CN116162640A - Escherichia coli Rosetta strain and application thereof in catalytic synthesis of alpha-arbutin - Google Patents
Escherichia coli Rosetta strain and application thereof in catalytic synthesis of alpha-arbutin Download PDFInfo
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Abstract
The invention discloses an escherichia coli Rosetta strain for biosynthesis of alpha-arbutin, wherein FruA, cscK and Pgi and a sucrose phosphorylase SmsP recombinant gene are inserted into the genome of the escherichia coli Rosetta strain, so that a sucrose phosphorylase SmsP protein product is anchored on the surface of escherichia coli cells. Also discloses the application of the corresponding transposase plasmid and CRISPR plasmid and the strain in the catalytic synthesis of alpha-arbutin. The invention uses escherichia coli Rosetta (DE 3) as a chassis cell, enhances the genes of FruA, cscK and Pgi in fructose metabolic pathway by using CRISPR transposition technology, and enhances the metabolic pathway of fructose which is a reaction accompanying product. The sucrose phosphorylase is anchored on the surface of the escherichia coli by utilizing an enzyme anchoring technology, so that the sucrose phosphorylase can grow on the surface of the escherichia coli and catalyze sucrose and hydroquinone in the external environment to be converted into alpha-arbutin and fructose.
Description
Technical Field
The invention relates to an escherichia coli Rosetta strain and application thereof in catalytic synthesis of alpha-arbutin.
Background
Alpha-arbutin, also called 4-hydroxyphenyl-alpha-D-glucopyranoside, is a class of glycoside compounds, is formed by linking glucosyl and hydroquinone through glycosidic bonds, is a glycoside form of hydroquinone, and is mainly found in plant pericarp and leaf in nature. However, the obtained method is seldom extracted from plants directly, and generally only can transfer reaction by enzyme of different microorganisms, and one molecule of hydroquinone and one molecule of glucose are combined to form single alpha-arbutin. The arbutin has oxidation resistance and strong inhibition effect on tyrosinase, can inhibit melanin generation, can be used as a skin whitening agent in commerce, and is widely applied to the cosmetic industry. The current synthesis methods mainly comprise a chemical synthesis method and a biological conversion method. However, the chemical method for synthesizing the alpha-arbutin has the problems of poor stereoselectivity of the product, severe reaction conditions, production of a large amount of byproducts, environmental pollution and the like. The bioconversion method which is more in line with the development concept of green and environment protection is a hot spot of the current research, and has the advantages of mild reaction conditions, strong reaction specificity, small environmental pollution, high substrate utilization rate, high catalytic efficiency and the like. The bioconversion method is to transfer gene fragments of a key enzyme sucrose phosphorylase (Sucrose Phosphorlase, SP) into escherichia coli cells through a vector to enable the gene fragments to be expressed efficiently through a DNA recombination technology, so that a large amount of sucrose phosphorylase is obtained, the enzyme can form glycosidic bonds in alpha-arbutin by utilizing energy released when glycosidic bonds in sucrose are broken, the catalytic activity of the enzyme is independent of auxiliary factors, and the conversion rate of the alpha-arbutin is high. However, the conventional enzymatic method for catalyzing sucrose and hydroquinone to produce alpha-arbutin and fructose has low reaction efficiency and slow reaction, so that the hydroquinone is easy to oxidize, and the synthesis efficiency and yield of the alpha-arbutin are hindered.
Disclosure of Invention
The invention mainly aims to obtain an escherichia coli Rosetta strain which can be used for efficiently catalyzing the synthesis of alpha-arbutin.
The invention discloses a transposase plasmid, which comprises coding genes of TnsA, tnsB, tnsC, tniQ, cas, cas7 and Cas8 proteins.
Preferably, the sequence is shown as SEQ ID No. 1.
The invention also discloses a CRISPR plasmid which comprises crRNA, LE, RE, fruA, cscK, pgi and a sucrose phosphorylase SmsP recombinant gene.
Preferably, the crRNA targets IS1, IS2, IS5, IS6 and IS8 sites.
Preferably, the sequence is shown in SEQ ID No. 2.
The invention also discloses an escherichia coli Rosetta strain for biosynthesis of alpha-arbutin, wherein FruA, csCk and Pgi and a sucrose phosphorylase SmsP recombinant gene are inserted into the genome of the escherichia coli Rosetta strain, so that a sucrose phosphorylase SmsP protein product is anchored on the surface of escherichia coli cells, and simultaneously, the expression of CsA and CscB genes related to sucrose metabolism in the genome of escherichia coli Rosetta (DE 3) is reduced.
Preferably, the transposase plasmid and the CRISPR plasmid are co-transformed into escherichia coli Rosetta strain.
Preferably, the strain is subjected to mutagenesis and subjected to growth acclimatization under conditions of high concentration sucrose and hydroquinone.
Preferably, the mutagenesis mode is one or a combination of more of plasma mutagenesis, microwave mutagenesis, ionizing radiation mutagenesis, ultraviolet mutagenesis, diethyl sulfate mutagenesis and nitrosoguanidine mutagenesis;
the domestication method is that the strain after mutagenesis is cultivated in a high-concentration sucrose and hydroquinone cultivation environment, the strain with the highest growth speed is selected, then mutagenesis is carried out, the strain is cultivated in the high-concentration sucrose and hydroquinone cultivation environment, and the mutagenesis-cultivation process is repeated until the growth speed of the strain after mutagenesis in the target concentration sucrose and hydroquinone cultivation environment reaches the growth speed of the normal environment before mutagenesis.
The invention also discloses application of the escherichia coli Rosetta strain in catalytic synthesis of alpha-arbutin.
Preferably, the substrates synthesized are sucrose and hydroquinone.
The invention also discloses a sucrose phosphorylase SmsP recombinant protein, and the amino acid sequence of the protein is shown as SEQ ID NO. 5.
The nucleotide sequence of the coding gene of the sucrose phosphorylase SmsP recombinant protein is shown as SEQ ID NO. 6
The invention has the beneficial effects that:
the invention uses escherichia coli Rosetta (DE 3) as chassis cells, and the escherichia coli Rosetta (DE 3) strain is a sucrose metabolism defect strain, so that the metabolic pathway of sucrose as a reaction substrate is reduced. First, using mutagenesis and acclimatization methods, chassis cells are obtained that are tolerant to high concentrations of sucrose and hydroquinone. Meanwhile, the FruA, cscK and Pgi genes in the fructose metabolic pathway are enhanced by using the CRISPR transposition technology, so that the metabolic pathway of the fructose which is a reaction accompanying product is enhanced. The sucrose phosphorylase is anchored on the surface of the escherichia coli by utilizing an enzyme anchoring technology, so that the sucrose phosphorylase can grow on the surface of the escherichia coli and catalyze sucrose and hydroquinone in the external environment to be converted into alpha-arbutin and fructose. The method for synthesizing the alpha-arbutin has the advantages of high catalytic efficiency, small loss, high speed and the like, and is very suitable for the industrialized production of the alpha-arbutin.
Drawings
Fig. 1: the invention modifies the principle schematic diagram of escherichia coli to synthesize alpha-arbutin.
Fig. 2: the efficiency of synthesizing alpha-arbutin by the traditional expression method is compared with that of synthesizing alpha-arbutin by a surface display method.
Fig. 3: and (5) comparing the efficiency of synthesizing the alpha-arbutin before and after domestication.
Fig. 4: schematic representation of transposase plasmid structure.
Fig. 5: schematic of CRISPR plasmid structure.
Fig. 6: the invention is compared with the traditional method (comparative example 1) for synthesizing alpha-arbutin by using escherichia coli.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention.
The materials or instruments used in the following examples, if not specifically described, were available from conventional commercial sources.
As shown in FIG. 1, the principle of the invention is that in the E.coli domesticated strain with high sucrose and hydroquinone concentration, fructose metabolization is over-expressed, and the reaction efficiency is increased. And meanwhile, the sucrose phosphorylase Smsp is anchored on the surface of escherichia coli, so that the stability of the Smsp and the catalysis efficiency of the Smsp on a substrate in a culture medium are improved.
Example 1
Traditional Smsp vector construction: the amino acid sequence of the unmodified Smsp gene is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4. The unmodified Smsp gene was constructed on pET-28a vector, transferred to a strain of ROSETTA (DE 3) (Shanghai Biotechnology Co., ltd., cat. No. EC 1010), plated on a kanamycin plate containing 50mg/l, and incubated overnight at 37 ℃. Selecting a monoclonal, adding 1000ml of liquid culture medium, shaking and culturing at 30 ℃ and 1mM IPTG until the OD600 value reaches 0.6, adding 500g/L sucrose, 70g/L hydroquinone and 0.1g/L iron powder into the mixture, maintaining the pH at 7.0 and at 30 ℃ and 150rpm, and reacting for 24 hours. The HPLC detection of the yield of alpha-arbutin is shown in FIG. 2.
Surface display Smsp vector construction: the amino acid sequence of the recombinant Smsp gene which can be displayed on the surface of the escherichia coli after transformation is shown as SEQ ID NO. 5, and the nucleotide sequence is shown as SEQ ID NO. 6. The recombinant Smsp gene after transformation was constructed on pET-28a vector, transferred to a strain of ROSETTA (DE 3) (Shanghai Biotechnology Co., ltd., cat# EC 1010), spread on kanamycin plate containing 50mg/l, and cultured overnight at 37 ℃. Selecting a monoclonal, adding 1000ml of liquid culture medium, shaking and culturing at 30 ℃ and 1mM IPTG until the OD600 value reaches 0.6, adding 500g/L sucrose, 70g/L hydroquinone and 0.1g/L iron powder into the mixture, maintaining the pH at 7.0 and at 30 ℃ and 150rpm, and reacting for 24 hours. The HPLC detection of the yield of alpha-arbutin is shown in FIG. 2.
Example 2
The activity of the strain is affected by considering that high concentration sucrose and hydroquinone cause larger osmotic pressure and oxidation pressure on E.coli cells. We performed acclimatization of chassis ROSETTA (DE 3) strain for high sucrose and high hydroquinone resistance. After the strain ROSETTA (DE 3) was subjected to mutagenesis in an ARTP mutagenesis instrument for 20s, the strain was spread on a non-antibiotic medium containing 100g/L sucrose and 10g/L hydroquinone, and the strain was placed in a 37℃incubator for cultivation until a monoclonal antibody was grown. Three monoclonal clones with the fastest growth vigor are picked into a non-resistant culture medium, shake-cultured until the OD600 value reaches 0.2, subjected to mutagenesis for 20s in an ARTP mutagenesis instrument, coated on the non-resistant culture medium containing 200g/L sucrose and 20g/L hydroquinone, and placed into a 42 ℃ incubator for culture until the monoclonal clones are grown. Three monoclonal clones with the fastest growth vigor are picked into a non-resistance culture medium containing 200g/L sucrose and 20g/L hydroquinone, shake-cultured until the OD600 value reaches 0.2, subjected to mutagenesis for 20s in an ARTP mutagenesis instrument, coated on the non-resistance culture medium containing 300g/L sucrose and 30g/L hydroquinone, and placed into a 42 ℃ incubator for culture until the monoclonal clones are grown. The mutagenesis culture was continued according to the above conditions, and the concentration of sucrose and hydroquinone was increased for each culture until a concentration of 500g/L sucrose and 70g/L hydroquinone was reached. And taking monoclonal, and preparing the monoclonal into competence by using a competence kit of the biological organism when the monoclonal is cultured in a non-antibiotic culture medium to an OD value of 0.8.
As in example 1, we transformed recombinant Smsp plasmid into competence to catalyze synthesis of α -arbutin. The results are shown in FIG. 3.
Example 3
In order to reduce the use of antibiotics in the process of industrially producing alpha-arbutin, we integrate the recombinant Smsp gene which can be displayed on the surface of escherichia coli after modification on the escherichia coli genome by utilizing CRISPR transposition, and enhance the metabolic pathway of fructose.
Construction of the transposase plasmid: the TnsA, tnsB, tnsC, tniQ, cas, cas7 and Cas8 protein genes are inserted behind the arabinose operon, constituting a transposase plasmid. The schematic diagram is shown in FIG. 4, and the nucleotide sequence is shown in SEQ ID NO. 1. Plasmids were synthesized in Guangzhou Ai Ji organism.
Construction of CRISPR plasmid: the recombinant gene of crRNA, LE, RE, fruA, cscK, pgi and sucrose phosphorylase SmsP IS inserted behind the tetracycline operon, wherein crRNA targets IS1, IS2, IS5, IS6 and IS8 sites to form CRISPR plasmid, the schematic diagram IS shown in figure 5, and the nucleotide sequence IS shown in SEQ ID NO. 2. The amino acid sequence of the modified sucrose phosphorylase SmsP recombinant gene is shown in SEQ ID NO. 5, the nucleotide sequence is shown in SEQ ID NO. 6, and the modified sucrose phosphorylase SmsP can be anchored on the cell surface. Plasmids were synthesized in Guangzhou Ai Ji organism.
The two plasmids were co-transferred to the selected sucrose-resistant hydroquinone-ROSETTA (DE 3) strain, which was plated on streptomycin and kanamycin plates at 50mg/l and incubated overnight at 37 ℃. Selecting monoclonal, adding liquid culture medium containing corresponding antibiotics, shake culturing until OD600 value reaches 0.4, adding 10mM arabinose and 0.1ug/L tetracycline, shake culturing at 25deg.C for 2 days. Streaking was performed on LB plates containing the corresponding antibiotics, and incubated overnight at 37 ℃. Selecting monoclonal, adding liquid culture medium containing corresponding antibiotics, shake culturing until OD600 value reaches 0.8. Selecting monoclonal with correct sequence, fully culturing in a non-resistant culture medium, streaking on the non-resistant culture medium, selecting the monoclonal to culture in the non-resistant culture medium, streptomycin and kanamycin culture medium respectively, and selecting the strain which grows in the non-resistant culture medium and does not grow in the streptomycin and kanamycin culture medium, namely the strain with lost plasmid. 1000ml of liquid culture medium is added, shake culture is carried out at 30 ℃ until the OD600 value reaches 0.6, the final concentration of 500g/L sucrose, 70g/L hydroquinone and 0.1g/L iron powder are added, the pH is maintained at 7.0, the temperature is 30 ℃, and the reaction is carried out at 150rpm for 24 hours. HPLC detects the yield of alpha-arbutin. The result is shown in figure 6, and the modified strain has higher efficiency and yield in catalyzing and synthesizing alpha-arbutin.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (12)
1. A transposase plasmid, characterized by: the transposase plasmid comprises genes encoding TnsA, tnsB, tnsC, tniQ, cas, cas7 and Cas8 proteins.
2. The transposase plasmid of claim 1, wherein: the sequence is shown as SEQ ID No. 1.
3. A CRISPR plasmid, characterized in that said CRISPR plasmid comprises crRNA, LE, RE, fruA, cscK and Pgi and a sucrose phosphorylase SmsP recombinant gene.
4. A CRISPR plasmid according to claim 3, characterized in that its sequence is as shown in SEQ ID No. 2.
5. An escherichia coli Rosetta strain for biosynthesis of alpha-arbutin, characterized in that the recombinant genes of FruA, cscK and Pgi and sucrose phosphorylase SmsP are inserted into the genome of the escherichia coli Rosetta strain, such that the sucrose phosphorylase SmsP protein product is anchored on the surface of the escherichia coli cells.
6. The escherichia coli Rosetta strain according to claim 5, wherein the transposase plasmid according to claim 1 or 2 and the CRISPR plasmid according to claim 3 or 4 are co-transformed into the escherichia coli Rosetta strain.
7. The escherichia coli Rosetta strain according to claim 5 or 6, wherein the strain is subjected to mutagenesis and subjected to growth acclimation treatment under high concentration sucrose and hydroquinone conditions.
8. The escherichia coli Rosetta strain according to claim 7, wherein the mutagenesis is performed by one or more of plasma mutagenesis, microwave mutagenesis, ionizing radiation mutagenesis, ultraviolet mutagenesis, diethyl sulfate mutagenesis and nitrosoguanidine mutagenesis;
the domestication method is that the strain after mutagenesis is cultivated in a high-concentration sucrose and hydroquinone cultivation environment, the strain with the highest growth speed is selected, then mutagenesis is carried out, the strain is cultivated in the high-concentration sucrose and hydroquinone cultivation environment, and the mutagenesis-cultivation process is repeated until the growth speed of the strain after mutagenesis in the target concentration sucrose and hydroquinone cultivation environment reaches the growth speed of the normal environment before mutagenesis.
9. Use of the escherichia coli Rosetta strain according to any one of claims 5-8 for the catalytic synthesis of α -arbutin.
10. Use according to claim 9, characterized in that the substrates synthesized are sucrose and hydroquinone.
11. A sucrose phosphorylase SmsP recombinant protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 5.
12. A recombinant gene of sucrose phosphorylase SmsP is characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO. 6.
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| CN116042684A (en) * | 2023-01-30 | 2023-05-02 | 态创生物科技(广州)有限公司 | Coli and application thereof in catalytic synthesis of psicose |
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| CN116042684A (en) * | 2023-01-30 | 2023-05-02 | 态创生物科技(广州)有限公司 | Coli and application thereof in catalytic synthesis of psicose |
| CN116042684B (en) * | 2023-01-30 | 2023-10-27 | 态创生物科技(广州)有限公司 | Coli and application thereof in catalytic synthesis of psicose |
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