Escherichia coli Rosetta strain and its application in the catalytic synthesis of α-arbutin

Abstract

This invention discloses an *E. coli* Rosetta strain for the biosynthesis of α-arbutin. Recombinant genes for FruA, CscK, Pgi, and sucrose phosphorylase SmsP are inserted into the genome of the *E. coli* Rosetta strain, anchoring the sucrose phosphorylase SmsP protein product to the surface of *E. coli* cells. The invention also discloses the corresponding transposase plasmids and CRISPR plasmids, as well as the application of the strain in the catalytic synthesis of α-arbutin. This invention utilizes *E. coli* Rosetta (DE3) as the chassis cell and uses CRISPR transposition technology to enhance the FruA, CscK, and Pgi genes in the fructose metabolic pathway, thereby enhancing the metabolic pathway of the reaction's accompanying product, fructose. Enzyme anchoring technology is used to anchor sucrose phosphorylase to the surface of *E. coli*, allowing the sucrose phosphorylase to grow on the *E. coli* surface and catalyze the conversion of sucrose and hydroquinone from the external environment into α-arbutin and fructose.

Description

Technical Field

[0001] This invention relates to an Escherichia coli Rosetta strain and its application in the catalytic synthesis of α-arbutin. Background Technology

[0002] α-Arbutin, also known as 4-hydroxyphenyl-α-D-glucopyranoside, is a glycoside compound composed of a glucose unit and hydroquinone linked by a glycosidic bond. It is the glycoside form of hydroquinone and is mainly found in plant peels and leaves in nature. However, it is rarely obtained directly from plants; it is generally obtained through enzymatic transfer reactions using different microorganisms, combining one molecule of hydroquinone with one molecule of glucose to form a single α-arbutin. Arbutin has antioxidant properties and a strong inhibitory effect on tyrosinase, which can inhibit melanin production. Commercially, it is used as a skin whitening agent and is widely used in the cosmetics industry. Currently, the main synthetic methods are chemical synthesis and biotransformation. However, chemical synthesis of α-arbutin suffers from poor stereoselectivity, harsh reaction conditions, the generation of numerous byproducts, and environmental pollution. Biotransformation, which aligns better with green and environmentally friendly development principles, is currently a research hotspot. Biotransformation offers advantages such as mild reaction conditions, high reaction specificity, low environmental pollution, high substrate utilization, and high catalytic efficiency. Biotransformation is a method that uses DNA recombination technology to transfer a gene fragment of the key enzyme sucrose phosphorylase (SP) into *E. coli* cells via a vector, enabling its efficient expression and yielding large quantities of sucrose phosphorylase. This enzyme utilizes the energy released when glycosidic bonds in sucrose break to form glycosidic bonds in α-arbutin, and its catalytic activity is independent of cofactors, resulting in a high conversion rate of α-arbutin. However, commonly used enzymatic methods for catalyzing the reaction between sucrose and hydroquinone to produce α-arbutin and fructose are inefficient and slow, making hydroquinone prone to oxidation and hindering the synthesis efficiency and yield of α-arbutin. Summary of the Invention

[0003] The main objective of this invention is to obtain an Escherichia coli Rosetta strain that can be used to efficiently catalyze the synthesis of α-arbutin.

[0004] This invention discloses a transposase plasmid containing the encoding genes for TnsA, TnsB, TnsC, TniQ, Cas6, Cas7 and Cas8 proteins.

[0005] Preferably, its sequence is as shown in SEQ ID: No.1.

[0006] The present invention also discloses a CRISPR plasmid containing crRNA, LE, RE, FruA, CscK, Pgi, and the sucrose phosphorylase SmsP recombinant gene.

[0007] Preferably, the crRNA targets the IS1, IS2, IS5, IS6 and IS8 sites.

[0008] Preferably, its sequence is as shown in SEQ ID: No.2.

[0009] The present invention also discloses an Escherichia coli Rosetta strain for α-arbutin biosynthesis, wherein recombinant genes FruA, CscK, Pgi and sucrose phosphorylase SmsP are inserted into the genome of the aforementioned Escherichia coli Rosetta strain, so that the sucrose phosphorylase SmsP protein product is anchored on the surface of Escherichia coli cells, while reducing the expression of CscA and CscB genes related to sucrose metabolism in the genome of Escherichia coli ROSETTA(DE3).

[0010] Preferably, the transposase plasmid and the CRISPR plasmid are co-transfected into Escherichia coli Rosetta strain.

[0011] Preferably, the strain is first subjected to mutagenesis and then subjected to growth acclimatization treatment under conditions of high concentrations of sucrose and hydroquinone.

[0012] Preferably, the mutagenesis method is one or a combination of plasma mutagenesis, microwave mutagenesis, ionizing radiation mutagenesis, ultraviolet mutagenesis, diethyl sulfate mutagenesis, and nitrosoguanidine mutagenesis;

[0013] The domestication method involves culturing the mutated strains in a culture environment with higher concentrations of sucrose and hydroquinone, selecting the strain with the fastest growth rate, mutating it again, and then culturing it in a culture environment with higher concentrations of sucrose and hydroquinone. This mutagenesis-culturing process is repeated until the growth rate of the mutated strains in a culture environment with the target concentration of sucrose and hydroquinone reaches the growth rate of the strains in the normal environment before mutagenesis.

[0014] The present invention also discloses the application of the aforementioned Escherichia coli Rosetta strain in the catalytic synthesis of α-arbutin.

[0015] Preferably, the substrates for synthesis are sucrose and hydroquinone.

[0016] The present invention also discloses a recombinant protein of sucrose phosphorylase SmsP, the amino acid sequence of which is shown in SEQ ID NO:6.

[0017] The nucleotide sequence of the gene encoding the above-mentioned sucrose phosphorylase SmsP recombinant protein is shown in SEQ ID NO:5.

[0018] The beneficial effects of this invention are:

[0019] This invention utilizes *Escherichia coli* Rosetta (DE3) as chassis cells. First, chassis cells capable of tolerating high concentrations of sucrose and hydroquinone are obtained through mutagenesis and domestication. Simultaneously, CRISPR transposon technology is used to enhance the FruA, CscK, and Pgi genes in the fructose metabolic pathway, thereby enhancing the metabolic pathway of the reaction byproduct fructose. Enzyme anchoring technology is used to anchor sucrose phosphorylase on the surface of *E. coli*, allowing the sucrose phosphorylase to grow on the *E. coli* surface and catalyze the conversion of sucrose and hydroquinone from the external environment into α-arbutin and fructose. This method for synthesizing α-arbutin has advantages such as high catalytic efficiency, low loss, and fast speed, making it highly suitable for the industrial production of α-arbutin. Attached Figure Description

[0020] Figure 1: Schematic diagram of the principle of synthesizing α-arbutin by modifying Escherichia coli according to the present invention.

[0021] Figure 2: Comparison of the efficiency of α-arbutin synthesis between traditional expression method and surface display method.

[0022] Figure 3: Comparison of α-arbutin synthesis efficiency before and after domestication.

[0023] Figure 4: Schematic diagram of transposase plasmid structure.

[0024] Figure 5: Schematic diagram of CRISPR plasmid structure.

[0025] Figure 6: Comparison of the production capacity of α-arbutin synthesized using Escherichia coli in this invention and the traditional method (Comparative Example 1). Detailed Implementation

[0026] The present invention will be further described below with reference to the embodiments, but the description of the embodiments does not limit the scope of protection of the present invention in any way.

[0027] Unless otherwise defined, 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 pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0028] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.

[0029] like Figure 1As shown, the principle of this invention is to overexpress fructose metabolism in E. coli strains with high sucrose and hydroquinone tolerance, thereby increasing reaction efficiency. Simultaneously, sucrose phosphorylase Smsp is anchored on the surface of E. coli, improving Smsp's stability and catalytic efficiency against substrates in the culture medium.

[0030] Example 1

[0031] Construction of the traditional Smsp vector: The amino acid sequence of the unmodified Smsp gene is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4. The unmodified Smsp gene was constructed into the pET-28a vector, transferred to ROSETTA(DE3) strain (Shanghai Weidi Biotechnology Co., Ltd., catalog number EC1010), and plated on a plate containing 50 mg / L kanamycin. The plate was incubated overnight at 37°C. Single colonies were picked, and 1000 ml of liquid medium was added. The culture was carried out at 30°C with shaking and 1 mM IPTG until the OD600 value reached 0.6. Then, 500 g / L sucrose, 70 g / L hydroquinone, and 0.1 g / L iron powder were added to a final concentration. The pH was maintained at 7.0, and the reaction was carried out at 30°C and 150 rpm for 24 h. The yield of α-arbutin was detected by HPLC, and the results are shown below. Figure 2 As shown.

[0032] Construction of the surface-displaying Smsp vector: The amino acid sequence of the modified recombinant Smsp gene that can be displayed on the surface of *E. coli* is shown in SEQ ID NO:5, and the nucleotide sequence is shown in SEQ ID NO:6. The modified recombinant Smsp gene was constructed into the pET-28a vector, transferred to *ROSETTA(DE3)* strain (Shanghai Weidi Biotechnology Co., Ltd., catalog number EC1010), and plated on a plate containing 50 mg / L kanamycin. The plate was incubated overnight at 37°C. Single colonies were picked, and 1000 ml of liquid medium was added. The culture was incubated at 30°C with shaking at 1 mM IPTG until the OD600 value reached 0.6. Then, 500 g / L sucrose, 70 g / L hydroquinone, and 0.1 g / L iron powder were added to a final concentration. The pH was maintained at 7.0, and the reaction was carried out at 30°C and 150 rpm for 24 h. The yield of α-arbutin was detected by HPLC, and the results are shown below. Figure 2 As shown.

[0033] Example 2

[0034] Considering that high concentrations of sucrose and hydroquinone exert significant osmotic and oxidative stress on *E. coli* cells, affecting strain activity, we acclimated the chassis ROSETTA (DE3) strain to high sucrose and hydroquinone levels. After mutagenesis for 20 seconds in an ARTP mutagen, the ROSETTA (DE3) strain was spread onto antibiotic-free medium containing 100 g / L sucrose and 10 g / L hydroquinone, and incubated at 37°C until single colonies emerged. The three fastest-growing single colonies were selected and transferred to antibiotic-free medium, shaken until the OD600 reached 0.2, then mutagenesis was performed again in an ARTP mutagen for 20 seconds. Finally, the colonies were spread onto antibiotic-free medium containing 200 g / L sucrose and 20 g / L hydroquinone, and incubated at 42°C until single colonies emerged. The three fastest-growing monoclonal clones were selected and placed in antibiotic-free medium containing 200 g / L sucrose and 20 g / L hydroquinone. After shaking and incubation until the OD600 value reached 0.2, they were mutagenized for 20 s in an ARTP mutagenizer. Then, they were spread onto antibiotic-free medium containing 300 g / L sucrose and 30 g / L hydroquinone and incubated at 42°C until monoclonal clones emerged. Mutagenesis was continued under the same conditions, with the concentrations of sucrose and hydroquinone increased each time, until a concentration of 500 g / L sucrose and 70 g / L hydroquinone was reached. Monoclonal clones were then cultured in antibiotic-free medium until the OD value reached 0.8, and then prepared into competent cells using a competent cell kit from Sangon Biotech.

[0035] Similar to Example 1, we transferred the modified recombinant Smsp plasmid into competent cells to catalyze the synthesis of α-arbutin. The results are shown in [Figure 1]. Figure 3 .

[0036] Example 3

[0037] To reduce antibiotic use during the industrial production of α-arbutin, we used CRISPR transposition to integrate a modified recombinant Smsp gene, which can be displayed on the surface of E. coli, into the E. coli genome and enhance the fructose metabolic pathway.

[0038] Construction of transposase plasmids: The TnsA, TnsB, TnsC, TniQ, Cas6, Cas7, and Cas8 protein genes were inserted after the arabinose operon to form transposase plasmids. See schematic diagram below. Figure 4 The nucleotide sequence is shown in SEQ ID NO:1. The plasmid was synthesized by Guangzhou Aiji Biotechnology.

[0039] Construction of CRISPR plasmids: The recombinant genes crRNA, LE, RE, FruA, CscK, Pgi, and sucrose phosphorylase SmsP are inserted after the tetracycline operon. The crRNA targets the IS1, IS2, IS5, IS6, and IS8 sites to form the CRISPR plasmid. See the schematic diagram below. Figure 5The 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:6, and the nucleotide sequence is shown in SEQ ID NO:5. The modified sucrose phosphorylase SmsP can anchor to the cell surface. The plasmid was synthesized by Guangzhou Aiji Biotechnology.

[0040] Two plasmids were co-transformed into the selected sucrose-tolerant hydroquinone strain ROSETTA(DE3), and plated on plates containing 50 mg / L streptomycin and kanamycin, and incubated overnight at 37°C. Single colonies were picked, and liquid medium containing the corresponding antibiotics was added. The culture was shaken until the OD600 value reached 0.4, then 10 mM arabinose and 0.1 μg / L tetracycline were added, and the culture was incubated at 25°C with shaking for 2 days. LB agar plates containing the corresponding antibiotics were streaked and incubated overnight at 37°C. Single colonies were picked, and liquid medium containing the corresponding antibiotics was added. The culture was shaken until the OD600 value reached 0.8. Single colonies with correct sequencing were picked, and after sufficient culture in antibiotic-free medium, they were streaked on antibiotic-free medium. Single colonies were then cultured in antibiotic-free, streptomycin, and kanamycin media respectively. Strains that grew in antibiotic-free medium but did not grow in streptomycin and kanamycin media were identified as strains with plasmid loss. Add 1000 ml of liquid culture medium and incubate at 30°C with shaking until the OD600 value reaches 0.6. Then add 500 g / L sucrose, 70 g / L hydroquinone, and 0.1 g / L iron powder to a final concentration, maintaining the pH at 7.0, and react at 30°C and 150 rpm for 24 h. The yield of α-arbutin was determined by HPLC. Results are shown below. Figure 6 The modified strain exhibits higher efficiency and yield in the catalytic synthesis of α-arbutin.

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An *Escherichia coli* Rosetta strain for the biosynthesis of α-arbutin, characterized in that, The recombinant genes FruA, CscK, Pgi, and sucrose phosphorylase SmsP were inserted into the genome of *E. coli* strain Rosetta, causing the sucrose phosphorylase SmsP protein product to anchor on the surface of *E. coli* cells. The transposase plasmid and CRISPR plasmid were co-transfected into *E. coli* strain Rosetta, wherein the sequence of the transposase plasmid is shown in SEQ ID No. 1, and the sequence of the CRISPR plasmid is shown in SEQ ID No.

2.

2. The application of the Escherichia coli Rosetta strain according to claim 1 in the catalytic synthesis of α-arbutin.

3. The application according to claim 2, characterized in that... The substrates for synthesis were sucrose and hydroquinone.

Images