CN110964764B - Method for reducing NAD analogue by using methanol - Google Patents

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 the reduced NAD analogue or the deuterated reduced analogue, and can also provide reduced coenzyme for the enzymatic reaction consuming the reduced NAD analogue, and the reduced NAD analogue can be used as the coenzyme for the enzymatic reduction reactions such as 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 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, wherein the NAD analogue can be used as the 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. The coenzymes can be used for producing chiral chemicals and preparing isotope 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 for transferring reducing power, 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. ACS Catal,2017,7, 1977-1983).

Several NAD analogues with good biocompatibility have been reported. For example, nicotinamide Cytosine Dinucleotide (NCD), nicotinamide 5-fluorocytosine dinucleotide (NFCD), nicotinamide 5-chlorocytosine dinucleotide (NClCD), nicotinamide 5-bromocytosine dinucleotide (NBrCD), and nicotinamide 5-methylcytosine dinucleotide (NMeCD) (Ji DB, et al J Am Chem Soc,2011,133,20857-20862, ji DB, et al Sci China Chem,2013,56, 296-300). Meanwhile, several enzymes recognizing NAD analogues have been reported, such as NADH oxidase from Enterococcus faecalis (NOX, genbank S45681), D-lactate dehydrogenase (DLDH, genbank CAA 47255) V152R mutant, malic enzyme (ME, genbank P26616) L310R/Q401C mutant, and malic dehydrogenase (MDH, genbank CAA 68326) L6R mutant.

Using NAD analogs and enzymes that recognize them, more cost-effective biocatalytic systems can be constructed (catalytic dictionary, jiidenbin et al, 2012,33, 530-535). 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 transporting NCD into the cell, where DLDH-V152R can use NCD to reduce pyruvate to lactate (Wang L, et al ACS Catal,2017,7, 1977-1983).

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 utilize methanol to 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 achieving efficient utilization of one carbon resource (Muller JE, et al. Metab Eng,2015,28,190-201, zhang W, et al. RSC adv,2017,7, 4083-4091).

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 CN 104946706A) utilizes phosphite dehydrogenase to reduce NAD analog to NADH with the formation of by-product phosphate using phosphorous acid as a substrate. Compared with a phosphate compound which is a product generated by catalyzing a phosphorous acid compound by phosphorous acid dehydrogenase, the methanol dehydrogenase has abundant methanol source 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. Metab Eng,2017,39, 49-59). 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.

Disclosure of Invention

The invention relates to an enzyme catalytic reduction method of coenzyme NAD (nicotinamide adenine dinucleotide) analogues, which is characterized in that methanol is used as a reducing agent, and an enzyme capable of utilizing the methanol is used as a catalyst to convert the NAD analogues into corresponding reduction states. 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 methanol as a reducing agent, taking enzyme capable of utilizing methanol as a catalyst, and reacting for 2-120min at 10-40 ℃ in a buffer system with pH of 5-8 to obtain the reduced NAD analogue.

NAD analogs include NCD, NFCD, NClCD, NBrCD, NMeCD, nicotinamide Guanine Dinucleotide (NGD), nicotinamide Thymine Dinucleotide (NTD), and Nicotinamide Uracil Dinucleotide (NUD), which have the following chemical structures:

the NAD analogues to which the present invention relates are prepared by reference methods (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 more than two of methanol dehydrogenase BsMDH (UniProtKB/Swiss-Prot P42327.1, https:// www.ncbi.nlm.nih.gov/protein/P42327.1) derived from Bacillus stearothermophilus or methanol dehydrogenase BmMDH (UniProtKB/Swiss-Prot: P31005.3, https:// www.ncbi.nlm.nih.gov/protein/P31005.3) derived from Bacillus methanoolicus, such as BsMDH-Y171G (BsMDH-Y171G represents that the amino acid at the position 171 of methanol dehydrogenase MDH is changed from Y to G, other same reasoning), bsMDH-Y171G/A238N, bsMDH-N BsMDA/A240K, bsMDH-K243Y 243, bsMDH-M243, bsMDH 240, bsH-K, bsMDH-M219, bmH-M240K, bsMDH-Q210, bmMDH-Q210/M210, bmMDH-D210/Q, bmMDH-D210 and BmMDH-D210/Q. 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 Chem Soc,2011,133, 20857).

The NAD analogue related to the invention contains nicotinamide mononucleotide unit, and the reduction state of the unit is 1, 4-dihydronicotinamide mononucleotide, like NAD. Therefore, the NAD analogue reduced product has stronger absorption in the ultraviolet spectral region near 340nm and a molar extinction coefficient epsilon ₃₄₀ About 6220M ^-1 ·cm ^-1 (Ji DB, et al. Creation of bioorganic redox systems depends on inorganic amino defluorination. J Am Chem Soc.2011,133, 20857-20862). The present invention utilizes this property to analyze NAD analog reduction processes. The conditions for quantifying the NAD analogue and the reduced product thereof 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 0.5mL/min. Each sample was tested for 20min. 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 substrate methanol is one or the combination of two of methanol and deuterated methanol.

The reduced products of the NAD analogue prepared can be used as coenzymes 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 a reduced state of NAD analogue 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 4 mu g/mL-1500 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 0.01mM-20mM (preferably 2mM-20mM, more preferably 2mM-15 mM), and the final concentration of the methanol is 0.4mM-100mM (preferably 10mM-100mM, more preferably 10mM-90 mM).

When the methanol dehydrogenase is used for reducing NAD analogue to provide reduced coenzyme for ME-L310R/Q401C, DLDH-V152R or saccharomyces cerevisiae alcohol dehydrogenase, a buffer system with pH of 5-8 is adopted, and the reaction temperature is 10-40 ℃.

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 (Accession NO. NC-003070) or NTT4 (Haferkamp I, et al. Nature,2004,432, 622-625); 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 analogs 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, 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, a reduced state of deuterated NAD analogs can be obtained for the preparation of high-purity deuterium-substituted biocatalytic products.

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, NFCD, NBrCD, NClCD, NMeCD, NGD, NTD and NUD) were prepared by reference methods (Ji DB, et al. Sci China Chem,2013,56, 296-300). The NAD analogue was made up into a 20mM solution with water for use.

1mM NAD analogue substrate and 4mM methanol were dissolved in 1mL of a 50mM Tris-HCl buffer solution at pH 7.5, mixed well, 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 Zorbax 150 mM. Times.3.0 mM (3.5 μm), the mobile phase was 5mM tetrabutylammonium sulfate, and the flow rate was 0.5mL/min. Each sample was tested for 20min. The detection wavelengths are 260nm (the cofactor and the reduced coenzyme thereof have stronger absorption at 260 nm) and 340nm (the reduced coenzyme has stronger light absorption 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 in the absence of enzyme.

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

Methanol dehydrogenase BsMDH (UniProtKB/Swiss-Prot P42327.1) 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 detection indicates that the sample loses the activity of catalytically reducing NAD to NADH.

The NAD analogues NCD, NFCD, NBrCD, NClCD, NMeCD, NGD, NTD and NUD are reacted according to the following method one by one: 1mM NAD analogue, 4mM methanol and 80. Mu.g of inactivated methanol dehydrogenase BsMDH were dissolved in 1mL of a 50mM concentration Tris-HCl buffer solution at pH 7.5, mixed, reacted at 30 ℃ for 2 hours, and 20. Mu.L thereof was analyzed.

All the samples of the reaction were analyzed by the method of comparative example 1, and were found to have no characteristic peak at 340nm, and only a characteristic peak at 260nm which was the same as the retention time of the NAD analogue was detected. 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

NAD and its analogues NCD, NFCD, NBrCD, NClCD, NMeCD, NGD, NTD or NUD are individually subjected to NAD analogue-methanol dehydrogenase combination with methanol dehydrogenase BsMDH, bsMDH-Y171G/A238N, bsMDH-N240A/A243K, bsMDH-K242Y/A243M, bsMDH-I196Q, bsMDH-D195E/A238V, bmMDH-V210S, bmMDH-N123S, bmMDH-V210T/M219K, bmMDH-D212E/M219R, bmMDH-Q217E, bmMDH-D153T/V210S, and reacted as follows: 1mM NAD or the like, 4mM methanol and 80. Mu.g methanol dehydrogenase were dissolved in 1mL of 50mM HEPES buffer solution at pH 7.5, mixed, reacted at 30 ℃ for 20min, and 20. Mu.L thereof was analyzed.

According to the analysis method of the comparative example 1, the samples show 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 epsilon of reduced products due to NAD analogues ₃₄₀ About 6220M ^-1 ·cm ^-1 The 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 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 is scaled up and can be used to prepare reduced NAD analogs. The preparation process is described by taking NUDH as an example. 20mM NUD, 25mM methanol and 5mg methanol dehydrogenase BsMDH-Y171G were dissolved in 10mL of a 50mM sodium phosphate buffer solution at pH 5.7, mixed, and reacted at 30 ℃ for 80min. 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 above white powder sample to high resolution mass spectrometry to determine the accurate molecular weight (M + H) ⁺ 643.1026, compared to the theoretical molecular weight of NUDH (C) ₂₀ H ₂₉ N ₄ O ₁₆ P ₂ ⁺ 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 to give NUDH in reduced form in about 35% yield. The results show that methanol dehydrogenase BsMDH-Y171G and phosphite dehydrogenase rsPDH-I151R can catalyze corresponding substrates to produce NAD analogues with approximate yield.

Example 3: methanol is used as a reducing agent, and methanol dehydrogenase is used for catalytic reduction of NAD (nicotinamide adenine dinucleotide) analogue

0.1mM NBrCD, 0.4mM methanol and 80. Mu.g methanol dehydrogenase BsMDH-Y171G/A238N were dissolved in 1mL of a 50mM PIPES buffer solution at 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 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.

The same amounts of NBrCD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R were used to produce NAD analogs as in example 3, yielding NBrCDH with a concentration of 43. Mu.M, i.e., a yield of 43%. The results show that methanol dehydrogenase BsMDH-Y171G/A238N and phosphite dehydrogenase rsPDH-I151R can catalyze corresponding substrates to produce NAD analogues, and the reaction yield catalyzed by methanol dehydrogenase BsMDH-Y171G 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 deuteromethanol and 40. Mu.g methanol dehydrogenase BsMDH-N240A/A243K were dissolved in 1mL of a 50mM MES buffer solution at pH 5.0, mixed, reacted at 10 ℃ for 120min, and 20. Mu.L thereof was 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 61%.

Subjecting the sample to high resolution mass spectrometry to determine the accurate molecular weight (M-H) ^- 641.1118 and NCD ² Theoretical molecular weight of H (C) ₂₀ H ₂₇ ² HN ₅ O ₁₅ P ₂ ^- 641.1125) indicating that a deuterated NCD reduced product is obtained.

The results of example 4 indicate that methanol dehydrogenase can catalytically reduce NAD analogs to the corresponding deuterated reduced products using deuterated methanol as the reducing agent.

The same amounts of NCD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R/E213C were used to produce the deuterated NAD analog according to the procedure of example 4, with the product NCDH concentration reaching 0.38mM, i.e., a yield of 38%. The methanol dehydrogenase BsMDH-N240A/A243K 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 ME-L310R/Q401C was purified for use by the reference (Ji DB, et al. 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 reaction catalyzed by malic enzyme ME-L310R/Q401C is as follows: pyruvic acid + CO ₂ + 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 + CO ₂ → 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 the system, NAD analogue is regenerated and circulated, and CO is fixed ₂ The effect of (2) has certain potential. The reaction system using methanol as a substrate can introduce carbon from methanol into a cell metabolism system while regenerating the reduced NAD analogue, and simultaneously realize the substance metabolism and the energy metabolism of the substrate. A representative experimental procedure is as follows:

with a Tris-HCl buffer system of 50mM, pH 5.0, a reaction system of 100. Mu.L had a composition of: 4.0mM methanol, 50mM pyruvate, 0.01mM NCD, 1.0mM MnCl ₂ 10mM sodium bicarbonate, 0.05mg/mL BsMDH-K242Y/A243M, and 0.06mg/mL ME-L310R/Q401C. The reaction was carried out at 10 ℃ for 120min, and 900. Mu.L of acetonitrile in water (acetonitrile: water = 4) 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-protected (50 mm. Times.4 mm) columns (200 mm. Times.4 mm) were used for ion Pac AS11-HC anion exchange analysis. The analysis conditions are as follows: the mobile phase was 24mM NaOH, flow rate 1mL/min, column temperature: 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 reactions, 4 additional sets of control experiments were set up, each lacking one of methanol, NCD, bsMDH-K242Y/A243M or ME-L310R/Q401C, and analysis revealed that these reactions did not produce malic acid. According to the stoichiometric relation of the reaction, the NCD is recycled for 360 times.

In the above reaction, 1 set of experiments was also set, and deuterated methanol was used instead of methanol, and other components and conditions were the same, and analysis found that the concentration of pyruvic acid in the reaction solution was reduced to 46.3mM and 3.5mM malic acid was produced, indicating that this system can use deuterated methanol as a reducing agent, and achieve an efficiency equivalent to methanol as a 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 systems 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. Shows that the catalytic activity of the system of methanol dehydrogenase, malic enzyme ME-L310R/Q401C and NAD analogue for 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 DLDH-V152R was purified for use according to the literature (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 + NFCDH → D-lactate + NFCD. The reaction catalyzed by methanol dehydrogenase is: methanol + NFCD → Formaldehyde + NFCDH. 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 MES buffer system of 50mM, pH 8.0, 100. Mu.L of the reaction system consisted of: 4.0mM methanol, 4.0mM pyruvate, 0.1mM NFCD, 0.05mg/mL BsMDH-I196Q, and 0.06mg/mL DLDH-V152R. After reaction at 40 ℃ for 10min, 900. Mu.L of acetonitrile/water mixture (acetonitrile: water = 4).

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction mixture contained 0.5mM 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 in a near quantitative manner, and high raw material utilization efficiency is achieved. According to the stoichiometric relation of the reaction, NFCD is recycled 33 times.

Lactate was prepared by the method of example 6 using the phosphite dehydrogenase rsPDH-I151R, 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 detection showed that the reaction solution contained 0.6mM of phosphorous acid, 3.0mM of D-lactic acid, and 0.8mM of pyruvic acid. The catalytic activity of the system of methanol dehydrogenase, D-lactate dehydrogenase DLDH-V152R and NAD analogue for preparing lactic acid by reduction of pyruvic acid is higher than that of the system of phosphite dehydrogenase rsPDH-I151R, D-lactate dehydrogenase DLDH-V152R and NAD analogue.

Example 7: 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 NTDH, 0.1mg/mL alcohol dehydrogenase derived from Saccharomyces cerevisiae (purchased from Sigma, cat. No.: A3263). The reaction was followed with a spectrophotometer at 340nm of UV wavelength at 30 ℃. After 30min of reaction, the NTDH in the system was reduced to 0.8mM. 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 8: methanol dehydrogenase-mediated intracellular reduction NAD analogue and application thereof

The identified methanol dehydrogenase of the NAD analogue, the oxidoreductase which prefers the NAD analogue and the NAD analogue transporter can be simultaneously expressed in a host 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. 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 (Palmieri F, et al.J Biol Chem,2009,284, 31249-31259) and can transport NCD. The gene of AtNDT2 expressing the transporter is expressed from the gapAP 1 promoter (Charpentier B, et al. J Bacteriol,1994,176, 830-839). The gene encoding BsMDH-D195E/A238V 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 engineering plasmid is introduced into E.coli XZ654 to obtain an engineering strain E.coli GXJ 007. Inducing engineering strain E.coli GXJ 007 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 25 ℃ and 200rpm for 48h to reach the thallus density OD _600nm The cells were collected by centrifugation at 2000 Xg for 6min at 4.5.

Washing the resuspended thallus with MOPS medium with pH 7.5, and determining the thallus density OD _600nm Adjusted to 9. To the above-mentioned engineering bacterium suspension, 10mM sodium bicarbonate, 10mM pyruvic acid, 5mM methanol, and 0.1mM NCD were added, and the mixture was anaerobically reacted for 4 hours in a shaker at 200rpm at 16 ℃,30 ℃ and 42 ℃, and then the reaction was terminated by adding 100. Mu.L acetonitrile aqueous mixture (acetonitrile: water = 4).

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 1.8mM 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 in the whole cell catalysis process, methanol dehydrogenase BsMDH-D195E/A238V provides NCDH for ME-L310R/Q401C by oxidizing methanol to catalyze the reduction and carboxylation of pyruvate into malic acid, so that the yield of the malic acid is increased from 1.9mM to 4.3mM. 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 via oxidative methanol, which are used as coenzymes by ME-L310R/Q401C for reduction reactions during whole cell catalysis, as a means to regulate the metabolic strength of malate in microorganisms by providing redox potential.

According to the method of example 8, the rsPDH-I151R/E213C gene and ME-L310R/Q410C gene were controlled by the isopropyl thiogalactose (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 shown that the catalytic efficiency of the above-mentioned catalytic system involving methanol dehydrogenase BsMDH-D195E/A238V is slightly higher at 30 ℃ and comparable at 16 ℃ and 42 ℃ to that of the similar catalytic system involving phosphite dehydrogenase rsPDH-I151R/E213C.

Example 9: 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 lactic acid-producing strain of Escherichia coli XZ654 (Zhang X, et al. Applenviron Microbiol,2011,77, 427-434) is 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 coding BmMDH-V210T/M219K and the gene coding DLDH-V152R are controlled by an 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 engineering plasmid.

The engineering plasmid is introduced into E.coli XZ654 to obtain an engineering strain E.coli GXJ 008. Inducing engineering bacteria E.coli 008 to express the three functional proteins in LB culture medium, adding 100 μ g/mL ampicillin and 1mM IPTG, culturing in 25 deg.C 200rpm shaker for 48h to OD _600nm The cells were collected by centrifugation at 2000 Xg for 6min at 4.5.

The resuspended cells were washed with M9 medium pH 8.0 and the density OD was determined _600nm Adjusted to 9. 10mM pyruvic acid, 5mM methanol and 0.1mM NUD were added to the above engineering bacterium suspension, and the mixture was anaerobically reacted for 3 hours in a shaker at 200rpm at 30 ℃ to terminate the reaction by adding 100. Mu.L acetonitrile aqueous solution (acetonitrile: water = 4).

As a result of analysis by the ion chromatography system in accordance with the method in 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 addition of either methanol or NUD, the lactic acid concentrations were 0.9mM, 0.9mM and 0.6mM, respectively.

Experimental results show that in the whole-cell catalysis process, methanol dehydrogenase BmMDH-V210T/M219K provides NUDH for DLDH-V152R by oxidizing methanol, catalyzes reduction of pyruvate into lactate, and enables the yield of lactate to be increased from 0.6mM to 4.9mM. 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, and the corresponding engineered strains were constructed and the contents of the respective components were determined by the same experiment and analysis. 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 ℃. The efficiency of the catalytic system involved in methanol dehydrogenase BmMDH-V210T/M219K is close to that of the catalytic system involved in phosphite dehydrogenase rsPDH-I151R/I218F.

Example 10: 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 BmMDH-D212E/M219R and the gene coding for DLDH-V152R are controlled by a lac promoter induced by isopropyl thiogalactoside (IPTG), 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 009. Coli GXJ 009 engineering bacteria E.coli is induced in LB medium to express the above two functional proteins, 100. Mu.g/mL ampicillin and 1mM IPTG are added into the medium, and cultured in a shaker at 25 deg.C and 200rpm for 48h to OD _600nm The cells were centrifuged at 2000 Xg for 6min at 4.5, and the cells were collected, washed with Tris-Cl at a concentration of 50mM, pH 7.5, and resuspended to OD _600nm Adjusted to 9, cells were permeabilized according to the literature method (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 toluene, shaking at 30 ℃ and 200rpm for 30min, and then left at 4 ℃ for 1h. 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 5.0 to obtain permeabilized cells.

10mM pyruvic acid, 5mM methanol, and 0.1mM NCD were added to the above permeabilized engineered bacterial suspension resuspended in Tris-Cl at a concentration of 50mM and pH 5.0, and anaerobic reaction was performed for 0.5h at 30 ℃ on a shaker at 200 rpm. To 100. Mu.L of the sample, 900. Mu.L of acetonitrile/water mixture (acetonitrile: water = 4).

When analyzed by an ion chromatography system according to the method of example 5, the reaction solution was found to contain 2.1mM of methanol, 2.6mM of lactic acid and 7.1mM of 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 in the whole cell catalysis process, methanol dehydrogenase BmMDH-D212E/M219R provides NCDH for DLDH-V152R by oxidizing methanol, catalyzes the reduction of pyruvate to generate lactic acid, and increases the yield of the lactic acid from 0.3mM to 2.6mM. 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 via oxidation of methanol during whole-cell catalysis, which is used as a coenzyme by DLDH-V152R for reduction reactions, as a means to regulate the metabolic strength of lactate in microorganisms by providing redox potential.

According to the method of example 10, the rsPDH-I151R gene and ME-L310R/Q401C gene 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. As a result, 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 BmMDH-D212E/M219R is higher than that of the similar catalytic system involved in phosphite dehydrogenase rsPDH-I151R.

Example 11: methanol dehydrogenase-mediated permeable Lactococcus lactis (Lactococcus lactis) AS1.2829 intracellular reduction NAD analogue 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 BmMDH-Q217E 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, 224-228.).

The engineering plasmid is introduced into lactococcus lactis to obtain an engineering strain L.lactis GXJ 010. Using a mixture of 10g/L sucrose, 10g/L yeast extract, 10g/L peptone and 10g/L KH at pH 6.8 ₂ PO ₄ 2g/L of MgSO 2 ₄ Inducing the engineering bacteria L.lactis GXJ 010 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 carrying out OD (OD) on the thallus density _600nm Adjusted to 9. The cells were permeabilized according to the method of example 10, in the following manner: thawing 5mL of frozen cells in a water bath at room temperature, adding 5mM EDTA and 1% toluene by volume, performing a temperature bath for 30min in a shaker at 30 ℃ and 200rpm, and then standing for 1h at 4 ℃. 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 1h. To 100. Mu.L of the reaction solution, 900. Mu.L of acetonitrile/water mixture (acetonitrile: water = 4).

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 for reduction, and that the amount of accumulated lactic acid is increased by 13.5-fold compared to control experiments without addition of formic acid and NFCD, and thus can be used as a means to regulate the metabolic strength of lactic acid in microorganisms by providing redox.

Example 12: methanol dehydrogenase mediated permeable 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 BmMDH-D153T/V210S and the gene coding 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. Culturing engineering bacteria S.cerevisiae GXJ 011 with YEPD culture medium containing 20g/L glucose, 10g/L yeast extract and 20g/L peptone at pH 6.0 to express the above two functional proteins, and culturing in shaker at 25 deg.C and 200rpm for 48 hr to thallus density OD _600nm Centrifugation 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 of cell density _600nm Adjusted 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 NGD were added to the above permeabilized engineered bacterial suspension resuspended in 50mM Tris-Cl, pH 7.5, and anaerobic reaction was performed for 1 hour at 30 ℃ in a shaker at 200 rpm. The reaction was terminated by adding 900. Mu.L of acetonitrile in water (acetonitrile: water = 4).

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 addition of either methanol or NGD, 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 via methanol oxidation during whole cell catalysis of saccharomyces cerevisiae, and that when DLDH-V152R is used as a coenzyme in reduction reactions, the accumulation of lactate is increased by 9.3 times compared to control experiments without methanol and NGD, and thus can be used as a means to regulate the metabolic strength of lactate in microorganisms by providing redox.

Example 13: 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 BmMDH-V210S 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 012, adding lactose 15g/L, yeast extract 10g/L, and (NH) 1g/L at pH 4.8 ₄ ) ₂ SO ₄ 3g/L KH ₂ PO ₄ 0.5g/L MgSO ₄ 0.6g/L of CaCl ₂ 0.05g/L of FeSO ₄ ·7H ₂ O, 0.0016g/L MnSO ₄ ·H ₂ O, 0.0014g/L ZnSO ₄ ·7H ₂ O, 0.0037g/L CoCl ₂ ·6H ₂ The engineered bacterium T.reesei GXJ 012 was induced to express the two functional proteins, cultured at 25 deg.C in a shaker at 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 (acetonitrile: water = 4).

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 1.5mM formic acid, 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 is used as a coenzyme for reduction, and that the amount of accumulated lactate is increased 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 14: 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 BmMDH-N123S and the gene coding DLDH-V152R are respectively controlled by a promoter GPD, a promoter PGK, a terminator Hspt and a terminator Tnos, and the two expression cassettes are integrated on a pZPK vector to obtain an engineering plasmid.

Toruloides GXJ 013 was obtained by introducing the above-mentioned engineered plasmid into Rhodosporidium toruloides through ATMT transformation, the above-mentioned two functional proteins were expressed by culturing the engineered strain GXJ 013 in YEPD medium containing 20g/L glucose, 10g/L yeast extract and 20g/L peptone at pH 6.0, culturing the cells in a shaker at 28 ℃ and 200rpm for 48h, centrifuging at 2000 Xg for 6min, collecting the cells, washing the resuspended cells with Tris-Cl at a concentration of 50mM and pH 7.5, and adjusting the cell density 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 (acetonitrile: water = 4).

The analysis by the ion chromatography system according to the method of example 5 revealed that the reaction solution contained 1.2mM formic acid, 3.0mM 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 oxidation of methanol during torula rhodozyma catalysis, and that DLDH-V152R used as a coenzyme for reduction increases the accumulation of lactate by 5-fold compared to control experiments without methanol and NTD, and thus can be used as a means to regulate the metabolic strength of lactate in microorganisms by providing redox.

Claims (8)

1. A method of reducing an NAD analog, comprising: using methanol as a reducing agent, using enzyme of the methanol as a catalyst, reacting for 2-120min at 10-40 ℃ in a buffer system with pH of 5-8, catalytically reducing the NAD analogue to obtain a reduced NAD analogue, and oxidizing the methanol into formaldehyde;

the reduced NAD analogue is one or more than two of NCD, NFCD, NClCD, NBrCD, NMeCD, NGD, NTD and NUD, and the chemical structures of the reduced NAD analogue are as follows:

the enzyme capable of using methanol is derived fromBacillus stearothermophilusIs BsMDH or is derived fromBacillus methanolicusThe mutant of methanol dehydrogenase BmMDH of (1), wherein the methanol dehydrogenase BmMDH is UniProtKB/Swiss-Prot P42327.1, methanol dehydrogenase BmMDH UniProtKB/Swiss-Prot: P31005.3; the mutant is one or more than two of methanol dehydrogenase mutant BsMDH-Y171G, bsMDH-Y171G/A238N, bsMDH-N240A/A243K, bsMDH-K242Y/A243M, bsMDH-I196Q, bsMDH-D195E/A238V, bmMDH-V210S, bmMDH-N123S, bmMDH-V210T/M219K, bmMDH-D212E/M219R, bmMDH-Q217E or BmMDH-D153T/V210S.

2. A method of reducing an NAD analogue according to claim 1, characterized in that: when the NAD analogue is NCD, methanol dehydrogenase mutant capable of utilizing methanol is one or more than two of BsMDH-D195E/A238V, bmMDH-V210T/M219K or BmMDH-Q217E; when the NAD analogue is NFCD, the methanol dehydrogenase mutant capable of utilizing methanol is one or more than two of BmMDH-N123S, bmMDH-V210T/M219K, bmMDH-Q217E or BmMDH-D153T/V210S; when the NAD analogue is NClCD, the methanol dehydrogenase mutant capable of utilizing methanol is one or more than two of BsMDH-D195E/A238V, bmMDH-N123S or BmMDH-Q217E; when the NAD analogue is NBrCD, the methanol dehydrogenase mutant capable of utilizing methanol is one or two of BmMDH-V210S or BmMDH-D212E/M219R; when the NAD analogue is NMeCD, the methanol dehydrogenase mutant capable of utilizing methanol is one or two of BmMDH-N123S or BmMDH-V210T/M219K; when the NAD analogue is NUD, methanol dehydrogenase mutant capable of utilizing methanol is one or more than two of BsmMDH-I196Q, bmmMDH-V210S or BmmMDH-D153T/V210S; when the NAD analogue is NTD, the methanol dehydrogenase mutant capable of utilizing methanol is one or two of BsMDH-D195E/A238V or BmMDH-V210T/M219K; when the NAD analogue is NGD, the methanol dehydrogenase mutant capable of utilizing methanol is one or more of BmMDH-N123S, bmMDH-D212E/M219R or BmMDH-D153T/V210S.

3. A method of reducing an NAD analogue according to claim 1, characterized in that: the reducing agent is one or the combination of two of methanol and deuterated methanol in any ratio.

4. A method of reducing an NAD analogue according to claim 1, characterized in that: the final concentration of the methanol dehydrogenase mutant in the buffer system is 4 mu g/mL-1500 mu g/mL, the final concentration of the NAD analogue is 0.01mM-20mM, and the final concentration of the methanol is 0.4mM-100mM; the buffer system is one or more than two of phosphate buffer, tris-HCl buffer, HEPES buffer, MES buffer and PIPES buffer.

5. A method of reducing an NAD analogue according to claim 1, characterized in that: the obtained reduced NAD analogue can be used as coenzyme by other enzymes for reduction reaction;

the other enzymes are one or more than two of malic enzyme ME-L310R/Q401C for catalyzing reduction of pyruvic acid into malic acid, lactate dehydrogenase DLDH-V152R for catalyzing reduction of pyruvic acid into lactic acid, and saccharomyces cerevisiae alcohol dehydrogenase for catalyzing reduction of acetaldehyde into ethanol.

6. A method of reducing an NAD analogue as claimed in claim 5, wherein: when the reduced NAD analogue is ME-L310R/Q401C, DLDH-V152R or Saccharomyces cerevisiae alcohol dehydrogenase ADH to provide reduced coenzyme, a buffer system with pH of 5-8 is adopted, and the reaction temperature is 10-40 ℃.

7. A method of reducing an NAD analog according to claim 1, characterized in that: the enzyme which can utilize methanol is expressed in the cells of the microorganism, NAD analogue and methanol are transported into the cells, and NAD analogue reduction reaction is carried out in the cells.

8. A method of reducing an NAD analogue according to claim 1 or 7, characterized in that: the methanol dehydrogenase mutant is replaced by a microbial cell with an NAD analogue transporter and expressing the methanol dehydrogenase mutant, and the microbial cell, the NAD analogue and methanol are added into the buffer system to react to generate a reduced NAD analogue; the microbial cells are: one or more of Escherichia coli, lactococcus lactis, saccharomyces cerevisiae, rhodotorula toruloides or Trichoderma reesei.