogenase 4C1(T197A/C256I/H260S/E261P/S381N) provides NCDH to DLDH-V152R by oxidizing formate in the whole cell catalytic process, catalyzes the reduction of pyruvate to lactate, and increases the yield of lactate from 0.6mM to 4.9 mM. There was no significant increase in lactic acid production with the addition of formic acid or NCD alone.

Example 10 demonstrates that intracellular formate dehydrogenase can provide reduced NCD by oxidizing formate in whole cell catalysis, which is used as a coenzyme by DLDH-V152R in reduction reactions as a means of regulating the metabolic strength of lactate in microorganisms by providing redox.

According to the method of example 10, the gene expressing rsPDH-I151R/I218F and the gene expressing DLDH-V152R were controlled by an Isopropylthiogalactose (IPTG) induced lac promoter to construct corresponding engineered strains, and the contents of the respective components were determined by the same experimental and analytical methods. As a result, the reaction mixture contained 0.1mM formic acid, 4.8mM lactic acid and 4.6mM pyruvic acid at 30 ℃. The efficiency of the catalytic system involved by formate dehydrogenase 4C1(T197A/C256I/H260S/E261P/S381N) is close to that of the catalytic system involved by phosphite dehydrogenase rsPDH-I151R/I218F.

Example 11: formate dehydrogenase mediated permeable intracellular reduction NAD analogue and application thereof

The formate dehydrogenase of the NCD identified and the oxidoreductase of the NCD preferred can be simultaneously expressed in the host cell to form a NAD analog-dependent NCD biocatalytic system. This biocatalytic system is initiated when formate compounds and NCD in the culture medium enter the host cell.

The gene coding A2(C256I/E261P/S381I) and the gene coding DLDH-V152R are controlled by an isopropyl thiogalactoside (IPTG) induced lac promoter, and the two expression cassettes are cloned on the same plasmid by replacing the lacZ gene of pUC18 to obtain an engineering plasmid.

The engineering plasmid is introduced into E.coli XZ654 to obtain an engineering strain E.coli GXJ 03. Inducing engineering bacteria E.coli GXJ 03 to express the two functional proteins in LB culture medium, adding ampicillin 100 μ g/mL and IPTG 1mM, and culturing in shaker at 25 deg.C and 200rpmCulturing for 48h to obtain thallus density OD_600nmCentrifugation at 2000 Xg for 6min at 4.5 to collect the cells, washing the resuspended cells with Tris-Cl at 50mM, pH7.5, and determining the cell density OD_600nmAdjusted to 9, the cells were permeabilized according to the literature (Zhang W, et al. Bioreduction with efficacy recycling of NADPHby supplemented microorganisms. appl. Environ Microbiol,2009,75,687-694) by thawing 5mL of frozen cells in a water bath at room temperature, adding 5mM EDTA and 1% by volume toluene, 30 ℃ and 30min in a shaker at 200rpm, and then left at 4 ℃ for 1 h. The supernatant containing EDTA and toluene was removed by centrifugation at 2000g for 6min, washed twice with 50mM Tris-Cl at pH7.5, and then resuspended in 5mL of 50mM Tris-Cl at pH 5.0 to obtain permeabilized cells.

10mM pyruvic acid, 5mM formic acid, and 0.1mM NCD were added to the above suspension of the permeabilized engineered bacteria resuspended in Tris-Cl at a concentration of 50mM and pH 5.0, and the mixture was anaerobically reacted for 0.5h at 30 ℃ in a shaker at 200 rpm. A100. mu.L sample was taken and added to 900. mu.L of a mixture of acetonitrile, methanol and water (acetonitrile: methanol: water: 4:1) to terminate the reaction.

When analyzed by an ion chromatography system according to the method of example 5, the reaction solution contained 1.1mM formic acid, 3.6mM lactic acid and 6.1 mM. Pyruvic acid

In the control experiments with addition of formic acid and NCD alone and without formic acid and NCD, the lactic acid concentrations were 0.6mM, 0.4mM and 0.3mM, respectively.

The experimental result shows that the formate dehydrogenase A2(C256I/E261P/S381I) provides NCDH for DLDH-V152R by oxidizing formic acid in the whole cell catalytic process, and catalyzes reduction of pyruvate to generate lactate, so that the yield of the lactate is increased from 0.3mM to 3.6 mM. There was no significant increase in malic acid production with the addition of formic acid or NCD alone.

Example 11 demonstrates that intracellular formate dehydrogenase can provide reduced NAD analogs via oxidation of formate in whole cell catalytic processes, which is used as a coenzyme by DLDH-V152R in reduction reactions as a means of regulating the metabolic strength of lactate in microorganisms by providing redox.

According to the method of example 11, the gene expressing rsPDH-I151R and the gene of ME-L310R/Q401C were controlled by an Isopropylthiogalactose (IPTG) -induced lac promoter to construct corresponding engineered strains, and the contents of the respective components were determined by the same experimental and analytical methods. As a result, the reaction solution contained 2.4mM formic acid, 2.5mM lactic acid and 7.8mM pyruvic acid at 30 ℃. The efficiency of the catalytic system involved by formate dehydrogenase A2(C256I/E261P/S381I) is higher than that of the similar catalytic system involved by phosphite dehydrogenase rsPDH-I151R.

Example 12: formate dehydrogenase mediated reduction NAD analogue in permeabilized Lactococcus lactis (Lactococcus lactis) AS1.2829 cell and application thereof

The formate dehydrogenase of the identified NAD analogue, and the oxidoreductase which prefers the NAD analogue, can be simultaneously expressed in lactococcus lactis to form a NAD analogue-dependent biocatalytic system. The biocatalytic system is activated when formate compounds and NAD analogs in the culture medium enter the host cell.

The genes encoding 4C4(V198I/C256I/P260S/E261P/S381N/H224C and the gene encoding DLDH-V152R are controlled by a constitutive expression promoter P32, and the two expression cassettes are passed through a P32 expression cassette (GUCHTE MV, et al.construction of a lacoccal expression vector: expression of hem eg white lysozyme in Lactococcus lactis subsp.Lactis.apple Enviromicrobiol, 1989,55, 224. 228.) in place of pMG36E to obtain engineered plasmids.

And introducing the engineering plasmid into lactococcus lactis to obtain an engineering strain L.lactis GXJ 04. Using a mixture of 10g/L sucrose, 10g/L yeast extract, 10g/L peptone and 10g/L KH at pH 6.8₂PO₄2g/L of MgSO 2₄Inducing the engineering bacteria L.lactis GXJ 04 to express the two functional proteins by using a culture medium of 5mg/L erythromycin, culturing the two functional proteins in a shaker at 25 ℃ and 200rpm for 48h until the thallus density is 4.5, centrifuging at 2000 Xg for 6min to collect the thallus, washing and re-suspending the thallus by using Tris-Cl with the concentration of 50mM and the pH value of 7.5, and performing OD (OD) on the thallus density_600nmAdjusted to 9. The cells were permeabilized according to the method of example 9, in the following manner: thawing 5mL of frozen cells in water bath at room temperature, adding 5mM EDTA and 1% toluene by volume, performing warm bath at 30 deg.C and 200rpm in a shaker for 30min, and standing at 4 deg.C for 1 h. Centrifuging at 2000g for 6min to remove EDTA and tolueneThe supernatant was washed twice with 50mM Tris-Cl (pH 7.5) and then resuspended in 5mL of 50mM Tris-Cl (pH 7.5) to obtain permeabilized cells.

10mM pyruvate and 5mM formic acid were added to the above-mentioned permeabilized engineered bacterial suspension resuspended in 50mM Tris-Cl, pH 7.5. 0.1mM NTD, in a shaker at 200rpm at 30 ℃ for 1 h. To 100. mu.L of the reaction mixture was added 900. mu.L of an acetonitrile-methanol-water mixture (acetonitrile: methanol: water: 4:1) to terminate the reaction.

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 1.9mM formic acid, 2.8mM lactic acid and 7.1mM pyruvic acid.

In the control experiments with addition of formic acid and NTD alone and without formic acid and NTD, the lactic acid concentrations were 0.3mM, 0.4mM and 0.2mM, respectively.

Example 12 demonstrates that intracellular formate dehydrogenase can provide reduced NAD analog by oxidation of formate in whole cell catalysis of lactococcus lactis, and that DLDH-V152R used as a coenzyme in reduction increases the accumulation of lactate by 13.5-fold compared to control experiments without addition of formate and NTD, and thus can be used as a means to regulate the metabolic strength of lactate in microorganisms by providing redox.

Example 13: formate dehydrogenase-mediated permeabilization Saccharomyces cerevisiae (Saccharomyces cerevisiae) BY4741 intracellular NAD (nicotinamide adenine dinucleotide) analogue reduction and application thereof

The formate dehydrogenase of the identified NAD analogue, and the oxidoreductase which prefers the NAD analogue, can be simultaneously expressed in Saccharomyces cerevisiae cells to form a NCD-dependent biocatalytic system. The biocatalytic system is activated when formate compounds and NAD analogs in the culture medium enter the host cell.

The gene coding 3A3(V198I/C256I/P260S/E261P/S381N/S383F) and the gene coding DLDH-V152R are controlled by a TEF constitutive promoter and a CYC1 terminator, and the two expression cassettes are integrated into a P416 yeast episomal shuttle expression vector to obtain an engineering plasmid.

And introducing the engineering plasmid into saccharomyces cerevisiae to obtain an engineering strain S. Using a pH 6.0 solution containing 20g/LInducing engineering bacteria S.cerevisiae GXJ 05 to express the two functional proteins by using YEPD culture medium containing glucose, 10g/L yeast extract and 20g/L peptone, and culturing in a shaker at 30 ℃ and 200rpm for 48h to obtain thallus density OD_600nmCentrifugation at 2000 Xg for 6min to collect the cells at 4.5, washing the resuspended cells with Tris-Cl at 50mM, pH7.5, and OD_600nmAdjusted to 9. The cells were permeabilized by the method described in example 9 to obtain permeabilized cells.

10mM pyruvic acid, 5mM formic acid, and 0.1mM NCD were added to the above suspension of the permeabilized engineered bacteria resuspended in 50mM Tris-Cl, pH7.5, and the mixture was anaerobically reacted for 1 hour in a shaker at 30 ℃ and 200 rpm. To 100. mu.L of this mixture was added 900. mu.L of acetonitrile-methanol-water mixture (acetonitrile: methanol: water: 4:1) to terminate the reaction.

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 0.6mM formic acid, 4.3mM lactic acid and 6.1mM pyruvic acid.

In the control experiments with addition of only one of formic acid and NCD and without formic acid and NCD, the lactic acid concentrations were 0.4mM, 0.6mM and 0.4mM, respectively.

Example 13 demonstrates that intracellular formate dehydrogenase can provide NCDH by oxidizing formate and is used as a coenzyme by DLDH-V152R in reduction reactions during whole cell catalysis of Saccharomyces cerevisiae, and the accumulation of lactate is increased by 9.8 times compared with the control experiment without addition of formate and NCD, thus serving as a means for regulating the metabolic strength of lactate in microorganisms by providing redox.

Example 14: formate dehydrogenase mediated intracellular reduction NAD analogue of Trichoderma reesei (Trichoderma reesei) and application thereof

The formate dehydrogenase of the identified NAD analogue and the oxidoreductase which prefers the NAD analogue can be simultaneously expressed in the Trichoderma reesei cell to form a NAD analogue-dependent biocatalytic system. The biocatalytic system is activated when formate compounds and NAD analogs in the culture medium enter the host cell.

The gene coding 4B4(T197C/C256I/H260S/E261P/S381N) and the gene coding DLDH-V152R are controlled by a promoter Pcbh1 and a terminator Tcbh1, and the two expression cassettes are integrated on a pCAMBIA1300 vector to obtain an engineering plasmid.

Introducing the above engineering plasmid into Trichoderma reesei to obtain engineering strain T.reesei GXJ 06, adding yeast extract containing lactose 15g/L, yeast extract 10g/L, and (NH) 1g/L at pH 4.8₄)₂SO₄3g/L KH₂PO₄0.5g/L MgSO₄0.6g/L of CaCl₂0.05g/L of FeSO₄·7H₂O, 0.0016g/L MnSO₄·H₂O, 0.0014g/L ZnSO₄·7H₂O, 0.0037g/L CoCl₂·6H₂The engineering bacteria T.reesei GXJ 06 is induced by the culture medium of O to express the two functional proteins, cultured in a shaker at 25 ℃ and 200rpm for 48h, centrifuged at 2000 Xg for 6min to collect the bacteria, washed with Tris-Cl with the concentration of 50mM and the pH value of 7.5 to resuspend the bacteria, and the density of the bacteria is adjusted to 3g dry weight/L of the bacteria. The cells were permeabilized by the method described in example 11 to obtain permeabilized cells.

10mM pyruvic acid, 5mM formic acid, and 0.1mM NCD were added to the above Tris-Cl resuspended permeable engineered bacterial suspension at a concentration of 50mM and pH7.5, and the mixture was anaerobically reacted for 2 hours in a shaker at 30 ℃ and 200 rpm. A100. mu.L sample was taken and added to 900. mu.L of a mixture of acetonitrile, methanol and water (acetonitrile: methanol: water: 4:1) to terminate the reaction.

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 1.2mM formic acid, 3.6mM lactic acid and 6.4mM pyruvic acid.

In the control experiments with and without addition of formic acid and NCD, the lactic acid concentrations were 1.2mM, 0.9mM and 0.6mM, respectively.

Example 14 demonstrates that intracellular formate dehydrogenase can provide reduced NAD analogs via oxidation of formate during whole cell catalysis of trichoderma reesei, and that DLDH-V152R, used as a coenzyme in reduction reactions, increases the amount of lactate accumulated by 5-fold compared to control experiments without addition of formate and NCD, and thus can be used as a means to regulate the metabolic strength of lactate in microorganisms by providing redox.

Example 15: intracellular reduction NAD analogue of Rhodosporidium toruloides (Rhodosporidium toruloides) mediated by formate dehydrogenase and application thereof

The formate dehydrogenase of the identified NAD analogue and the oxidoreductase which prefers the NAD analogue can be simultaneously expressed in the rhodosporidium toruloides to form a NAD analogue-dependent biocatalytic system. The biocatalytic system is initiated when formate and NAD analogs in the culture medium enter the host cell.

The gene coding 3C4(A199C/E261P/S381N) and the gene coding DLDH-V152R are controlled by a promoter GPD, a promoter PGK, a terminator Hspt and a terminator Tnos respectively, and the two expression cassettes are integrated on a pZPK vector to obtain an engineering plasmid.

Toruloides GXJ 07 is obtained by transforming the above engineering plasmid into Rhodosporidium toruloides through ATMT, the two functional proteins are expressed by culturing the engineering bacterium GXJ 07 in YEPD culture medium containing 20g/L glucose, 10g/L yeast extract and 20g/L peptone with pH 6.0, culturing in a shaker at 28 ℃ and 200rpm for 48h, centrifuging at 2000 Xg for 6min to collect the thallus, washing the resuspended thallus with Tris-Cl with concentration of 50mM and pH7.5, and adjusting the thallus density to 3g dry thallus weight/L. The cells were permeabilized by the method described in example 11 to obtain permeabilized cells.

10mM pyruvic acid, 5mM formic acid, and 0.1mM NCD were added to the above Tris-Cl resuspended permeable engineered bacterial suspension at a concentration of 50mM and pH7.5, and the mixture was anaerobically reacted for 2 hours in a shaker at 30 ℃ and 200 rpm. The reaction was terminated by adding 900. mu.L of acetonitrile in water (acetonitrile: water. RTM.4: 1) to 100. mu.L of the sample.

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 1.2mM formic acid, 3.6mM lactic acid and 6.0mM pyruvic acid.

In the control experiments with and without addition of formic acid and NCD, the lactic acid concentrations were 1.2mM, 0.7mM and 0.6mM, respectively.

Example 15 demonstrates that intracellular formate dehydrogenase can provide NCDH by oxidizing formate in the process of catalysis by Rhodosporidium toruloides, and DLDH-V152R is used as coenzyme for reduction reaction, and the accumulation amount of lactate is increased by 5 times compared with the control experiment without addition of formate and NCD, thus can be used as a way to regulate the metabolic strength of lactate in the microorganism by providing redox.

Example 16: crystal analysis of formate dehydrogenase mutant

The formate dehydrogenase mutants a2 and 3A3 were subjected to crystal analysis with NCD, respectively, to obtain a crystal structure containing the ligand NCD.

The purified protein was screened by sitting-drop method using a crystallization screening kit from Hampton Research and Wizard. The crystal culture conditions for diffraction of the mutant A2 were 0.1M ammonium sulfate, 0.1M Tris (pH 7.0), 10% polyethylene glycol monoethyl ether 5000, protein concentration 6mg/mL, and temperature 30 ℃. The crystallization conditions for mutant 3A3 were 0.4M sodium acetate, 0.3M sodium methionate, 0.1M MOPS (pH 8.0) 50% w/v polyethylene glycol 8000. The crystal diffraction is performed by a Shanghai synchrotron radiation device (SSRF), and the beam line BL18U 1. 5mM NCD was added to the crystal-immersed mother liquor, and then the low-temperature treatment and data collection were performed. The resulting crystal structure is shown in fig. 1 or fig. 2.

Formate dehydrogenase derived from Pseudomonas sp.101, NCBI protein database number P33160.3, containing the complete amino acid sequence from position 1 to 401 as follows:

sequence listing

<110> institute of chemistry and physics, large connection of Chinese academy of sciences

<120> a method for reducing NAD analog using formic acid

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>401

<212>PRT

<213> formate dehydrogenase (Pseudomonas sp. 101)

<400>1

Met Ala Lys Val Leu Cys Val Leu Tyr Asp Asp Pro Val Asp Gly Tyr

15 10 15

Pro Lys Thr Tyr Ala Arg Asp Asp Leu Pro Lys Ile Asp His Tyr Pro

20 25 30

Gly Gly Gln Thr Leu Pro Thr Pro Lys Ala Ile Asp Phe Thr Pro Gly

35 40 45

Gln Leu Leu Gly Ser Val Ser Gly Glu Leu Gly Leu Arg Lys Tyr Leu

50 55 60

Glu Ser Asn Gly His Thr Leu Val Val Thr Ser Asp Lys Asp Gly Pro

65 70 75 80

Asp Ser Val Phe Glu Arg Glu Leu Val Asp Ala Asp Val Val Ile Ser

85 90 95

Gln Pro Phe Trp Pro Ala Tyr Leu Thr Pro Glu Arg Ile Ala Lys Ala

100 105 110

Lys Asn Leu Lys Leu Ala Leu Thr Ala Gly Ile Gly Ser Asp His Val

115 120 125

Asp Leu Gln Ser Ala Ile Asp Arg Asn Val Thr Val Ala Glu Val Thr

130 135 140

Tyr Cys Asn Ser Ile Ser Val Ala Glu His Val Val Met Met Ile Leu

145 150 155 160

Ser Leu Val Arg Asn Tyr Leu Pro Ser His Glu Trp Ala Arg Lys Gly

165170 175

Gly Trp Asn Ile Ala Asp Cys Val Ser His Ala Tyr Asp Leu Glu Ala

180 185 190

Met His Val Gly Thr Val Ala Ala Gly Arg Ile Gly Leu Ala Val Leu

195 200 205

Arg Arg Leu Ala Pro Phe Asp Val His Leu His Tyr Thr Asp Arg His

210 215 220

Arg Leu Pro Glu Ser Val Glu Lys Glu Leu Asn Leu Thr Trp His Ala

225 230 235 240

Thr Arg Glu Asp Met Tyr Pro Val Cys Asp Val Val Thr Leu Asn Cys

245 250 255

Pro Leu His Pro Glu Thr Glu His Met Ile Asn Asp Glu Thr Leu Lys

260 265 270

Leu Phe Lys Arg Gly Ala Tyr Ile Val Asn Thr Ala Arg Gly Lys Leu

275 280 285

Cys Asp Arg Asp Ala Val Ala Arg Ala Leu Glu Ser Gly Arg Leu Ala

290 295 300

Gly Tyr Ala Gly Asp Val Trp Phe Pro Gln Pro Ala Pro Lys Asp His

305 310 315 320

Pro Trp Arg Thr Met Pro Tyr Asn Gly Met Thr Pro His Ile Ser Gly

325330 335

Thr Thr Leu Thr Ala Gln Ala Arg Tyr Ala Ala Gly Thr Arg Glu Ile

340 345 350

Leu Glu Cys Phe Phe Glu Gly Arg Pro Ile Arg Asp Glu Tyr Leu Ile

355 360 365

Val Gln Gly Gly Ala Leu Ala Gly Thr Gly Ala His Ser Tyr Ser Lys

370 375 380

Gly Asn Ala Thr Gly Gly Ser Glu Glu Ala Ala Lys Phe Lys Lys Ala

385 390 395 400

Val

Claims (9)

1. A method of reducing an NAD analog with formic acid, comprising: taking one or more formic acid compounds in any ratio of formic acid, formate, deuterated formic acid and deuterated formate as a reducing agent, and taking enzyme of the reducing agent as a catalyst to mix with the NAD analogue to obtain the reduced NAD analogue through reaction; the catalyst is one or more of genetically engineered mutated enzymes, namely formate dehydrogenase pseFDH mutant E (V198/C256/P260/E261/S381, mutant A (C256/E261/S381), mutant 3C (A199/E261/S381), mutant 2A (A199/E261/S381), mutant 4C (V198/C256/P260/E261/S381/H224), mutant 4C (T197/C256/H260/E261/S381), mutant 4B (T197/C256/H260/E261/S381), mutant 4A (T197/C256/H260/E261/S381) and 3A (V198/C256/P260/E261/S381/S383).

2. The method of claim 1, wherein: the reduced NAD analogue is one or more than two of Nicotinamide Cytosine Dinucleotide (NCD), Nicotinamide Thymine Dinucleotide (NTD) and Nicotinamide Uracil Dinucleotide (NUD), and the chemical structure of the reduced NAD analogue is as follows:

3. the method of claim 1, wherein: the enzyme capable of utilizing a reducing agent formic acid compound is an active protein having a function of catalytically reducing an NAD analogue to a corresponding reduced state by using the formic acid compound as a reducing agent.

4. The method of claim 1, wherein: the reaction is carried out in a buffer solution of pH3-9 (preferably pH4-8, more preferably pH7.5) at a reaction temperature of 10-40 deg.C (preferably 15-40, more preferably 25 deg.C); the final concentration of the enzyme at the initiation of the reaction is 4. mu.g/mL-500. mu.g/mL (preferably 20. mu.g/mL-400. mu.g/mL, more preferably 100. mu.g/mL), the final concentration of the NAD analogue is 0.01mM-100mM (preferably mM, more preferably 0.2mM), and the final concentration of the formic acid compound is 0.4mM-25mM (preferably 0.9mM-18mM, more preferably 10 mM); the buffer system is one or more than two of phosphate buffer solution, Tris-HCl buffer solution, HEPES buffer solution, MES buffer solution, PIPES buffer solution and acetic acid-sodium acetate buffer system.

5. The method of claim 1, wherein: the reduced NAD analogs are useful as coenzymes for other enzymes, including but not limited to: malic enzyme ME-L310R/Q401C, D-lactate dehydrogenase DLDH-V152R, Saccharomyces cerevisiae alcohol dehydrogenase, hydroxy butanone dehydrogenase, D-xylose dehydrogenase.

6. The method of claim 1, further characterized by: the enzyme that can utilize the formate compound is expressed in the cells of the microorganism, and the NAD analog and the formate compound enter the cells, and the reaction proceeds in the cells.

7. The method of claim 1 or 6, wherein: the reaction is carried out intracellularly, and microorganisms expressing phosphite dehydrogenase and used for intracellular reduction of NAD analogs include, but are not limited to, prokaryotic microorganisms and eukaryotic microorganisms.

8. The method of claim 7, wherein: the prokaryotic microorganism includes but is not limited to one or two of Escherichia coli or lactococcus lactis.

9. The method of claim 7, wherein: the eukaryotic microorganism includes but is not limited to one or more than two of saccharomyces cerevisiae, trichoderma reesei or rhodotorula toruloides.

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