CN116769748A - 5-aminolevulinic acid synthetase mutant and escherichia coli producing B12 precursor ALA - Google Patents

5-aminolevulinic acid synthetase mutant and escherichia coli producing B12 precursor ALA Download PDF

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CN116769748A
CN116769748A CN202310831702.3A CN202310831702A CN116769748A CN 116769748 A CN116769748 A CN 116769748A CN 202310831702 A CN202310831702 A CN 202310831702A CN 116769748 A CN116769748 A CN 116769748A
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aminolevulinic acid
ala
mutant
escherichia coli
gene
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CN116769748B (en
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夏小乐
刘喜荣
杜爽
蔡永超
冯静
曾春玲
谢斯思
高玲
罗意
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Hunan Xinhexin Biological Medicine Co ltd
Jiangnan University
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Hunan Xinhexin Biological Medicine Co ltd
Jiangnan University
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Abstract

The invention discloses a 5-aminolevulinic acid synthetase mutant and escherichia coli producing a B12 precursor ALA, and belongs to the technical field of biology. The invention modifies key enzyme 5-aminolevulinic acid synthetase for ALA synthesis based on isothermal compression coefficient disturbance engineering of high-pressure molecular dynamics simulation to obtain a mutant with improved specific activity, and then synthesizes ALA by multi-gene combined expression fermentation in escherichia coli, in particular, two key genes succinyl-CoA synthetase gene SUC and polyphosphate kinase gene PPK from escherichia coli MG1655 and key gene 5-aminolevulinic acid synthetase gene mutant from rhodobacter capsulatus are introduced into receptor cells, thereby establishing a synthesis path of ALA in the receptor cells and laying a foundation for developing health-care food rich in vitamin B12.

Description

5-aminolevulinic acid synthetase mutant and escherichia coli producing B12 precursor ALA
Technical Field
The invention relates to a 5-aminolevulinic acid synthetase mutant and escherichia coli producing a B12 precursor ALA, belonging to the technical field of biology.
Background
Vitamin B12, also known as cobalamin, is one of the smallest but structurally complex molecules recognized in the field of bioscience, and is the only water-soluble vitamin containing metal ions, which has wide application in medicine and cosmetics, plays an important cofactor role in fat biochemical reactions, protein synthesis and regulation, and it has now been found that only microorganisms have the ability to synthesize cobalamin through aerobic and anaerobic pathways.
5-aminolevulinic acid (ALA) is an important intermediate for the in vivo synthesis of tetrapyrroles (precursors of vitamin B12, chlorophyll and heme). ALA is synthesized by two routes, namely C4 and C5, wherein the C5 route is mainly found in higher plants, algae and various bacteria, and is obtained by three enzymatic reactions of glutamic acid. The C4 pathway is the condensation of two precursor substances glycine and succinyl-CoA, four carbon compounds, in a 1:1 ratio, under the catalysis of ALA synthase (ALAS, encoded by the hemA gene) to form ALA. The aerobic and anaerobic vitamin B12 synthesis pathway can be divided into four parts: synthesis of ALA, synthesis of corrin ring component, construction of lower axial ligand and synthesis of cobalamin. Industrial microbial fermentation is used as an alternative to the initially established chemical synthesis of vitamin B12, which requires at least 60 steps.
Industrial production of vitamin B12 is mainly carried out by fermentation of microorganisms such as Pseudomonas denitrificans (Pseudomonas denitrificans), propionibacterium freudenreichii (Propionibacterium freichi) and Sinorhizobium meliloti (Sinorhizobium meliloti). In recent years, escherichia coli constructed by synthetic biology and metabolic engineering techniques can be used for producing vitamin B12, and the escherichia coli has the advantages of fast growth, mature genetic operation tools and the like and is widely applied. For example, in 2003, lee et al used hemA from a slow-growing rhizobium sojae to transfer into E.coli BL21 (DE 3), optimizing the C, N ratio in the medium and increasing the yield to 3.8g/L. In 2004, kang et al introduced hemA derived from rhodobacter capsulatus into E.coli BL21 (DE 3), and measured ALA yield of 2.8g/L. In 2008, choi et al transferred hemA gene derived from Rhodopseudomonas palustris KUGB306 into E.coli BL21 (DE 3) via pGEX-KG vector for expression, succinic acid, glycine and glucose were added into the medium, and the ALA yield was 5.2g/L. In 2009, lin et al transferred the hemA gene from Agrobacterium radiobacter zju-0121 into E.coli Rosetta (DE 3) via pET28a vector, and ALA yields as high as 7.3g/L were measured under optimal fermentation conditions. In 2013, zhang et al cloned hemA and hemO genes from Rhodopseudomonas palustris and successfully expressed in E.coli, and measured ALA yields in the fermentation broths were 5.7g/L and 6.3g/L, respectively. In 2014, lou et al were introduced into E.coli Rosetta (DE 3) using hemA from rhodobacter capsulatus, and ALA yield was 8.8g/l measured under optimal culture conditions. However, as shown in patent CN102206606a, the modified escherichia coli is fermented in a 3L fermentation tank for 56 hours, and the final ALA yield only reaches 4.13g/L, so that the effect of the microorganism on synthesizing the vitamin B12 precursor ALA needs to be further improved.
Disclosure of Invention
In order to solve the problems, the invention provides a 5-aminolevulinic acid synthetase mutant with obviously improved specific enzyme activity, and a method for efficiently preparing a precursor substance ALA of vitamin B12 is developed based on the mutant, so that a foundation is laid for developing health-care food rich in vitamin B12.
The first object of the present invention is to provide a 5-aminolevulinic acid synthase mutant obtained by mutating arginine at position 151 of a 5-aminolevulinic acid synthase having an amino acid sequence as shown in SEQ ID NO.1 into isoleucine (R151I).
It is a second object of the present invention to provide a gene encoding the above-mentioned 5-aminolevulinic acid synthase mutant.
A third object of the present invention is to provide a recombinant plasmid carrying the above gene.
It is a fourth object of the present invention to provide host cells expressing the above-described mutants of 5-aminolevulinic acid synthase.
Further, the host cell is a bacterial, fungal, plant cell or animal cell.
It is a fifth object of the present invention to provide the use of the above-described 5-aminolevulinic acid synthase mutants, genes, recombinant plasmids or host cells for the preparation of 5-aminolevulinic acid or vitamin B12.
A sixth object of the present invention is to provide a recombinant E.coli producing 5-aminolevulinic acid, which heterologously expresses the above-mentioned 5-aminolevulinic acid synthase mutant.
Further, the recombinant E.coli also overexpresses succinyl-CoA synthetase and polyphosphate kinase.
Further, the amino acid sequence of the succinyl-CoA synthetase is shown as SEQ ID NO. 2; the amino acid sequence of the polyphosphate kinase is shown as SEQ ID NO. 3.
Furthermore, E.coli BL21 (DE 3) was used as starting strain.
A seventh object of the present invention is to provide the use of the recombinant E.coli described above for the preparation of 5-aminolevulinic acid or vitamin B12.
An eighth object of the present invention is to provide a method for producing 5-aminolevulinic acid, comprising the step of fermentation production using the recombinant E.coli as described above.
Further, glucose is used as a substrate for fermentation production.
The invention has the beneficial effects that:
the invention reforms the key enzyme ALAS (5-aminolevulinic acid synthetase) for ALA synthesis through isothermal compression coefficient disturbance engineering based on high-pressure molecular dynamics simulation, and obtains mutants with improved specific enzyme activity. And then, a polygene combined expression engineering technology is used for introducing a 5-aminolevulinic acid synthesis key enzyme gene into an escherichia coli expression system, and an ALA biosynthesis path is established in a receptor cell body, so that high-level production of ALA is realized, and escherichia coli is easy to realize high-density large-scale culture, thereby greatly reducing the production cost. Finally, the OD600 reaches 23 after fermentation in a 5L fermentation tank for 32 hours, and the ALA yield reaches 28 g.L -1 。
Drawings
FIG. 1 is a schematic representation of the synthesis of 5-aminolevulinic acid via the C4 pathway.
FIG. 2 shows the specific enzyme activities of the wild type and mutant.
FIG. 3 is a graph showing the yield of ALA produced by shake flask fermentation of a wild strain and a mutant strain.
FIG. 4 is a graph showing the yield of ALA produced by fermentation in a 5L fermenter of a wild strain and a mutant strain.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The materials and methods involved in the following examples are as follows:
(1) Sequence:
the wild type amino acid sequence of the 5-aminolevulinic acid synthetase is shown as SEQ ID NO. 1.
The amino acid sequence of succinyl-CoA synthetase is shown as SEQ ID NO. 2.
The amino acid sequence of the polyphosphate kinase is shown as SEQ ID NO. 3.
(2) Culture medium:
seed medium (g.L-1): yeast extract 5.0, peptone 10.0, sodium chloride 5.0, ph7.0.
Fermentation medium (g.L-1): yeast extract 2.0, ammonium sulfate 16.0, potassium dihydrogen phosphate 3.0, disodium hydrogen phosphate dodecahydrate 16.0, magnesium sulfate heptahydrate 1.0, manganese sulfate monohydrate 0.01, mgSO 4 ·7H 2 O and MnSO 4 ·H 2 O was sterilized separately and 0.09M glycine and 0.1M succinic acid were added as reaction substrates. Antibiotics were added according to different resistance requirements at concentrations of 100. Mu.g.mL-1 for ampicillin, 50. Mu.g.mL-1 for kanamycin and 50. Mu.g.mL-1 for spectinomycin.
(3) The detection method of the ALA product comprises the following steps:
1mL of the bacterial liquid is taken and centrifuged for 1min under the condition of 12000 r.min < -1 >, and the extracellular supernatant is taken for ALA concentration measurement. Preparing a reaction system: 400. Mu.L of sodium acetate buffer, 400. Mu.L of supernatant or supernatant diluent and 100. Mu.L of acetylacetone were added in this order, and after thorough mixing, the mixture was reacted in a boiling water bath (100 ℃ C.) for 15 minutes. After taking out, cooling to room temperature, adding 900 mu LModiiedEhrlich's reagent (adding 16mL of 70% perchloric acid into 2g of p-dimethylaminobenzaldehyde, fixing the volume to 100mL with glacial acetic acid), fully mixing for 15min, measuring the absorbance of the reaction solution at 540nm by using an ultraviolet spectrophotometer, and calculating the ALA concentration according to a standard curve.
Example 1
According to the sequence of escherichia coli MG1655 succinyl-CoA synthetase gene and polyphosphate kinase gene ppk published in Genebank, designing proper primers, taking escherichia coli MG1655 genome DNA as a template, and cloning algae haematochrome synthesis key enzyme genes, the gene and the ppk by adopting a genetic engineering method;
and (3) applying a polygene combination expression engineering method, respectively adding BamHI and HindIII enzyme cutting sites at two ends of the ppk gene, and inserting the BamHI and HindIII enzyme cutting sites into a first multicloning site of the pACYC-dure plasmid to obtain a recombinant vector pACYC-dure-ppk. NdeL and XhoI restriction sites are added at two ends of the gene suc respectively, and the gene is cloned into another polyclonal site of pACYC-dur-ppk, so that a recombinant vector pACYC-dur-ppk-suc is obtained.
Example 2
1. Virtual screening of high stability and active mutants
The method comprises the steps of screening out a high-plasticity area which is positioned on the surface of a protein and far away from an active center through isothermal compression coefficient disturbance of high-pressure molecular dynamics simulation (namely, applying gradient pressure to a 5-aminolevulinic acid synthase system, calculating isothermal compression coefficients under different pressures, selecting an area with large isothermal compression coefficient fluctuation as the high-plasticity area under a pressurizing disturbance mode), calculating Dynamic Flexibility Index (DFI) of amino acid on the high-plasticity area of the 5-aminolevulinic acid synthase, screening out the amino acid with the% DFI of more than 0.2, then calculating Dynamic Coupling Index (DCI) of the amino acid with the active center (H138-H143-E184), screening out the amino acid with the DCI of more than 0.8, comparing the amino acid with an amino acid sequence of the 5-aminolevulinic acid synthase reported by Genbank, and mutating the amino acid as a conservative amino acid. 7 mutants K150E, R151I, G192L, R374V, V390F, V390W, L395F were obtained.
2. Construction of Single-Point mutant recombinant plasmid
Primers shown in Table 1 were designed. And carrying out full plasmid PCR amplification by taking plasmid pET-28a-RphemA as an original template, and constructing to obtain a mutant recombinant plasmid. The PCR reaction system was 50. Mu.L: ddH 2 O18. Mu.L; 2xMaxBuffer 25. Mu.L; dNTPMix (10 mM) 1. Mu.L; 1 μl of pET-28 a-RphhemA template; 2. Mu.L of each of the upstream and downstream primers (10 mM); phantaMaxSuper-FidelityDNAPloymerase 1. Mu.L. PCR reaction conditions: 95 ℃ for 30s; 15s at 95 ℃, 15s at 68 ℃, 5min at 72 ℃,30 cycles; stored at 72℃for 5min and at 4 ℃. After the reaction is finished, the PCR product is digested by DpnI enzyme, the digested product is transferred into E.coliJM109, and finally recombinants with correct sequence are amplified and cultured, plasmids are extracted, and transferred into E.coliBL21 (DE 3) and stored at the temperature of minus 20 ℃.
TABLE 1PCR primers
3. Characterization of the enzymatic Properties of the mutants
The 5-aminolevulinic acid synthetase gene is subjected to protein expression by adopting an escherichia coli expression system, wherein a selected plasmid is pET-28a, and escherichia coli E.coli BL21 (DE 3) is taken as a host.
E.coliBL21 (DE 3) transformant carrying mutant recombinant plasmid is selected for small-scale expansion culture, and bacterial liquid PCR and sequencing verification are carried out. After verification, E.coli BL21 (DE 3) of the 9 mutant recombinant plasmids were subjected to expansion culture and induced expression, and after the induction culture was completed, the cells were collected, washed with 20mM phosphate buffer (pH 7.4) for one time to remove the remaining medium as much as possible, resuspended in this buffer, and subjected to cell disruption using an ultrasonic disruption instrument (450 w,5s/5s,25 min). After the crushing, centrifuging the crushed solution at 4 ℃ (10000 r.min < -1 >) for 1h, collecting the supernatant, and filtering the supernatant by a 0.22 mu m microporous filter to obtain crude enzyme solution.
The crude enzyme solution of the 9 mutant 5-aminolevulinic acid synthetases was purified using a nickel ion affinity column (1 mLHistTrapFF) packed with Ni-NTA and an AKTA protein purifier as follows: (1) equilibration column: the column (20 volumes) was equilibrated with ultrapure water and then equilibrated with a final concentration of imidazole of 20mM in the binding solution (20 volumes). (2) sample loading: and (3) adopting a sample injection pump to automatically sample, and loading the crude enzyme liquid at a flow rate of 1 mL/min. (3) elution: washing 10 column volumes with buffer solution with imidazole final concentration of 20mM to remove part of the impurity protein, washing 30 column volumes with eluent with imidazole final concentration of 500mM, collecting the eluted product under the target peak and labeling. (4) column regeneration: due to the loss of nickel ions in the purification process, after the purification is finished, the nickel column is regenerated by the pre-prepared regeneration solution, so that the next use is convenient. (5) And performing SDS-PAGE verification and enzyme activity detection on the purified and collected enzyme solution.
The enzyme activity of the product 5-ALA obtained by the reaction was measured by a chromogenic method. After induction, the thalli are subjected to ultrasonic wall breaking, the wall is placed at 12000rpm for 5min, 1000 mu L of supernatant is taken and 1000 mu L of reaction buffer solution is added: 50mM Tris-HC1, pH=7.5, 0.2mM succinyl CoA,0.1mM pyridoxal phosphate (PLP), were mixed and reacted at 37℃for 20min, respectively. The reaction was stopped by adding 500. Mu.L of 10% trichloroacetic acid at the end of the reaction time. After the reaction was completed, the mixture was centrifuged at 12000rpm for 5 minutes, and the supernatant was transferred to another tube. To the supernatant, 2mL of sodium acetate (pH 4.6) at a concentration of 2mo1/L, 500. Mu.L of acetylacetone was added, followed by mixing, boiling for 15min, and cooling to room temperature. The enzyme activity was calculated by adding 2mL of modified Ehrich's reagent, and measuring the absorbance at 554nm after 15min of reaction at room temperature. Definition of enzyme activity: the amount of enzyme required for converting the substrate to 1nmol of 5-ALA was converted at 37℃and pH7.5 for 1 min. The results of the enzyme activities are shown in FIG. 2.
Example 3
The wild-type recombinant strain constructed in example 2 and 7 mutant recombinant strains were subjected to shake flask fermentation. When ALA is produced by shake flask fermentation, 0.1 mmol.L final concentration is required to be added -1 IPTG induces the expression of exogenous genes, and the shake flask fermentation yields of wild type and mutant are shown in FIG. 3.
Example 5
In order to further evaluate and verify the ALA production capability of the recombinant engineering bacteria (recombinant bacteria containing mutant R151I) with the optimal shake flask fermentation effect, the strain is subjected to amplification culture in a 5L fermentation tank, and cell growth, carbon source consumption and ALA synthesis conditions are analyzed.
In a 5L tank, the concentration of the recombinant strain is greatly improved compared with that of shake flask culture, the OD600 reaches 23 at most, and more and denser thalli are provided for ALA production; with the consumption of glucose as a carbon source and the increase of the bacterial body quantity, the ALA yield steadily increases along with time, reaches the peak of 28 g.L-1 at about 32h, and is improved by about 255.5 percent compared with the yield of a wild strain. The fermentation yield of the wild type and mutant at 32h of fermentation is shown in FIG. 4.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (10)

1. A mutant 5-aminolevulinic acid synthase, characterized by: is obtained by mutating arginine at position 151 of 5-aminolevulinic acid synthetase with an amino acid sequence shown as SEQ ID NO.1 into isoleucine.
2. A gene encoding the 5-aminolevulinic acid synthase mutant according to claim 1.
3. A recombinant plasmid carrying the gene of claim 2.
4. A host cell expressing the 5-aminolevulinic acid synthase mutant of claim 1.
5. Use of a 5-aminolevulinic acid synthase mutant according to claim 1, a gene according to claim 2, a recombinant plasmid according to claim 3 or a host cell according to claim 4 for the preparation of 5-aminolevulinic acid or vitamin B12.
6. A recombinant escherichia coli producing 5-aminolevulinic acid, characterized by: the recombinant escherichia coli heterologously expresses the 5-aminolevulinic acid synthetase mutant as defined in claim 1.
7. The recombinant E.coli according to claim 6, wherein: the recombinant E.coli also overexpresses succinyl-CoA synthetase and polyphosphate kinase.
8. Use of the recombinant escherichia coli of claim 6 or 7 for the preparation of 5-aminolevulinic acid or vitamin B12.
9. A process for producing 5-aminolevulinic acid, characterized by: comprising the step of fermentation production using the recombinant E.coli of claim 6 or 7.
10. The method according to claim 9, wherein: and fermenting and producing by taking glucose as a substrate.
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