Synthesis of hydroxysalidin and recombinant plasmid composition, engineering bacteria and application thereof

Abstract

The application belongs to the field of industrial microorganism and fermentation technology, and particularly relates to synthesis of hydroxyl rhodiolin, a recombinant plasmid composition, an engineering bacterium and application thereof. The recombinant plasmid composition is composed of a recombinant plasmid PDWS1121 and a recombinant plasmid PDWS0874. In the application, the raip gene and the kivD gene are constructed into the plasmid PDWS1121, and the plasmid PDWS0874 containing a glucose-based transferase UGT gene is introduced into Saccharomyces cerevisiae PLVC to obtain a recombinant Saccharomyces cerevisiae strain RSL101 for producing hydroxyl rhodiolin, and then efficient production of hydroxyl rhodiolin is realized. The method for producing hydroxyl rhodiolin is simple and feasible, and has strong product synthesis capacity, so that the production cost can be greatly reduced and economic benefits can be improved.

Description

Technical Field

[0001] This invention belongs to the field of industrial microbiology and fermentation technology, specifically relating to the synthesis of hydroxyrhodioloside and its recombinant plasmid composition, engineered bacteria and applications. Background Technology

[0002] Hydroxysalidroside, chemical formula C 14 H 20 O8, a glycoside of hydroxytyrosol, is also a hydroxylated product of rhodioloside. Rhodioloside possesses multiple regulatory mechanisms, including hypoglycemic effects, anti-oxidative stress, and inhibition of vascular smooth muscle proliferation, making Rhodiola rosea increasingly popular in the treatment of various diseases. Hydroxytyrosol, based on its multi-hydroxyl structure, shows broad application prospects in antioxidation, tumor prevention / treatment, anti-fatigue, and anti-aging. Hydroxyrhodioloside, as a derivative of rhodioloside and hydroxytyrosol, has similar pharmacological activities to rhodioloside, and even exhibits better performance due to the additional hydroxyl group, but its specific efficacy still needs further exploration. Existing studies have shown that it has anti-breast cancer activity and can effectively reduce the proliferation of breast cancer cells. Meanwhile, the aglycone of hydroxyrhodioloside, hydroxytyrosol, is a natural polyphenol compound with strong antioxidant activity, mainly existing in the form of esters in the fruit and leaves of olives. The two hydroxyl groups attached to the benzene ring of hydroxytyrosol are its main antioxidant active groups, and its inherent antioxidant activity can prevent the occurrence of various diseases. In addition, hydroxytyrosol can eliminate free radicals in the body, restore the health of human organs, prevent brain aging, and delay aging. Research on hydroxyrhodioloside can further expand the derivative library of this drug by developing downstream products of popular drug molecules, which will help provide new ideas for drug use and drug development.

[0003] Common methods for preparing hydroxyrhodioloside include chemical synthesis and biosynthesis. Biosynthesis of hydroxyrhodioloside utilizes microorganisms such as bacteria and fungi, using glucose or biomass hydrolysate as a carbon source, through microbial fermentation. Compared to chemical synthesis, the biggest advantage of biosynthesis is the availability and low cost of raw materials, and it is also environmentally friendly, aligning with green manufacturing practices. The fermentation strain is a key element in the biosynthesis of hydroxyrhodioloside. Although there are few reports on specific strains, the technical research on the biosynthesis of rhodioloside and hydroxytyrosol is relatively mature.

[0004] In the prior art, invention patents with patent numbers / application numbers ZL201410115011.4, ZL201610395330.4, ZL201610408741.2, ZL201610361309.2, ZL201711479443.3, CN201811116170.0, CN201910911304.6, CN202011466963.2, and CN202111307645.6 all disclose recombinant microorganisms and construction methods for producing rhodioloside. The strains used mainly include *Saccharomyces cerevisiae*, *Escherichia coli*, and *Eurotium cristatum*. Among these, *Saccharomyces cerevisiae*, due to its eukaryotic cell characteristics, has become a potential strain for the biosynthesis of glycosides such as rhodioloside. However, the production level of the aforementioned recombinant microorganisms is still far from industrial-scale and needs further improvement. Furthermore, none of the aforementioned patents describe how to further synthesize hydroxyrhodioloside.

[0005] Chinese invention patent CN201510242626.8 discloses a method for overexpressing the E. coli-derived monooxygenase gene cluster HpaBC in E. coli to synthesize hydroxytyrosol de novo using glucose as a substrate. The drawbacks of this method are that hydroxytyrosol is toxic to the bacteria, and the expression level of the exogenous protein HpaBC is low; therefore, the efficiency of hydroxytyrosol production is difficult to improve and insufficient to support the subsequent glycosylation process. Meanwhile, patent CN109295113A also discloses a method for producing hydroxytyrosol by fermenting tyrosine, generating 0.74±0.088mM hydroxytyrosol in E. coli using 1mM L-DOPA. Chinese invention patent CN201711054680.5 discloses a method for producing tyrosol and hydroxytyrosol via a heterologous metabolic pathway. This method efficiently expresses aminotransferase, ketoacid decarboxylase, and alcohol dehydrogenase in the host to produce tyrosol, which is then converted to hydroxytyrosol by 4-hydroxyphenylacetic acid hydroxylase. The disadvantages of this method are the need for the addition of expensive coenzymes pyridoxal phosphate (PLP) and reduced coenzyme I (NADH), and a significant reduction in the efficiency of adding a hydroxyl group to the benzene ring, resulting in a very low yield of hydroxytyrosol.

[0006] Therefore, given the shortcomings of current methods, there is an urgent need for a simple and convenient method for producing hydroxyrhodioloside, which can efficiently synthesize hydroxyrhodioloside and improve economic benefits. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide an RSL101 engineered bacterium for high production of hydroxyrhodioloside. This engineered bacterium contains recombinant plasmid PDWS1121 carrying the Raip gene and kivD gene and recombinant plasmid PDWS0874 carrying the UGT gene. Using basic Saccharomyces cerevisiae culture medium as raw material, and with the addition of L-DOPA, it can produce high levels of hydroxyrhodioloside.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The RSL101 engineered bacterium for high-yield production of hydroxyrhodioloside was prepared by transforming recombinant plasmids PDWS1121 and PDWS0874 into the starting bacteria. The recombinant plasmid PDWS1121 carries the Raip gene and the kivD gene, and the recombinant plasmid PDWS0874 carries the UGT gene. The nucleotide sequences of the Raip gene, the kivD gene, and the UGT gene are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0010] Due to the broad substrate scope of the enzyme, this application discovered that raip, as an L-lysine α-oxidase, not only has a catalytic effect on the oxidation of lysine, but also exhibits oxidative activity for L-DOPA. Simultaneous action with the α-keto acid decarboxylase kivD can realize the synthetic pathway from L-DOPA to hydroxytyrosol (see details). Figure 1 This invention further combines glucosyltransferase UGT to achieve the biosynthesis of hydroxyrhodioloside.

[0011] Preferably, the recombinant plasmid PDWS1121 is a vector carrying complete coding sequences of the Raip gene and the kivD gene.

[0012] Preferably, the recombinant plasmid PDWS0874 is a vector carrying the complete coding sequence of the UGT gene.

[0013] Furthermore, the recombinant plasmid PDWS0874 carries the UGT gene after a directed mutation.

[0014] Furthermore, the starting strain is Saccharomyces cerevisiae INVSC1, purchased from Baoguang Biotechnology.

[0015] Furthermore, the method for constructing the recombinant plasmid PDWS1121 is as follows:

[0016] 1) Primers raiP-F and raiP-R were designed and amplified using the Raip gene as a template to obtain the Raip gene fragment with a sequence as shown in SEQ ID NO.1, which is 1488 bp in length; the vector pESC and the Raip gene fragment were double-digested with XbaI and XhoI, respectively, and the Raip gene fragment was inserted between the restriction sites of the vector to obtain the recombinant plasmid pESC-raip;

[0017] 2) Primers kivD-F and kivD-R were designed to amplify the kivD gene using it as a template, and the kivD gene fragment with the sequence shown in SEQ ID NO.2 was obtained, with a length of 1954 bp.

[0018] 3) The plasmid pESC-raip obtained in step 1) and the kivD gene fragment obtained in step 2) were digested with BamHI and HindIII, respectively, and the kivD gene fragment was inserted between the restriction sites of the vector to construct the recombinant plasmid PDWS1121.

[0019] The nucleotide sequences of the primers raiP-F, raiP-R, kivD-F, and kivD-R are shown in SEQ ID NO.4-SEQ ID NO.7, respectively.

[0020] Furthermore, the method for constructing the recombinant plasmid PDWS0874 is as follows:

[0021] 1) Design primers UGT-F and UGT-R, and amplify the UGT gene using the UGT gene as a template to obtain the UGT gene fragment with the sequence shown in SEQ ID NO.3, which is 1426 bp in length;

[0022] 2) The vector pESC-LEU and the UGT gene fragment were digested with BamHI and HindIII, respectively, and the UGT gene fragment was inserted between the restriction sites of the vector to construct the recombinant plasmid PDWS0874.

[0023] The nucleotide sequences of the primers UGT-F and UGT-R are shown in SEQ ID NO.8-SEQ ID NO.9, respectively.

[0024] Furthermore, sequencing confirmed that the Raip gene fragment with the sequence shown in SEQ ID NO.1 is indeed the Raip gene, which matches the MG423617.1 gene in the NCBI gene bank.

[0025] Furthermore, sequencing confirmed that the kivD gene fragment shown in SEQ ID NO.2 is indeed the kivD gene, which matches the AJ746364.1 gene in the NCBI gene bank.

[0026] The second objective of this invention is to provide a method for producing hydroxyrhodioloside by fermenting L-DOPA using the RSL101 engineered bacteria. This method is simple, convenient, and highly feasible, and can achieve efficient production of hydroxyrhodioloside.

[0027] To achieve the above objectives, the present invention adopts the following technical solution:

[0028] The method for producing hydroxyrhodioloside by fermenting L-DOPA using the RSL101 engineered bacteria involves culturing the RSL101 engineered bacteria in a catalytic medium, adding lactose to induce exogenous gene expression after 8 hours of culture, introducing O2 and adding L-DOPA after 10 hours of fermentation to obtain the hydroxyrhodioloside.

[0029] Furthermore, the culture is carried out at 250 RPM and 30°C for 8 hours.

[0030] Furthermore, the fermentation conditions are: fermentation temperature of 30℃, pH = 6, and fermentation time of 72h-96h.

[0031] As a preferred option, the fermentation time is 96 hours.

[0032] Furthermore, the concentration of L-DOPA is 5-10 g / L.

[0033] Preferably, the concentration of the substrate L-DOPA is 10 g / L.

[0034] A third objective of this invention is to provide the application of the RSL101 engineered bacteria in the preparation of hydroxyrhodioloside and / or in the genetically engineered fermentation production of hydroxyrhodioloside.

[0035] The fourth objective of this invention is to provide an application of the raip gene in the synthesis of hydroxytyrosol and / or hydroxyrhodioloside.

[0036] The technical solution of this invention is as follows: Using *Saccharomyces cerevisiae* strain INVSC1 purchased from Baoguang Biotechnology as the starting strain PLVC, a recombinant *Saccharomyces cerevisiae* strain capable of high-yield production of hydroxytyrosol in synthetic culture medium by co-expressing L-α-oxidase (raip) and α-keto acid decarboxylase (kivD) genes was obtained. Further, tyrosol glycoside-derived tyrosol glycoside-UGT was introduced, characterized by its highly efficient catalytic activity towards hydroxytyrosol after targeted mutation. Thus, a recombinant *Saccharomyces cerevisiae* strain capable of producing hydroxytyrosol using basic *Saccharomyces cerevisiae* culture medium as raw material and with the addition of L-DOPA was obtained.

[0037] The technical concept of this invention is as follows: the L-lysine α-oxidase (raip) gene and the α-keto acid decarboxylase (kivD) gene are constructed into plasmid PDWS1121, which is introduced into Saccharomyces cerevisiae PLVC along with plasmid PDWS0874 containing the glucosyltransferase UGT gene, to obtain a recombinant Saccharomyces cerevisiae strain RSL101 that produces hydroxyrhodioloside, thereby achieving the purpose of producing hydroxyrhodioloside.

[0038] The beneficial effects of this invention are as follows:

[0039] The strains and plasmids of this invention can convert L-tyrosine to hydroxyrhodioloside without affecting the growth of the strain. Experimental results show that the recombinant strain grows in a synthetic medium supplemented with levodopa and produces high yields of hydroxyrhodioloside. After 96 hours of shake-flask fermentation, consuming 10 g / L of levodopa, it produces 8.64 g / L of hydroxyrhodioloside, achieving a yield of 86.4%. Compared with other currently published technical routes, the Raip enzyme used in the route described in this invention is used for the first time in the synthesis of hydroxytyrosol, and the hydroxytyrosol produced is superior to that of other currently published technical routes. The UGT glucosyltransferase has undergone gene mutation, resulting in a 20.5% increase in yield. Attached Figure Description

[0040] Figure 1 A schematic diagram of the production pathway for hydroxyrhodioloside;

[0041] Figure 2 This is a schematic diagram illustrating the construction of the PDWS1121 plasmid;

[0042] Figure 3 This is a schematic diagram illustrating the construction of the PDWS0874 plasmid;

[0043] Figure 4 This is a comparison chart of growth curves;

[0044] Figure 5 A comparison of the yields of hydroxyrhodioloside with different concentrations of L-DOPA as substrate;

[0045] Figure 6 Curves showing the yield of hydroxyrhodioloside and the residual amount of L-DOPA at different fermentation times;

[0046] Figure 7 Line graph showing the effect of different pH values ​​on the yield of hydroxyrhodioloside. Detailed Implementation

[0047] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0048] In this embodiment of the invention, unless otherwise specified, all chemicals were purchased from Aladdin and restriction endonucleases were purchased from Takara Ltd.

[0049] In this embodiment of the invention, the culture medium is prepared as follows:

[0050] (1) LB medium: Each liter of water contains 5g yeast extract, 10g tryptone and 10g NaCl. Solid medium is liquid medium with 1.5% agar powder added. To increase the selectivity of the medium, the concentration of ampicillin (Amp) and kanamycin (Kan) is 100μg / mL.

[0051] (2) Yeast double-antibiotic medium: LEU-URA medium powder SD (purchased from Proton), with an additional 2% agar powder added during plate screening. L-DOPA was added during galactose induction, with the galactose addition amount being 3%.

[0052] (3) Catalytic medium: 20% glucose, 10% peptone, 0.50% yeast extract, 0.05% magnesium sulfate, and 0.05% glycine. L-DOPA was added during galactose induction, with the amount of galactose added being 3%.

[0053] In this embodiment of the invention, plasmids PDWS1121 and PDWS0874, which were successfully constructed in Example 1, were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing to verify the correctness of the nucleotide sequences.

[0054] In this embodiment of the invention, the starting strain, Saccharomyces cerevisiae INVSC1, was purchased from Protech Biotechnology Co., Ltd.

[0055] Example 1

[0056] According to such Figure 1 The production route for hydroxyrhodioloside shown includes the following steps:

[0057] 1. Construction of recombinant plasmid PDWS1121

[0058] A schematic diagram of the construction of plasmid PDWS1121 is shown below. Figure 2 As shown, the plasmid PDWS1121 was constructed by combining the L-lysine α-oxidase (raip) gene and the α-keto acid decarboxylase (kivD) gene, specifically including the following steps:

[0059] 1) Design the following primers:

[0060] raip-F: 5′-GGAATTCCATATGGAGCACCTGGCAGACTGTCTGG-3′;

[0061] raip-R: 5′-CCGCTCGAGCAGTTCGTCCTCTGGTATG-3′.

[0062] Using the raip gene as a template, a 1488 bp fragment was amplified, the sequence of which is shown in SEQ ID NO.1, and was used to construct the plasmid PDWS1121. Sequencing confirmed that the fragment was indeed the raip gene, which matched the MG423617.1 gene in the NCBI gene library. Then, the vector pESC-URA and the raip gene fragment were double-digested with XbaI and XhoI, respectively, and the raip gene fragment was inserted between the restriction sites of the vector to obtain the recombinant plasmid pESC-raip.

[0063] 2) Design the following primers:

[0064] kivD-F: 5′-CGCGGATCCGCGCTAGAGTTTTCTTTAGTCAT-3′

[0065] kivD-R: 5′-CCCAAGCTTGGGTAGGAGCATAGGATA-3′

[0066] Using the kivD gene as a template, a 1954 bp fragment was amplified, the sequence of which is shown in SEQ ID NO.2, and was used to construct the plasmid PDWS1121. Sequencing confirmed that the fragment was indeed the kivD gene, which matched the AJ746364.1 gene in the NCBI gene library. Then, the plasmid pESC-raip and the kivD gene fragment were double-digested with BamHI and HindIII, respectively, and the kivD gene fragment was inserted between the restriction sites of the vector to construct the recombinant plasmid PDWS1121.

[0067] 2. Enzyme digestion analysis of recombinant plasmid PDWS1121

[0068] 1) Plasmid DNA was extracted using the alkaline lysis method. The raip gene was amplified and digested with XbaI and XhoI, reacting at 37°C for 3 hours. The digestion reaction system is shown in Table 1.

[0069] Table 1. Enzyme digestion reaction system

[0070] raip gene fragment 28μL 10×CutSmart Buffer 5μL restriction enzyme XbaI 1μL restriction enzyme XhoI 1μL <![CDATA[ddH2O]]> 15μL Total volume 50μL

[0071] 2) Ligation of pESC-URA and raip. The enzyme-digested raip gene and pESC-URA vector were ligated and reacted at 22℃ for 1 h. The ligation reaction system is shown in Table 2.

[0072] Table 2. Connection Reaction System

[0073]

[0074]

[0075] 3) Double digestion of pESC-raip. The plasmid pESC-raip containing the raip gene obtained in step 2) was double-digested with BamHI and HindIII. The reaction was carried out at 37°C for 3 hours. The digestion reaction system is shown in Table 3.

[0076] Table 3. Enzyme digestion reaction system

[0077] <![CDATA[ddH2O]]> 13μL pESC-raip 30μL 10×CutSmart Buffer 5μL restriction enzyme BamHI 1μL restriction enzyme HindIII 1μL Total volume 50μL

[0078] 4) Double digestion of the kivD gene. The kivD gene was amplified and double-digested with BamHI and HindIII. The reaction was carried out at 37°C for 3 hours. The reaction system is shown in Table 4.

[0079] Table 4. Reaction System

[0080] kivD gene fragment 28μL 10×CutSmart Buffer 5μL restriction enzyme BamHI 1μL restriction enzyme HindIII 1μL <![CDATA[ddH2O]]> 15μL Total volume 50μL

[0081] 5) Ligation of pESC-raip and kivD. The enzyme-digested kivD gene was ligated to the pESC-raip vector and reacted at 22℃ for 1 hour to obtain the engineered plasmid PDWS1121. The ligation reaction system is shown in Table 5.

[0082] Table 5. Connection Reaction System

[0083] pESC-raip 12μL kivD clips 5μL T4 10× DNA ligase buffer 2μL T4 DNA ligase 1μL Total volume 20μL

[0084] 6) Screening of PDWS1121 positive clones

[0085] Step 5) After the ligation reaction is complete, PDWS1121 transformation is performed. Single colonies growing on the transformed plate are picked and inoculated into LB liquid medium containing kanamycin resistance. The culture is carried out at 37°C and 250 rpm for 10-12 hours. Plasmids are extracted and digested with BamHI and HindIII. Two clear bands are obtained: one for the pESC-raip vector and the other for the kivD gene. The pESC-raip vector is recovered by gel extraction and further digested with XhoI and XbaI, yielding two clear bands: one for the pESC vector and the other for the raip gene. This preliminarily verifies the successful cloning of PDWS1121.

[0086] 3. Construction of recombinant plasmid PDWS0874

[0087] A schematic diagram of the construction of plasmid PDWS0874 is shown below. Figure 3 As shown, it includes the following steps:

[0088] 1) Primer design:

[0089] UGT-F: 5′-GCGGATCCGCCAAATTCCTCAGAGGGTGA-3′;

[0090] UGT-R: 5′-CCAAGCTTGGGAGATAACAGATAAT-3′.

[0091] The UGT gene, mutated in the laboratory, was amplified to obtain a fragment of 1426 bp, the sequence of which is shown in SEQ ID NO.3, and was used to construct the plasmid PDWS0874. Then, the vector pESC-LEU and the UGT gene fragment were digested with BamHI and HindIII, respectively, and the UGT gene fragment was inserted between the restriction sites of the vector to construct the recombinant plasmid PDWS0874.

[0092] 4. Enzyme digestion analysis of recombinant plasmid PDWS0874

[0093] 1) Extract plasmid DNA using the alkaline lysis method. Amplify the UGT gene and digest it with BamHI and HindIII. Incubate at 37°C for 3 hours. The enzyme digestion reaction system is shown in Table 6.

[0094] Table 6. Enzyme digestion reaction system

[0095] UGT gene 35μL 10×CutSmart Buffer 5μL restriction enzyme BamHI 1μL restriction enzyme HindIII 1μL <![CDATA[ddH2O]]> 8μL Total volume 50μL

[0096] After the enzyme digestion reaction was carried out at 37°C for 3 hours, the degree of digestion was examined by electrophoresis. The digested plasmid was recovered using a DNA recovery kit. After digestion, the sample was loaded and electrophoresed for confirmation.

[0097] 2) Double digestion of the UGT gene. Take 35 μL of the UGT3 gene fragment and digest it with BamHI and HindIII. Mix well and centrifuge slightly to allow the solution to pool at the bottom of the tube. Incubate at 37°C for 3 hours. The digestion reaction system is shown in Table 7.

[0098] Table 7. Enzyme digestion reaction system

[0099]

[0100]

[0101] 3) Ligation of pESC-LEU and UGT. The UGT fragment was mixed with the prepared pESC-LEU vector in the following system and then ligated, as shown in Table 8. The ligation reaction was carried out at 22℃ for 1 h, and the resulting engineered plasmid PDWS0874 was obtained and used for transformation reactions.

[0102] Table 8. Connection Reaction System

[0103] pESC-LEU 10μL UGT clips 7μL T4 10× DNA ligase buffer 2μL T4 DNA ligase 1μL Total volume 20μL

[0104] (4) Screening of PDWS0874 positive clones. The DNA ligation product was transformed, and the transformation experimental steps were the same as those in "6) Screening of PDWS1121 positive clones". After single colony culture, plasmids were extracted in small quantities using a plasmid extraction kit. The plasmids were analyzed by double digestion with BamHI and HindIII. The recombinant plasmids that were verified by enzyme digestion were sequenced to ensure that the cloned gene sequence was correct.

[0105] 5. Transformation

[0106] (1) Take 100 μL of INVSC1 competent cells thawed on ice, add 2 μL of pre-cooled target plasmid, 10 μL of carrier DNA (98℃, 5 min, rapid ice bath, repeat once), and 500 μL of PEG / LiAc, and mix by pipetting several times. Incubate in a 30℃ water bath for 30 min (invert 6-8 times at 15 min to mix).

[0107] (2) Place the tube in a 42℃ water bath for 15 minutes (invert 6-8 times at 7.5 minutes to mix).

[0108] (3) Centrifuge at 5000 rpm for 40 s and discard the supernatant. Resuspend in 400 μL of ddH2O and centrifuge for 30 s and discard the supernatant.

[0109] (4) Resuspend in 50 μL of ddH2O, spread on plasmid for screening, and incubate at 29℃ for 48-96 h.

[0110] (5) Spread 100 μL of bacterial culture evenly onto an Ampicillin plate containing a final concentration of 100 μg / mL. Invert the plate and incubate overnight at 37°C. Using a sterile pipette tip or toothpick, pick a single colony into 20 μL of LB medium and mix well. Take 1 μL directly as a PCR template. Inoculate the remaining bacterial culture of PCR-positive colonies into LB medium containing appropriate antibiotics and incubate overnight. Extract plasmids for subsequent identification.

[0111] 6. Growth characteristics and stability analysis of recombinant bacteria

[0112] 100 μL of the double-plasmid transformed bacterial solution was evenly spread onto a yeast double-antibiotic medium plate, inverted, and incubated overnight at 37°C. Thirty such colonies were picked and inoculated onto a double-antibiotic selection plate; bacteria without plasmids on this plate could not grow. The original strain INVSC1 did not exhibit resistance, and this genetically engineered bacterium could grow normally on the double-antibiotic selection plate, indicating that the recombinant plasmid had been successfully transferred into the original strain. After 24 hours of incubation, 26 colonies grew on the plate, demonstrating that the bacterium remained highly stable under no selective pressure, with a stability of approximately 87%.

[0113] 7. Growth rate analysis of recombinant bacteria RSL101

[0114] A single colony of RSL101 from a plate culture was picked and inoculated into 20 mL of catalytic medium containing 100 μg / mL uracil. The culture was incubated at 30°C and 250 rpm for 12 hours. Then, a 1% inoculum was inoculated into 200 mL of catalytic medium containing 100 μg / mL uracil and incubated for another 12 hours. The culture was then transferred to a 1 L fermenter, and samples were taken every 6 hours to determine the OD value of the cells. 600 Value, see details Figure 4 It can be seen that after 10 hours, the bacteria are in the logarithmic growth phase, with a rapid growth rate and increased biomass. After 30 hours, the OD of the bacteria... 600 The value has reached its maximum value of 150.

[0115] 8. Optimization of fermentation conditions

[0116] To explore optimal fermentation conditions, the effects of fermentation temperature, pH, and substrate concentration on the yield of fermented hydroxyrhodioloside were investigated. The results are as follows: Figures 5-7 As shown, the optimal fermentation conditions under the existing conditions are a temperature of 30℃, pH=6, and a fermentation time of 96h. When the substrate L-DOPA concentration is 5g / L, the yield increases to 4.02g / L compared to the yield before optimization of 3.68g / L, indicating an improvement in yield.

[0117] 9. Exploration of Industrial Applications

[0118] To further explore ways to increase production, 5OD 600 The seed culture of engineered strain RSL101 was added to 5L of catalytic medium and cultured in a 10L reactor at 250 RPM and 30℃ for 8 hours. Lactose was then added to induce exogenous gene expression. After 10 hours, O2 was introduced, and 10g / L L-DOPA was added. The sample at this point was taken as the 0h sample. Based on the time-yield graph, the yield within 72 hours was selected for analysis. At the fermentation endpoint, the yield of hydroxyrhodioloside reached 8.64g / L.

Claims

1. An engineered strain, RSL101, for high production of hydroxyrhodioloside, characterized in that, Recombinant plasmids PDWS1121 and PDWS0874 were transformed into the starting bacteria to obtain the RSL101 engineered strain. Recombinant plasmid PDWS1121 carries the Raip gene and the kivD gene, and recombinant plasmid PDWS0874 carries the UGT gene. The nucleotide sequences of the Raip gene, the kivD gene, and the UGT gene are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The starting bacteria was *Saccharomyces cerevisiae* INVSC1. The method for constructing the recombinant plasmid PDWS1121 is as follows: 1) Primers raiP-F and raiP-R were designed and amplified using the Raip gene as a template to obtain the Raip gene fragment with the sequence shown in SEQ ID NO.1; the vector pESC-URA and the Raip gene fragment were double-digested with XbaI and XhoI, respectively, and the Raip gene fragment was inserted between the restriction sites of the vector to obtain the recombinant plasmid pESC-raip; the nucleotide sequences of raiP-F and raiP-R are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; 2) Primers kivD-F and kivD-R were designed and amplified using the kivD gene as a template to obtain the kivD gene fragment with a length of 1954 bp, as shown in SEQ ID NO.2; the nucleotide sequences of kivD-F and kivD-R are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively. 3) The plasmid pESC-raip obtained in step 1) and the kivD gene fragment obtained in step 2) were digested with BamHI and HindIII, respectively. The kivD gene fragment was inserted between the restriction sites of the vector to construct the recombinant plasmid PDWS1121. The method for constructing the recombinant plasmid PDWS0874 is as follows: 1) Primers UGT-F and UGT-R were designed and amplified using the UGT gene as a template to obtain the UGT gene fragment with the sequence shown in SEQ ID NO.3, which is 1426 bp in length; the nucleotide sequences of UGT-F and UGT-R are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively; 2) The vector pESC-LEU and the UGT gene fragment were digested with BamHI and HindIII, respectively. The UGT gene fragment was then inserted between the restriction sites of the vector to construct the recombinant plasmid PDWS0874.

2. A method for producing hydroxyrhodioloside by fermenting L-DOPA using the RSL101 engineered bacteria as described in claim 1, characterized in that, The RSL101 engineered bacteria were cultured in a catalytic medium. After 8 hours of culture, lactose was added to induce the expression of the exogenous gene. After 10 hours, O2 was introduced and L-DOPA was added to ferment and obtain the hydroxyrhodioloside.

3. The method according to claim 2, characterized in that, The culture was carried out at 250 RPM and 30°C for 8 hours.

4. The method according to claim 2, characterized in that, The fermentation conditions are: fermentation temperature of 30℃, pH=6, and fermentation time of 72h-96h.

5. The method according to claim 2, characterized in that, The concentration of L-DOPA is 5-10 g / L.

6. The use of the RSL101 engineered bacteria according to claim 1 in the preparation of hydroxyrhodioloside.

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