CN116064344B - Construction method and application of recombinant escherichia coli for producing hydroxytyrosol by fermentation - Google Patents

Abstract

The invention relates to a technical improvement of biosynthesis of hydroxytyrosol, in particular to a construction method and application of recombinant escherichia coli for producing hydroxytyrosol by fermentation, and a way for synthesizing hydroxytyrosol from a simple carbon source is realized; culturing recombinant E.coli BLAH carrying plasmid pRSFDuet-HpaBC-LAAD-ARO10-ADH and plasmid pETDuet-AROG-TYRA and recombinant E.coli BLCK carrying plasmids pRSFDuet-1 and pETDuet-1 in LB medium; inoculating the strain into TB culture solution according to the inoculation amount of 0.5-2%, performing expansion culture, adding 0.1-0.3 mM inducer for induction culture, continuously culturing at 30 ℃ and 220r/min for product accumulation, and detecting the hydroxytyrosol yield by liquid chromatography every 8 hours. As the simplest model organism, the background is clear, the growth is quick, the large-scale fermentation culture is easy, and the cost is low; and the key genes in the endogenous synthesis tyrosine pathway of the escherichia coli are over-expressed, and the heterologous genes for synthesizing the hydroxytyrosol from the tyrosine are introduced into the escherichia coli host for accumulating the tyrosine, so that the pathway for synthesizing the hydroxytyrosol from a simple carbon source is constructed.

Description

Construction method and application of recombinant escherichia coli for producing hydroxytyrosol by fermentation

Technical Field

The invention relates to a technical improvement of biosynthesis of hydroxytyrosol, in particular to a construction method and application of recombinant escherichia coli for producing hydroxytyrosol by fermentation.

Background

Hydroxytyrosol (Hydroxytyrosol, HT), also known as 3, 4-dihydroxyphenethyl alcohol (3, 4-Dihydroxyphenylethanol), has a molecular weight of 154.164 and a molecular formula of C ₈H₁₀O₃, is a natural polyphenol compound, and has various biological and pharmacological activities. The hydroxytyrosol has strong antioxidant effect, and can transfer the hydrogen of the phenolic hydroxyl to active oxygen, so that the damage of hydrogen peroxide in human red blood cells to DNA and cell membranes can be protected, free radicals generated under ultraviolet irradiation can be removed, and the photodamage of skin can be protected. Hydroxytyrosol has antiinflammatory activity, can reduce gene expression related to inflammation, has antitumor effect, can promote apoptosis and antibacterial activity of cancer cells by activating molecular signal transduction, has effect of inhibiting microbial activity, and has antibacterial effect on various strains in intestinal tract. Therefore, hydroxytyrosol has wide application in cosmetics, pharmaceutical industry and health food. Currently commercialized hydroxytyrosol is mainly extracted from natural plants or obtained by chemical synthesis. In nature, hydroxytyrosol is present in large amounts in olive leaves and fruits, and can be obtained by hydrolysis of oleuropein and extraction from olive oil or waste water from the olive oil production process.

Despite the abundance and low cost of olive related sources of raw materials, the method of extraction from plants still has the following drawbacks: the operation process is complex, the recovery rate is low, the energy consumption is high, the strong acidity is realized, and the time is long. The hydroxytyrosol can also be synthesized by a chemical method, but the method has the problems of higher substrate cost, complex process, severe conditions, environmental pollution, solvent residues and the like, and is difficult to produce in batches. Therefore, the hydroxyl tyrosol with high added value is efficiently produced by using cheap raw materials through a biosynthesis way, which is beneficial to promoting the industrialized development of the hydroxyl tyrosol.

The biosynthesis of hydroxytyrosol has been studied: santos et al used lactic acid bacteria to degrade oleuropein to synthesize hydroxytyrosol, tested the degradation efficiency of six bacteria on oleuropein under aerobic and anaerobic conditions, and found that the most effective strain was Lactobacillus plantarum 6907, the degradation rate on oleuropein can reach 90%, but the conversion rate of oleuropein into hydroxytyrosol is only 30%; allouche and the like utilize serratia marcescens and pseudomonas aeruginosa to synthesize hydroxytyrosol through tyrosol isomers, calcium alginate hydrogel is used as a carrier, a microorganism solid phase loading technology is applied to carry out immobilization on serratia marcescens and pseudomonas aeruginosa, and then the two immobilized cells catalyze tyrosol to generate hydroxytyrosol, and the result shows that the carrier can use 4g/L tyrosol solution as a substrate to generate hydroxytyrosol, and the product conversion rate reaches 82%; brooks et al use Pseudomonas putida expressing tyrosinase activity to catalyze tyrosol to synthesize hydroxytyrosol, and the yield reaches 77%; bouallagui and the like utilize a microorganism solid phase loading technology to fixedly load the resting cells of the pseudomonas aeruginosa in the calcium alginate beads on the calcium alginate hydrogel, 5g/L tyrosol can be used as a substrate to generate hydroxytyrosol, and the product conversion rate reaches 86 percent. In the research of the biosynthesis of hydroxytyrosol, tyrosol is used as a substrate, so that the cost is high, and mass production is difficult. Satoh et al studied the way of biosynthesis of hydroxytyrosol using L-tyrosine as substrate, tyrosine was hydroxylated by mouse-derived tyrosine hydroxylase in the presence of cofactor tetrahydrobrowning protein, but tetrahydrobrowning protein is not currently available commercially, thus severely limiting the biocatalytic efficiency of key enzymes in this way, and studied the production of hydroxytyrosol using glucose as a raw material, with a final hydroxytyrosol content of 12.3mg/L. Li et al synthesized intermediate tyrosol with L-tyrosine as substrate, and then further synthesized hydroxytyrosol with a yield close to 50%. Chen et al studied two mixed pathways for producing hydroxytyrosol using tyrosine as substrate, the synthesis capacity was significantly improved when both pathways acted simultaneously compared to each individual pathway, and the final hydroxytyrosol yield was 1.89g/L by fed-batch fermentation.

Therefore, it can be seen from the above that the prior art method for biosynthesis of hydroxytyrosol is feasible, but how to realize high yield of hydroxytyrosol in the biosynthesis mode is still a technical problem to be solved.

Disclosure of Invention

The invention aims to overcome the existing defects, and provides a construction method and application of recombinant escherichia coli for producing hydroxytyrosol by fermentation, so as to realize a way of synthesizing hydroxytyrosol from a simple carbon source.

In order to solve the technical problems, the invention provides the following technical scheme: the construction method of recombinant escherichia coli for producing hydroxytyrosol by fermentation comprises the following steps of firstly, obtaining HpaBC and LAAD genes and linearization pRSFDuet-1 vector fragments by PCR amplification, and fusing the fragments by homologous recombination to obtain plasmid pRSFDuet-HpaBC-LAAD; PCR amplification to obtain ARO10, ADH gene and pRSFDuet-HpaBC-LAAD vector fragment, and fusion of these fragments by homologous recombination to obtain plasmid pRSFDuet-HpaBC-LAAD-ARO10-ADH; PCR amplification to obtain AROG and TYRA genes and linearized pETDuet-1 vector fragments, and fusing the fragments through homologous recombination to obtain plasmid pETDuet-AROG-TYRA;

Step two, the plasmid pRSFDuet-HpaBC-LAAD-ARO10-ADH and the plasmid pETDuet-AROG-TYRA are jointly transferred into competent cells of the escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli BLAH, and the plasmid pRSFDuet-1 and pETDuet-1 are jointly transferred into competent cells of the escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli BLCK.

In order to solve the technical problems, the invention provides the following technical scheme: the use of recombinant E.coli for fermentative production of hydroxytyrosol, the recombinant E.coli according to claim 1 being used for fermentative production of hydroxytyrosol.

Preferably, 6 enzymes are expressed by recombinant E.coli: hydroxylase, L-alpha-amino acid deaminase, alpha-keto acid decarboxylase, alcohol dehydrogenase, 2-dehydro-3-deoxyphosphoheptylaldehyde aldolase and chorismate mutase/benzoate dehydrogenase.

Preferably, the recombinant E.coli production method comprises the following steps,

Step one, culturing recombinant escherichia coli BLAH carrying plasmids pRSFDuet-HpaBC-LAAD-ARO10-ADH and pETDuet-AROG-TYRA and recombinant escherichia coli BLCK carrying plasmids pRSFDuet-1 and pETDuet-1 in an LB culture medium;

And step two, inoculating the strain into TB culture solution according to the inoculum size of 0.5-2% for expansion culture, then adding an inducer of 0.1-0.3 mM for induction culture, continuously culturing at 30 ℃ and 220r/min for product accumulation, and detecting the hydroxytyrosol yield by liquid chromatography every 8 hours.

The invention has the beneficial effects that: the escherichia coli is used as a zymogen for producing hydroxytyrosol by fermentation and is used as the simplest model organism, the background is clear, the growth is quick, the large-scale fermentation culture is easy, and the cost is low; and the key genes in the endogenous synthesis tyrosine pathway of the escherichia coli are over-expressed, and the heterologous genes for synthesizing the hydroxytyrosol from the tyrosine are introduced into the host of the escherichia coli for accumulating the tyrosine, so that the pathway for synthesizing the hydroxytyrosol from a simple carbon source is constructed, and the amplified production is carried out at the level of a 5-L fermentation tank, thereby realizing the de-novo synthesis of the hydroxytyrosol in the escherichia coli, remarkably improving the yield of the hydroxytyrosol and laying a foundation for the large-scale industrial production and application of the hydroxytyrosol.

Drawings

FIG. 1 is a diagram of the enzymatic synthesis of hydroxytyrosol pathway for HpaBC, LAAD, ARO, ADH, AROG and TYRC according to an embodiment of the present invention;

FIG. 2 is a graph showing the time profile of production of hydroxytyrosol by recombinant E.coli strain BLAH in a 5-L fermenter according to an example of the invention;

FIG. 3 is a HPLC verification chart of a catalytic reaction of recombinant E.coli strain BLAH with glucose as substrate for 48h according to an example of the present invention.

Detailed Description

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market; the related experiments are conventional experiments unless otherwise specified.

The sources of the materials are as follows: coli strains BL21 (DE 3) and JM109 are commercially available, E.coli strain BL21 (DE 3) is used for expression of all genes in the present invention, JM109 is used for vector construction. Coli expression vectors pETDuet-1, pRSFDuet-1 were derived from Novagen, PRIMERSTAR high-fidelity DNA polymerase, restriction enzymes, and E.coli competent preparation kit purchased from Bao Ri doctor Material technology (Beijing) Co. Plasmid extraction kit, DNA purification kit and gel recovery kit, ampicillin, kanamycin, isopropyl-beta-D-thiogalactoside (IPTG) were purchased from Shanghai bioengineering Co., ltd. Yeast powder, peptone in this study were purchased from Oxoid Co., ltd. All chemical reagents were purchased from the national drug group and were all analytically pure and above.

The LB medium consists of: 10g/L peptone, 5g/L yeast powder, 5g/L NaCl, and the balance of double distilled water, and sterilizing at 121 ℃ for 20min.

The composition of the TB medium is: 12g/L peptone, 24g/L yeast powder, 2.31g/L KH2PO4, 12.54g/L K2HPO4, 0.4% glycerol, and the balance double distilled water, and sterilizing the culture medium at 121deg.C for 20min.

Example 1 construction of recombinant E.coli BLAH and BLCK;

1. Acquisition of the target gene: the target gene sequence is shown in SEQ ID NO 1-SEQ ID NO 7, and gene synthesis is carried out by Tianlin biotechnology (Shanghai) limited company; the target gene is amplified by PRIMESTAR DNA Polymerase high-fidelity enzyme, the PCR system is a 50 μl standard system, and the amplification conditions are as follows: pre-denaturing at 95 ℃ for 30s, annealing at 58 ℃ for 45s, extending at 72 ℃ for 1min for 30s, performing 25 cycles, extending at 72 ℃ for 10min, performing nucleic acid electrophoresis on the PCR product, purifying, connecting the PCR product with a carrier, converting the PCR product into competent cells of Escherichia coli JM109, picking up positive transformants, extracting plasmids, and sending the plasmids to Suzhou gold intelligent biotechnology company for sequencing;

2. preparation of E.coli competence: dipping a small amount of bacterial liquid from the frozen tube bacteria by using a sterile inoculating loop, streaking on a non-resistant LB solid culture medium plate, and placing the plate at 37 ℃ for inverted culture for 12 hours; after colonies growing on the plates, single colonies are picked by a sterile inoculating loop and transferred into a 50mL conical flask filled with 10mL LB liquid medium, and the colonies are cultured at 37 ℃ and 180r/min overnight; activating strains in 10mL of LB liquid medium, inoculating 10 mu L of bacterial liquid into a 250mL conical flask containing 50mL of LB liquid medium by using a sterile pipettor, culturing until OD600 is about 0.6, and then placing the conical flask containing bacterial liquid and a 50mL sterile centrifuge tube on ice for 30min for precooling; transferring the bacterial liquid into a 50m L centrifuge tube after precooling is finished, centrifuging for 5min at 8000r/min and 4 ℃ and collecting bacterial bodies; removing the supernatant, removing the residual culture medium with a liquid transfer device as much as possible, adding 2000 mu L of Solution A, slowly blowing and sucking to uniformly disperse the thalli in the Solution A, centrifuging at 8000r/min and 4 ℃ for 1min; removing the supernatant, carefully removing residual Solution A by a pipette, adding 2000 mu L of Solution B, gently blowing and sucking by using Solution B, and subpackaging 100 mu L of resuspended bacterial liquid by each 1.5mL EP tube to obtain competent cells;

3. Construction of recombinant plasmid pRSFDuet-HpaBC-LAAD-ARO10-ADH: respectively taking sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 as templates, hpaBC-F1/HpaBC-R1 and HpaBC-F2/HpaBC-R2 as primers, and obtaining HpaBC genes through overlapping and extending of two genes; amplifying by using a sequence shown in SEQ ID NO. 3 as a template and LAAD-F/LAAD-R as a primer to obtain a LAAD gene; carrying out amplification by taking pRSFDuet-1 plasmid as a template and pRSFDuet-F1/pRSFDuet-R1 as a primer to obtain a linearization vector pRSFDuet-1; carrying out three-segment homologous recombination connection on the two gene segments and the linearization vector segment, converting JM109 competent cells, and extracting plasmids to obtain recombinant plasmids pRSFDuet-HpaBC-LAAD; amplifying by using a sequence shown in SEQ ID NO. 4 as a template and ARO10-F/ARO10-R as a primer to obtain an ARO10 gene; amplifying by using a sequence shown in SEQ ID NO. 5 as a template and ADH-F/ADH-R as a primer to obtain an ADH gene; amplifying by using pRSFDuet-HpaBC-LAAD plasmid as a template and pRSFDuet-F2/pRSFDuet-R2 as a primer to obtain a linearization vector pRSFDuet-HpaBC-LAAD; carrying out three-fragment homologous recombination connection on the obtained ARO10 gene, ADH gene and pRSFDuet-HpaBC-LAAD linearization vector, converting JM109 competent cells, and extracting plasmids to obtain recombinant plasmids pRSFDuet-HpaBC-LAAD-ARO10-ADH;

4. Constructing a recombinant plasmid pETDuet-AROG-TYRA: amplifying by using a sequence shown in SEQ ID NO. 6 as a template and AROG-F/AROG-R as a primer to obtain a AROG gene; amplifying by taking the pETDuet-1 plasmid as a template and pETDuet-F1/pETDuet-R1 as a primer to obtain a linearization vector pETDuet-1; carrying out homologous recombination connection on the AROG gene fragment and the linearization vector fragment obtained in the above way, converting JM109 competent cells, and extracting plasmids to obtain recombinant plasmids pETDuet-AROG; amplifying by using a sequence shown in SEQ ID NO. 7 as a template and TYRA-F/TYRA-R as a primer to obtain TYRA genes; amplifying by using pETDuet-AROG plasmid as a template and pETDuet-F2/pETDuet-R2 as a primer to obtain a linearization vector pETDuet-AROG; carrying out homologous recombination connection on the TYRA gene and the pETDuet-AROG linearization vector, converting into competent cells of escherichia coli JM109, and extracting plasmids to obtain recombinant plasmids pETDuet-AROG-TYRA;

5. Obtaining recombinant escherichia coli BLAH and BLCK, namely jointly transforming escherichia coli BL21 competent cells by the two recombinant plasmids pRSFDuet-HpaBC-LAAD-ARO10-ADH and pETDuet-AROG-TYRA obtained in the above way; the transformation method is as follows: placing the freshly prepared competent cells on ice for 5min; in an ultra clean bench, 10. Mu.L of DNA for transformation is added into a 1.5mL EP tube filled with competent cells by a sterile pipette, gently sucked and mixed evenly, and then placed on ice for 30min; placing in a water bath at a temperature of 42 ℃ for heat shock for 90s, and then placing on ice for 5min; adding 900 mu L of LB liquid medium into the 1.5mL EP tube in an ultra-clean workbench by using a sterile pipette, and resuscitating for 1h by using a shaking table at 37 ℃; centrifugally collecting bacterial cells cultured in an EP tube, reserving 200 mu L of culture medium, blowing and sucking, uniformly mixing, and screening in a solid culture medium containing two antibiotics of ampicillin and kanamicin to obtain a positive clone BLAH escherichia coli recombinant strain; and transforming the competent cells of the escherichia coli BL21 with the plasmids pRSFDuet-1 and pETDuet-1 together, and screening the competent cells in a culture medium containing two antibiotics of ampicillin and kanamicin to obtain a positive clone BLCK escherichia coli recombinant strain;

table 1: primers for PCR amplification

EXAMPLE 2 shake flask fermentation of recombinant E.coli BLAH and BLCK

1. Strains BLAH and BLCK were streaked on a solid LB medium plate and grown in a 37℃incubator for 12 hours, then single colonies were picked up and cultured in 10mL of LB liquid medium containing 50mg/L kanamycin and 100mg/L ampicillin for 12 hours at 37℃to obtain a seed solution. Transferring the seed solution into 50mL of TB medium added with 50mg/L kanamycin and 100mg/L ampicillin according to 2% transferring amount (1 mL), carrying out shaking culture at 37 ℃ until OD600 reaches 0.7-0.8, adding IPTG with final concentration of 0.1-0.3 mM for induction, culturing at 25-30 ℃ for 24 hours, and sampling and detecting the hydroxytyrosol content in the fermentation liquor at regular intervals;

2. The High Performance Liquid Chromatography (HPLC) detection method of hydroxytyrosol comprises the steps of firstly carrying out sample treatment after sampling, carrying out metal bath treatment on fermentation liquor at 100 ℃ for 15min, centrifuging at 8000r/min for 1min, collecting supernatant, and carrying out microporous filter membrane filtration treatment on the supernatant; conditions for the detection by liquid chromatography were as follows: the mobile phase is 0.1% (w/v) formic acid, pure methanol=80:20 (v/v), the flow rate is 1mL/min, the chromatographic column is a Thermo SCIENTIFIC C column (4.6X250 mm,5 μm), the column temperature is 40 ℃, the wavelength of an ultraviolet detector is 280nm, and the sample injection amount is 10 mu L;

EXAMPLE 3 5-L tank fermentation of recombinant E.coli BLAH and BLCK

Amplifying and culturing the recombinant escherichia coli in the embodiment 2 by a 5-L fermentation tank, transferring a seed culture solution into a TB culture medium with 2L liquid loading capacity according to the inoculation amount of 5-8% (v/v), controlling the fermentation temperature to be 37 ℃, controlling the pH value to be between 6.8 and 7.2, and increasing the initial rotation speed to 300r/min step by step after 3 hours to 500-600 r/min, wherein the volume of the feed culture medium is 300mL for each 1L fermentation system; when the thalli are cultured until the OD600 is about 15-20, adding IPTG with the final concentration of about 0.1-0.3 mM, and carrying out induction expression for 48 hours at the temperature of 25-30 ℃, and sampling and detecting the hydroxytyrosol content in the fermentation liquor at regular intervals;

The fermentation in the 5-L fermentation tank can monitor relevant parameters such as pH, DO and the like in the fermentation process in real time, and compared with shaking flask fermentation, the fermentation of BLAH in the 5-L fermentation tank is beneficial to industrial production, the result is shown in figure 2, the yield of hydroxytyrosol is continuously accumulated along with the increase of time, the growth starts slowly after 40 hours, and the final fermentation obtains 20.06g/L hydroxytyrosol in 48 hours.

The above is a preferred embodiment of the present invention, and a person skilled in the art can also make alterations and modifications to the above embodiment, therefore, the present invention is not limited to the above specific embodiment, and any obvious improvements, substitutions or modifications made by the person skilled in the art on the basis of the present invention are all within the scope of the present invention.

Claims (4)

1. The construction method of the recombinant escherichia coli for producing the hydroxytyrosol by fermentation is characterized by comprising the following steps of firstly, obtaining HpaBC and LAAD genes and linearization pRSFDuet-1 vector fragments by PCR amplification, and fusing the fragments by homologous recombination to obtain plasmid pRSFDuet-HpaBC-LAAD; PCR amplification to obtain ARO10, ADH gene and pRSFDuet-HpaBC-LAAD vector fragment, and fusion of these fragments by homologous recombination to obtain plasmid pRSFDuet-HpaBC-LAAD-ARO10-ADH; PCR amplification to obtain AROG and TYRA genes and linearized pETDuet-1 vector fragments, and fusing the fragments through homologous recombination to obtain plasmid pETDuet-AROG-TYRA; the sequence of HpaBC gene is SEQ ID NO.1 and SEQ ID NO.2, the sequence of LAAD gene is SEQ ID NO.3, the sequence of ARO10 gene is SEQ ID NO.4, the sequence of ADH gene is SEQ ID NO.5, the sequence of AROG gene is SEQ ID NO.6, and the sequence of TYRA gene is SEQ ID NO.7;

Step two, the plasmid pRSFDuet-HpaBC-LAAD-ARO10-ADH and the plasmid pETDuet-AROG-TYRA are jointly transferred into competent cells of the escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli BLAH.

2. The use of recombinant E.coli for fermentative production of hydroxytyrosol, characterized in that the recombinant E.coli according to claim 1 is used for fermentative production of hydroxytyrosol.

3. Use of recombinant escherichia coli for the fermentative production of hydroxytyrosol according to claim 2, wherein 6 enzymes are expressed by the recombinant escherichia coli: hydroxylase, L-alpha-amino acid deaminase, alpha-keto acid decarboxylase, alcohol dehydrogenase, 2-dehydro-3-deoxyphosphoheptylaldehyde aldolase and chorismate mutase/benzoate dehydrogenase.

4. The use of recombinant E.coli produced by fermentation according to claim 2, wherein the recombinant E.coli production method comprises the steps of,

Step one, culturing recombinant escherichia coli BLA H carrying plasmids pRSFDuet-HpaBC-LAAD-ARO10-ADH and plasmids pETDuet-AROG-TYRA in an LB culture medium;

And step two, inoculating the strain into TB culture solution according to the inoculum size of 0.5-2% for expansion culture, then adding an inducer of 0.1-0.3 mM for induction culture, continuously culturing at 30 ℃ and 220r/min for product accumulation, and detecting the hydroxytyrosol yield by liquid chromatography every 8 hours.