CN111826409A

CN111826409A - Method for reducing NAD analogue by using methanol

Info

Publication number: CN111826409A
Application number: CN201910303618.8A
Authority: CN
Inventors: 赵宗保; 王俊婷
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2019-04-16
Filing date: 2019-04-16
Publication date: 2020-10-27

Abstract

The invention discloses a method for reducing NAD analogue by methanol and application thereof. In the method, the reducing agent is methanol, the catalyst is methanol dehydrogenase capable of utilizing the methanol, and the NAD analogue is converted into a reduction state while the methanol dehydrogenase oxidizes the methanol. The method can be used for producing reduced NAD analogues or deuterated reduced analogues, and can also provide reduced coenzyme for enzymatic reactions consuming the reduced NAD analogues, and the reduced NAD analogues can be used as the coenzyme for enzymatic reduction reactions catalyzed by malic enzyme ME-L310R/Q401C, D-lactate dehydrogenase DLDH-V152R, saccharomyces cerevisiae alcohol dehydrogenase and the like, and are beneficial to wide application of the NAD analogues. 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 using methanol

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 the catalysis of enzyme by using methanol 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 a series of 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. Since NADH can be consumed by various pathways in the metabolic network, the efficiency of the target pathway's utilization of reducing power is affected. When NAD analogues are used for transferring reducing force, the analogues can only be recognized by mutant oxidoreductases, so that the target oxidoreduction process is regulated at the coenzyme level, and the analogue has important significance for biological catalysis and synthetic biology research (Ji DB, et al J Am Chem Soc,2011,133, 20857-20862; Wang L, et al ACCcatal, 2017,7, 1977-1983).

Several NAD analogues with good biocompatibility have been reported. Such as Nicotinamide Cytosine Dinucleotide (NCD), Nicotinamide Thymine Dinucleotide (NTD), Nicotinamide Uracil Dinucleotide (NUD) (Ji DB, et al, JAm Chem Soc,2011,133, 20857-20862; Ji DB, et al, Sci China Chem,2013,56, 296-300). Also, some enzymes recognizing NAD analogues have been reported, such as NADH oxidase (NOX, Genbank S45681) from Enterococcus faecalis, D-lactate dehydrogenase (DLDH, Genbank CAA47255) V152R mutant, malic enzyme (ME, Genbank P26616) L310R/Q401C mutant, and malic dehydrogenase (MDH, Genbank CAA68326) L6R mutant.

Using NAD analogs and enzymes that recognize them, more cost-effective biocatalytic systems can be constructed (catalytic dictionary, Edinbin et al, 2012,33, 530-. By selecting proper NAD analogues and recognizing enzymes thereof, the crude enzyme solution can be used for reaction to achieve the effect of pure enzyme catalysis, and the control of a complex biocatalytic conversion system at the coenzyme level is realized. Currently, regulation of intracellular metabolic reactions using NAD analogs has been achieved, and specific biocatalytic regulation has been achieved by intracellular transport of NCD, which is available for the reduction of pyruvate to lactate by DLDH-V152R (Wang L, et al ACS Catal,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. Methanol dehydrogenase can convert methanol into formaldehyde, and formaldehyde can be converted into formate by catalysis of formaldehyde dehydrogenase endogenous to cells in microbial cells or enter a riboketone monophosphate pathway, a tetrahydromethopterin pathway and the like to generate metabolites and reducing power necessary for cell life activities, thereby realizing effective utilization of a carbon resource (Muller JE, equivalent. Metab Eng,2015,28, 190-.

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 methanol 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 a phosphate compound which is a product generated by catalyzing a phosphorous acid compound by phosphorous acid dehydrogenase, a methanol dehydrogenase substrate has rich methanol sources and low price, and is a carbon compound with a larger prospect, and methanol can be reduced by the methanol dehydrogenase by utilizing microbial cells to finally generate a high value-added compound (Whitaker WB, et al. Therefore, the regeneration of NAD analogue by methanol dehydrogenase reduction is a new reduction method combining one-carbon resource utilization and NAD analogue regeneration, and carbon conversion and energy transfer are realized by one-carbon compound methanol while NAD analogue reduction is realized.

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

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 one or two of methanol and deuterated methanol combined substrate in any proportion as a reducing agent and taking enzyme of the reducing agent as a catalyst. These reduced states of 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 analogue by methanol, which is characterized in that: taking a substrate of one or two of methanol and deuterated methanol combined in any proportion as a reducing agent, taking an enzyme capable of utilizing methanol as a catalyst, and reacting for 2-120min in a buffer system with pH4-9 at the reaction temperature of 15-40 ℃, at the final concentration of 1-600 [ mu ] g/mL of the enzyme, at the final concentration of 0.001-20 mM of the NAD analogue and at the final concentration of 1-1000 mM of the methanol compound to obtain the reduced NAD analogue. The buffer system comprises but is not limited to one or more than two of phosphate buffer, Tris-HCl buffer, HEPES buffer, MES buffer, PIPES buffer and acetic acid-sodium acetate buffer system.

NAD analogs include NCD, Nicotinamide Thymine Dinucleotide (NTD), and Nicotinamide Uracil Dinucleotide (NUD), which have the following chemical structure:

the NAD analogue related to the invention is prepared by reference method (Ji DB, et al. Sci China Chem,2013,56, 296-300).

The methanol dehydrogenase used in the invention is an active protein which takes methanol as a reducing agent and catalyzes and reduces NAD analogue into a corresponding reduction state. These enzymes are mutants of methanol dehydrogenase BsMDH (NCBI protein database No. P42327.1, containing complete amino acid sequences from 1 to 339) derived from Bacillus stearothermophilus (the mutation sites are represented by amino acid numbers and amino acid names before and after mutation, for example, V237T indicates that the 237 th amino acid is mutated from V to T, and the rest sites are similar), and include mutants BsMDH-V237T/N240E, BsMDH-V237T/N240E/K241A, BsMDH-Y171/171R/V237T/N240E/K241A, BsMDH-Y171R/I196V/V237T/N240E/K241A. V237T represents the mutation of the 237 th amino acid from V to T, N240E represents the mutation of the 240 th amino acid from N to E, and the like. Expression and purification of these enzymes was performed according to literature methods for expression of other oxidoreductases in E.coli (Ji DB, et al. J. Am ChemSoc,2011,133,20857).

The NAD analogs of the present invention contain nicotinamide mononucleotide units, as do NAD, the reduced form of which is 1, 4-dihydronicotinamide mononucleotide. Therefore, the NAD analogue reduced product has stronger absorption in the ultraviolet spectral region near 340nm and molar extinction coefficient₃₄₀About 6220M^-1·cm^-1(Ji DB, et al. creation of bioorchol redox systems pending on a nicotinamide flucytoside. J Am ChemSoc.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 Zorbax150mM X3.0 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 the reduced state of a cyclic NAD analog. By the technique of the present invention, the reducing power of methanol is transferred and stored in the NAD analog reduced state to facilitate selective reduction of other substrates.

The buffer system used comprises one or more than two of phosphate buffer, Tris-HCl buffer, HEPES buffer, MES buffer and PIPES buffer, wherein the final concentration of the methanol dehydrogenase is 1 mu g/mL-600 mu g/mL (preferably 100 mu g/mL-500 mu g/mL, more preferably 100 mu g/mL-300 mu g/mL), the final concentration of the NAD analogue is 0.001mM-20mM (preferably 0.01mM-20mM, more preferably 0.1mM-10mM), and the final concentration of the methanol is 1mM-1000mM (preferably 10mM-1000mM, more preferably 100mM-900 mM).

Reducing NAD analogs using the methanol dehydrogenase to include, but not limited to, malic enzyme ME (NCBI No. NP-415996.1) mutant ME-L310R/Q401C which catalyzes the reduction of pyruvate; when the lactate dehydrogenase DLDH (GeneBank No. CAA47255.1, PDB ID:2DLD) mutant DLDH-V152R, V152R/I177R/A212G or V152R/I177S/A212D or saccharomyces cerevisiae alcohol dehydrogenase catalyzing reduction of pyruvate provides reduced coenzyme, a buffer system with pH4-9 is adopted, and the reaction temperature is 15-40 ℃.

The methanol-utilizing enzyme is expressed in the cells of the microorganism, and the NAD analogue can be transferred into the cells through an NAD transporter AtNDT2(Access NO. NC-003070) or NTT4(Haferkamp I, et al. Nature,2004,432, 622-; methanol permeates into the cells and the NAD analog reduction reaction proceeds inside the cells.

The microbial cells expressing methanol 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, Rhodotorula rubra or Trichoderma reesei and the like.

The invention has the advantages and beneficial effects that: methanol as a reducing agent is low in price and abundant in reserves, and is a carbon resource with a great prospect. The reaction of enzyme catalysis methanol can be used in a biological reaction system, and methanol is converted into chemicals with high added values by means of microbial fermentation and the like, so that the selective transfer of reducing power and the effective utilization of carbon are realized. In addition, by using deuterated methanol, the reduced state of the deuterated NAD analogue can be obtained and used for preparing high-purity deuterium-substituted biocatalytic products.

Description of the drawings:

FIG. 1 is a crystal structure of BsMDH-Y171R/I196E/V237T/N240E/K241A complex;

FIG. 2 is a crystal structure of BsMDH-Y171R/V237T/N240E/K241A 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 methanol with NAD analogs in the absence of enzymes

NAD analogs (NCD, NTD and NUD) were prepared according to the literature methods (Ji DB, et al. Sci China Chem,2013,56, 296-300). The NAD analogue was made up to a 20mM concentration in water for use.

1mM NAD analogue substrate and 8mM methanol were dissolved in 1mL Tris-HCl buffer solution with a concentration of 50mM and pH 7.5, mixed, reacted at 30 ℃ for 2 hours, and 20. mu.L thereof was analyzed.

The NAD analogue substrate and its reduced product were detected by HPLC. The liquid chromatograph was Agilent 1100, the analytical column was Zorbax150 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 reaction samples had no characteristic peak at 340nm, and only a characteristic peak at 260nm was detected, which was identical to the retention time of the NAD analogue. Indicating that methanol cannot directly reduce the NAD analog without the enzyme.

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

Methanol dehydrogenase BsMDH (NCBI protein database No. P42327.1, containing complete amino acid sequences from 1 to 339) from Bacillus stearothermophilus was heated in a water bath at 98 ℃ for 60min for use. The reference describes a method for measuring NADH (Guo Q, et al biochemistry,2016,55,2760-2771), in which NAD is used as a substrate, and the detection shows that the sample loses the activity of catalytically reducing NAD into NADH.

The NAD analogs NCD, NTD and NUD were reacted one by one as follows: 1mM NAD analogue, 8mM methanol and 80. mu.g of inactivated methanol dehydrogenase BsMDH were dissolved in 1mL of Tris-HCl buffer solution of 50mM concentration, pH 7.5, mixed, reacted at 30 ℃ for 2 hours, and 20. mu.L thereof was analyzed.

All samples of the reaction were found to have no characteristic peak at 340nm and only a characteristic peak at 260nm was detected which was identical to the retention time of the NAD analogue, as analyzed by the method of comparative example 1. Indicating that the heat-inactivated enzyme is unable to catalyze the reduction of the NAD analog by methanol.

Example 1: methanol is used as a reducing agent, and methanol dehydrogenase is used for catalytic reduction of NAD analogue

The NAD and its analogues NCD, NTD or NUD are combined with methanol dehydrogenase BsMDH, BsMDH-V237T, BsMDH-V237T/N240E, BsMDH-V237T/N240E/K241A, BsMDH-Y171R/V237T/N240E/K241A, BsMDH-Y171R/I196V/V237T/N240E/K241A one by one to react according to the following method: 1mM NAD or the like, 8mM methanol and 80. mu.g methanol dehydrogenase were dissolved in 1mL HEPES buffer solution of 50mM concentration, pH 7.5, mixed well, reacted at 30 ℃ for 20min, and 20. mu.L thereof was analyzed.

According to the analysis method of the 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 the methanol dehydrogenase can catalyze methanol 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). It can be seen that BsMDH has a low overall catalytic activity, and several other methanol dehydrogenases have good activity. Suitable methanol dehydrogenases can be selected depending on the NAD analog. The efficiencies of mutants BsMDH-Y171R/I196E/V237T/N240E/K241A and BsMDH-Y171R/V237T/N240E/K241A for reducing NCD are 96% and 93%, respectively, which are improved compared with the catalytic efficiencies (89%, 46% and 83%, respectively, patent application number 201811154784.8) of mutants BsMDH-I196Q, BsMDH-K242Y/A243M and BsMDH-D195E/A238V obtained in the previous patents. The mutant BsMDH-V237T/N240E/K241A has an NUD reduction efficiency of 91 percent, which is higher than the activity of the mutant obtained in the previous patent on NUD (the conversion rate of the mutant BsMDH-I196Q on NUD is 89 percent at most, and the patent application number is 201811154784.8). The reduction efficiency of the mutant BsMDH-V237T/N240E to NTD is 85 percent, which is higher than the activity of the mutant obtained in the previous patent to NTD (BsMDH-D195E/A238V has the highest conversion rate to NTD, which is 69 percent, and the patent application number is 201811154784.8). furthermore, the catalytic efficiency of the methanol dehydrogenase mutant related to the invention to NTD and NUD is higher than that of the existing mutant on the whole (see the patent, the Chinese patent application number is 201811154784.8).

The results of example 1 show that methanol dehydrogenase can effectively catalyze methanol to reduce the NAD analogue originally involved in the invention to prepare the corresponding reduced product. Combining the results of example 1, comparative example 1 and comparative example 2 demonstrates that active methanol dehydrogenase plays an irreplaceable role in reducing NAD analogs using methanol as the reducing agent.

TABLE 1 Experimental results for methanol dehydrogenase catalyzing reduction of NAD and its analogs

Example 2: preparation of reduced NAD analogs

The reaction system of example 1 was scaled up and used to prepare reduced NAD analogs. The preparation process is described by taking NUDH as an example. 20mM NUD, 20mM methanol and 5mg methanol dehydrogenase BsMDH-V237T were dissolved in 10mL sodium phosphate buffer solution of 50mM concentration and pH 5.7, and the mixture was mixed and reacted at 30 ℃ for 80 min. Directly freeze-drying after reaction, concentrating to total volume of about 4mL, separating with formic acid type anion exchange resin column, collecting product with ultraviolet wavelength of 340nm, and freeze-drying to obtain white powder 5.6mg with yield of about 44%.

Subjecting the white powder sample to high resolution mass spectrometry to obtain accurate molecular weight (M + H)⁺643.1026, theoretical molecular weight of NUDH (C)₂₀H₂₉N₄O₁₆P₂ ⁺643.1054), indicating that the reduced product NUDH was obtained.

NAD analogs were produced using the same amounts of NUD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R as in example 2, giving NUDH in reduced form in about 35% yield. The results show that methanol dehydrogenase BsMDH-V237T and phosphite dehydrogenase rsPDH-I151R both catalyze the corresponding substrates to produce NAD analogues with similar yield.

Example 3: methanol is used as a reducing agent, and methanol dehydrogenase is used for catalytic reduction of NAD analogue

0.1mM NUD, 0.8mM methanol and 80. mu.g methanol dehydrogenase BsMDH-V237T/N240E were dissolved in 1mL of 50mM PIPES buffer pH 8.0, mixed, reacted at 40 ℃ for 3min, and 20. mu.L 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 NBrCDH produced reached 60. mu.M, i.e., the yield reached 60%.

The results of example 1 and example 3 were combined to show that methanol dehydrogenase is used as a reducing agent in the catalytic reduction of NAD analogues, and that the NAD analogues can be reduced.

NAD analogues were produced using the same amounts of NUD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R as in example 3, giving a concentration of NUDH of 43. mu.M, i.e.a yield of 40%. The results show that methanol dehydrogenase BsMDH-V237T/N240E and phosphite dehydrogenase rsPDH-I151R both catalyze the corresponding substrates to produce NAD analogues, and the reaction yield catalyzed by methanol dehydrogenase BsMDH-V237T is higher than that catalyzed by phosphite dehydrogenase rsPDH-I151R.

Example 4: catalytic reduction of NAD analogue by methanol dehydrogenase with deuterated methanol as reducing agent

1mM NCD, 4mM deuterium-substituted methanol and 40. mu.g methanol dehydrogenase BsMDH-V237T/N240E/K241A were dissolved in 1mL MES buffer solution of 50mM concentration and pH 5.0, mixed, reacted at 10 ℃ for 120min, and 20. mu.L was collected and analyzed.

According to the analysis method of comparative example 1, the sample shows a characteristic absorption peak at 340 nm. The product NCDH concentration reached 0.61mM, i.e., the yield was 60%.

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 methanol dehydrogenase can catalytically reduce NAD analogs to the corresponding deuterated reduced products using deuterated methanol 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 0.38mM, i.e.a yield of 38%. The methanol dehydrogenase BsMDH-V237T/N240E/K241A and rsPDH-I151R/E213C can catalyze corresponding substrates to produce NAD analogues, and the reaction yield catalyzed by the methanol dehydrogenase rsPDH-I151R/E213C is higher than that catalyzed by the phosphite dehydrogenase rsPDH-I151R/E213C.

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

The malic enzyme (NCBI No. NP-415996.1) mutant ME-L310R/Q401C was purified for use by the reference (Ji DB, et al. J Am Chem Soc,2011,133,20857-20862) method. 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 methanol dehydrogenase is: methanol + NCD → Formaldehyde + NCDH. The two reactions were combined and the net reaction was: methanol + pyruvic acid + CO₂→ malic acid + formaldehyde. Therefore, the system consisting of methanol dehydrogenase and malic enzyme can reduce and carboxylate pyruvic acid to malic acid by using methanol as a reducing agent. In this system, NAD analogs are recycled and CO fixation is achieved₂The effect of (2) has certain potential. In a reaction system using methanol as a substrate, carbon from the methanol is introduced into a cell metabolism system while the reduced NAD analogue is regenerated, and the substance metabolism and the energy metabolism of the substrate are realized simultaneously. A representative experimental procedure is as follows:

with 50mM Tris-HCl buffer system, pH 5.0, a 100. mu.L reaction system consisted of: 800mM methanol, 50mM pyruvate, 0.1mM NCD, 1.0mM MnCl₂10mM sodium bicarbonate, 0.05mg/mL BsMDH-Y171R/V237T/N240E/K241A and 0.06mg/mL ME-L310R/Q401C. The reaction was carried out at 10 ℃ for 120min, and 900. mu.L of acetonitrile/water mixture (volume ratio, acetonitrile: water: 4:1) was added to terminate the reaction (Rabinowitz JD, et al. anal Chem,2007,79, 6167-6173).

The content of malic acid, pyruvic acid and methanol 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 methanol, 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 methanol, NCD, BsMDH-Y171R/V237T/N240E/K241A 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.

When the reaction is carried out, deuterated methanol is used for replacing methanol, other components and conditions are the same, and analysis shows that the concentration of pyruvic acid in the reaction liquid is reduced to 46.3mM, and 3.5mM malic acid is generated, which indicates that the system can use deuterated methanol as a reducing agent and achieve the efficiency equivalent to that of methanol as the reducing agent.

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 methanol 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 by methanol dehydrogenase, D-lactate dehydrogenase DLDH-V152R and NAD analogue system

D-lactate dehydrogenase (GeneBank No. CAA47255.1, PDB ID:2DLD) mutant DLDH-V152R was purified for use according to the reference (Ji DB, et al. J Am Chem Soc,2011,133, 20857-20862). D-lactate dehydrogenase DLDH-V152R prefers NAD analogs, requiring reduced analogs as cofactors.

The reaction catalyzed by lactate dehydrogenase is: pyruvate + NTCDH → D-lactate + NTCD. The reaction catalyzed by methanol dehydrogenase is: methanol + NTCD → formaldehyde + NTCDH. The two reactions were combined and the total reaction was: methanol + pyruvate → D-lactic acid + formaldehyde. Therefore, the system comprising methanol dehydrogenase and D-lactate dehydrogenase can reduce pyruvic acid to D-lactate using methanol 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 50mM, pH 8.0 MES buffer system, 100. mu.L of the reaction system consisted of: 8mM methanol, 4.0mM pyruvate, 0.1mM NTCD, 0.05mg/mL BsMDH-Y171R/I196V/V237T/N240E/K241A and 0.06 mg/mLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDLDV 152R. After 10min at 40 ℃, 900 μ L of acetonitrile/water mixture (volume ratio, acetonitrile: water: 4:1) was added to terminate the reaction.

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction mixture contained 0.6mM methanol, 3.3mM D-lactic acid and 0.6mM pyruvic acid.

Experimental results show that the system utilizes methanol as a reducing agent to reduce pyruvic acid to lactic acid nearly quantitatively, and high raw material utilization efficiency is achieved. According to the stoichiometric relation of the reaction, NTCD regeneration and recycling are carried out 33 times.

Lactate was prepared by pyruvate reduction catalyzed by methanol dehydrogenase mutant BsMDH-I196Q (see earlier patent, Chinese patent application No. 201811154784.8), D-lactate dehydrogenase DLDH-V152R, and NAD analog system according to the method of example 6. Using the same reaction system and analysis method, the results of the tests showed that the reaction solution contained 0.6mM methanol, 3.0mM D-lactic acid, and 0.8mM pyruvic acid. The catalytic activity of the methanol dehydrogenase mutant BsMDH-Y171R/I196V/V237TN240E/K241A, the D-lactate dehydrogenase DLDH-V152R and the NAD analogue system for preparing lactic acid by catalyzing reduction of pyruvic acid is higher than that of the methanol dehydrogenase mutant BsMDH-I196Q, the D-lactate dehydrogenase DLDH-V152R and the NAD analogue system.

Example 7: preparation of lactic acid by catalyzing reduction of pyruvate by methanol dehydrogenase, D-lactate dehydrogenase DLDH-V152R and NAD analogue 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 8mM methanol, 2.9mM D-lactic acid and 0.9mM pyruvic acid. It was demonstrated that the system containing mutant BsMDH-Y171R/I196E/V237T/N240E/K241A can reduce pyruvic acid to lactic acid almost quantitatively with methanol as a reducing agent under the condition of pH 9.0, and thus, the utilization efficiency of raw materials is high. But the conversion efficiency was lower than that of the reaction system at pH 8.0.

Example 8: preparation of lactic acid by catalyzing reduction of pyruvate by methanol dehydrogenase, D-lactate dehydrogenase DLDH-V152R and NAD analogue 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 6mM methanol, 1.7mM D-lactic acid and 2.2mM pyruvic acid. It was demonstrated that a system containing BsMDH-Y171R/I196E/V237T/N240E/K241A can reduce pyruvic acid to lactic acid in a near quantitative manner using methanol as a reducing agent under a pH of 4.0, and that the raw material utilization efficiency is high. But the conversion efficiency was lower than in the reaction systems of pH 8.0 and pH 9.0.

Example 9: alcohol dehydrogenase catalyzes aldehyde reduction reaction using reduced NAD analog

The reduced analog NTDH was prepared according to the method of example 2 and was ready for use.

A sodium phosphate buffer system with 20mM and pH 7.5 is adopted, and the composition of a 500-mu-L reaction system is as follows: 3.0mM acetaldehyde, 2.0mM TDH, 0.1mg/mL alcohol dehydrogenase derived from Saccharomyces cerevisiae (purchased from Sigma, cat # A3263). The reaction was followed with a spectrophotometer at a UV wavelength of 340nm at 30 ℃. After 30min of reaction, the NTDH in the system was reduced to 0.8 mM. At the same time, 1.1mM ethanol was produced in the system.

In the control experiment system without adding the saccharomyces cerevisiae alcohol dehydrogenase, the concentration of NTDH is not obviously changed after 30min of reaction. The results of example 7 demonstrate that reduced NAD analogs can be used as coenzymes by redox enzymes to catalyze reduction reactions.

Example 10: methanol dehydrogenase-mediated intracellular reduction NAD analogue and application thereof

The methanol dehydrogenase of the identified NAD analog, the oxidoreductase that prefers NAD analogs, and the NAD analog transporter can be simultaneously expressed in a host to form a NAD analog-dependent biocatalytic system. The biocatalytic system is activated when methanol and NAD analogs in the culture medium enter the host cell. Thus, using the methanol dehydrogenase-mediated intracellular reduction of NAD analogs, extracellular reducing forces can be selectively delivered to intracellular target redox reactions. The construction of malic acid-producing engineered strains of Escherichia coli XZ654(Zhang X, et al. appl Environ Microbiol,2011,77,427-434) is described below as an example.

The NAD transporter AtNDT2(Accession NO. NC-003070) has a broader substrate spectrum (PalmieriF, et al. J Biol Chem,2009,284,31249-31259) and can transport NCD. The gene of AtNDT2 expressing the transporter was expressed under the control of the gapAP 1 promoter (Charpentier B, et al. J Bacteriol,1994,176, 830-839). The gene encoding BsMDH-Y171R/I196E and the gene encoding ME-L310R/Q401C were controlled by the isopropyl thiogalactose (IPTG) -induced lac promoter, and the three expression cassettes were cloned into the same plasmid by replacing the LacZ gene of pUC18 to obtain an engineered plasmid.

The above-mentioned engineering plasmid was introduced into e.coli Bl21(DE3) to obtain an engineering strain e.coli WJT 007. Inducing engineering strain E.coli WJT007 in LB culture medium to express the above three functional proteins, adding 50. mu.g/mL ampicillin and 0.1mM IPTG into the culture medium, culturing in a shaker at 25 deg.C and 200rpm for 48h to OD_600nmThe cells were collected by centrifugation at 2000 Xg for 6min at 4.5.

The suspended cells were washed with MOPS medium at pH 7.5, and the cell density OD was determined_600nmAdjusted to 9. 10mM sodium bicarbonate, 10mM pyruvic acid, 8mM methanol and 0.1mM NCD are added into the engineering bacteria suspension, anaerobic reaction is carried out for 4h in a shaking table at 200rpm and at the temperature of 16 ℃, 30 ℃ and 42 ℃ respectively, 100 mu L of acetonitrile is added into 900 mu L of acetonitrile water mixed solution (acetonitrile: water is 4:1), and the reaction is stopped.

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 2mM methanol, 2.8mM malic acid, and 7.1mM pyruvic acid at 16 ℃. The reaction solution contained 0.2mM methanol, 4.3mM malic acid, and 5.4mM pyruvic acid at 30 ℃. The reaction solution at 42 ℃ contained 0.5mM methanol, 3.8mM malic acid, and 5.6mM pyruvic acid.

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

The experimental results show that methanol dehydrogenase BsMDH-Y171R/I196E catalyzes the reductive carboxylation of pyruvate to malate by oxidizing methanol to provide NCDH to ME-L310R/Q401C, increasing the malate production from 1.9mM to 4mM during whole-cell catalysis. The malic acid yield did not increase significantly when only methanol was added, and did not increase when only NCD was added.

Example 8 demonstrates that intracellular methanol dehydrogenase can provide reduced NAD analogs by oxidizing methanol during whole-cell catalysis, which is used as a coenzyme by ME-L310R/Q401C in reduction reactions as a means of regulating the metabolic strength of malate in microorganisms by providing redox.

According to the method of example 8, 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. It is demonstrated that the catalytic system involving methanol dehydrogenase BsMDH-Y171R/I196E has a slightly higher catalytic efficiency at 30 ℃ and an equivalent catalytic efficiency at 16 ℃ and 42 ℃ compared with the similar catalytic system involving phosphite dehydrogenase rsPDH-I151R/E213C.

Example 11: methanol dehydrogenase-mediated intracellular reduction NAD analogue and application thereof

The methanol dehydrogenase of the identified NAD analog, the oxidoreductase that prefers NAD analogs, and the NAD analog transporter can be simultaneously expressed in a host to form a NAD analog-dependent biocatalytic system. The construction of an engineered strain for producing lactic acid by engineering Escherichia coli XZ654(Zhang X, et al. appl Environ Microbiol,2011,77,427-434) will be described below as an example.

The NAD transporter NTT4(Haferkamp I, et al. Nature,2004,432,622-625.) can transport NGD. The three genes expressing the transporter NTT4 are expressed under the control of gapAP 1 promoter. The gene encoding BsMDH-Y171R/V237T and the gene encoding DLDH-V152R were controlled by the isopropyl thiogalactoside (IPTG) induced lac promoter, and the three expression cassettes were cloned on the same plasmid by replacing the LacZ gene of pUC18 to obtain an engineered plasmid.

The above-mentioned engineering plasmid was introduced into e.coli Bl21(DE3) to obtain the engineering strain e.coli WJT 008. The three functional proteins are expressed by inducing engineering bacteria E.coli 008 in LB culture medium, adding 50 ug/mL ampicillin and 0.1mM IPTG, and culturing in a shaker at 25 deg.C and 200rpm 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 methanol and 0.1mM NUD 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-water mixed solution (volume ratio, acetonitrile: water is 4:1) is taken 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 0.1mM methanol, 4.9mM lactic acid and 4.7mM pyruvic acid at 30 ℃.

In the control experiments with and without the addition of either methanol or NUD, the lactic acid concentrations were 0.9mM, 0.9mM and 0.6mM, respectively.

The experimental results show that methanol dehydrogenase BsMDH-Y171R/V237T catalyzes the reduction of pyruvate to lactate by oxidizing methanol to provide NUDH to DLDH-V152R in a whole cell catalytic process, and the yield of lactate is increased from 0.6mM to 4.9 mM. There was no significant increase in lactic acid production with the addition of methanol or NUD alone.

Example 9 demonstrates that intracellular methanol dehydrogenase can provide reduced NAD analogs by oxidizing methanol during 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 9, 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 of phosphorous acid, 4.8mM of lactic acid and 4.6mM of pyruvic acid at 30 ℃. It is shown that the catalytic system involved in methanol dehydrogenase BsMDH-Y171R/V237T is close to the catalytic system involved in phosphite dehydrogenase rsPDH-I151R/I218F.

Example 12: methanol dehydrogenase mediated permeable intracellular reduction NAD analogue and application thereof

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

The gene coding for BsMDH-V237T/N240V/K241A and the gene coding for 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 engineered plasmid.

The above engineered plasmid was introduced into e.coli XZ654 to obtain the engineered strain e.coli WJT 009. Coli WJT 009 engineering bacterium induced in LB medium to express the above two functional proteins, adding 100. mu.g/mL ampicillin and 1mM IPTG into the medium, and culturing in a shaker at 25 deg.C and 200rpm for 48h to OD_600nmCentrifugation at 2000 Xg for 6min at 4.5 to collect the cells, washing the resuspended cells with Tris-Cl at 50mM, pH 7.5, and determining the cell density OD_600nmAdjusted to 9, the cells were permeabilized according to the literature (Zhang W, et al. 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 of toluene, shaking at 30 ℃ and 200rpm for 30min, 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 pH 7.5, and then resuspended in 5mL of 50mM Tris-Cl at pH 5.0 to obtain permeabilized cells.

10mM pyruvic acid, 5mM methanol and 0.1mM NCD were added to the above-mentioned permeabilized engineered bacterial suspension resuspended in Tris-Cl at a concentration of 50mM and pH 5.0, and anaerobic reaction was carried out for 0.5h 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.

When analyzed by an ion chromatography system according to the method of example 5, the reaction solution was found to contain 2mM methanol, 2.6mM lactic acid and 7.1mM pyruvic acid.

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

The experimental result shows that methanol dehydrogenase BsMDH-V237T/N240V/K241A provides NCDH for DLDH-V152R by oxidizing methanol in the whole cell catalytic process, catalyzes reduction of pyruvate to generate lactate, and increases the yield of the lactate from 0.3mM to 2.6 mM. There was no significant increase in malic acid production with the addition of methanol or NCD alone.

Example 10 demonstrates that intracellular methanol dehydrogenase can provide reduced NAD analogs by oxidizing methanol during whole-cell catalysis, which is used as a coenzyme by DLDH-V152R for 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 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. The results showed that the reaction solution at 30 ℃ contained 2.4mM of phosphorous acid, 2.5mM of lactic acid and 7.8mM of pyruvic acid. The efficiency of the catalytic system involved in methanol dehydrogenase BsMDH-V237T/N240V/K241A is higher than that of the similar catalytic system involved in phosphite dehydrogenase rsPDH-I151R.

Example 13: methanol dehydrogenase mediated reduction of NAD analogue in permeabilized Lactococcus lactis (Lactococcus lactis) AS1.2829 cell and application thereof

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

The gene encoding BsMDH-Y171R and the gene encoding DLDH-V152R were controlled by the constitutive expression promoter P32, and both expression cassettes were used to obtain engineered plasmids by replacing the P32 expression cassette of pMG36e (GUCHTE MV, et al. appl Environ Microbiol,1989,55, 224-.

The engineering plasmid is introduced into lactococcus lactis to obtain engineering strain L.lactis WJT 010. 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 WJT 010 to express the two functional proteins by a culture medium of 5mg/L erythromycin, culturing in a shaker at 25 ℃ and 200rpm for 48h until the thallus density is 4.5, centrifuging at 2000 Xg for 6min to collect thallus, washing and re-suspending the thallus by 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 10, 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. The supernatant containing EDTA and toluene was removed by centrifugation at 2000g for 6min, 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 pyruvic acid and 5mM methanol were added to the above permeation-engineered bacterial suspension resuspended in 50mM Tris-Cl, pH 7.5. 0.1mM NFCD, in a shaker at 200rpm at 30 ℃ for 1 h. To 100. mu.L of the reaction mixture was added 900. mu.L of a mixture of acetonitrile and water (acetonitrile: 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 methanol, 2.7mM lactic acid and 7.1mM pyruvic acid.

In the control experiments with and without methanol and NFCD added, the lactic acid concentrations were 0.4mM, 0.4mM and 0.2mM, respectively.

Example 11 demonstrates that intracellular methanol dehydrogenase can provide reduced NAD analog by oxidizing methanol during whole-cell catalysis of lactococcus lactis, and that DLDH-V152R is used as a coenzyme in reduction reactions, and that the accumulation of lactic acid is increased by 13.5-fold compared to control experiments without methanol and NFCD, and thus can be used as a means to regulate the metabolic strength of lactic acid in microorganisms by providing redox.

Example 14: methanol dehydrogenase-mediated permeabilization Saccharomyces cerevisiae (Saccharomyces cerevisiae) BY4741 intracellular reduction NAD analogue and application thereof

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

The gene coding for BsMDH-Y171R/I196R/V237T/N240E and the gene coding for DLDH-V152R are controlled by TEF constitutive promoter and 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.cerevisiae WJT 011. Culturing engineering bacteria S.cerevisiae WJT 011 with YEPD culture medium containing 20g/L glucose, 10g/L yeast extract and 20g/L peptone at pH6.0 to express the above two functional proteins, and culturing in a shaker at 25 deg.C 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, pH 7.5, and OD_600nmAdjusted to 9. The cells were permeabilized by the method described in example 11 to obtain permeabilized cells.

10mM pyruvic acid, 5mM methanol and 0.1mM NUD were added to the above-mentioned permeabilized engineered bacterial suspension resuspended in 50mM Tris-Cl, pH 7.5, and anaerobic reaction was carried out for 1 hour in a shaker at 30 ℃ and 200 rpm. The reaction was terminated by adding 900. mu.L of acetonitrile/water mixture (volume ratio, acetonitrile: water: 4:1) to 100. mu.L of acetonitrile/water mixture.

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 0.6mM methanol, 3.7mM lactic acid and 6.1mM pyruvic acid.

In the control experiments with and without the addition of either methanol or NUD, the concentrations of lactic acid were 0.4mM, 0.6mM and 0.4mM, respectively.

Example 12 demonstrates that intracellular methanol dehydrogenase can provide reduced NAD analogs by oxidizing methanol during whole-cell catalysis of saccharomyces cerevisiae, and that DLDH-V152R is used as a coenzyme for reduction reactions, with an accumulation of lactate that is 9.3-fold higher than that of control experiments without methanol and NUD, and thus can be used as a means to regulate the metabolic strength of lactate in microorganisms by providing redox.

Example 15: methanol dehydrogenase mediated intracellular reduction NAD analogue of Trichoderma reesei (Trichoderma reesei) and application thereof

The methanol 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 methanol and NAD analogs in the culture medium enter the host cell.

The gene coding BsMDH-Y171R/I196V/V237T/N240E/K241A 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 engineered plasmid into Trichoderma reesei to obtain engineered strain T.reesei WJT 012, adding 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 engineered bacterium T.reesei WJT 012 was induced by culture medium O to express the above two functional proteins, cultured in a shaker at 25 ℃ and 200rpm for 48h, centrifuged at 2000 Xg for 6min to collect the cells, washed with Tris-Cl at 50mM and pH 7.5 to resuspend the cells, and the cell density was adjusted to 3g dry cell weight/L. The cells were permeabilized by the method described in example 11 to obtain permeabilized cells.

10mM pyruvic acid, 5mM methanol and 0.1mM NCD were added to the above Tris-Cl resuspended permeable engineered bacterial suspension at a concentration of 50mM and pH 7.5, 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/water mixture (volume ratio, acetonitrile: water: 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 1.5mM methanol, 3.3mM lactic acid and 6.4mM pyruvic acid.

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

Example 13 demonstrates that intracellular methanol dehydrogenase can provide reduced NAD analog by oxidizing methanol during whole cell catalysis of trichoderma reesei, and that DLDH-V152R used as a coenzyme in reduction reactions increases the accumulation of lactate by 5.5-fold compared to control experiments without methanol and NCD, and thus can be used as a means to regulate the metabolic strength of lactate in microorganisms by providing redox.

Example 16: intracellular reduction NAD analogue of Rhodosporidium toruloides (Rhodosporidium toruloides) mediated by methanol dehydrogenase and application thereof

The methanol 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 activated when methanol and NAD analogs in the culture medium enter the host cell.

The gene coding BsMDH-Y171R/N240E and the gene coding DLDH-V152R are controlled by a promoter GPD, a promoter PGK, a terminator Hspt and a promoter Tnos respectively, and the two expression cassettes are integrated on a pZPK vector to obtain an engineering plasmid.

Toruloides WJT 013, which was an engineered strain obtained by transforming the above engineered plasmid into Rhodosporidium toruloides by ATMT, was cultured in YEPD medium containing 20g/L glucose, 10g/L yeast extract and 20g/L peptone at pH6.0 and WJT 013 expressing the above two functional proteins, cultured in a shaker at 28 ℃ and 200rpm for 48 hours, centrifuged at 2000 Xg for 6min to collect the cells, washed with Tris-Cl at a concentration of 50mM and pH 7.5 to resuspend the cells, and the cell density was adjusted to 3g dry cell weight/L. The cells were permeabilized by the method described in example 11 to obtain permeabilized cells.

10mM pyruvic acid, 5mM methanol and 0.1mM NTD were added to the above Tris-Cl resuspended permeable engineered bacterial suspension at a concentration of 50mM and pH 7.5, 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/water mixture (volume ratio, acetonitrile: water: 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 methanol, 3.0mM lactic acid and 6.0mM pyruvic acid.

In the control experiments with and without the addition of either methanol or NTD, the lactic acid concentrations were 1.2mM, 0.7mM and 0.6mM, respectively.

Example 14 demonstrates that intracellular methanol dehydrogenase can provide reduced NAD analog by oxidizing methanol during torula rhodozyma catalysis, and that DLDH-V152R is used as a coenzyme for reduction, and that the amount of accumulated lactic acid is increased by 5-fold compared to control experiments without methanol and NTD, and thus can be used as a means for regulating the metabolic strength of lactic acid in microorganisms by providing redox.

Example 17: crystal analysis of methanol dehydrogenase mutant

The methanol dehydrogenase mutants BsMDH-Y171R/I196E/V237T/N240E/K241A and BsMDH-Y171R/V237T/N240E/K241A 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 conditions for crystal culture for diffraction were 0.1M ammonium sulfate, 0.1M Tris (pH 7.0), 10% polyethylene glycol monoethyl ether 5000, protein concentration 6mg/mL, temperature 30 ℃.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.

Derived from methanol dehydrogenase, NCBI protein database No. P42327.1, containing the complete amino acid sequence from position 1 to 339 as follows:

MKAAVVNEFKKALEIKEVERPKLEEGEVLVKIEACGVCHTDLHAAHGDWPIKPKLPLIPGHEGVGIVVEVAKGVKSIKVGDRVGIPWLYSACGECEYCLTGQETLCPHQLNGGYSVDGGYAEYCKAPADYVAKIPDNLDPVEVAPILCAGVTTYKALKVSGARPGEWVAIYGIGGLGHIALQYAKAMGLNVVAVDISDEKSKLAKDLGADIAINGLKEDPVKAIHDQVGGVHAAISVAVNKKAFEQAYQSVKRGGTLVVVGLPNADLPIPIFDTVLNGVSVKGSIVGTRKDMQEALDFAARGKVRPIVETAELEEINEVFERMEKGKINGRIVLKLKED。

sequence listing

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

<120> a method for reducing NAD analog using methanol

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>339

<212>PRT

<213> methanol dehydrogenase BsMDH (Bacillus stearothermophilus)

<400>1

Met Lys Ala Ala Val Val Asn Glu Phe Lys Lys Ala Leu Glu Ile Lys

1 5 10 15

Glu Val Glu Arg Pro Lys Leu Glu Glu Gly Glu Val Leu Val Lys Ile

20 25 30

Glu Ala Cys Gly Val Cys His Thr Asp Leu His Ala Ala His Gly Asp

35 40 45

Trp Pro Ile Lys Pro Lys Leu Pro Leu Ile Pro Gly His Glu Gly Val

50 55 60

Gly Ile Val Val Glu Val Ala Lys Gly Val Lys Ser Ile Lys Val Gly

65 70 75 80

Asp Arg Val Gly Ile Pro Trp Leu Tyr Ser Ala Cys Gly Glu Cys Glu

8590 95

Tyr Cys Leu Thr Gly Gln Glu Thr Leu Cys Pro His Gln Leu Asn Gly

100 105 110

Gly Tyr Ser Val Asp Gly Gly Tyr Ala Glu Tyr Cys Lys Ala Pro Ala

115 120 125

Asp Tyr Val Ala Lys Ile Pro Asp Asn Leu Asp Pro Val Glu Val Ala

130 135 140

Pro Ile Leu Cys Ala Gly Val Thr Thr Tyr Lys Ala Leu Lys Val Ser

145 150 155 160

Gly Ala Arg Pro Gly Glu Trp Val Ala Ile Tyr Gly Ile Gly Gly Leu

165 170 175

Gly His Ile Ala Leu Gln Tyr Ala Lys Ala Met Gly Leu Asn Val Val

180 185 190

Ala Val Asp Ile Ser Asp Glu Lys Ser Lys Leu Ala Lys Asp Leu Gly

195 200 205

Ala Asp Ile Ala Ile Asn Gly Leu Lys Glu Asp Pro Val Lys Ala Ile

210 215 220

His Asp Gln Val Gly Gly Val His Ala Ala Ile Ser Val Ala Val Asn

225 230 235 240

Lys Lys Ala Phe Glu Gln Ala Tyr Gln Ser Val Lys Arg Gly Gly Thr

245 250255

Leu Val Val Val Gly Leu Pro Asn Ala Asp Leu Pro Ile Pro Ile Phe

260 265 270

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

275 280 285

Arg Lys Asp Met Gln Glu Ala Leu Asp Phe Ala Ala Arg Gly Lys Val

290 295 300

Arg Pro Ile Val Glu Thr Ala Glu Leu Glu Glu Ile Asn Glu Val Phe

305 310 315 320

Glu Arg Met Glu Lys Gly Lys Ile Asn Gly Arg Ile Val Leu Lys Leu

325 330 335

Lys Glu Asp

Claims

1. A method of reducing an NAD analog, comprising: taking a substrate of one or two of methanol and deuterated methanol combined in any proportion as a reducing agent, taking an enzyme of the reducing agent as a catalyst, mixing with the NAD analogue, reacting to obtain a reduced NAD analogue, and oxidizing the methanol into formaldehyde; the catalyst is a mutant of wild methanol dehydrogenase Bacillusstearothermophilus, DSM 2334(ADH 2334) which is modified by genetic engineering; the enzyme capable of utilizing methanol is one or more of BsMDH-V237, BsMDH-V237/N240, BsMDH-V237/N240/K241, BsMDH-Y171/V237/N240/K241, BsMDH-Y171/V237, BsMDH-Y171/I196, BsMDH-Y171/I196/V237/N240/K241, BsMDH-Y171, BsMDH-Y171/N240, BsMDH-Y171/I196/V237/N240/K241, BsMDH-V237/N240/K241, BsMDH-Y171/I196/V237/N240.

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

3. the method of claim 1, further characterized by: the enzyme capable of utilizing methanol is an active protein which takes methanol as a reducing agent and catalyzes and reduces NAD analogues into corresponding reduction states.

4. A method of reducing an NAD analog according to claim 1, further characterized by: in a buffer solution with the pH value of 4-9, the reaction temperature is 15-40 ℃, the initial final concentration of enzyme is 1 mu g/mL-600 mu g/mL, the initial concentration of NAD analogue is 0.001mM-20mM, and the initial concentration of methanol is 0.4mM-1000mM, wherein the buffer system comprises one or more than two of phosphate buffer solution, Tris-HCl buffer solution, HEPES buffer solution, MES buffer solution and PIPES buffer solution.

5. A method of reducing an NAD analog according to claim 1, further characterized by: the obtained reduced NAD analogue can be used as coenzyme by other enzymes for reduction reaction; such other enzymes include, but are not limited to, one or more of the following: malic enzyme ME (NCBI No. NP-415996.1) mutant ME-L310R/Q401C, lactate dehydrogenase DLDH (GeneBank No. CAA47255.1, PDB ID:2DLD) mutant DLDH-V152R, V152R/I177R/A212G or V152R/I177S/A212D; saccharomyces cerevisiae alcohol dehydrogenase catalyzing the reduction of acetaldehyde to ethanol, hydroxybutanone dehydrogenase catalyzing the reduction of diacetyl to hydroxybutanone, malate dehydrogenase (PDB id 1EMD) mutant MDH-L6R catalyzing the oxidation of malate to oxaloacetate.

6. A method of reducing an NAD analogue as claimed in claim 1 or 6 further characterized by: the enzyme that can utilize methanol is expressed in the cells of the microorganism, while the NAD analog and methanol are transported into the cells, and the NAD analog reduction reaction is carried out in the cells.

7. The method of claim 1 or 6, wherein: the reaction is carried out intracellularly, and the microorganisms expressing one or more of malic enzyme, lactate dehydrogenase, and the like 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.