An engineered Yarrowia lipolytica strain for synthesizing pterostilbene and its construction method and application

Abstract

The present application relates to an engineered bacterium of Yarrowia lipolytica for synthesizing pterostilbene, and a construction method and application thereof, and belongs to the technical field of biosynthesis of natural products. The engineered bacterium for synthesizing pterostilbene of the present application expresses tyrosine ammonia lyase TAL, p-coumarate coenzyme A ligase 4CL, stilbene synthase STS and caffeic acid-O-methyltransferase COMT; and knocks out the ku70 gene and the ku80 gene. The present application knocks out the ku70 and ku80 genes, integrates the coding genes of TAL, 4CL, STS and COMT into Yarrowia lipolytica, and can use glucose as a substrate, utilize the endogenous metabolic pathway of Yarrowia lipolytica to obtain the precursor tyrosine and malonyl-CoA of pterostilbene, and a series of enzyme reactions obtained by the heterologous metabolic pathway to achieve the biosynthesis of pterostilbene.

Description

Technical Field

[0001] The present application relates to the technical field of biosynthesis of natural products, and in particular to an engineered Yarrowia lipolytica for synthesizing pterostilbene, and a construction method and application thereof. Background Art

[0002] Pterostilbene (i.e. (E)-3,5-dimethoxy-4'-hydroxystilbene) is a stilbene compound obtained from seed plants such as grapes, blueberries, blackberries and Pterocarpus scapularis. Modern pharmacology shows that pterostilbene has anti-tumor, antioxidant, anti-aging, anti-diabetic, anti-inflammatory, antibacterial and lipid-lowering effects. Pterostilbene is also a methylated derivative of resveratrol, but its bioavailability is higher and more stable than resveratrol. Compared with resveratrol, pterostilbene is more easily absorbed, has a good biopharmaceutical response and more stable metabolism, and exhibits stronger antioxidant capacity. According to research experiments by many medical research institutions at home and abroad, pterostilbene can simultaneously show excellent melanin inhibition, tyrosinase inhibition, anti-multiple free radicals, antioxidant, UV resistance, anti-inflammatory and anti-glycation effects. It is known as a comprehensive and leading natural active ingredient for whitening and anti-aging, and has become the most advanced and high-end raw material for whitening and anti-aging cosmetics and health products today.

[0003] In the prior art, the engineered bacteria for biosynthesis of pterostilbene mainly use resveratrol or 4-coumaric acid as substrate raw materials to synthesize pterostilbene. Resveratrol and 4-coumaric acid as substrates have the disadvantages of high cost, low substrate utilization, and low pterostilbene yield. In addition, the poor water solubility of resveratrol is not conducive to the industrial production of pterostilbene. Therefore, it is urgent to develop an engineered bacteria with low production cost, high substrate utilization and high yield. Summary of the invention

[0004] The purpose of the present application is to overcome the shortcomings of the prior art and provide an engineered Yarrowia lipolytica strain that synthesizes pterostilbene using glucose as a substrate. The engineered strain has the advantages of high pterostilbene yield and substrate utilization and low production cost.

[0005] To achieve the above purpose, the technical solution adopted by this application is:

[0006] In a first aspect, the present application provides an engineered Yarrowia lipolytica for synthesizing pterostilbene, wherein the engineered Yarrowia lipolytica expresses tyrosine ammonia lyase TAL, p-coumarate CoA ligase 4CL, stilbene synthase STS, and caffeic acid-O-methyltransferase COMT;

[0007] The engineered bacteria knock out the ku70 gene and the ku80 gene.

[0008] The present application knocks out the ku70 and ku80 genes, and integrates the coding genes of tyrosine ammonia lyase TAL, p-coumarate CoA ligase 4CL, stilbene synthase STS and caffeic acid-O-methyltransferase COMT into Yarrowia lipolytica, so that glucose can be used as a substrate to synthesize tyrosine precursor and malonyl-CoA through the endogenous metabolic pathway of Yarrowia lipolytica, and the obtained tyrosine reacts with tyrosine ammonia lyase to obtain p-coumaric acid, p-coumaric acid reacts with p-coumaric acid-CoA ligase to obtain p-coumaric acid-CoA, p-coumaric acid-CoA reacts with three malonyl-CoAs under the action of dipropylene glycol synthase to obtain resveratrol, and resveratrol reacts with caffeic acid-O-methyltransferase to obtain pterostilbene.

[0009] The ku70 and ku80 genes of Yarrowia lipolytica are closely related to its non-homologous end joining (NHEJ). Knocking out the ku70 and ku80 genes can inhibit the repair mechanism of NHEJ, thereby improving the repair efficiency of homologous recombination (HR) and further improving the integration efficiency of the expressed gene cluster.

[0010] As a preferred embodiment of the Yarrowia lipolytica engineered bacteria of the present application, the tyrosine ammonia lyase TAL is derived from Rhodosporidium toruloides;

[0011] The p-coumarate-CoA ligase 4CL is derived from Arabidopsis thaliana;

[0012] The stilbene synthase STS is derived from wine grapes;

[0013] The caffeic acid-O-methyltransferase COMT is derived from Arabidopsis thaliana.

[0014] The tyrosine ammonia lyase TAL derived from Rhodosporidium toruloides is a bifunctional phenylalanine / tyrosine ammonia lyase. The use of this enzyme can combine the biosynthetic pathways of phenylalanine and tyrosine pathways to increase the production of resveratrol, a precursor of pterostilbene.

[0015] Coumarate CoA ligase 4CL from Arabidopsis thaliana is the most efficient one in synthesizing downstream products among various coumarate CoA ligases from different sources; caffeic acid-O-methyltransferase from Arabidopsis thaliana has a more comprehensive and efficient ability to synthesize pterostilbene, which can further increase the yield of pterostilbene.

[0016] As a preferred embodiment of the Yarrowia lipolytica engineered bacteria of the present application, the coding sequence of the tyrosine ammonia lyase TAL is shown in SEQ ID NO.1;

[0017] The coding sequence of p-coumarate-CoA ligase 4CL is shown in SEQ ID NO.2;

[0018] The coding sequence of the stilbene synthase STS is shown in SEQ ID NO.3;

[0019] The coding sequence of the caffeic acid-O-methyltransferase COMT is shown in SEQ ID NO.4.

[0020] As a preferred embodiment of the Yarrowia lipolytica engineered bacteria described in the present application, the ku70 gene sequence is shown as SEQ ID NO.5; the ku80 gene sequence is shown as SEQ ID NO.6.

[0021] As a preferred embodiment of the Yarrowia lipolytica engineered bacteria described in the present application, the engineered bacteria is deposited in the Guangdong Provincial Microbiological Culture Collection Center, the preservation date is May 31, 2024, and the preservation number is GDM CC No: 64708.

[0022] In a second aspect, the present application provides a method for constructing the above-mentioned Yarrowia lipolytica engineered bacteria, which comprises the following steps:

[0023] A1. Knock out the ku70 gene in Yarrowia lipolytica using gene editing technology to obtain engineered bacteria A;

[0024] A2, using gene editing technology to knock out the ku80 gene in the engineered bacteria A obtained in step A1 to obtain engineered bacteria B;

[0025] A3, transferring the pterostilbene integrated expression gene cluster into the engineered bacteria B obtained in step A2 to obtain an engineered bacteria C, wherein the engineered bacteria C is an engineered Yarrowia lipolytica bacteria that synthesizes pterostilbene.

[0026] As a preferred embodiment of the construction method described in the present application, in step A3, the pterostilbene integrated expression gene cluster is mainly prepared by the following steps:

[0027] B1. Using homologous recombination technology, the promoter and terminator of Yarrowia lipolytica, the coding gene of tyrosine ammonia lyase TAL, and the linearized vector were assembled into the RtTAL expression gene cluster;

[0028] B2, using homologous recombination technology to assemble the promoter and terminator carried by Yarrowia lipolytica, the encoding gene of p-coumarate coenzyme A ligase 4CL, and the linearized vector into an At4CL expression gene cluster;

[0029] B3, using homologous recombination technology to assemble the promoter and terminator carried by Yarrowia lipolytica, the coding gene of stilbene synthase STS, and the linearized vector into a VvSTS expression gene cluster;

[0030] B4, using homologous recombination technology, the promoter and terminator of Yarrowia lipolytica, the coding gene of caffeic acid-O-methyltransferase COMT, and the linearized vector were assembled into an AtCOMT expression gene cluster;

[0031] B5. Assemble the RtTAL expression gene cluster obtained in step B1, the At4CL expression gene cluster obtained in step B2, the VvSTS expression gene cluster obtained in step B3 and the AtCOMT expression gene cluster obtained in step B4 into the RtTAL-At4CL-VvSTS-AtCOMT expression gene cluster by homologous recombination technology. The obtained RtTAL-At4CL-VvSTS-AtCOMT expression gene cluster is the pterostilbene integrated expression gene cluster.

[0032] As a preferred embodiment of the construction method described in the present application, it includes at least one of the following (I)-(II):

[0033] (I) In steps B1-B4, the promoter carried by the Yarrowia lipolytica is at least one of pTEF, pEX P and pGPD; the terminator carried by the Yarrowia lipolytica is at least one of mig1t, lip2t and xpr2t;

[0034] (II) In steps B1-B4, the coding gene sequences of tyrosine ammonia lyase TAL, p-coumarate coenzyme A ligase 4CL, stilbene synthase STS and caffeic acid-O-methyltransferase COMT are all codon-optimized using Yarrowia lipolytica as the host strain.

[0035] As a preferred embodiment of the construction method described in the present application, in step A3, the pterostilbene integration expression gene cluster and the pCAS1yl-rDNA plasmid are co-transfected into the engineering bacteria B, and the rDNA gene sequence in the pCAS1yl-rDNA plasmid is shown in SEQ ID NO.7.

[0036] In a third aspect, the present application provides the use of the above-mentioned Yarrowia lipolytica engineered bacteria in the synthesis of pterostilbene.

[0037] In a fourth aspect, the present application provides a method for synthesizing pterostilbene, which is obtained by fermenting the above-mentioned Yarrowia lipolytica engineered bacteria and a culture medium containing glucose in a bioreactor.

[0038] The present application provides a method for synthesizing pterostilbene, which is prepared by inducing an engineered bacterium GY003 to use glucose as a substrate through a series of biochemical reactions. The engineered bacterium GY 003 used in the synthesis process can synthesize tyrosine synthesized by an endogenous pathway into p-coumaric acid, which is converted into p-coumaric acid coenzyme A through a reaction, and p-coumaric acid coenzyme A and malonyl coenzyme A synthesized by an endogenous pathway form resveratrol under the action of dipropylene glycol synthase, and resveratrol is further reacted to obtain pterostilbene, and the shake flask fermentation yield of the obtained pterostilbene can reach 150 mg / L.

[0039] Compared with the prior art, the beneficial effects of this application are:

[0040] (1) The Yarrowia lipolytica engineered bacteria for synthesizing pterostilbene in the present application knocks out the ku70 and ku80 genes, and integrates the coding genes of tyrosine ammonia lyase TAL, p-coumarate CoA ligase 4CL, stilbene synthase STS and caffeic acid-O-methyltransferase COMT into Yarrowia lipolytica, so that tyrosine precursor and malonyl-CoA can be synthesized through the endogenous metabolic pathway of Yarrowia lipolytica using glucose as a substrate, the obtained tyrosine reacts with tyrosine ammonia lyase to obtain p-coumaric acid, p-coumaric acid reacts with p-coumaric acid CoA ligase to obtain p-coumaric acid CoA, p-coumaric acid CoA reacts with three malonyl-CoAs under the action of dipropylene glycol synthase to obtain resveratrol, and resveratrol reacts with caffeic acid-O-methyltransferase to obtain pterostilbene.

[0041] (2) The present application provides a method for synthesizing pterostilbene, wherein pterostilbene is prepared by inducing an engineered bacterium GY003 to use glucose as a substrate through a series of biochemical reactions. The engineered bacterium GY003 used in the synthesis process can synthesize tyrosine synthesized by an endogenous pathway into p-coumaric acid, which is converted into p-coumaric acid coenzyme A through a reaction, and p-coumaric acid coenzyme A and malonyl coenzyme A synthesized by an endogenous pathway form resveratrol under the action of dipropylene glycol synthase, and resveratrol is further reacted to obtain pterostilbene, and the shake flask fermentation yield of the obtained pterostilbene can reach 150 mg / L.

[0042] (3) The engineered bacteria of the present application directly biosynthesize pterostilbene using glucose as a substrate, which has the advantages of reducing costs, improving efficiency, and realizing industrial production of pterostilbene compared with the existing technology, while being green, environmentally friendly, safe, stable, and free of chemical pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the plasmid map of pCAS1yl-ku70 in Example 1 of this application;

[0044] Figure 2 This is the plasmid map of pUC19-ku70-up-down in Example 1 of this application;

[0045] Figure 3 This is the plasmid map of pCAS1yl-ku80 in Example 2 of this application;

[0046] Figure 4 This is the plasmid map of pUC19-ku80-up-down in Example 2 of this application;

[0047] Figure 5 This is the plasmid map of pUC19-pEXP-RtTAL-lip2t in Example 3 of this application;

[0048] Figure 6 This is the plasmid map of pUC19-pGPD-At4CL-xpr2t in Example 3 of the present application;

[0049] Figure 7 This is the plasmid map of pUC19-pTEF-VvSTS-mig1t in Example 3 of this application;

[0050] Figure 8 This is the plasmid map of pUC19-pTEF-AtCOMT-mig1t in Example 3 of this application;

[0051] Fig. 9 This is the pUC19-rDNA-up-down plasmid map in Example 3 of this application;

[0052] Fig.10 This is the plasmid map of pCAS1yl-rDNA in Example 3 of this application;

[0053] Fig.11 This is the plasmid map of pUC19-rDNA-up-RtTAL-At4CL-rDNA-down in Example 3 of the present application;

[0054] Fig.12 This is the plasmid map of pUC19-rDNA-up-RtTAL-At4CL-VvSTS-rDNA-down in Example 3 of the present application;

[0055] Fig.13 This is the plasmid map of pUC19-rDNA-up-RtTAL-At4CL-VvSTS-AtCOMT-rDNA-down in Example 3 of the present application;

[0056] Fig.14 The whole process of pterostilbene biosynthesis by the engineering bacteria GY003 in the effect example of this application;

[0057] Fig.15 This is a liquid chromatogram of pterostilbene synthesized by the engineering bacteria GY003 in the effect example of this application;

[0058] Fig.16 This is the mass spectrum of pterostilbene synthesized by the engineering bacteria GY003 in the effect example of this application. DETAILED DESCRIPTION

[0059] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with specific embodiments.

[0060] Unless otherwise specified, the materials, reagents, etc. used in the embodiments, comparative examples and experimental examples can be obtained from commercial sources.

[0061] Escherichia coli DH5α (hereinafter referred to as DH5α) is used for vector construction and can be obtained from commercial channels.

[0062] Yarrowia lipolytica Po1g (hereinafter referred to as Po1g) was constructed according to the following reference: Madzak C, Tréton B, Blanchin-Roland S. Strong hybrid promoters and integrative expression / secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J Mol Microbiol Biotechnol. 2000, 2, 207-216.

[0063] Plasmids pCAS1yl-trp and pUC19 were provided by Hangzhou Baosai Biotechnology Co., Ltd.

[0064] High-fidelity PCR mix was purchased from Sangon Biotech (Shanghai) Co., Ltd. with a catalog number of B639300. The PCR amplification system is shown in Table 1 , and the amplification conditions are shown in Table 2 .

[0065] Table 1 PCR amplification system

[0066]

[0067] Table 2 PCR amplification conditions

[0068] step Temperature(℃) time 1 98 30s 2 98 10 3 (Tm+3) 5s 4 72 5-10s / kb 5 72 30s

[0069] Plasmid extraction kit, DNA purification kit, gel recovery kit, and bacterial genomic DNA extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd.

[0070] LB medium (for DH5α growth): 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, sterilized at 121°C for 20 min; if prepared as solid culture medium, add 15-20 g / L agar powder.

[0071] SOC medium (for DH5α transformation): 20 g / L tryptone, 5 g / L yeast extract, 0.5 g / L sodium chloride, 10 mL 250 mM potassium chloride solution, 10 mL 20 mM glucose solution, 10 mL 10 mM magnesium chloride, sterilize at 121°C for 20 min; if prepared as solid culture medium, add 15-20 g / L agar powder.

[0072] YPD medium (for growth of Yarrowia lipolytica): 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, sterilized at 121°C for 20 min.

[0073] SD medium (for Yarrowia lipolytica screening): 20g / L glucose, 6.7g / L amino acid-free yeast basic nitrogen source, 0.7g / L defective medium (leucine-free / tryptophan-free / uracil-free), 20g / L agar powder. Glucose needs to be sterilized separately, and the remaining components are mixed and sterilized at 121°C for 20min. If prepared as SD / ΔLeu medium, 100mg / L uracil and 50mgL tryptophan must also be added.

[0074] In the following examples, the culture, storage and competent transformation methods of DH5α are as follows:

[0075] 1. Cultivation: Select the strain and streak or spread it on a solid LB plate with corresponding resistance, then invert and culture it in a constant temperature incubator at 37°C for 12-16 hours, perform colony PCR identification or seal it with sealing film and store it inverted in a refrigerator at 4°C;

[0076] Pick a freshly recovered E. coli single clone from the LB solid medium plate, inoculate it into a certain volume of LB liquid medium, add corresponding resistance as needed, and culture it at 37°C and 220rpm for 12-16h;

[0077] 2. Storage: Take 500 μL of fresh overnight culture seed solution, add it to a cryotube containing 500 μL of 50% sterilized glycerol, and store it in a -80℃ ultra-low temperature refrigerator;

[0078] 3. Transformation: Add the transformation system to DH5α competent medium melted on ice, ice bath for 30min, heat shock at 42℃ for 90s, ice bath again for 2min, then add 900μL SOC medium preheated at 37℃, and culture at 37℃, 220rpm for 45min; take 100μL of bacterial solution and add it to LB solid medium containing corresponding antibiotics, culture at 37℃ for 12-16h, pick a certain number of clones from the plate for colony PCR identification, and streak them onto the corresponding plates, and culture at 37℃ for 12-16h for use.

[0079] Homologous recombination technology IIOne Step Cloning Kit (single fragment) or Cl The ligation was carried out according to the instructions of MultiS One Step Cloning Kit (multi-fragment). The ligation system used is shown in Table 3. The ligation conditions were 37°C for 30 min. IIOne Step Cloning Kit, MultiS One Step Cloning Kit was provided by Nanjing Novozyme Biotechnology Co., Ltd., with catalog numbers C112 and C113;

[0080] The plasmid or expression gene cluster assembled by homologous recombination technology is transferred into DH5α, and cloned PCR amplification and agarose gel electrophoresis are performed using corresponding primers. After positive transformants are identified, they are stored or activated according to the above method for future use;

[0081] 2×Rapid Taq Master Mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd. with the catalog number P222-01. The PCR amplification system is shown in Table 4 , and the amplification conditions are shown in Table 5 ;

[0082] Cloning PCR method: pick a single transformant into the PCR reaction solution and react; agarose gel electrophoresis is performed according to the operation in "Molecular Biology Experiment".

[0083] Table 3 Homologous recombination ligation system

[0084] Reagents Dosage 5×CE II / MultiS Buffer 2μL Exnase II / MultiS 1μL Vector DNA 0.03pmol Fragmented DNA 0.03 / 0.06pmol <![CDATA[ddH2O]]> Make up to 10 μL

[0085] Table 4 Cloning PCR amplification system

[0086] Reagents Dosage 2× Rapid Taq Master Mix 25μL 10 μM upstream primer 2μL 10 μM downstream primer 2μL <![CDATA[ddH2O]]> Make up to 50 μL

[0087] Table 5 Cloning PCR amplification conditions

[0088] step Temperature(℃) time 1 95 3min 2 95 15s 3 (Tm) 15s 4 72 15s / kb 5 72 5min

[0089] In the following examples, the activation, culture, preservation, and competent cell preparation and transformation methods of Yarrowia lipolytica are as follows:

[0090] 1. Activation and culture: Take out the strain from the -80℃ refrigerator, pick the bacterial liquid and streak it into YPD or SD solid culture medium, incubate it at 30℃ for 24-36h, pick a single colony with good growth and inoculate it into YPD or SD / ΔLeu liquid culture medium, and culture it at 30℃ and 250rpm for 16-24h to obtain Yarrowia lipolytica liquid;

[0091] 2. Storage method: Same as that of DH5α;

[0092] 3. Preparation and transformation of competent cells: Follow the instructions of the Zymogen Frozen-EZ Yeast Transformation Kit II, and modify some steps as follows:

[0093] (1) For DNA used for transformation: extract the plasmid from 10 mL of bacterial solution containing the plasmid, and elute it with no more than 50 μL of ddH2O. Determine the concentration using an ultra-micro spectrophotometer to control the DNA concentration to be above 400 ng / μL; extract the plasmid from 5 mL of bacterial solution containing the integrated fragment plasmid, and determine the concentration using a spectrophotometer. Take 10 μg of the plasmid and perform double digestion with restriction endonuclease Not I at 37°C. Recover the target fragment from the gel and elute it with no more than 20 mL of ddH2O to obtain a linearized integrated fragment, the concentration of which is generally controlled to be above 400 ng / μL and stored at -20°C for future use;

[0094] (2) Preparation of competent cells: Pick a freshly activated single clone and place it in 10 mL of YPD culture medium. Culture it at 30°C and 250 rpm for 12-16 h. 600 When the value reaches 0.6-1.0, prepare competent cells according to the instructions of the kit. The competent cells are packaged in 20 μL per tube and stored at -80°C after being wrapped in paper towels;

[0095] (3) Transformation: Perform transformation according to the instructions of the kit. During transformation, the DNA fragment, plasmid and competent medium are mixed in a ratio of 400 ng:400 ng:20 μL in 200 μL solution III and incubated;

[0096] (4) Identification of transformants: Pick an appropriate amount of bacteria from the transformation plate and resuspend them in a PCR tube containing 50 μL of 20 mM NaOH (0.08%). Use a sterile pipette tip to pick up a small amount of the bacteria to be tested and pipette them into the tube to mix them evenly. Place them in a 95°C water bath for 10 min to rupture the cell wall and remove them. After cooling on ice for 2-5 min, store them at -20°C for 10 min, centrifuge them at 12,000 rpm for 10 min, transfer them to a clean 1.5 mL EP tube, and take 1 μL of the supernatant as a template for colony PCR identification (the colony PCR method is the same as the above-mentioned PCR amplification method);

[0097] (5) Loss of pCAS1yl plasmid in engineered bacteria: Pick a positive single clone and culture it overnight in 10 mL YPD liquid medium for 16 h. After appropriate dilution, take 100 μL and spread it on a YPD plate and culture it at 30°C for 2-3 days. Pick a larger single clone and streak it on a SD plate and SD / ΔLeu plate at the same time. Continue to culture it at 30°C for 2 days. The engineered bacteria that have successfully lost the pCAS1yl plasmid can be obtained based on the growth conditions of the two plates.

[0098] Pterostilbene standard was purchased from Sigma with the catalog number P1499.

[0099] Example 1

[0100] This embodiment provides an engineering bacterium GY001 for synthesizing pterostilbene and a construction method thereof, wherein the construction method comprises the following steps:

[0101] 1.1 Using pCAS1yl-trp plasmid as template, PCR amplification was performed using primer pairs pCAS1yl-F / pCAS1yl-ku70-R and pCAS1yl-ku70-F / pCAS1yl-R, respectively. After gel recovery, pCAS1yl-ku70-1 and pCAS1yl-ku70-2 fragments were obtained. The pCAS1yl-ku70 plasmid was obtained by homologous recombination technology assembly, transformation, identification, and plasmid extraction (see the plasmid map for details). Figure 1 );

[0102] 1.2 Using pUC19 plasmid (empty vector) as template, use primer pair pUC19-tong-F / pUC19-tong-R to perform PCR amplification to obtain linearized pUC19 fragment;

[0103] 1.3 Using Po1g genome as template, PCR amplification was performed using primer pairs ku70-1-NotI-F / ku70-up-down-R and ku70-down-F / ku70-1-NotI-R, and ku70up and ku70down fragments were obtained after gel recovery;

[0104] 1.4 The linearized pUC19 fragment obtained in step 1.2 and the ku70up and ku70down fragments obtained in step 1.3 were assembled, transformed, identified, and plasmids were extracted by homologous recombination technology to obtain the pUC19-ku70-up-down plasmid (see the plasmid map) Figure 2 );

[0105] 1.5 The pCAS1yl-ku70 plasmid obtained in step 1.1 and the pUC19-ku70-up-down plasmid obtained in step 1.4 were digested with NotⅠ to obtain linearized pCAS1yl-ku70 plasmid and linearized pUC 19-ku70-up-down plasmid respectively;

[0106] 1.6 The linearized pCAS1yl-ku70 plasmid, pUC19-ku70-up-down plasmid and pCAS1yl-gRNA plasmid obtained in step 1.5 were transformed into Po1g, and the positive transformants were identified by colony PCR. The obtained positive transformants were the engineered bacteria GY001 with the ku70 gene knocked out;

[0107] The above primer sequences are shown in Table 6.

[0108] Table 6 Primers used in the construction of engineering bacteria GY001

[0109]

[0110] Example 2

[0111] This embodiment provides a pterostilbene engineered bacterium GY002 and a construction method thereof, wherein the construction method comprises the following steps:

[0112] 2.1 Using the pCAS1yl-trp plasmid as a template, PCR amplification was performed using primer pairs pCAS1yl-F / pCAS1yl-ku80-R and pCAS1yl-ku80-F / pCAS1yl-R, respectively. After gel recovery, pCAS1yl-ku80-1 and pCAS1yl-ku80-2 fragments were obtained. The pCAS1yl-ku80 plasmid was obtained by homologous recombination technology assembly, transformation, identification, and plasmid extraction (see the plasmid map for details). Figure 3 );

[0113] 2.2 Obtain the linearized pUC19 fragment according to the method of step 1.2 in Example 1;

[0114] 2.3 Using Po1g genome as template, PCR amplification was performed using primer pairs ku80-1-NotI-F / ku80-up-down-R and ku80-down-F / ku80-1-NotI-R, and ku80up and ku80down fragments were obtained after gel recovery;

[0115] 2.4 The linearized pUC19 fragment obtained in step 2.2 and the ku80up and ku80down fragments obtained in step 2.3 were assembled, transformed, identified, and plasmids were extracted by homologous recombination technology to obtain the pUC19-ku80-up-down plasmid (see the plasmid map for details). Figure 4 );

[0116] 2.5 The pCAS1yl-ku80 plasmid obtained in step 2.1 and the pUC19-ku80-up-down plasmid obtained in step 2.4 were used according to the method in step 1.5 of Example 1 to obtain a linearized pCAS1yl-ku80 plasmid and a linearized pUC19-ku80-up-down plasmid;

[0117] 2.6 The linearized pCAS1yl-ku80 plasmid and pUC19-ku80-up-down plasmid obtained in step 2.5 were transformed into the engineering bacteria GY001 obtained in Example 1, and the positive transformants were identified and screened by colony PCR. The obtained positive transformants were the engineering bacteria GY002 with the ku80 gene knocked out;

[0118] The primer sequences used above are shown in Table 7.

[0119] Table 7 Primers used in the construction of engineering bacteria GY002

[0120]

[0121] Example 3

[0122] This embodiment provides a pterostilbene engineered bacterium GY003 and a construction method thereof, wherein the construction method comprises the following steps:

[0123] 3.1 Tyrosine ammonia lyase TAL (hereinafter referred to as TAL, derived from Rhodosporidium toruloides), p-coumarate CoA ligase 4CL (hereinafter referred to as 4CL, derived from Arabidopsis thaliana), stilbene synthase STS (hereinafter referred to as STS, derived from wine grapes) and caffeic acid-O-methyltransferase COMT (hereinafter referred to as COMT, derived from Arabidopsis thaliana) were codon optimized for expression in Yarrowia lipolytica host, and the optimized sequences were respectively commissioned to be synthesized by GenScript Biotech Co., Ltd. to obtain plasmid pUC57-RtTAL containing the optimized TAL sequence, plasmid pUC57-At4CL containing the optimized 4CL sequence, plasmid pUC57-VvSTS containing the optimized STS sequence, and plasmid pUC57-AtCOMT containing the optimized COMT sequence; the optimized TAL sequence is shown in SEQ ID NO.1, the optimized 4CL sequence is shown in SEQ ID NO.2, the optimized STS sequence is shown in SEQ ID NO.3, and the optimized COMT is shown in SEQ ID NO.4;

[0124] 3.2 Obtain the linearized pUC19 fragment according to the method of step 1.2 in Example 1;

[0125] 3.3 Using Po1g genome as template, PCR amplification was performed with primer pairs EXP-F / EXP-R, lip2t-F / lip2t-R, GPD-F / GPD-R, xpr2t-F / xpr2t-R, TEF-F / TEF-R and mig1t-F / mig1t-R, and the promoters pEXP, pGPD, pTEF and terminators lip2t, xpr2t, mig1t were obtained after gel recovery;

[0126] 3.4 Using the pUC57-RtTAL plasmid obtained in step 3.1 as a template, use the primer pair RtTAL-F / RtTA LR to perform PCR amplification, and obtain the RtTAL fragment after gel recovery;

[0127] 3.5 The linearized pUC19 fragment obtained in step 3.2, the promoter pEXP and terminator lip2t obtained in step 3.3, and the RtTAL fragment obtained in step 3.4 were assembled, transformed, identified, and plasmids were extracted by homologous recombination technology to obtain the pUC19-pEXP-RtTAL-lip2t plasmid with the RtTAL expression gene cluster (see the plasmid map for details). Figure 5 );

[0128] 3.6 Using the pUC57-At4CL plasmid obtained in step 3.1 as a template, the primer pair At4CL-F / At4CL-R was used for PCR amplification, and the At4CL fragment was recovered from the gel; the linearized pUC19 fragment obtained in step 3.2, the promoter pGPD and terminator xpr2t obtained in step 3.3, and the At4CL fragment were prepared according to the method in step 3.5 to prepare the pUC19-pGPD-At4CL-xpr2t plasmid with the At4CL expression gene cluster (see the plasmid map for details). Figure 6 );

[0129] 3.7 Using the pUC57-VvSTS plasmid obtained in step 3.1 as a template, PCR amplification was performed using primers VvSTS-F / VvST SR, and the VvSTS fragment was recovered from the gel; the linearized pUC19 fragment obtained in step 3.2, the promoter pTEF and terminator mig1t obtained in step 3.3, and the VvSTS fragment were prepared according to the method in step 3.5 to form a pUC19-pTEF-VvSTS-mig1t plasmid with a VvSTS expression gene cluster (see the plasmid map for details). Figure 7 );

[0130] 3.8 Using the pUC57-AtCOMT plasmid obtained in step 3.1 as a template, the primer pair AtCOMT-F / AtCOMT-R was used for PCR amplification, and the AtCOMT fragment was recovered from the gel; the linearized pUC19 fragment obtained in step 3.2, the promoter pTEF and terminator mig1t obtained in step 3.3, and the AtCOMT fragment were prepared according to the method in step 3.5 to prepare the pUC19-pTEF-AtCOMT-mig1t plasmid with the AtCOMT expression gene cluster (see the plasmid map for details). Figure 8 );

[0131] 3.9 PCR amplification was performed using the Po1g genome as a template using primers rDNA-1-NotI-F / rDNA-1-BEK-R and rDNA-2-BEK-F / rDNA-2-NotI-R. After gel recovery, rDNAup and rDNAdown fragments were obtained. The linearized pUC19 fragment obtained in step 3.2, rDNAup and rDNAdown fragments were prepared according to the method in step 3.5 to prepare pUC19-rDNA-up-down plasmid (see the plasmid map for details). Fig. 9 );

[0132] 3.10 Using the pCAS1yl-trp plasmid as a template, PCR amplification was performed using primer pairs pCAS1yl-F / pCAS1yl-rDNA AR and pCAS1yl-rDNA-F / pCAS1yl-R, respectively. After gel recovery, pCAS1yl-rDNA-1 and pCAS1yl-rDNA-2 fragments were obtained. The pCAS1yl-rDNA plasmid was prepared according to the method in step 3.5 (see the plasmid map for details). Fig.10 );

[0133] 3.11 Using the pUC19-PEXP-RtTAL-lip2t plasmid obtained in step 3.5 as a template, use the primer pair rDNA-BamHI-F / rDNA-BamHI-R to perform PCR amplification, and obtain PEXP-RtTAL-lip2t fragment 1 after gel recovery;

[0134] 3.12 The pUC19-rDNA-up-down plasmid obtained in step 3.9 was digested with BamHI to obtain a linearized pUC19-rDNA-up-down plasmid;

[0135] 3.13 Using the pUC19-pEXP-RtTAL-lip2t plasmid obtained in step 3.5 as a template, PCR amplification was performed using primer pair rD rDNA-BamHI-F / lip2t-GPD-R, and pEXP-RtTAL-lip2t fragment 2 was obtained after gel recovery; using the pUC19-pGPD-At4CL-xpr2t plasmid obtained in step 3.6, PCR amplification was performed using primer pair GPD-F / rDNA-BamHI-R, and pGPD-At4CL-xpr2t fragment was obtained after gel recovery;

[0136] 3.14 The linearized pUC19-rDNA-up-down plasmid obtained in step 3.12, the pEXP-RtTAL-lip2t fragment 2 obtained in step 3.13, and the pGPD-At4CL-xpr2t fragment were prepared according to the method in step 3.5 to form the pUC19-rDNA-up-RtTAL-At4CL-rDNA-down plasmid (see the plasmid map for details). Fig.11 );

[0137] 3.15 Using the pUC19-pTEF-VvSTS-mig1t plasmid obtained in step 3.7 as a template, PCR amplification was performed using primer pair UC19-EcoRI-F / UC19-EcoRI-R, and pTEF-VvSTS-mig1t fragment 1 was obtained after gel recovery;

[0138] 3.16 The pUC19-rDNA-up-RtTAL-At4CL-rDNA-down plasmid obtained in step 3.14 was digested with EcoRI to obtain a linearized pUC19-rDNA-up-RtTAL-At4CL-rDNA-down plasmid; the linearized pUC19-rDNA-up-RtTAL-At4CL-rDNA-down plasmid and the PTEF-VvSTS-mig1t fragment obtained in step 3.15 were prepared into a pUC19-rDNA-up-RtTAL-At4CL-Vv STS-rDNA-down plasmid according to the method of step 3.5 (see the plasmid map for details). Fig.12 );

[0139] 3.17 Using the pUC19-PTEF-VvSTS-mig1t plasmid obtained in step 3.7 as a template, PCR amplification was performed using primer pairs rDNA-BamHI-F / mig1t-TEF-R and TEF-F / rDNA-BamHI-R, and the PTEF-VvSTS-mig1t fragment 2 was obtained after gel recovery; using the pUC19-PTEF-AtCOMT-mig1t plasmid obtained in step 3.8 as a template, PCR amplification was performed using primer pairs rDNA-BamHI-F / mig1t-TEF-R and TEF-F / rDNA-BamHI-R, and the PTEF-AtCOMT-mig1t fragment was obtained after gel recovery;

[0140] 3.18 The pUC19-rDNA-up-RtTAL-At4CL-VvSTS-rDNA-down plasmid obtained in step 3.16 was digested with EcoRI to obtain a linearized pUC19-rDNA-up-RtTAL-At4CL-VvSTS-rDNA-down plasmid; the linearized pUC19-rDNA-up-RtTAL-At4CL-VvSTS-rDNA-down plasmid, the PTEF-VvSTS-mig1t fragment 2 obtained in step 3.17, and the PTEF-AtCOMT-mig1t fragment were prepared according to the method of step 3.5 to prepare the pUC19-rDNA-up-RtTAL-At4CL-VvSTS-AtCOMT-rDNA-down plasmid (see the plasmid map for details). Fig.13 );

[0141] 3.19 The pUC19-rDNA-up-RtTAL-At4CL-VvSTS-AtCOMT-rDN A-down plasmid obtained in step 3.18 was double-digested with NotⅠ to obtain linearized pUC19-rDNA-up-RtTAL-At4CL-Vv The STS-AtCOMT-rDNA-down plasmid and the pCAS1yl-rDNA plasmid obtained in step 3.10 are co-transformed into the engineered bacteria GY002 obtained in Example 2, and the obtained transformants are subjected to colony PCR amplification, and the PCR amplification products are subjected to agarose gel electrophoresis detection and sequencing. The obtained sequence is consistent with the expected sequence, and the obtained positive transformant is determined to be the engineered bacteria GY003. The engineered bacteria GY003 can be used for synthesizing pterostilbene after the pCAS1yl plasmid loss treatment is performed on the engineered bacteria GY003. The engineered bacteria GY003 is the Yarrowia lipolytica engineered bacteria for biosynthesis of pterostilbene. The engineered bacteria GY003 is deposited in the Guangdong Provincial Microbiological Culture Collection Center, with a deposit date of May 31, 2024, a deposit number of GDMCC No: 64708, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province;

[0142] The primer sequences used in the above steps are shown in Table 8.

[0143] Table 8 Primers used in the construction of engineering bacteria GY003

[0144]

[0145]

[0146] Effect example

[0147] The engineered bacteria GY003 for synthesizing pterostilbene was fermented in a shake flask and the pterostilbene content was detected by liquid chromatography. The specific scheme is as follows:

[0148] The engineered bacteria GY003 was inoculated into YPD medium and fermented in a shake flask at 30°C and 250rpm for 36h to obtain a culture. The process of GY003 biosynthesis of pterostilbene is shown in Fig.14 ;

[0149] The culture obtained above was extracted with an equal volume of ethyl acetate, the ethyl acetate was blown dry and resuspended with 800 μL of chromatography-grade methanol, centrifuged at 12000 rpm for 10 min, 500 μL was taken and filtered through a 0.22 μm filter membrane and then detected by liquid chromatography;

[0150] The liquid chromatography parameters are as follows: the chromatograph is Agilent 1260 Infinity II, the detector is a differential index detector, the chromatographic column is Aminex HPX-87H (Bio-Rad), the mobile phase is acetonitrile and water containing 0.05% trifluoroacetic acid, the flow rate is 1 mL / min, the column temperature is 30°C, the injection volume is 10 μL, the program is: 20%-100% acetonitrile for 20 min, 100% acetonitrile for 5 min, the flow rate is 1 mL / min; the internal standard method is used to determine the yield of pterostilbene, and the liquid chromatogram is shown in Fig.15 , it was calculated that the pterostilbene content obtained by GY003 shake flask fermentation was 150 mg / L.

[0151] The prepared GY003 culture samples were subjected to liquid chromatography-mass spectrometry, and electrospray ionization (ESI) was performed in positive ion mode using an ion trap mass spectrometer coupled to an ESI (electrospray ionization) source using a Thermo U3000-LTQ XL (Thermo Scientific, USA) system. The results are shown in Fig.16 :

[0152] The liquid chromatography parameters were as follows: 10 μL of sample was injected into a C18 column (2.1×150 mm; 2.5 μm particle size); the mobile phase for gradient elution was acetonitrile (0.1% formic acid) as component A and water (0.1% formic acid) as component B, with a flow rate of 0.3 mL / min. The gradient elution program started with 5% A, increased A to 100% in the next 15 min, maintained at 100% A for 5 min, and returned to the initial condition (5% A) in the next 5 min;

[0153] The mass spectrometry parameters were as follows: the ion source was ESI source, sensitivity mode, capillary voltage was (+) 3.0 kV, (-) 2.5 kV, ion source temperature was 100 °C, cone gas flow rate was 50 L / h, degassing flow rate was 1000 L / h, collision voltage was (low) 6 V, (high) 10-60 V, cone voltage was 30 V;

[0154] Mass spectra such as Fig.16 As shown, the [M+H] + The ion peak m / z=257.1234, that is, the molecular weight of the product is 256.1234, and it can be determined that the obtained product is pterostilbene.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. An engineered strain of Yarrowia lipolytica, characterized in that The engineered Yarrowia lipolytica bacteria are deposited in Guangdong Microbiological Culture Collection Center on May 31, 2024, and the deposit number is GDMCC No: 64708.

2. The method for constructing the Yarrowia lipolytica engineered bacteria according to claim 1, characterized in that: The following steps are involved: A1. Knock out the ku70 gene in Po1g of Yarrowia lipolytica using gene editing technology to obtain engineered bacteria A; A2, using gene editing technology to knock out the ku80 gene in the engineered bacteria A obtained in step A1 to obtain engineered bacteria B; A3. The pterostilbene integrated expression gene cluster is transferred into the engineered bacteria B obtained in step A2 to obtain an engineered bacteria C, wherein the engineered bacteria C is an engineered bacteria of Yarrowia lipolytica for synthesizing pterostilbene; the pterostilbene integrated expression gene cluster is composed of tyrosine ammonia lyase TAL shown in SEQ ID NO.1, coumaric acid coenzyme A ligase 4CL shown in SEQ ID NO.2, stilbene synthase STS shown in SEQ ID NO.3 and caffeic acid-O-methyltransferase COMT shown in SEQ ID NO.

4.

3. The construction method according to claim 2, characterized in that: In step A3, the pterostilbene integrated expression gene cluster is prepared by the following steps: B1. Using homologous recombination technology, the promoter and terminator of Yarrowia lipolytica, the coding gene of tyrosine ammonia lyase TAL, and the linearized vector were assembled into the RtTAL expression gene cluster; B2, using homologous recombination technology to assemble the promoter and terminator carried by Yarrowia lipolytica, the encoding gene of p-coumarate coenzyme A ligase 4CL, and the linearized vector into an At4CL expression gene cluster; B3, using homologous recombination technology to assemble the promoter and terminator carried by Yarrowia lipolytica, the coding gene of stilbene synthase STS, and the linearized vector into a VvSTS expression gene cluster; B4, using homologous recombination technology, the promoter and terminator of Yarrowia lipolytica, the coding gene of caffeic acid-O-methyltransferase COMT, and the linearized vector were assembled into an AtCOMT expression gene cluster; B5. Assemble the RtTAL expression gene cluster obtained in step B1, the At4CL expression gene cluster obtained in step B2, the VvSTS expression gene cluster obtained in step B3 and the AtCOMT expression gene cluster obtained in step B4 into the RtTAL-At4CL-VvSTS-AtCOMT expression gene cluster by homologous recombination technology. The obtained RtTAL-At4CL-VvSTS-AtCOMT expression gene cluster is the pterostilbene integrated expression gene cluster.

4. The construction method according to claim 3, characterized in that: Including at least one of the following (I)-(II): (I) In steps B1-B4, the promoter carried by the Yarrowia lipolytica is at least one of pTEF1, pEXP1 and pGPD2; the terminator carried by the Yarrowia lipolytica is at least one of mig1t, lip2t and xpr1t; (II) In steps B1-B4, the coding gene sequences of tyrosine ammonia lyase TAL, p-coumarate coenzyme A ligase 4CL, stilbene synthase STS and caffeic acid-O-methyltransferase COMT are all codon-optimized using Yarrowia lipolytica as the host strain.

5. The construction method according to claim 3, characterized in that: In step A3, the pterostilbene integration expression gene cluster and the pCAS1yl-rDNA plasmid are co-transformed into the engineering bacteria B, and the rDNA gene sequence in the pCAS1yl-rDNA plasmid is shown in SEQ ID NO.

7.

6. Use of the engineered Yarrowia lipolytica bacteria as claimed in claim 1 in the synthesis of pterostilbene.

7. A method for synthesizing pterostilbene, characterized in that: The Yarrowia lipolytica engineered bacteria according to claim 1 is fermented with a culture medium containing glucose in a bioreactor.

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