Modification and application of 5-aminolevulinic acid synthetase

Abstract

The invention discloses modification of 5-aminolevulinic acid synthetase and application of the 5-aminolevulinic acid synthetase, and the 5-aminolevulinic acid synthetase has relatively high activity and stability in a 5-ALA synthesis process and can effectively relieve substrate inhibition. According to the invention, the I361 site in 5-ALA synthetase is subjected to saturated mutation modification, and a mutant with enhanced activity and substrate inhibition removal effects is screened out. The mutant enzyme can maintain relatively high catalytic efficiency in the presence of substrate inhibition, so that the synthesis yield of 5-ALA is remarkably increased. The enzyme mutant disclosed by the invention has a wide application prospect in industrial production, is particularly suitable for the production process of large-scale biosynthesis of 5-ALA, can effectively reduce the production cost, improve the product yield and reduce the environmental pollution, and provides technical support for wide application of 5-ALA.

Description

Technical Field

[0001] The present invention relates to the modification of 5-aminolevulinic acid synthase and its application, and belongs to the field of biotechnology. Background Art

[0002] 5-Aminolevulinic acid (5-ALA) is an important biological molecule. As a precursor for the biosynthesis of tetrapyrrole compounds such as heme, chlorophyll and vitamin B12, 5-ALA has important application value and broad application prospects in medicine, health care, animal health and plant nutrition due to its key metabolic nodes in the body and the important physiological functions of its downstream metabolites. As a second-generation photosensitizer, 5-ALA has been used in 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 humans and livestock and poultry, and enhance the vitality and immunity of the body. Due to its biodegradable and non-toxic and residue-free 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. At present, 5-ALA is mainly produced through chemical synthesis. However, the high complexity and high pollution of the chemical synthesis process limit the scale of its industrial production. Currently, no domestic company can even provide products on a scale of 100 kilograms at a time. Multi-step catalytic reactions and low product yields also further increase the production cost of the product, thereby limiting its large-scale application and promotion in various fields.

[0003] There are two 5-ALA biosynthetic pathways in the body, namely the C4 pathway and the C5 pathway, which use succinyl-CoA and glycine and glutamate as substrates, respectively, and synthesize 5-ALA through one-step or three-step enzymatic reactions. The key enzymes in the synthesis pathway have good enzymatic properties and intracellular activity, can quickly catalyze the conversion of substrates into target products, and continuously introduce metabolic flux into the synthesis pathway of products, which is the key to the construction of efficient cell factories. ALAS, the key enzyme of the C4 pathway, is a complex enzyme with a centrosymmetric tetrameric structure. The activity of the enzyme depends on two important cofactors: succinyl-CoA (CoA-Succinyl) and pyridoxal phosphate (PLP). The substrate is glycine, which first binds to PLP to form PLG (phosphoglycine), then binds to succinyl-CoA and finally synthesizes 5-ALA through the catalytic action of the enzyme. ALAS is not only subject to feedback inhibition by heme, but also has the problem of protein instability. Although the protein structure of ALAS has been resolved and reported a long time ago, there are relatively few studies on the modification of key enzymes in the 5-ALA synthesis pathway. Therefore, modifying ALAS, improving the catalytic activity of the enzyme and improving its substrate inhibition effect can provide a feasible solution for the large-scale, low-cost production of 5-ALA and has broad application prospects. Summary of the invention

[0005] In view of the problem of low biosynthetic yield of 5-ALA, the present invention provides a 5-ALA synthase with higher catalytic efficiency and stronger substrate inhibition release ability, thereby improving the synthesis efficiency and production yield of 5-ALA.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The present invention uses alanine scanning technology to evaluate the effect 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 that affect the activity and stability of 5-ALA synthase. Saturation mutation is performed on the key site I361 to screen out mutants with better activity and relief of substrate inhibition effect.

[0008] The present invention uses site-directed mutagenesis technology to design mutation primers, constructs a mutation library through PCR amplification, and replaces the I361 site with another 19 amino acids, namely: I361A to I361Y. The gene of each mutant is cloned into a suitable expression vector and transformed into a host cell, and the recombinant ALAS enzymes of different mutants are obtained by inducing protein expression and purifying.

[0009] The invention verifies through experiments that the ability of the mutant I361V to synthesize 5-ALA is 2.5 times higher than that of the wild type, showing that a relatively high catalytic efficiency is maintained under the condition of substrate inhibition.

[0010] The present invention uses molecular dynamics simulation to analyze the structural changes of the I361V mutant and finds that the loop flexibility of the mutant in the import and export pocket region of succinyl-CoA is increased, which is beneficial to the formation of the substrate channel and the release of the product, thereby improving the turnover number and activity of the enzyme.

[0011] Beneficial effects of the present invention:

[0012] The present invention significantly improves the catalytic activity and substrate inhibition release ability of the enzyme by mutating the I361 site of the 5-ALA synthase. The ability of the variant I361V to synthesize 5-ALA is 2.5 times higher than that of the wild type. These improvements improve production efficiency, shorten production cycles, reduce production costs, and provide an economical and feasible solution for the industrial production of 5-ALA, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the ALAS reaction mechanism;

[0014] Figure 2 It is the enzyme activity map of the screening site.

[0015] Figure 3 This is the PLS fitting situation

[0016] Figure 4 This is the 5-fold cross validation fit

[0017] Figure 5 I361 saturation mutation

[0018] Figure 6 The molecular dynamics simulation results and structure diagram

[0019] Figure 7 Substrate inhibition at different concentrations DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with embodiments and drawings:

[0021] Example 1: Quantitative detection of product 5-ALA

[0022] Take 20 μL of 5-ALA sample, add 200 μL of 1 mol / L sodium acetate buffer solution and 20 μL of acetylacetone. Heat the mixture in a boiling water bath for 15 min to ensure 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 developer. After stabilizing the reaction for 2 min, use a spectrophotometer to measure the absorbance (A554) at a wavelength of 554 nm to quantitatively analyze the content of 5-ALA.

[0023] Example 2: Site screening

[0024] The role of non-conservative amino acid residues near the active site of the ALAS enzyme in the enzyme catalysis process was explored through alanine scanning technology. Non-conservative amino acid residues F375, I361, T83, I86, S277 and R374 in the ALAS enzyme sequence were selected and replaced with alanine through site-directed mutagenesis to evaluate the changes in the enzyme activity of these mutants. The experimental results are as follows Figure 2 , I361V has a significant effect on enzyme activity, providing important information for synthetase modification.

[0025] Example 3: Machine learning assisted mechanism analysis

[0026] Machine learning assisted mechanism analysis

[0027] In this study, PyPEF was used to fit the saturation mutation dataset of I361V ( Figure 3 ). We used PLS as a regression method. Five amino acid indices were directly obtained. We can observe that two key points have a certain influence on the structure. After combining the orthogonal experiment of 5-Fold, we determined that the amino acid indices CHOP780212 and MIYS990101 are relatively accurate in fitting the results ( Figure 4 ).

[0028] Example 4: Saturation mutation

[0029] The present invention selects the I361 site in the ALAS enzyme for saturation mutation, generates a mutant library of the site, and evaluates the effects of different amino acid substitutions on the catalytic activity of the ALAS enzyme through high-throughput screening and functional analysis. Specifically, the I361 site will be replaced with all 20 natural amino acids. Using the method of synthetic 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 Escherichia coli, and the recombinant ALAS enzymes I361A to I361Y of different mutants are obtained by inducing protein expression and purifying. The expression of the mutants was detected by SDS-PAGE to ensure that the mutants were successfully expressed in Escherichia coli and reached a level that can be used for enzyme activity detection. The mutation results are shown as follows. Figure 5 As shown, the data in the figure reflect the activity of each mutant in synthesizing 5-ALA relative to the wild type (WT). The results showed that multiple mutants showed higher activity than the wild type, among which the activity of the I361V mutant was the most significantly improved, and its ability to synthesize 5-ALA was 2.5 times higher than that of the wild type. This discovery is of great significance for understanding the catalytic mechanism of the ALAS enzyme and guiding future enzyme engineering.

[0030] Example 5: I361V structural analysis

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

[0032] After RMSF analysis, I361V has a certain impact on ARG372-PHE375. The loop flexibility of this region increases. This region constitutes the inlet and outlet pocket of succinyl-CoA. The increase in RMSF in this region is conducive to the swing of the loop region and the formation of a larger substrate channel opening. Specifically, the volume of the channel changes from Upgrade to This facilitates the entry and exit of the coenzyme, and the product is more easily released from the channel. Therefore, the turnover number and activity are improved to a certain extent.

[0033] At the same time, the binding energy of the enzyme with succinyl-CoA changes greatly, with the wild type binding energy with succinyl-CoA being -839.884 kJ / mol and the I361C binding energy being -982.948 kJ / mol. The variant provides a tighter binding configuration during the binding of succinyl-CoA to PLG, which promotes the rapid progress of the reaction.

[0034] Example 6: Relationship between wild-type and mutant substrates and enzymatic reaction rates

[0035] like Figure 7 The figure shows the relationship between the enzymatic reaction rate of the wild type (WT) and the mutant (I361V) and the glycine substrate. It can be seen from the figure that at low concentrations of glycine, the activities of WT and I361VV are similar, but as the glycine concentration increases, the activity of WT increases faster. The activity of WT reaches a plateau when the glycine concentration is about 2mM, indicating that the enzyme may have reached a saturated state, that is, all enzyme active sites are occupied by substrates. The activity of I361V increases more slowly when the glycine concentration increases, but it shows better stability and catalytic properties than WT at high substrate concentrations.

Claims

1. 5-aminolevulinic acid synthase modification and application thereof, characterized in that: The influence of non-conservative amino acid residues near the active site of ALAS enzyme on the catalytic activity of ALAS enzyme was evaluated by alanine scanning technology, the key sites affecting the activity and stability of 5-ALA synthetase were determined, and saturation mutation was performed on the key sites. The I361 site in 5-ALA synthetase was transformed by saturation mutation, and mutants with better activity and relief of substrate inhibition effect were screened out. The mutants can maintain a high catalytic efficiency under the condition of substrate inhibition, thereby significantly improving the synthesis yield of 5-ALA.

2. The method according to claim 1, characterized in that The non-conservative amino acid residues replaced by alanine scanning technology are: F375, I361, T83, I86, S277, R374, and the I361 site is determined to be a key site affecting the activity and stability of 5-ALA synthase.

3. The method according to claim 2, characterized in that Through site-directed mutagenesis technology, mutation primers were designed, and a mutation library was constructed by PCR amplification. The gene of each mutant was cloned into the XX expression vector, and the I361 site was replaced with another 19 amino acids, namely: I361A~I361Y.

4. The method according to claim 2 or 3, characterized in that: The mutants obtained by saturation mutagenesis showed that the ability of variant I361V to synthesize 5-ALA was increased by 2.5 times compared with the wild type.

5. A recombinant DNA molecule, characterized in that Contains the coding sequence of the modified enzyme according to claim 3.

6. A host cell, characterized in that The method comprises the recombinant DNA molecule of claim 3 and is capable of expressing the modified enzyme in the production process of 5-ALA.

Images