阅读说明：本技术 一种生物催化还原丙酮酸产d-乳酸的方法 (Method for producing D-lactic acid by biological catalytic reduction of pyruvic acid ) 是由 赵宗保 刘玉雪 王雪颖 于 2019-04-16 设计创作，主要内容包括：本发明公开了一种生物催化还原丙酮酸产D-乳酸的方法及其应用。该方法中,利用还原型烟酰胺胞嘧啶二核苷酸为还原剂,D-乳酸脱氢酶为催化剂,催化还原丙酮酸产D-乳酸。该方法可以与还原烟酰胺胞嘧啶二核苷酸的方法偶联,通过促进还原剂再生,推动D-乳酸积累。该偶联体系可以用于酶催化和生物转化,还原型烟酰胺胞嘧啶二核苷酸可作为还原剂选择性介导还原丙酮酸产D-乳酸,具有应用潜力和经济价值。(The invention discloses a method for producing D-lactic acid by biologically catalyzing and reducing pyruvic acid and application thereof. In the method, reduced nicotinamide cytosine dinucleotide is used as a reducing agent, D-lactate dehydrogenase is used as a catalyst, and pyruvic acid is catalytically reduced to produce D-lactic acid. The method can be coupled with a method for reducing nicotinamide cytosine dinucleotide, and promotes D-lactic acid accumulation by promoting the regeneration of a reducing agent. The coupling system can be used for enzyme catalysis and biotransformation, and the reduced nicotinamide cytosine dinucleotide can be used as a reducing agent to selectively mediate and reduce pyruvic acid to produce D-lactic acid, so that the coupling system has application potential and economic value.)

1. A method for producing D-lactic acid by biologically catalyzing and reducing pyruvic acid is characterized in that D-lactic acid is synthesized by enzyme catalysis by taking pyruvic acid as a substrate and reduced nicotinamide cytosine dinucleotide as a reducing agent; the enzyme is genetically engineered, namely one or more than two of D-lactate dehydrogenase (DLDH, Gnebank CAA47255) mutant DLDH-V152R/I177K/N213I, DLDH-V152R/N213E or DLDH-V152R/V210N/N213E.

2. The method of claim 1, wherein: the reduced nicotinamide cytosine dinucleotide can be obtained by catalyzing and reducing nicotinamide cytosine dinucleotide through oxidoreductase;

the oxidoreductases include one or more than two of phosphite dehydrogenase, methanol dehydrogenase, formate dehydrogenase and malic enzyme.

3. A method according to claim 1 or 2, characterized in that: the D-lactate dehydrogenase and the redox enzyme for regenerating reduced nicotinamide cytosine dinucleotide are obtained by coding corresponding DNA sequences.

4. A method according to claim 1 or 3, characterized by: the redox enzyme and D-lactate dehydrogenase of the regenerated reduced nicotinamide cytosine dinucleotide, and corresponding coding DNA sequences thereof are cloned in a protein expression vector for controllable expression.

5. The method according to claim 1, 3 or 4, wherein said redox enzyme for regenerating reduced nicotinamide cytosine dinucleotide and D-lactate dehydrogenase are produced by microbial cells carrying expression vectors for the corresponding proteins, and the crude enzyme solution is obtained by purifying the corresponding proteins or lysing the cells and used for in vitro enzymatic synthesis of D-lactate under the conditions: pH3.0-9.0 (preferably pH4-8, more preferably pH7.5), and temperature 20-55 deg.C (preferably 15-40, more preferably 25 deg.C).

6. The method of claim 1, wherein: expressing the proteins of claims 1 and 2 in the microorganism, and simultaneously expressing a mutant NadD-P22G/Y84A/C132D of nicotinamide mononucleotide adenyltransferase from E.coli, constructing an engineering strain for improving the yield of D-lactic acid.

Technical Field

The invention belongs to the technical field of biology, and relates to a method for producing D-lactic acid by reducing pyruvic acid through reduced Nicotinamide Cytosine Dinucleotide (NCDH) driven biocatalysis reduction and application thereof. The method can be coupled with a method for regenerating NCDH, and provides more reducing force to promote the accumulation of D-lactic acid by promoting the regeneration of a reducing agent. The coupled system can be used for enzyme catalysis and whole cell catalysis processes, and NCDH is used as a reducing agent to selectively mediate D-lactate dehydrogenase to catalytically reduce pyruvic acid to produce D-lactate.

Background

Nicotinamide Adenine Dinucleotide (NAD) and its reduced form (NADH) are used as carriers of hydrogen and electrons, and participate in various redox reactions in cells. In a complex metabolic network, cofactor engineering is usually used at the present stage to regulate a target metabolic pathway. Common strategies are 1) altering intracellular coenzyme synthesis, degradation, anabolism and the ability of different coenzyme molecules to interconvert, 2) altering the coenzyme preference of oxidoreductases, 3) expressing enzymes of renewable coenzymes in cells, such as glucose dehydrogenase and the like, and additionally adding substrates for the corresponding enzymes in the culture environment. However, as nad (h) is a common coenzyme, perturbation of intracellular nad (h) levels and different oxidation states often has unpredictable global effects on cell physiology, metabolism, etc. Therefore, in order to realize specific regulation of the target metabolic network, a method is needed to make the target metabolic network independent from the complex metabolic network, i.e. a bioorthogonal system needs to be redesigned (k.short, et al.drug discovery today.2002,7,872).

In a bioarthogonal system that relies on NAD analogs, NAD (h) analogs can be transported in this orthogonal metabolic pathway without affecting other metabolic pathways that utilize NAD (h), and likewise, NAD (h) in other metabolic pathways does not affect the bioorthogonal metabolic pathways. Therefore, the specific metabolic regulation of the bioorthogonal system can be carried out only by regulating the content of the NAD (H) analogue, thereby achieving the aim of improving the yield of the target pathway.

Among the reported NAD analogs, Nicotinamide Cytosine Dinucleotide (NCD) is a NAD analog with better biocompatibility (Ji, D., et al. creation of biological redox systems depending on nicotinic acid flucytosine dinuclotide. journal of the American chemical society.2011,133, 20857; Zhaozen et al, a method for reducing NAD analogs, application No. 201410117146.6). Currently, a method for biosynthesis of NCD has also been reported, in which nicotinamide mononucleotide and cytosine nucleoside triphosphate are used as substrates, and a mutant of nicotinamide mononucleotide adenylyltransferase is used as a catalyst to achieve biosynthesis of NCD at a pure enzyme level and in microbial cells. Several NCD-recognized enzymes have also been reported, such as malic enzyme (ME, Genbank P26616) L310R/Q401C mutant, NADH oxidase (NOX, Genbank S45681), phosphite dehydrogenase psPDH (Genbank069054) L151V/D213Q mutant and rPDH (Genbank AEQ29500) I151R or I151R/E213C mutant, D-lactate dehydrogenase (GnEBank CAA47255) V152R mutant, and formate dehydrogenase (pseFDH, UniProtKB/Swiss-Prot P33160.3) mutant pseeFDH 223S/L257R.

Using NAD analogs and enzymes that recognize them, more cost-effective biocatalytic systems can be constructed (Giidebin et al, catalysis of L-malic acid oxidative decarboxylation by artificial oxidase systems, catalytic journal, 2012,33, 530). For example, the cell lysate of Escherichia coli genetic engineering bacteria over expressing ME-L310R/Q401C and NOX can efficiently and selectively convert malic acid into pyruvic acid in the presence of NAD analogue; in the presence of NAD, pyruvate is further reduced to lactate by endogenous lactate dehydrogenase. Therefore, by selecting proper NAD analogues and recognizing enzymes thereof, a crude enzyme solution can be used for reaction to achieve the effect of pure enzyme catalysis, and a complex biocatalytic conversion system is controlled at the coenzyme level. Currently, regulation of intracellular metabolic reactions using NCD has been achieved by transporting NCD into the cell, producing reduced NCDH via catalytic reaction of rPDH-I151R, ME-L310R/Q401C achieving specific biocatalytic regulation by reducing pyruvate to malate using NCDH (Wang L, et al. synthetic factor-linked metabolic reagents for selective transduction. ACS Catalysis,2017,7, 1977).

D-lactic acid is an industrial chemical with wide application, and can be used for producing polylactic acid as a precursor substance. D-lactic acid can be synthesized by chemical method and biological method, but the chemical method has the problem of optical purity of the target product in the synthesis process, and the biological method can selectively synthesize the D-lactic acid. Biosynthesis of D-lactic acid is carried out mainly by using glucose, xylose or starch as substrates and using microorganisms engineering bacteria for biotransformation (Zhang, Y., et al. biosynthesis of D-lactic acid from lipid microbiological biology. Biotechnology letters, 2018,40, 1167). Although the redox level is unchanged during the conversion of glucose to lactate, NADH produced by the metabolism of glucose to pyruvate is involved in respiration, cell growth and other redox reactions in addition to the reduction of pyruvate to produce lactate. By using NCDH preferential D-lactate dehydrogenase and NCDH as a reducing agent, the reduction reaction from pyruvic acid to D-lactate can be selectively regulated and controlled, the synthesis efficiency and the yield of the D-lactate can be improved, and the application prospect is good.

Based on the background and the advantages, the invention utilizes the directed evolution method to obtain the mutant which takes NCDH as a reducing agent to selectively catalyze pyruvic acid to D-lactic acid.

Disclosure of Invention

The invention relates to a method for producing D-lactic acid by biologically catalyzing and reducing pyruvic acid, in particular to a method for synthesizing D-lactic acid by taking pyruvic acid as a substrate, NCDH as a reducing agent and D-lactic dehydrogenase as a catalyst. NCD can be reduced by chemical means to give NCDH, or by coupling with an NCD-preferred oxidoreductase to reduce NCD to NCDH. Therefore, the method can be applied to the fields of biological catalysis and biological conversion and has important value.

The D-lactate dehydrogenase related to the invention has NCD preference, is obtained by continuous mutation on the basis of a V152R mutant (the mutation site is represented by an amino acid sequence number and amino acid names before and after mutation, for example, V198I indicates that the 198 th amino acid is mutated from V to I, and other sites are similar) from Lactobacillus helveticus D-lactate dehydrogenase (DLDH, NCBI protein database number CAA47255 and contains 1-337 complete amino acid sequences), and comprises DLDH-V152R/V210N/N213E, DLDH-V152R/I177K/N213I and DLDH-V152R/N213E. V152R represents the mutation of amino acid 152 from V to R, and the like.

The reducing agent NCDH related to the invention is obtained by reducing NCD by a chemical method or is generated by reducing NCD by an oxidoreductase with a cofactor preference type, wherein the oxidoreductase comprises but is not limited to malic enzyme mutant ME-L310R/Q401C, phosphorous acid dehydrogenase mutant rPDH-I151R/P176R/M207A, formate dehydrogenase mutant pseFDH-H223S/L257R, and methanol dehydrogenase BmMDH (NCBI protein database number 31005.3) mutant BmMDH-D212E/M219R derived from Bacillus methanolica.

The genes expressing oxidoreductase and D-lactate dehydrogenase for NCDH regeneration of the present invention are constructed on an expression vector and constructed on the vector by the RF cloning method. Expression and purification of the pure enzyme were carried out according to the literature methods for expressing other oxidoreductases in E.coli (Ji DB, et al. creation of biological redox system dependent on a microbial enzyme hydrolysate. journal of the American chemical Society,2011,133,20857). The reaction is carried out in a buffer system, including but not limited to one or more of phosphate buffer, Tris-HCl buffer, HEPES buffer, MES buffer and MOPS buffer.

In the reaction system, a substrate corresponding to oxidoreductase is added during regeneration of NCDH, a substrate corresponding to phosphite dehydrogenase is phosphite, a substrate corresponding to malic enzyme is L-malate, a substrate corresponding to formate dehydrogenase is formate, and a substrate corresponding to methanol dehydrogenase is short-chain alcohol such as methanol or isoamyl alcohol. In the reaction, the reaction substrate for regenerating NCDH was used at a concentration of 1mM-25 mM.

The method of the invention can be carried out in a pure enzyme system or a crude enzyme liquid system. In the pure enzyme reaction system, the oxidoreductase for regenerating NCDH is used at a concentration of 4. mu.g/mL-500. mu.g/mL, and the D-lactate dehydrogenase is used at a concentration of 5. mu.g/mL-1000. mu.g/mL. In a crude enzyme solution reaction system, crude enzyme solution is obtained by cracking and expressing escherichia coli engineering bacteria of oxidoreductase and D-lactate dehydrogenase for reducing NCDH, centrifuging and taking supernatant. In the reaction system, NCD is used at a concentration of 0.01mM-2mM and pyruvic acid is used at a concentration of 0.1mM-20 mM. The reaction conditions are as follows: the pH value is 3.0-9.0, the temperature is 20-55 ℃, and the time is 0.2-30 h.

The NCD can be autonomously synthesized by expressing a mutant NadD-P22G/Y84A/C132D of the nicotinamide mononucleotide adenyltransferase in a microorganism and taking intracellular nicotinamide mononucleotide and cytosine nucleoside triphosphate as substrates. The reaction of NCDH driving D-lactate dehydrogenase to reduce pyruvate to produce D-lactate can be carried out in cells, and the cells of microorganisms include but are not limited to prokaryotic microorganisms such as Escherichia coli, lactococcus lactis and the like or eukaryotic microorganisms such as Saccharomyces cerevisiae and the like.

The invention has the advantages and beneficial effects that: the biocatalytic reduction condition is mild, the reaction efficiency is high, the product purity is high, and when the biocatalytic reduction method is applied to an endosome system, the reduction force can be used for selectively driving the synthesis of pyruvic acid to D-lactic acid, so that the yield and the yield of the D-lactic acid are effectively improved.

Drawings

FIG. 1 is a crystal structure diagram of DLDH-V152R/V210N/N213E-NCD complex;

FIG. 2 is a crystal structure diagram of DLDH-V152R/I177K/N213I-NCD complex;

FIG. 3 is a crystal structure of DLDH-V152R/N213E-NCD complex.

Detailed Description

The invention will be further illustrated by the following examples, which will be more readily understood by reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The NCD used in the present invention was prepared by the NCD reference method (Ji DB, et al. Synthesis of NAD analogstode biochemical system. Sci China Chem,2013,56,296) or by the NCD reference method (Zhao Zongbao et al, method for enzymatically synthesizing nicotinamide cytosine dinucleotide, CN 106884029A) by enzymatic synthesis by expressing a mutant of nicotinamide mononucleotide adenyltransferase derived from E.coli, NadD-P22G/Y84A/C132D.

In the present invention, the method for transformation of Escherichia coli refers to the method for electrotransformation in molecular cloning guide, and the method for transformation of Saccharomyces cerevisiae refers to the method for transformation of lithium acetate in the literature (Gietz, R.D., et al. Nature protocols.2007,2, 31).

In the invention, the detection method of the D-lactic acid comprises ion chromatography, an Agilent ion chromatograph, a chameleon software workstation, an IonPac AS 11-HC anion exchange analytical column (250mm multiplied by 4mm, Dionex) and an IonPac AG 11-HC anion exchange analytical column anion exchange protective column (50mm multiplied by 4mm, Dionex). Flow rate 1mL/min, column temperature: 30 ℃, sample introduction: 25 μ L. The isocratic method comprises the following steps: 10mM NaOH solution, assay 10 min. Concentration of sulfuric acid regeneration liquid: 30mM, nitrogen pressure: 40 psi.

Comparative example 1: reaction for catalytic reduction of pyruvate by D-lactate dehydrogenase without NCDH

1mM NCD was reduced with two equivalents of sodium dithionite, acetone precipitated to give reduced NCDH, which was prepared as a 5mM solution for further use.

1mM NCDH and 2mM pyruvic acid were dissolved in 1mL Tris-HCl buffer solution (50mM, pH7.5), D-lactate dehydrogenase DLDH-V152R/N213E was added to the solution to a final concentration of 50. mu.g/mL, the mixture was mixed well, and reacted at 37 ℃ for 2 hours, 100. mu.L was taken and 900. mu.L of acetonitrile/methanol/water mixture (volume ratio, acetonitrile: methanol: water: 4:1) was added to terminate the reaction.

The concentration of D-lactic acid in the reaction was measured by ion chromatography, and no characteristic peak was observed at 3.7min, and only a characteristic peak of pyruvic acid was detected. Indicating that D-lactate dehydrogenase cannot reduce pyruvate to produce D-lactate without NCDH.

Comparative example 2: reaction for producing D-lactic acid by catalytic reduction of pyruvic acid under enzyme inactivation condition

D-lactate dehydrogenase DLDH-V152R/N213E was heated in a water bath at 80 ℃ for 20min to inactivate the enzyme for use.

1mM NCDH and 2mM pyruvic acid were dissolved in 1mL 50mM Tris-HCl buffer solution, pH7.5, and then added with inactivated D-lactate dehydrogenase to a final concentration of 200. mu.g/mL, and the mixture was mixed well, reacted at 37 ℃ for 2 hours, and then 100. mu.L of the mixture was added with 900. mu.L acetonitrile/methanol/water mixture (volume ratio, acetonitrile: methanol: water: 4:1) to terminate the reaction.

The concentration of D-lactic acid in the reaction was measured by ion chromatography, and no characteristic peak was observed at 3.7min, and only a characteristic peak of pyruvic acid was detected. Indicating that D-lactate dehydrogenase inactivated by heating can not reduce pyruvic acid to produce D-lactic acid under the condition of NCDH.

Comparative example 3: d-lactate dehydrogenase for catalytically reducing pyruvic acid to produce D-lactic acid by using NADH as reducing agent

1mM NADH and 0.5mM pyruvic acid were dissolved in 1mL of 50mM Tris-HCl buffer solution, pH7.5, and then added with D-lactate dehydrogenase DLDH-V152R/N213E to a final concentration of 100. mu.g/mL, and the mixture was mixed well, reacted at 37 ℃ for 2 hours, 100. mu.L was taken, and 900. mu.L of acetonitrile/methanol/water mixture (volume ratio, acetonitrile: methanol: water: 4:1) was added to terminate the reaction.

The concentration of D-lactic acid and pyruvic acid in the reaction was measured by ion chromatography, and D-lactic acid was not detected, only pyruvic acid was detected, and the amount of pyruvic acid was not decreased. Indicating that the NCD-preferred D-lactate dehydrogenase is unable to reduce pyruvate to produce D-lactate in the presence of NADH.