Modification of 5-amino levulinic acid synthetase and uses thereof

Abstract

The application discloses 5-aminoacetylpropionic acid synthetic enzyme modification and application thereof, which has high activity and stability in the 5-ALA synthesis process, and can effectively remove substrate inhibition. The application carries out saturation mutation modification on I361 site in the 5-ALA synthetic enzyme, and screens out a mutant with enhanced activity and removed substrate inhibition effect. The mutant enzyme can maintain high catalytic efficiency under the condition of substrate inhibition, thereby significantly improving the synthesis yield of 5-ALA. The enzyme mutant of the application has wide application prospect in industrial production, is especially suitable for the production process of large-scale biosynthesis of 5-ALA, can effectively reduce the production cost, improve the product yield, simultaneously reduce environmental pollution, and provides technical support for the wide application of 5-ALA.

Description

TECHNICAL FIELD

[0001] The present application relates to 5-aminolevulinic acid synthetase modification and its application, belonging to the field of biotechnology BACKGROUND

[0002] 5-aminolevulinic acid (5-ALA) is an important biomolecule, as a precursor of heme, chlorophyll and vitamin B12, etc. four porphyrin compounds, due to the key metabolic node in the body and the important physiological function of its downstream metabolites, 5-ALA has important application value and broad application prospect in medicine, health care, animal health and plant nutrition, etc. As the second generation of photosensitizer, 5-ALA has been used for photodynamic therapy of skin diseases and photodynamic diagnosis and auxiliary resection of cancer since the 1990s. Based on the important role of heme and vitamin B12 in animal energy and material metabolism, exogenous supplementation of 5-ALA can promote the metabolism of human body and livestock and poultry, and enhance the vitality and immunity of the body. Because of its biodegradable and non-toxic and non-residual characteristics, 5-ALA is also used as a green and safe plant growth regulator to promote the growth of crops under adverse conditions and fruit coloring, etc. At present, 5-ALA is mainly produced by chemical synthesis method, however, the high complexity and high pollution of chemical synthesis process limit the scale of its industrial production, and multi-step catalytic reaction and low product yield further increase the production cost of the product, thus limiting its large-scale application and promotion in various fields.

[0003] There are two 5-ALA biosynthesis pathways in living organisms, namely C4 pathway and C5 pathway, which take succinyl coenzyme A and glycine, glutamic acid as substrates, respectively, and synthesize 5-ALA through one or three-step enzymatic reaction. The key enzyme of the synthesis pathway has good enzymatic properties and intracellular activity, which can quickly catalyze the substrate to convert into the target product, and continuously introduce the metabolic flow into the synthesis pathway of the product, which is the key to the construction of high-efficiency cell factory. The key enzyme ALAS of C4 pathway is a complex enzyme with a central symmetrical tetramer structure. The activity of the enzyme depends on two important cofactors: succinyl-coenzyme A (CoA-Succinyl) and phosphopyridoxyl (PLP). The substrate is glycine, which first combines with PLP to form PLG (phosphoglycine), then combines with succinyl-coenzyme A and is catalyzed by the enzyme to finally synthesize 5-ALA. ALAS is not only inhibited by heme feedback, but also has the problem of protein instability. Although the protein structure of ALAS has been resolved and reported very early, the research on the modification of the key enzyme of 5-ALA synthesis pathway is relatively less. Therefore, the modification of ALAS to improve the catalytic activity of the enzyme and improve its substrate inhibition effect can provide a feasible solution for large-scale and low-cost production of 5-ALA, and has wide application prospect. SUMMARY

[0004] In order to solve the problem of low biosynthesis yield of 5-ALA, the application provides a 5-ALA synthetase with higher catalytic efficiency and stronger substrate inhibition release capacity, so as to improve the synthesis efficiency and production yield of 5-ALA.

[0005] The technical scheme adopted in the application is as follows:

[0006] The application uses alanine scanning technology to evaluate the influence of non-conserved amino acid residues near the active site of ALAS enzyme on the catalytic activity of ALAS enzyme, and determines the key sites affecting the activity and stability of 5-ALA synthetase.

[0007] The application designs a mutant primer through site-directed mutagenesis technology, constructs a mutant library through PCR amplification, replaces the I361 site with another 19 kinds of amino acids, which are I361A-I361Y respectively, clones the gene of each mutant into a suitable expression vector, and transforms it into a host cell.

[0008] The application verifies through experiments that the ability of mutant I361V to synthesize 5-ALA is 2.5 times higher than that of the wild type, which shows that it maintains high catalytic efficiency under the condition of substrate inhibition.

[0009] The application analyzes the structural changes of I361V mutant through molecular dynamics simulation, finds that the Loop flexibility in the import and export pocket region of succinyl coenzyme A of the mutant is larger, which is beneficial to the formation of substrate channel and the release of product, so as to improve the turnover number and activity of the enzyme.

[0010] The application has the following beneficial effects:

[0011] The application significantly improves the catalytic activity and substrate inhibition release capacity of the enzyme through mutation of the I361 site of 5-ALA synthetase. The ability of variant I361V to synthesize 5-ALA is 2.5 times higher than that of the wild type. These improvements improve the production efficiency, shorten the production cycle, reduce the production cost, and provide an economic and feasible solution for the industrial production of 5-ALA, which has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 is the ALAS reaction mechanism;

[0013] Figure 2 is the enzyme activity graph of the screening site.

[0014] Figure 3 is the PLS fitting condition

[0015] Figure 4 is 5-fold cross-validation fitting case

[0016] Figure 5 is I361 saturation mutation

[0017] Figure 6 is molecular dynamics simulation results and structure diagram

[0018] Figure 7 is substrate inhibition under different concentrations DETAILED DESCRIPTION

[0019] The present application will be described in detail below with reference to examples and drawings:

[0020] Example 1: Quantitative detection of product 5-ALA

[0021] Take 20 μL of 5-ALA sample, add 200 μL of 1 mol / L sodium acetate buffer solution and 20 μL of acetylacetone to it. Heat the mixture in a boiling water bath for 15 min to ensure that the reaction is complete, then cool the reaction solution to room temperature. Take 100 μL of the cooled reaction solution and mix it with 100 μL of Ehrlich's color reagent, and after 2 min of stable reaction, use a spectrophotometer to measure the absorbance (A554) at a wavelength of 554 nm to quantitatively analyze the content of 5-ALA.

[0022] Example 2: Site screening

[0023] Through the alanine scanning technique, the role of non-conserved amino acid residues near the active site of ALAS enzyme in the enzyme catalytic process was explored. Non-conserved amino acid residues in the sequence of ALAS enzyme were selected

[0024] F375, I361, T83, I86, S277 and R374, etc., were replaced by alanine through site-directed mutagenesis, and the changes in enzyme activity of these mutants were evaluated. The experimental results are as follows Figure 2 I361V has a significant impact on enzyme activity, providing important information for the modification of synthetic enzymes.

[0025] Example 3: Machine learning assisted mechanism analysis

[0026] Machine learning assisted mechanism analysis

[0027] In this study, the PyPEF was used to fit the saturation mutation data set of I361V Figure 3). We adopted PLS as the regression method. Five amino acid indices were directly obtained. We can observe two key points to generate a certain impact on the structure. After combining the 5-fold orthogonal experiment, we determine that the fitting of the amino acid indices CHOP780212, MIYS990101 on the results is relatively accurate Figure 4

[0028] Example 4: Saturation mutagenesis

[0029] The I361 site in ALAS enzyme is selected for saturation mutagenesis, and a mutation library of this site is generated. The influence of different amino acid substitutions on the catalytic activity of ALAS enzyme is evaluated through high-throughput screening and functional analysis. Specifically, the I361 site will be replaced by all 20 natural amino acids. Using the method of synthesizing oligonucleotides, mutation primers for the I361 site are designed, and a mutation library is constructed by PCR amplification. The gene of each mutant is cloned into a suitable expression vector. The expression vector containing the mutant gene is transformed into E. coli, and protein expression is induced and purified to obtain recombinant ALAS enzymes I361A~I361Y of different mutants. The expression of the mutants is detected by SDS-PAGE to ensure that the mutants are successfully expressed in E. coli and reach a level that can be used for enzyme activity detection. The mutation results are shown in Figure 5 The data in the figure reflect the activity of each mutant in synthesizing 5-ALA relative to the wild type (WT). The results show that multiple mutants show higher activity than the wild type, among which the I361V mutant has the most significant activity increase, and its ability to synthesize 5-ALA is 2.5 times higher than that of the wild type. This finding is of great significance for understanding the catalytic mechanism of ALAS enzyme and guiding future enzyme engineering.

[0030] Example 5: I361V structure analysis

[0031] Analysis using fast molecular dynamics simulation Figure 6 ) and at selected pH 7.4, with the addition of physiological concentrations of 0.9% NaCl ions, after the steepest descent and simulated annealing minimization to eliminate conflicts, the solute, GAFF2 and AM1BCC for ligands, TIP3P for water, were simulated using the AMBER14 force field. At a temperature of 298K and a pressure of 1atm (NPT ensemble). After checking the change of solute RMSD with simulation time, the 6ns was considered as the equilibrium time and was excluded from further analysis.

[0032] ​I361V has some effect on ARG372-PHE375. The loop flexibility in this region is increased. This region forms the entrance and exit pocket for succinyl-CoA. The increased RMSF in this region allows for more wobble in the loop region, creating a larger opening for the substrate channel. Specifically, the channel volume is increased from 1.5 A3 to 2.1 A3. Increased to This allows for easier entry and exit of coenzyme and easier release of product from the channel. Therefore, the turnover number and activity are increased.

[0033] The binding energy of the enzyme to succinyl-CoA also changes significantly. The wild type has a binding energy of -839.884 kJ / mol to succinyl-CoA, while I361C has a binding energy of -982.948 kJ / mol. The tighter binding of succinyl-CoA to the variant during the binding process promotes a faster reaction.

[0034] Example 6: Relationship between wild type and mutant substrate and enzyme reaction rate

[0035] As shown in Figure 6, the relationship between the enzyme reaction rate and the glycine substrate for the wild type (WT) and the mutant (I361V) is shown. As can be seen from the figure, the activity of WT and I361V are similar at low concentrations of glycine, but as the concentration of glycine increases, the activity of WT increases more rapidly. The activity of WT reaches a plateau at a glycine concentration of about 2 mM, indicating that the enzyme can have reached saturation, i.e., all of the enzyme active sites are occupied by substrate. The activity of I361V increases more slowly as the concentration of glycine increases, but shows better stability and catalytic properties than WT at high substrate concentrations. Figure 7 As shown in Figure 6, the relationship between the enzyme reaction rate and the glycine substrate for the wild type (WT) and the mutant (I361V) is shown. As can be seen from the figure, the activity of WT and I361V are similar at low concentrations of glycine, but as the concentration of glycine increases, the activity of WT increases more rapidly. The activity of WT reaches a plateau at a glycine concentration of about 2 mM, indicating that the enzyme can have reached saturation, i.e., all of the enzyme active sites are occupied by substrate. The activity of I361V increases more slowly as the concentration of glycine increases, but shows better stability and catalytic properties than WT at high substrate concentrations.

Claims

1. A method for modifying 5-aminolevulinic acid synthase, characterized in that, The effects of non-conserved amino acid residues near the active site of ALAS enzyme on the catalytic activity of ALAS enzyme were evaluated by alanine scanning technology. The key site affecting the activity and stability of 5-ALA synthase was identified as the I361 site, and the I361 site was mutated by I361V. The amino acid sequence of the I361V mutant is shown in SEQ ID No.

1.

2. A recombinant DNA molecule, characterized in that, The 5-aminolevulinic acid synthase mutant is coded as shown in SEQ ID No.

1.

3. A host cell, characterized in that, The device comprises the recombinant DNA molecule of claim 2 and is capable of expressing the 5-aminolevulinic acid synthase mutant during the production of 5-ALA, wherein the host cell is a non-plant cell.

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