Construction method and application of recombinant yarrowia lipolytica with high hydroxytyrosol yield

Abstract

The invention provides a construction method and application of recombinant yarrowia lipolytica with high hydroxytyrosol yield, and belongs to the field of bioengineering. The recombinant Yarrowia lipolytica capable of producing the hydroxytyrosol at the high yield is named as a Yarrowia lipolytica XJ-HT strain, and after the recombinant Yarrowia lipolytica XJ-HT strain is subjected to fed-batch fermentation, the hydroxytyrosol can be synthesized at a relatively high level. The construction method of the recombinant yarrowia lipolytica is easy to operate, and efficient synthesis of hydroxytyrosol can be achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and particularly relates to a construction method and application of a recombinant Yarrowia lipolytica with high yield of hydroxytyrosol. BACKGROUND

[0002] Hydroxytyrosol, scientific name: 3,4-dihydroxyphenylethanol, mainly exists in plants olives, and is considered to be one of the strongest natural antioxidants known in the world. Studies have shown that hydroxytyrosol has various biological activities, antioxidant: hydroxytyrosol has outstanding free radical scavenging ability and oxidative stress inhibition effect, and its antioxidant efficiency is significantly better than that of common antioxidants, which makes it have important application value in the field of functional cosmetics and dietary supplements; cardiovascular protection: hydroxytyrosol shows significant effect in the prevention and treatment of cardiovascular diseases such as atherosclerosis and hypertension; promote bone health: regulate bone metabolism related signal pathways, and have a positive effect on preventing bone system diseases such as osteoporosis; anti-aging: can eliminate free radicals in the body, restore the health status of human viscera and organs, and delay aging; anti-tumor: promote recovery in later stages of cancer and improve the effect of chemotherapy. Hydroxytyrosol is considered to be the key of the “Mediterranean” diet, and is known as “liquid platinum” and “Mediterranean nectar”. At present, hydroxytyrosol has passed the safety certification of the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA), and is widely used in food, medicine and cosmetics and other fields.

[0003] Traditionally, hydroxytyrosol is mainly extracted from olives or olive processing wastewater, but this production method is heavily dependent on plant resources, and the operation process is complex, the yield is low, the energy consumption is high, the acidity is strong, and the time is long. On the other hand, the chemical synthesis of hydroxytyrosol is mainly through lithium aluminum hydride, TMSCHNH / sodium borohydride, tetra-n-butyl borane amine / iodomethane, and excess sodium borohydride to reduce 3,4-dihydroxyphenylacetic acid or to oxidize tyrosol with H2O2 under the catalysis of clay loaded with aluminum and iron (see Chinese patent CN102344344A). The above routes have poor raw material stability and high cost, and are not suitable for industrial production. In addition, since hydroxytyrosol is an important raw material for food, medicine and health products, the product obtained by chemical method is not popular with people.

[0004] Compared with plant extraction method and chemical synthesis, microbial fermentation method has the advantages of short growth cycle, all-weather production, low cost, mild conditions and less environmental pollution, and is a research hotspot. At present, the chassis cells for producing hydroxytyrosol by microorganisms mainly concentrate in Saccharomyces cerevisiae and Escherichia coli. However, Saccharomyces cerevisiae will have glucose effect, and Escherichia coli is easy to be contaminated by phages, which restricts the large-scale production of hydroxytyrosol. As a non-conventional oleaginous yeast, Yarrowia lipolytica has a natural Ehrlich pathway and a high-flux pentose phosphate pathway, and is a Crabtree-negative yeast very suitable for industrial production, so it is an economic and efficient way to produce hydroxytyrosol by Yarrowia lipolytica. In the prior art, there is a lack of microorganisms capable of efficiently producing hydroxytyrosol. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a construction method and application of recombinant Yarrowia lipolytica for high-yield hydroxytyrosol in view of the deficiencies of the prior art.

[0006] To solve the above technical problems, the present application discloses a construction method and application of recombinant Yarrowia lipolytica for high-yield hydroxytyrosol. The specific technical solutions are as follows: The present application provides a construction method of recombinant Yarrowia lipolytica for high-yield hydroxytyrosol. The recombinant Yarrowia lipolytica expresses double-component riboflavin-dependent monooxygenase oxidase encoding genes HPAB , double-component riboflavin-dependent monooxygenase reductase encoding genes HPAC , DAHP synthase mutant encoding genes AroG S180F and double-function shikimate mutase / prephenate dehydrogenase mutant encoding genes TyrA M53I / A354V ; wherein the HPAB , HPAC , AroG S180F and TyrA M53I / A354V are all derived from Escherichia coli Escherichia coli . Preferably, the nucleotide sequences of the HPAB , HPAC , AroG S180F and TyrA M53I / A354V are obtained after codon optimization, and the sequences are shown in SEQ ID No. 1~4, respectively.

[0007] Wherein, the HPAB and HPACThe expression is driven by a growth-dependent promoter. Preferably, the growth-dependent promoter comprises UP12, the nucleotide sequence of which is shown as SEQ ID No. 10.

[0008] The recombinant Yarrowia lipolytica is further modified by any one or a combination of a1~a7: a1, expressing a phosphoketolase encoding gene from Leuconostoc citreum Leuconostoc mesenteroides ; LmPK ; a2, expressing a phosphotransacetylase encoding gene from Clostridium kluyveri Clostridium kluyveri ; CkPTA ; a3, expressing an aromatic aldehyde synthase encoding gene from Petroselinum crispum Petroselinum crispum ; PcAAS ; a4, expressing a hemoglobin encoding gene from Vitreoscilla Vitreoscilla stercoraria ; VsVHB ; the VsVHB can significantly improve the oxygen uptake capacity of the host under low oxygen conditions, and improve cell growth and oxygen-containing compound production.

[0009] a5, overexpressing an endogenous phenylpyruvate decarboxylase encoding gene from Yarrowia lipolytica Yarrowia lipolytica ; YlARO10 ; a6, knocking out a prephenate dehydratase encoding gene PHA2 ; a7, knocking out an anthranilate synthase encoding gene TRP3.

[0010] The nucleotide sequence is obtained after codon optimization, and the sequences are shown as SEQ ID No. 5~8, respectively; the LmPK, CkPTA, PcAAS, VsVHB gene number is YALI1_D08884g; the YlARO10 gene number is YALI1_B22549g; the PHA2 gene number is YALI1_D13963g. TRP3 Preferably, the present application provides a construction method of a recombinant Yarrowia lipolytica, which expresses

[0011] , HPAB , HPAC , AroG S180F and TyrA M53I / A354V , the HPAB , HPAC , AroG S180F and TyrAM53I / A354V The nucleotide sequences are shown in SEQ ID No. 1~4 respectively. HPAB and HPAC Expression was driven by the growth-dependent promoter UP12, the nucleotide sequence of which is shown in SEQ ID No. 10; the recombinant Yersinia lipolyticis also underwent all of the following modifications a1~a7: a1, expressing *Leuconostoc mesenteroides* L. mesenteroides The source of the phosphate transketolase encoding gene LmPK ; a2, expressing Clostridium krychnifolium C. kluyveri The source of the phosphoryltransferase encoding gene CkPTA ; a3, meaning parsley P. crispum The source of the aromatic aldehyde synthase encoding gene PcAAS ; a4, expressing *Oscillatoria hygroscopica* V. stercoraria Hemoglobin encoding gene of origin VsVHB ; a5, overexpression of Yersinia lipophila Y. lipolytica Endogenous phenylpyruvate decarboxylase encoding gene YlARO10 ; a6, Knockout of the gene encoding prebenzoic acid dehydratase PHA2 ; a7, knockout of the gene encoding anthranilate synthase TRP3 ; The aforementioned LmPK, CkPTA, PcAAS, VsVHB The nucleotide sequences are as shown in SEQ ID No. 5~8 respectively; YlARO10 The gene number is YALI1_D08884g; the aforementioned PHA2 The gene number is YALI1_B22549g; the aforementioned TRP3 The gene number is YALI1_D13963g.

[0012] More preferably, the recombinant Yersinia lipophila originates from a strain that has been knocked out. KU70 Yeast lipolysis Yarrowia lipolytica Po1f. Po1f was purchased from the American Culture Collection (ATCC) under the number ATCC MYA-2613.

[0013] The coding gene is integrated into Yersinia lipophila. Yarrowia lipolyticaThe integration site on the genome is selected from any one or more of the A08 site, 26s rDNA site, IntA site, IntB site, IntC site, IntD site, IntE site, IntF site, lip1 site, SCP2 site, or YlSCD site. Preferably, the integration site is the A08 site, IntA site, IntB site, IntC site, IntD site, IntE site, and IntF site of Yarrowia lipolytica.

[0014] In a second aspect, the present application provides the recombinant Yarrowia lipolytica constructed by the construction method of the first aspect.

[0015] In a third aspect, the present application provides the use of the recombinant Yarrowia lipolytica of the second aspect in the fermentation production of hydroxytyrosol. The recombinant Yarrowia lipolytica is inoculated into a fermentation medium, and then cultured at 20-30 ℃ for 120-144 h to obtain hydroxytyrosol. Preferably, the culture is performed at 28-30 ℃ for 5 days, and the fermentation medium comprises glucose 50-100 g / L, yeast extract 5-20 g / L, and peptone 10-30 g / L.

[0016] Advantages: Compared with the prior art, the recombinant Yarrowia lipolytica of the present application Yarrowia lipolytica The XJ-HT strain expresses the bifunctional riboflavin-dependent monooxygenase-encoding gene, DAHP synthase mutant-encoding gene, bifunctional chorismate mutase / prephenate dehydrogenase mutant-encoding gene, phosphotransketolase-encoding gene, phosphotransacetylase-encoding gene, aromatic aldehyde synthase-encoding gene, and hemoglobin-encoding gene, overexpresses the phenylpyruvate decarboxylase-encoding gene, and knocks out the prephenate dehydratase-encoding gene and anthranilate synthase-encoding gene, and uses the characterized Yarrowia lipolytica endogenous growth-dependent promoter to control the expression of the bifunctional riboflavin-dependent monooxygenase-encoding gene, and experiments prove that the recombinant Yarrowia lipolytica realizes the efficient synthesis of hydroxytyrosol after fed-batch fermentation 。 BRIEF DESCRIPTION OF DRAWINGS

[0017] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0018] Figure 1This is a metabolic diagram of hydroxytyrosol synthesis in this invention. Ac-CoA: Acetyl-CoA; AcP: Acetyl phosphate; CHA: Cranialized acid; DAHP: 3-deoxy-D-arabinohepenulose-7-phosphate; DHQ: 3-dehydroquinine; DHS: 3-dehydroshikimate; EPSP: 5-enolpyruvate-shikimate-3-phosphate; E4P: Erythrose-4-phosphate; F1,6BP: Fructose-1,6-bisphosphate; F6P: Fructose-6-phosphate; G3P: Glyceraldehyde-3-phosphate; G6P: Glucosamine 6-phosphate Sugar; Hydroxytyrosol; PEP: Phosphoenolpyruvate; PPA: Prephenylacetic acid; PYR: Pyruvate; RU5P: Ribulose-5-phosphate; R5P: Ribulose-5-phosphate; SHIK: Shikimic acid; S3P: Shikimic acid-3-phosphate; S7P: Sedum heptanose-7-phosphate; Tyrosol: Tyrosol; X5P: Xylulose-5-phosphate; 4HPAA: 4-Hydroxyphenylacetaldehyde; 4HPP: 4-Hydroxyphenylpyruvate.

[0019] Figure 2 For recombinant plasmid pUC-A08- EcAROG S180F The plasmid map.

[0020] Figure 3 The recombinant plasmid pUC-HUH-IntA-UP12- EcHpaB / C The plasmid map.

[0021] Figure 4 Comparison of hydroxytyrosol (HT) content in the fermentation production of recombinant strains 1-12 (YL-1~YL-12). Detailed Implementation

[0022] The present invention will be further described below through specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the genes mentioned are synthesized by Suzhou Genewiz Biotechnology Co., Ltd. unless otherwise specified.

[0023] In the following examples, the formulations of YPD medium, YPD plates containing 5-fluoroorotic acid, screening medium SD-Leu, screening medium SD-Ura, and *Yersinia lipolytica* (YPD) are described. Yarrowia lipolytica For detailed information on Po1f, please refer to Chinese Patent CN120082582A.

[0024] For detailed information on the A08 site integration plasmid, IntA site integration plasmid, IntB site integration plasmid, IntC site integration plasmid, IntD site integration plasmid, IntE site integration plasmid, IntF site integration plasmid, pUC57-HisG-Ura-HisG plasmid, and pUC57-Leu plasmid described in the following embodiments, please refer to Chinese Patent CN120082582A.

[0025] The biosynthetic pathway of hydroxytyrosol described in this application is as follows: Figure 1 As shown.

[0026] Example 1: Amplification of Gene Elements and Preparation of Target Plasmids (I) Preparation of target gene According to Escherichia coli ( E. coli Genes encoding the two-component riboflavin-dependent monooxygenase oxidase component from this source. EcHPAB (GenBank accession number: CAQ34705.1) and reductase-encoding gene EcHPAC The nucleotide sequence of (GenBank accession number: AAR11356.1) was used to synthesize codon-optimized versions by a commissioned company. EcHPAB and EcHPAC (SEQ ID No. 1-2), and inserted into plasmid pUC57, to obtain plasmid pUC57- EcHPAB / C According to Escherichia coli (E. coli) E.coli The DAHP synthase mutant gene from which this is derived. EcAROG S180F The nucleotide sequence of (GenBank accession number: EET3970042.1) was used to synthesize codon-optimized versions by a commissioned company. EcAROG S180F (SEQ ID No. 3), and inserted into plasmid pUC57, to obtain plasmid pUC57- EcAROG S180F According to Escherichia coli (E. coli) E.coli The gene encoding a bifunctional branched acid mutase / prephenylate dehydrogenase mutant from [source missing]. EcTYRA M53I / A354V The nucleotide sequence of (GenBank accession number: CP084207.1) was used to synthesize codon-optimized versions by a commissioned company. EcTYRA M53I / A354V (SEQ ID No. 4), and inserted into plasmid pUC57, to obtain plasmid pUC57- EcTYRA M53I / A354V According to Leuconostoc mesenteroides (… L. mesenteroides Phosphotransketase encoding gene from ) LmPK and Clostridium kuristeum ( C. kluyveriPhosphotransacetase encoding gene from ) CkPTA The nucleotide sequence was used to commission a company to synthesize codon-optimized versions. LmPK (SEQ ID No. 5) and CkPTA (SEQ ID No. 6), and inserted into plasmid pUC57, to obtain plasmid pUC57- LmPK - CkPTA According to parsley ( P. crispum The gene encoding aromatic aldehyde synthase from ( ) PcAAS The nucleotide sequence of (GenBank accession number: AAA33860.1) was used to synthesize codon-optimized versions by a commissioned company. PcAAS (SEQ ID No. 7), and inserted into plasmid pUC57, to obtain plasmid pUC57- PcAAS According to *Vibrio hystericus* (… V. stercoraria Hemoglobin encoding gene from which the source is located. VsVHB The nucleotide sequence of (GenBank accession number: UZP69255.1) was used to synthesize codon-optimized versions by a commissioned company. VsVHB (SEQ ID No. 8), and inserted into plasmid pUC57, to obtain plasmid pUC57- VsVHB.

[0027] by Y. lipolytica Po1f Using Δku70 genomic DNA as a template, corresponding primer pairs were designed, and the target gene encoding phenylpyruvate decarboxylase was obtained by PCR amplification. YlARO10 (Gene ID: YALI1_D08884g), gene encoding cladosoilase. YlARO7 (Gene ID: YALI1_E20751g) and the gene encoding prephenylacetic acid dehydrogenase YlTYR1 (Gene number: YALI1_F23441g), and the gene encoding the branching acid mutase mutant was obtained through point mutation. YlARO7 G139S , YlARO7 G139S The nucleotide sequence is shown in SEQ ID No. 9.

[0028] by Y. lipolytica Using Po1fΔku70 genomic DNA as a template, corresponding primer pairs were designed, and the endogenous promoter P of Yersinia lipolytica was obtained by PCR amplification. TEFin P EXP and the terminator T lip2t T cyc1t T xpr2t T pex10t .

[0029] (II) Construction of recombinant plasmids 1. Recombinant plasmid pUC-A08- EcAROG S180F Construction Recombinant plasmid pUC-A08- EcAROG S180F Using the A08 site integration plasmid as a backbone, [the plasmid] was inserted between the upstream and downstream homologous arms of A08. EcAROG S180F Expression Box (P) TEFin - EcAROG S180F -T xpr2t For details, see [link / details]. Figure 2 .by Y. lipolytica Using Po1f Δku70 genomic DNA as a template, primers in Table 1 were used to amplify the following sequences: EcAROG S180F Expression box promoter P TEFin and Termination T xpr2t Using plasmid pUC57- EcAROG S180F Using the template, amplification was performed using the primers in Table 1. EcAROG S180F Genes were analyzed. The PCR amplification system consisted of: 25 μL Primer STAR Max Premix, 1 μL template, 2 μL primer F, 2 μL primer R, and 20 μL distilled water. Primer STAR Max Premix was purchased from Baori Biotechnology (Beijing) Co., Ltd. The PCR program was: 98 °C denaturation for 10 s, 55 °C annealing for 5 s, and 72 °C extension (extension time = target fragment length / 1 kb, in min), repeated 30 times. Fragments were purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit. The A08 site integration plasmid was purified using restriction endonucleases. SnaB I. After enzyme digestion, the linearized A08 site integrated plasmid was recovered by gel extraction.

[0030] The linearized A08 site integration plasmid and the plasmid constructed in this embodiment were used. EcAROG S180F The components in the gene expression cassette (promoter P) TEFin ,Gene EcAROG S180F and Termination T xpr2t Using the ClonExpress MultiS One StepCloning Kit, one-step cloning can be achieved. EcAROG S180FThe gene expression cassette was inserted between the upstream and downstream homologous arms of the integration plasmid at the A08 site, resulting in a circular recombinant vector. This circular recombinant vector was transformed into *E. coli* DH5α competent cells, and positive recombinant plasmids pUC-A08- were obtained through ampicillin-resistant LB plate selection and colony PCR and sequencing verification. EcAROG S180F Using restriction endonucleases Not Ⅰ For plasmid pUC-A08- EcAROG S180F After enzyme digestion, linearized pUC-A08 was recovered by agarose gel electrophoresis. EcAROG S180F Plasmids are used for yeast transformation.

[0031] Table 1. Construction of recombinant plasmid pUC-A08- EcAROG S180F Primers used

[0032] 2. Recombinant plasmid pUC-HUH-IntA-TEFin- EcHPAB / C Construction Recombinant plasmid pUC-HUH-IntA-TEFin- EcHPAB / C Using the IntA site integration plasmid as a backbone, inserts were placed between the upstream and downstream homologous arms of IntA. EcHPAB Gene expression cassette (P TEFin - EcHPAB -T lip2t )and EcHPAC Gene expression cassette (P TEFin - EcHPAC -T cyc1t Its construction method is similar to that of the recombinant plasmid pUC-A08- EcAROG S180F Similar, the difference lies in... EcAROG S180F The expression box was replaced with EcHPAB / C Expression box, i.e., each element ( EcHPAB The promoter P of the gene expression cassette TEFin ,Gene EcHPAB, EcHPAB Gene expression cassette terminator T lip2t , EcHPAC Gene expression cassette promoter P TEFin ,Gene EcHPAC and EcHPAC Gene expression cassette terminator T cyc1t The primers and templates used for amplification differ; see Table 2 for details. The IntA site integration plasmid is then amplified using restriction endonucleases. Pac I. After enzyme digestion, the linearized IntA site integration plasmid was recovered via gel extraction. The expression cassette P... TEFin - EcHPAB-T lip2t , P TEFin - EcHPAC -T cyc1t and linearization IntA site integration plasmid by one-step cloning and verification to obtain positive recombinant plasmid pUC-HUH-IntA-TEFin- EcHPAB / C.

[0033] Table 2 Construction of recombinant plasmid pUC-HUH-IntA-TEFin- EcHPAB / C Primers used

[0034] 3. Construction of recombinant plasmid pUC-HUH-IntA-UP12- EcHPAB / C Recombinant plasmid pUC-HUH-IntA-UP12- EcHPAB / C was constructed in the same way as the construction of recombinant plasmid pUC-HUH-IntA-TEFin- EcHPAB / C , the only difference being that the promoter TEFin in the expression cassette was replaced by the promoter UP12 (nucleotide sequence as shown in SEQ ID No. 10), i.e. the recombinant plasmid comprises EcHPAB gene expression cassette P UP12 - EcHPAB -T lip2t (including EcHPAB the promoter P of the gene expression cassette UP12 , the gene EcHPAB , EcHPAB the terminator T of the gene expression cassette lip2t ) and EcHPAC gene expression cassette P UP12 - EcHPAC -T cyc1t (including EcHPAC the promoter P of the gene expression cassette UP12 , the gene EcHPAC and EcHPAC the terminator T of the gene expression cassette cyc1t ), wherein the primer sequences for amplifying each element are shown in Table 3, EcHPAC the terminator T of the gene expression cassette cyc1t is the same as in Table 2. The recombinant plasmid pUC-HUH-IntA-UP12- EcHPAB / C used plasmid map is shown in Figure 3 .

[0035] Table 3 Primers used for constructing recombinant plasmid pUC-HUH-IntA-UP12- EcHPAB / C

[0036] ​​4. Recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 Construction Recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 Using the IntB site integration plasmid as a backbone, [the plasmid] was inserted between the upstream and downstream homologous arms of IntB. YlTYR1 Gene expression cassette (P EXP - YlTYR1 -T xpr2t )and YlARO7 Gene expression cassette (P TEFin - YlARO7 -T lip2t Its construction method is similar to that of the recombinant plasmid pUC-A08- EcAROG S180F Similar, the difference lies in... EcAROG S180F The expression box was replaced with YlTYR1 Expression box and YlARO7 Expression box, i.e., each element ( YlTYR1 promoter P EXP ,Gene YlTYR1 and YlTYR1 Termination T xpr2t , YlARO7 promoter P TEFin ,Gene YlARO7 and YlARO7 Termination T lip2t The primers and templates used for amplification differ; see Table 4 for details. The IntB site integration plasmid is then amplified using restriction endonucleases. Pac I. After enzyme digestion, the linearized IntB site integration plasmid was recovered via gel extraction. The expression cassette P... EXP - YlTYR1 -T xpr2t P TEFin - YlARO7 -T lip2t The linearized IntB site integration plasmid was cloned and validated in one step to obtain the positive recombinant plasmid pUC-HUH-IntB-. YlTYR1 - YlARO7.

[0037] 5. Recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 G139S Construction Recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 G139S Using the IntB site integration plasmid as a backbone, [the plasmid] was inserted between the upstream and downstream homologous arms of IntB. YlTYR1 Gene expression cassette (PEXP - YlTYR1 -T xpr2t )and YlARO7 G139S gene expression cassette (P TEFin - YlARO7 G139S -T lip2t ). The construction method is similar to that of recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 , except that the YlARO7 gene is replaced by the YlARO7 G139S gene, i.e. the primers and templates used for amplification of each element are different, as shown in Table 5, wherein, YlTYR1 the amplification sequences of each element of the YlARO7 G139S gene expression cassette are the same as those in Table 4.The YlARO7 G139S gene is amplified using primers No. 2 and 3 in Table 5 as templates, to obtain fragment 1 and fragment 2. After ligation of fragment 1 and fragment 2, the complete mutated Pac EXP - YlTYR1 -T xpr2t , P TEFin - YlARO7 G139S -T lip2t and the linearized IntB site integration plasmid are used to obtain the recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 G139S 。

[0038] Table 4 Construction of recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 primers used

[0039] Table 5 Construction of recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 G139S primers used

[0040] 6. Construction of recombinant plasmid pUC-HUH-IntB- EcTYRA M53I / A354V Recombinant plasmid pUC-HUH-IntB- EcTYRA ​M53I / A354V Using the IntB site integration plasmid as a backbone, [the plasmid] was inserted between the upstream and downstream homologous arms of IntB. EcTYRA M53I / A354V Gene expression cassette (P TEFin - EcTYRA M53I / A354V -T lip2t Its construction method is similar to that of the recombinant plasmid pUC-A08- EcAROG S180F Similar, the difference lies in... EcAROG S180F The expression box was replaced with EcTYRA M53I / A354V Expression boxes, i.e., each element (promoter P) TEFin ,Gene EcTYRA M53I / A354V and Termination T lip2t The primers and templates used for amplification differ; see Table 6 for details. The IntB site integration plasmid is then amplified using restriction endonucleases. Pac I. After enzyme digestion, the linearized IntB site-integrated plasmid was recovered via gel extraction and incorporated into the expression cassette P. TEFin - EcTYRA M53I / A354V -T lip2t The recombinant plasmid pUC-HUH-IntB- was obtained through one-step cloning. EcTYRA M53I / A354V 。

[0041] Table 6. Construction of recombinant plasmid pUC-HUH-IntB- EcTYRA M53I / A354V Primers used

[0042] 7. Recombinant plasmid pUC-HUH-IntE- LmPK - CkPTA Construction Recombinant plasmid pUC-HUH-IntE- LmPK - CkPTA Using the IntE site integration plasmid as a backbone, inserts were placed between the upstream and downstream homologous arms of IntE. LmPK Gene expression cassette (P EXP - LmPK -T cyc1t )and CkPTA Gene expression cassette (P TEFin - CkPTA -T pex10t Its construction method is similar to that of the recombinant plasmid pUC-A08- EcAROG S180F Similar, the difference lies in... EcAROG S180F The expression box was replaced withLmPK Expression box and CkPTA Expression boxes, i.e., each element (promoter P) EXP ,Gene LmPK Termination of sub-T cyc1t promoter P TEFin ,Gene CkPTA and Termination T pex10t The primers and templates used for amplification differ; see Table 7 for details. The IntE site integration plasmid is then amplified using restriction endonucleases. Pac I. After enzyme digestion, the linearized IntE site-integrated plasmid was recovered via gel extraction and incorporated into the expression cassette P. EXP - LmPK -T cyc1t and P TEFin - CkPTA -T pex10t The positive recombinant plasmid pUC-HUH-IntE- was obtained through one-step cloning and verification. LmPK - CkPTA.

[0043] 8. Recombinant plasmid pUC-HUH-IntD- PcAAS Construction Recombinant plasmid pUC-HUH-IntD- PcAAS Using the IntD site integration plasmid as a backbone, [the plasmid] was inserted between the upstream and downstream homologous arms of IntD. PcAAS Gene expression cassette (P TEFin -Pc AAS -T xpr2t Its construction method is similar to that of the recombinant plasmid pUC-A08- EcAROG S180F Similar, the difference lies in... EcAROG S180F The expression box was replaced with PcAAS Expression boxes, i.e., each element (promoter P) TEFin ,Gene PcAAS and Termination T xpr2t The primers and templates used for amplification differ; see Table 8 for details. The IntD site integration plasmid is then processed using restriction endonucleases. Pac I. After enzyme digestion, the linearized IntD site-integrated plasmid was recovered via gel extraction and incorporated into the expression cassette P. TEFin - PcAAS -T xpr2t The positive recombinant plasmid pUC-HUH-IntD- was obtained through one-step cloning and verification. PcAAS .

[0044] Table 7. Construction of recombinant plasmid pUC-HUH-IntE- LmPK - CkPTA Primers used

[0045] Table 8. Construction of recombinant plasmid pUC-HUH-IntD- PcAAS Primers used

[0046] 9. Recombinant plasmid pUC-HUH-IntF- VsVHB Construction Recombinant plasmid pUC-HUH-IntF- VsVHB Using the IntF site integration plasmid as a backbone, inserts were placed between the upstream and downstream homologous arms of IntF. VsVHB Gene expression cassette (P EXP - VsVHB -T cyc1t Its construction method is similar to that of the recombinant plasmid pUC-A08- EcAROG S180F Similar, the difference lies in... EcAROG S180F The expression box was replaced with VsVHB Expression boxes, i.e., each element (promoter P) EXP ,Gene VsVHB and Termination T cyc1t The primers and templates used for amplification differ; see Table 9 for details. The IntF site integration plasmid is then amplified using restriction endonucleases. Pac I. After enzyme digestion, the linearized IntF site-integrated plasmid was recovered via gel extraction and incorporated into the expression cassette P. EXP - VsVHB -T cyc1t The positive recombinant plasmid pUC-HUH-IntF- was obtained through one-step cloning and verification. VsVHB.

[0047] Table 9. Construction of recombinant plasmid pUC-HUH-IntF- VsVHB Primers used

[0048] 10. Recombinant plasmid pUC-HUH-IntC- YlARO10 Construction Recombinant plasmid pUC-HUH-IntC- YlARO10 Using the IntC site integration plasmid as a backbone, inserts were placed between the upstream and downstream homologous arms of IntC. YlARO10 Gene expression cassette (P TEFin - YlARO10 -T cyc1t Its construction method is similar to that of the recombinant plasmid pUC-A08- EcAROG S180F Similar, the difference lies in... EcAROG S180F The expression box was replaced with YlARO10Expression cassette, i.e. each element (promoter P TEFin , gene YlARO10 and terminator T cyc1t ) used primers and templates are different, see Table 10. The IntC site integration plasmid is digested with restriction enzyme Pac I, and the linearized IntC site integration plasmid is recovered from the gel. The expression cassette P TEFin - YlARO10 -T cyc1t is obtained by one-step cloning and verification. The positive recombinant plasmid pUC-HUH-IntC- YlARO10.

[0049] Table 10 Construction of recombinant plasmid pUC-HUH-IntC- YlARO10 primers used

[0050] 11. Construction of recombinant plasmid pUC-HUH-PHA2 The recombinant plasmid pUC-HUH-PHA2 is constructed by inserting the 1500 bp homologous arm upstream of the start codon of PHA2 site (gene number YALI1_B22549g) in the Pofl Δku70 genome (PHA2-up) at the restriction enzyme site, and inserting the 1500 bp homologous arm downstream of the stop codon of PHA2 site (PHA2-dn) at the restriction enzyme site, and inserting the hisG tag coding gene and orotidine-5'-phosphate decarboxylase coding gene expression cassette (containing the endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) between the upstream and downstream homologous arms, using pUC57-hisG-Ura-hisG as the backbone. EcoR I. The upstream homologous arm of PHA2 site PHA2-up is amplified using the template of Pofl Δku70 genome DNA and the primers in Table 11. The pUC57-hisG-Ura-hisG plasmid is digested with restriction enzyme Y. lipolytica I, and the linearized pUC57-hisG-Ura-hisG plasmid is recovered from the gel. One-step cloning is achieved using ClonExpress MultiS One Step Cloning Kit with the upstream homologous arm PHA2-up, to obtain a circular recombinant vector. The circular recombinant vector is transformed into E. coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-PHA2-up is obtained by ampicillin-resistant plate screening, colony PCR and sequencing verification. Kpn I. The upstream homologous arm of PHA2 site PHA2-up is amplified using the template of Pofl Δku70 genome DNA and the primers in Table 11. The pUC57-hisG-Ura-hisG plasmid is digested with restriction enzyme Y. lipolytica I, and the linearized pUC57-hisG-Ura-hisG plasmid is recovered from the gel. One-step cloning is achieved using ClonExpress MultiS One Step Cloning Kit with the upstream homologous arm PHA2-up, to obtain a circular recombinant vector. The circular recombinant vector is transformed into E. coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-PHA2-up is obtained by ampicillin-resistant plate screening, colony PCR and sequencing verification. Y. lipolytica I. The upstream homologous arm of PHA2 site PHA2-up is amplified using the template of Pofl Δku70 genome DNA and the primers in Table 11. The pUC57-hisG-Ura-hisG plasmid is digested with restriction enzyme Y. lipolytica I, and the linearized pUC57-hisG-Ura-hisG plasmid is recovered from the gel. One-step cloning is achieved using ClonExpress MultiS One Step Cloning Kit with the upstream homologous arm PHA2-up, to obtain a circular recombinant vector. The circular recombinant vector is transformed into E. coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-PHA2-up is obtained by ampicillin-resistant plate screening, colony PCR and sequencing verification. Y. lipolytica I. The upstream homologous arm of PHA2 site PHA2-up is amplified using the template of Pofl Δku70 genome DNA and the primers in Table 11. The pUC57-hisG-Ura-hisG plasmid is digested with restriction enzyme Y. lipolytica I, and the linearized pUC57-hisG-Ura-hisG plasmid is recovered from the gel. One-step cloning is achieved using ClonExpress MultiS One Step Cloning Kit with the upstream homologous arm PHA2-up, to obtain a circular recombinant vector. The circular recombinant vector is transformed into E. coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-PHA2-up is obtained by ampicillin-resistant plate screening, colony PCR and sequencing verification. EcoR I. The upstream homologous arm of PHA2 site PHA2-up is amplified using the template of Pofl Δku70 genome DNA and the primers in Table 11. The pUC57-hisG-Ura-hisG plasmid is digested with restriction enzyme Y. lipolytica I, and the linearized pUC57-hisG-Ura-hisG plasmid is recovered from the gel. One-step cloning is achieved using ClonExpress MultiS One Step Cloning Kit with the upstream homologous arm PHA2-up, to obtain a circular recombinant vector. The circular recombinant vector is transformed into E. coli DH5α competent cells, and positive recombinant plasmid pUC-HUH-PHA2-up is obtained by ampicillin-resistant plate screening, colony PCR and sequencing verification.

[0051] With Y. lipolytica Polf Δku70 genomic DNA as template, the homologous arm PHA2-dn downstream of the stop codon of PHA2 site was amplified using the primers in Table 11. The linearized pUC-HUH-PHA2-up plasmid was obtained by enzyme digestion of the plasmid pUC-HUH-PHA2-up with restriction enzyme I and gel recovery. Kpn I, and the PHA2-dn was cloned into the linearized pUC-HUH-PHA2-up plasmid to obtain the circular recombinant vector. The circular recombinant vector was transformed into E. coli DH5α competent cells, and the recombinant plasmid pUC-HUH-PHA2 was obtained by ampicillin-resistant plate screening, colony PCR and sequencing verification. The linearized pUC-HUH-PHA2 plasmid was obtained by enzyme digestion of the recombinant plasmid pUC-HUH-PHA2 with restriction enzyme Not I and gel recovery.

[0052] Table 11 Primers used for construction of the recombinant plasmid pUC-HUH-PHA2

[0053] 12. Construction of the recombinant plasmid pUC-HUH-TRP3 The recombinant plasmid pUC-HUH-TRP3 was constructed using pUC57-hisG-Ura-hisG as the backbone, and the homologous arm TRP3-up of 1500 bp upstream of the start codon of the TRP3 site (Gene ID: YALI1_D13963g) in Polf Δku70 genome was inserted at the I enzyme digestion site, and the homologous arm TRP3-dn of 1500 bp downstream of the stop codon of the TRP3 site was inserted at the I enzyme digestion site. EcoR Y. lipolytica The recombinant plasmid pUC-HUH-TRP3 was constructed using pUC57-hisG-Ura-hisG as the backbone, and the homologous arm TRP3-up of 1500 bp upstream of the start codon of the TRP3 site (Gene ID: YALI1_D13963g) in Polf Δku70 genome was inserted at the I enzyme digestion site, and the homologous arm TRP3-dn of 1500 bp downstream of the stop codon of the TRP3 site was inserted at the I enzyme digestion site. Kpn I, and the PHA2-dn was cloned into the linearized pUC-HUH-PHA2-up plasmid to obtain the circular recombinant vector. The circular recombinant vector was transformed into E. coli DH5α competent cells, and the recombinant plasmid pUC-HUH-PHA2 was obtained by ampicillin-resistant plate screening, colony PCR and sequencing verification. The linearized pUC-HUH-PHA2 plasmid was obtained by enzyme digestion of the recombinant plasmid pUC-HUH-PHA2 with restriction enzyme Not I and gel recovery. Y. lipolytica The endogenous promoter P TEFin , orotidine-5'-phosphate decarboxylase and terminator T xpr2t ) between the upstream and downstream homologous arms. The construction method of the recombinant plasmid pUC-HUH-TRP3 was the same as that of the pUC-HUH-PHA2 plasmid described above, except that the upstream and downstream homologous arms were amplified using the primers described in Table 12, and the upstream and downstream homologous arms obtained by amplification were connected to the pUC57-hisG-Ura-hisG backbone according to the method described in “11. Construction of the recombinant plasmid pUC-HUH-PHA2” above.

[0054] Table 12 Primers used for construction of the recombinant plasmid pUC-HUH-TRP3

[0055] Example 2. Construction of recombinant Y. lipolytica​ (I) Construction (expression) of recombinant bacteria 1-2 EcHPAB / C ) The recombinant plasmid pUC-HUH-IntA-TEFin- EcHPAB / C Import Y. lipolytica In Po1f Δku70, P TEFin - EcHPAB / C -T lip2t The expression cassette was integrated into the IntA site of the genome to obtain recombinant strain 1. Then, under 5-fluoroorotic acid selection pressure, a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection.

[0056] The recombinant plasmid pUC-HUH-IntA-UP12- EcHPAB / C Import Y. lipolytica In Po1f Δku70, P UP12 - EcHPAB / C -T lip2t The expression cassette was integrated into the IntA site of the genome to obtain recombinant bacteria 2. Then, under 5-fluoroorotic acid selection pressure, a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection.

[0057] The specific method for constructing recombinant bacteria 1 is as follows: (1) Y. lipolytica Po1f Δku70 was cultured overnight in YPD liquid medium, and competent cells were prepared using the Zymogen Frozen EZ Yeast Transformation Kit II from ZymoResearch Corporation. The linearized recombinant plasmid pUC-HUH-IntA-TEFin- was then applied. Ec HpaB / C conversion into Y. lipolytica Homologous recombination was performed in Po1f Δku70 competent cells. Positive clones were screened using SD-Ura selection medium and then identified by PCR.

[0058] (2) Positive clones identified by PCR were plated on YPD plates containing 5-fluoroorotic acid. Then, single colonies were streaked simultaneously on both YPD plates and SD-Ura plates containing 5-fluoroorotic acid. Single colonies that grew on YPD plates containing 5-fluoroorotic acid but failed to grow on SD-Ura plates were selected, indicating successful loss of the Ura selection marker, and recombinant strain 1 was obtained.

[0059] The construction method of recombinant bacteria 2 is the same as that of recombinant bacteria 1, except that the plasmid used for homologous recombination is changed accordingly.

[0060] (II) Construction (expression) of recombinant strain 3 EcAROGS180F ) The recombinant plasmid pUC-A08- EcAROG S180F The recombinant plasmid pUC-A08- EcAROG S180F The expression cassette is integrated into the genomic A08 site to obtain the recombinant strain 3. The specific method for constructing the recombinant strain 3 is as follows: The recombinant strain 2 is cultured overnight in YPD liquid medium, and the competent cells are prepared by using the Zymogen Frozen EZ Yeast Transformation Kit II of Zymo Research Corporation, and the linearized recombinant plasmid pUC-A08- EcAROG S180F The linearized recombinant plasmid pUC-A08-

[0061] (Three) Construction of recombinant strains 4-6 (expression YlTYR1 - YlARO7 , YlTYR1 - YlARO7 G139S or EcTYRA M53I / A354V ) The recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 The recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 The expression cassette is integrated into the genomic IntB site to obtain the recombinant strain 4, and then one HisG tag and Ura selection marker are lost under the selection pressure of 5-fluoroorotic acid, so as to be used for the next round of Ura selection.

[0062] The recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 G139S The recombinant plasmid pUC-HUH-IntB- YlTYR1 - YlARO7 G139S The expression cassette is integrated into the genomic IntB site to obtain the recombinant strain 5, and then one HisG tag and Ura selection marker are lost under the selection pressure of 5-fluoroorotic acid, so as to be used for the next round of Ura selection.

[0063] The recombinant plasmid pUC-IntB- EcTYRA M53I / A354V The recombinant plasmid pUC-IntB- EcTYRA M53I / A354VThe expression cassette is integrated into the genome at the IntB site to obtain recombinant bacteria 6, which then loses one HisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, so as to be used for the next round of Ura selection.

[0064] The construction method of the recombinant bacteria 4-6 is the same as that of the recombinant bacteria 1, except that the plasmid for homologous recombination and the host for transformation need to be changed accordingly.

[0065] (Four) Construction of recombinant bacteria 7-9 (expression LmPK - CkPTA 、 PcAAS 、and VsVHB ) The recombinant plasmid pUC-HUH-IntE- LmPK - CkPTA is introduced into the recombinant bacteria 6 that have lost Ura, LmPK - CkPTA The expression cassette is integrated into the genome at the IntE site to obtain recombinant bacteria 7, which then loses one HisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, so as to be used for the next round of Ura selection.

[0066] The pUC-HUH-IntD- PcAAS is introduced into the recombinant bacteria 7 that have lost Ura, PcAAS The expression cassette is integrated into the genome at the IntD site to obtain recombinant bacteria 8, which then loses one HisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, so as to be used for the next round of Ura selection.

[0067] The pUC-HUH-IntF- VsVHB is introduced into the recombinant bacteria 8 that have lost Ura, VsVHB The expression cassette is integrated into the genome at the IntF site to obtain recombinant bacteria 9, which then loses one HisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, so as to be used for the next round of Ura selection.

[0068] The construction method of the recombinant bacteria 7-9 is the same as that of the recombinant bacteria 1, except that the plasmid for homologous recombination and the host for transformation need to be changed accordingly.

[0069] (Five) Construction of recombinant bacteria 10 (overexpression YlARO10 ) The pUC-HUH-IntC- YlARO10 is introduced into the recombinant bacteria 9 that have lost Ura, YlARO10 The expression cassette is integrated into the genome at the IntC site to obtain recombinant bacteria 10, which then loses one HisG tag and Ura selection marker under 5-fluoroorotic acid selection pressure, so as to be used for the next round of Ura selection.

[0070] The construction method of recombinant strain 10 is the same as that of recombinant strain 1, except that the plasmid used for homologous recombination and the host used for transformation need to be changed accordingly.

[0071] (vi) Construction of recombinant bacteria 11-12 (knockout) PHA2 and TRP3 ) Linearized recombinant plasmid pUC-HUH- PHA2 Importing recombinant bacteria 10, which has lost its Ura component, into the culture medium... PHA2 Gene knockout yielded recombinant strain 11, which then lost a HisG tag and an Ura selection marker under 5-fluoroorotic acid selection pressure for use in the next round of Ura selection.

[0072] Linearized recombinant plasmid pUC-HUH- TRP3 Importing recombinant bacteria 11, which has lost its Ura component, into the culture medium... TRP3 Gene knockout yielded recombinant bacteria 12.

[0073] The construction method of recombinant bacteria 11-12 is the same as that of recombinant bacteria 1, except that the plasmid used for homologous recombination and the host used for transformation need to be changed accordingly.

[0074] Example 3: Application of recombinant Yersinia lipolytica in the production of hydroxytyrosol (a) Cultivation of engineered bacteria and extraction of products The initial strain was Yersinia lipophila (Yersinia lipophila). Yarrowialipolytica The recombinant strain 1-12 in Example 2 (Po1f Δku70) produced hydroxytyrosol. The specific method is as follows: The strain was activated and cultured in YPD liquid medium at 30 ℃ and 220 rpm for 16 h to obtain a seed culture. The seed culture was inoculated into 50 mL of fermentation medium at a 1% v / v inoculation rate and cultured at 30 ℃ and 220 rpm for 5 days to obtain the fermentation broth. The broth was centrifuged at 12000 rpm for 5 min, and the supernatant was used for liquid chromatography to determine the hydroxytyrosol concentration. The remaining sample was then dried in an oven (75 ℃) to constant weight for biomass measurement. The fermentation medium formulation was: 20 g / L peptone, 10 g / L yeast extract, and 60 g / L glucose, with water as the solvent.

[0075] (II) Qualitative and quantitative analysis of hydroxytyrosol 1. Hydroxytyrosol detection Detection conditions: UV detector, equipped with C18 reverse phase column, mobile phase A liquid: 5% (v / v) formic acid, mobile phase B liquid: 100% acetonitrile, mobile phase concentration gradient: 0~15 min, 85%-60% A liquid; 15~20 min, 60%-3% A liquid; 20~25 min, 3%-85% A liquid. Detection wavelength is 280 nm, column oven temperature 30 ℃, mobile phase flow rate 0.3 mL / min.

[0076] After 5 days of fermentation, Y. lipolytica No hydroxytyrosol was detected in Po1f Δku70 (YL-0), as Figure 4 As shown, the hydroxytyrosol concentrations of recombinant bacteria 1~12 (YL-1~YL-12) were 35.8, 189.3, 517.2, 581.9, 589.7, 732.4, 825.9, 957.3, 1021.6, 1282.8, 1539.1, 1681.7 mg / L, respectively. Among them, the hydroxytyrosol yield of recombinant bacteria 12 was the highest, reaching 1681.7 mg / L, i.e. 1681.7 mg of hydroxytyrosol was produced per liter of fermentation broth. Recombinant bacteria 12 was named Yarrowia lipolytica (Y. lipolytica) XJ-HT strain. Yarrowia lipolytica

[0077] Table 13 Hydroxytyrosol yield of initial bacteria and recombinant bacteria

[0078] Recombinant bacteria 12 was inoculated into 50 mL YPD medium at 30 ℃, 220 rpm for 24 h, and then the seed liquid was inoculated into a 5 L fermenter containing 2.5 L fermentation medium at an inoculation amount of 5% v / v. The dissolved oxygen was controlled at 20% for 0-48 h of fermentation, and at 0-5% for more than 48 h of fermentation. During the fermentation process, the pH value was constantly controlled at 5.5. At 40 h and 96 h of fermentation, each was fed once, and 800 mL of mixed aqueous solution containing 11.1 g / L ammonium sulfate and 900 g / L glucose was fed each time. The fermentation temperature was 28 ℃, and the culture time was 5 days. Among them, the composition of the fermentation medium was glucose 60 g / L, yeast extract 10 g / L and peptone 20 g / L. After 5 days of fermentation, the hydroxytyrosol yield of recombinant bacteria 12 reached the highest, reaching 13587.6 mg / L, i.e. 13587.6 mg of hydroxytyrosol was produced per liter of fermentation broth (Table 13).

[0079] ​The application provides a construction method and application of recombinant Yarrowia lipolytica with high yield of hydroxytyrosol. The method and approach for specifically realizing the technical scheme are various, and the above description is only a preferred embodiment of the application. It should be pointed out that, for ordinary skilled in the art, several improvements and refinements can be made without departing from the principle of the application, and the improvements and refinements should be regarded as the protection scope of the application. The components not explicitly described in the embodiment can be realized by the prior art.

Claims

1. A method for constructing a recombinant Yarrowia lipolytica with high production of hydroxytyrosol, characterized in that, The recombinant Yarrowia lipolytica expresses a bifunctional riboflavin-dependent monooxygenase-oxidase encoding gene HPAB a bifunctional riboflavin-dependent monooxygenase-reductase encoding gene HPAC a DAHP synthase mutant encoding gene AroG S180F and a bifunctional chorismate mutase / prephenate dehydrogenase mutant encoding gene TyrA M53I / A354V ; Wherein, the HPAB , HPAC , AroG S180F and TyrA M53I / A354V are derived from Escherichia coli Escherichia coli .

2. The construction method according to claim 1, characterized in that, The nucleotide sequences of the nucleic acid molecules of the application are shown in SEQ ID No. 1 to 4, respectively. HPAB , HPAC , AroG S180F and TyrA M53I / A354V The nucleotide sequences of the nucleic acid molecules of the application are shown in SEQ ID No. 1 to 4, respectively.

3. The construction method of claim 1, wherein, The HPAB and HPAC expression is driven by a growth-dependent promoter.

4. The construction method according to claim 3, characterized in that, The growth-dependent promoter comprises UP12, and the nucleotide sequence of UP12 is shown as SEQ ID No.

10.

5. The construction method of claim 1, wherein, The recombinant Yarrowia lipolytica is modified by any one or a combination of a1-a7 as follows: a1, leuconostoc mesenteroides Leuconostoc mesenteroides phosphoketolase encoding gene of origin LmPK ; a2, clostridium expression Clostridium kluyveri phosphotransacetylase encoding gene of origin CkPTA ; a3, parsley Petroselinum crispum gene encoding an aromatic aldehyde synthase from PcAAS ; a4, hemoglobin encoding gene from leucothrix Vitreoscilla stercoraria origin VsVHB ; a5, Yarrowia lipolytica overexpressing Yarrowia lipolytica endogenous phenylpyruvate decarboxylase encoding gene YlARO10 ; a6, knock-out of prebenzoic acid dehydratase encoding gene PHA2 ; a7, knockout of anthranilate synthase encoding genes TRP3.

6. The construction method of claim 5, wherein, The LmPK, CkPTA, PcAAS and VsVHB The nucleotide sequences are shown in SEQ ID No. 5~8 respectively; the YlARO10 The gene number is YALI1_D08884g; the PHA2 The gene number is YALI1_B22549g; the TRP3 The gene number is YALI1_D13963g.

7. The construction method of claim 1, wherein, The nucleotide sequences of the HPAB , HPAC , AroG S180F and TyrA M53I / A354V are shown in SEQ ID No. 1~4, respectively, the nucleotide sequence of the HPAB and HPAC is driven by the growth-dependent promoter UP12, and the nucleotide sequence of UP12 is shown in SEQ ID No. 10; the recombinant Yarrowia lipolytica has also been modified as follows: a1, leuconostoc mesenteroides Leuconostoc mesenteroides phosphoketolase encoding gene of lactic acid bacteria LmPK ; a2, clostridium expression Clostridium kluyveri phosphotransacetylase encoding gene of origin CkPTA ; a3, parsley Petroselinum crispum genes encoding aldehyde-forming dehydrogenases PcAAS ; a4, hemoglobin encoding gene from leucothrix Vitreoscilla stercoraria VsVHB ;​ a5, Yarrowia lipolytica overexpressing Yarrowia lipolytica endogenous phenylpyruvate decarboxylase encoding gene YlARO10 ; a6, knock-out of prebenzoic acid dehydratase encoding gene PHA2 ; a7, knockout of anthranilate synthase encoding genes TRP3 ; The LmPK, CkPTA, PcAAS, VsVHB The nucleotide sequences are shown in SEQ ID No. 5~8, respectively; the YlARO10 The gene number is YALI1_D08884g; the PHA2 The gene number is YALI1_B22549g; the TRP3 The gene number is YALI1_D13963g.

8. The recombinant Yarrowia lipolytica constructed by the construction method of any one of claims 1-7.

9. The application of the recombinant Yarrowia lipolytica of claim 8 in the fermentation production of hydroxytyrosol.

10. Use according to claim 9, characterized in that, The recombinant Yarrowia lipolytica is inoculated into a fermentation medium, and is subjected to fermentation culture at 20-30 ℃ for 120-144 h to obtain hydroxytyrosol. The recombinant Yarrowia lipolytica is inoculated into a fermentation medium, and is subjected to fermentation culture at 20-30 ℃ for 120-144 h to obtain hydroxytyrosol.

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