Recombinant yarrowia lipolytica with high yield of denosine as well as construction method and application of recombinant yarrowia lipolytica

Abstract

The invention relates to the field of bioengineering, in particular to recombinant yarrowia lipolytica with high yield of denosine as well as a construction method and application of the recombinant yarrowia lipolytica. According to the invention, Yarrowia lipolytica is taken as a chassis cell, a phenylalanine ammonialyase gene PtrPAL3, a coumaroyl-coenzyme A ligase gene Pc4CL1 and a stilbene synthetase gene PtSTS are inserted into a genome, and 3-deoxy-D-arabinohepturonate-7-phosphate synthetase gene ARO3 and an acetyl-coenzyme A carboxylase gene ACC1 are overexpressed, so that the Yarrowia lipolytica is obtained. The 4-hydroxyphenylpyruvate dioxygenase gene HPD and the phenylpyruvate decarboxylase gene ARO10 are knocked out, and the recombinant yarrowia lipolytica is constructed and obtained. After genetic modification, the strain has the capability of synthesizing the pinosine from the beginning, the fermentation yield reaches 260mg / L by using a glucose shake flask, and the yield of the pinosine fermented in a 5L fermentation tank reaches 1200mg / L. According to the method, high-efficiency production of the pinosine is realized by utilizing the yarrowia lipolytica for the first time, and the pinosine yield of the constructed recombinant yarrowia lipolytica is the highest level of microbial fermentation production at present.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a recombinant Yarrowia lipolytica with high yield of pinospermum officinale and a construction method and application thereof. Background Art

[0002] Pinus syringae is a stilbene substance, a natural secondary metabolite synthesized in pine plants through the phenylpropanoid metabolic pathway. Pinus syringae not only protects plants from bacteria and fungi, but also has good medicinal value, with antioxidant, anti-inflammatory and anti-cancer effects.

[0003] Pinus densiflorus can be prepared by plant extraction, chemical synthesis and biosynthesis. The plant extraction method mainly uses pine wood as raw material to purify pinus densiflorus. However, the content of pinus densiflorus in pine heartwood is relatively low, only 1 to 40 mg / m 3 . At the same time, the secondary metabolites of this raw material are complex in composition, containing a variety of flavonoids, stilbenes and other substances with similar structures, which increases the difficulty of separating the single component pine tin and also increases the economic cost. Among the chemical synthesis methods, the Wittig or Wittig-Horner reaction method is the most commonly used method for synthesizing pine tin. However, this method requires the use of toxic reagents and the reaction conditions are harsh. Therefore, it has become a trend to develop a low-energy, pollution-free, and low-cost method for preparing pine tin. In comparison, the synthesis of pine tin by microorganisms has the advantages of short cycle, renewable raw materials, mild reaction and efficient production. Therefore, the production of pine tin by microorganisms is an economical and efficient production method.

[0004] Yarrowia lipolytica has a unique metabolic pathway. In the cytoplasm of Yarrowia lipolytica, the sources of acetyl-CoA mainly come from two forms: (1) When the nitrogen source is insufficient but the carbon source is sufficient, the excess citrate in the TCA cycle is excreted from the mitochondria and converted into acetyl-CoA by ATP citrate lyase; (2) At the same time, acetyl-CoA is also produced by acetate catalyzed by acetyl-CoA synthetase (ACS). Therefore, Yarrowia lipolytica is rich in the key precursor acetyl-CoA, which can provide sufficient precursors for the synthesis of malonyl-CoA, compensating for the low yield of safflower oil caused by insufficient malonyl-CoA. At the same time, Yarrowia lipolytica is rich in NADPH and ATP and has a unique hydrophobic environment. At present, Yarrowia lipolytica is widely used in the production of various acetyl-CoA derivatives.

[0005] Therefore, the present invention utilizes Yarrowia lipolytica to biosynthesize pinospermum officinale, in order to solve the key problem of low yield of pinospermum officinale synthesis at present. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a recombinant Yarrowia lipolytica with high yield of pinostearin in view of the deficiencies of the prior art.

[0007] The technical problem that the present invention needs to solve is to provide a method for constructing the above-mentioned recombinant Yarrowia lipolytica.

[0008] The final technical problem to be solved by the present invention is to provide the application of the recombinant Yarrowia lipolytica.

[0009] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0010] A recombinant Yarrowia lipolytica with high yield of kaempferol is constructed by taking Yarrowia lipolytica as a chassis cell, inserting a phenylalanine ammonia lyase gene PtrPAL3, a p-coumaroyl-CoA ligase gene Pc4CL1 and a stilbene synthase gene PtSTS into the genome, overexpressing a 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene ARO3 and an acetyl-CoA carboxylase gene ACC1, and knocking out a 4-hydroxyphenylpyruvate dioxygenase gene HPD and a phenylpyruvate decarboxylase gene ARO10.

[0011] Wherein, the Yarrowia lipolytica is Yarrowia lipolytica Po1fΔku70.

[0012] The phenylalanine ammonia lyase gene PtrPAL3, the p-coumaroyl-CoA ligase gene Pc4CL1, and the stilbene synthase gene PtSTS are derived from Populus trichocarpa, Petroselinum crispum, and Pinus thunbergii, respectively.

[0013] Specifically, the phenylalanine ammonia lyase gene PtrPAL3, the p-coumaroyl-CoA ligase gene Pc4CL1 and the stilbene synthase gene PtSTS are codon-optimized genes.

[0014] Specifically, the nucleotide sequence of the phenylalanine ammonia lyase gene PtrPAL3 is the sequence shown in SEQ ID NO.1, or a nucleotide sequence with a homology greater than 90% and having an equivalent function for pinosyl alcohol synthesis; the nucleotide sequence of the p-coumaroyl-CoA ligase gene Pc4CL1 is the sequence shown in SEQ ID NO.2, or a nucleotide sequence with a homology greater than 90% and having an equivalent function for pinosyl alcohol synthesis; the nucleotide sequence of the stilbene synthase gene PtSTS is the sequence shown in SEQ ID NO.3, or a nucleotide sequence with a homology greater than 90% and having an equivalent function for pinosyl alcohol synthesis.

[0015] The 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene ARO3, the acetyl-CoA carboxylase gene ACC1, the 4-hydroxyphenylpyruvate dioxygenase gene HPD and the phenylpyruvate decarboxylase gene ARO10 are all derived from Yarrowia lipolytica.

[0016] Specifically, the nucleotide sequence of the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene ARO3 is shown in SEQ ID NO.4, the nucleotide sequence of the acetyl-CoA carboxylase gene ACC1 is shown in SEQ ID NO.5, the nucleotide sequence of the 4-hydroxyphenylpyruvate dioxygenase gene HPD is shown in SEQ ID NO.6, and the nucleotide sequence of the phenylpyruvate decarboxylase gene ARO10 is shown in SEQ ID NO.7.

[0017] The method for constructing the recombinant Yarrowia lipolytica is also within the scope of protection of the present invention.

[0018] The integration expression plasmids carrying PtrPAL3, Pc4CL1 and PtSTS genes, the integration expression plasmid carrying PtSTS gene, the integration expression plasmid carrying ARO3 gene, the integration expression plasmid carrying ACC1 gene, the HPD gene knockout plasmid and the ARO10 gene knockout plasmid are linearized and sequentially introduced into chassis cells to construct the recombinant Yarrowia lipolytica.

[0019] Wherein, the integration expression plasmid carrying the PtSTS gene is a multi-copy integration expression plasmid with a copy number ≥2.

[0020] Wherein, the integration expression plasmids are all inserted into the corresponding integration sites in the form of expression cassettes.

[0021] Specifically, the integration sites are the IntA site, IntD site, IntC site and IntF site of chassis cells.

[0022] Specifically, the expression cassette contains a promoter and a terminator; wherein the promoter is promoter P HP4D , the promoter P of Yarrowia lipolytica TEF , the promoter P of Yarrowia lipolytica TEFin Any one of the above, wherein the terminator is the terminator T of Yarrowia lipolytica xpr2t , terminator T of Yarrowia lipolytica lip2t , terminator T of Yarrowia lipolytica mig1t Any one of .

[0023] Furthermore, the construction method specifically comprises firstly introducing the integrated expression plasmid carrying the PtrPAL3, Pc4CL1 and PtSTS genes into the IntA site of the Yarrowia lipolytica genome (i.e., introducing the optimized PtrPAL3 gene, the optimized Pc4CL1 gene and the optimized PtSTS gene into the IntA site of the Yarrowia lipolytica genome at the same time) to obtain the recombinant Yarrowia lipolytica TAN1; and then introducing the integrated expression plasmid carrying the PtSTS gene into the IntD site of the recombinant Yarrowia lipolytica TAN1 (i.e., introducing the key enzyme stilbene synthase gene PtSTS into the recombinant Yarrowia lipolytica TAN1 with different copy numbers (≥2)). The recombinant Yarrowia lipolytica TAN2 is obtained by sequentially introducing an integration expression plasmid carrying the ARO3 gene and an integration expression plasmid carrying the ACC1 gene into the IntF and IntC sites of the recombinant Yarrowia lipolytica TAN2 (i.e., the ARO3 gene and the ACC1 gene are sequentially introduced into the IntF and IntC sites of the recombinant Yarrowia lipolytica TAN2), and the HPD gene and the ARO10 gene are knocked out using a plasmid for knocking out the HPD gene and a plasmid for knocking out the ARO10 gene to obtain the recombinant Yarrowia lipolytica TAN3, i.e., the recombinant Yarrowia lipolytica.

[0024] The application of the recombinant Yarrowia lipolytica in the biosynthesis of pinospermum officinale is also within the scope of protection of the present invention.

[0025] The formula of the biosynthetic fermentation medium is: 10-40 g / L peptone, 5-20 g / L yeast extract and 20-120 g / L glucose.

[0026] The fermentation conditions for the biosynthesis are as follows: fermentation temperature of 28-30°C, dissolved oxygen of 30-40%, pH of 5.0-6.0, stirring speed of 200-1000rpm, ventilation volume of 2-3vvm, fermentation time of 72-200h, and glucose concentration of 5-10g / L during fermentation.

[0027] Specifically, in a 5L fermentation tank, the recombinant Yarrowia lipolytica is used to ferment and synthesize pinosylvin, and effective accumulation of pinosylvin up to 1200 mg / L or more can be achieved in the fermentation liquid, which has an industrial application background.

[0028] Beneficial effects: The present invention uses Yarrowia lipolytica Po1f-Δku70 as a host, inserts phenylalanine ammonia lyase gene PtrPAL3, p-coumaroyl-CoA ligase gene Pc4CL1 and multi-copy stilbene synthase gene PtSTS into its genome, integrates and expresses 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene ARO3 and acetyl-CoA carboxylase gene ACC1, knocks out 4-hydroxyphenylpyruvate dioxygenase gene HPD and phenylpyruvate decarboxylase gene ARO10, and constructs a recombinant Yarrowia lipolytica capable of synthesizing sutrin from scratch. The recombinant Yarrowia lipolytica has the ability to synthesize sutrin from scratch after genetic modification. In shake flask fermentation, the sutrin production reaches 260 mg / L, and the sutrin production reaches 1200 mg / L in a 5L fermenter. The present invention realizes the high-efficiency production of pinus erythrogen by using Yarrowia lipolytica for the first time, reaches the highest level of biosynthesis of pinus erythrogen so far, and lays a foundation for the subsequent industrial biosynthesis of pinus erythrogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0030] Figure 1 Metabolic diagram of heterologous synthesis of sutsugastatin in Yarrowia lipolytica.

[0031] Figure 2 This is the structural diagram of the recombinant plasmid pUC-HUH-IntA-PtrPAL3-Pc4CL1-PtSTS.

[0032] Figure 3 This is the structural diagram of the recombinant plasmid pUC-HUH-IntD-PtSTS03.

[0033] Figure 4 This is the structural diagram of the recombinant plasmid pUC-HUH-IntF-ARO3.

[0034] Figure 5 This is the structural diagram of the recombinant plasmid pUC-HUH-IntC-ACC1.

[0035] Figure 6 This is the structural diagram of the recombinant knockout plasmid pUC-HUH-HPD.

[0036] Figure 7 This is the structural diagram of the recombinant knockout plasmid pUC-HUH-ARO10.

[0037] Figure 8 The HPLC spectrum and calibration curve of pinus erythritol and the intermediate product cinnamic acid. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0039] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0040] In the following examples, the Yarrowia lipolytica Po1f is from the American College of Microbiology and Biotechnology, with the number being ATCCMYA-2613.

[0041] In the following examples, the Yarrowia lipolytica Po1fΔku70 (i.e., MatA, Δku70∷hisG, leu2-270, ura3-302, xpr2-322, axp1-2) was constructed by knocking out the ku70 gene encoding non-homologous recombination from Yarrowia lipolytica Po1f (disclosed in KretzschmarA, et al., Current Genetics, 2013, 59(1-2):63-72, patent CN117778218A, patent CN117778219A).

[0042] In the following examples, the pUC57-hisG-Ura-hisG (i.e., pUC-HUH) plasmid, Ura expression cassette, IntA site integration plasmid, IntC site integration plasmid, IntF site integration plasmid and their construction process have been disclosed in patent CN117778218A and patent CN117778219A, and the IntD site integration plasmid and its construction process have been disclosed in patent CN117778219A. All the above plasmids and expression cassettes were donated by Professor Ji Xiaojun of Nanjing University of Technology.

[0043] Example 1: Amplification of genetic elements and preparation of target plasmids

[0044] 1. Source of target gene

[0045] The nucleotide sequences of phenylalanine ammonia lyase PtrPAL3 from Populus trichocarpa, p-coumaroyl-CoA ligase Pc4CL1 from Petroselinum crispu and stilbene synthase PtSTS from Pinus thunbergii were codon-optimized and inserted into plasmid pUC57 to obtain recombinant plasmids pUC57-PtrPAL3, pUC57-Pc4CL1 and pUC57-PtSTS. The optimized nucleotide sequences of phenylalanine ammonia lyase PtrPAL3, p-coumaroyl-CoA ligase Pc4CL1 and stilbene synthase PtSTS are shown in SEQ ID NO.1 to 3. The strong promoter HP4D was synthesized by General Biotechnology (Anhui) Co., Ltd. and inserted into plasmid pUC57 to obtain recombinant plasmid pUC57-HP4D.

[0046] 2. Construction of integration expression plasmid

[0047] 1. Construction of integration expression plasmid pUC-HUH-IntA-PtrPAL3-Pc4CL1-PtSTS

[0048] The integration expression plasmid pUC-HUH-IntA-PtrPAL3-Pc4CL1-PtSTS is based on the plasmid pUC57-hisG-Ura-hisG, which is inserted into the homology arm IntA-up upstream of the start codon and the homology arm IntA-down downstream of the stop codon of the IntA site on chromosome A of the Po1f genome of Yarrowia lipolytica, and the PtrPAL3 gene expression cassette (P TEF -PtrPAL3-T xpr2t ), Pc4CL1 gene expression cassette (P TEF -Pc4CL1-T xpr2t ) and PtSTS(P TEF -PtSTS-T lip2t ) gene expression cassette, Ura expression cassette (promoter TEFin , orotidine-5'-phosphate decarboxylase encoding gene and terminator T xpr2t The specific structure is shown in Figure 2 The specific construction process is as follows:

[0049] (1) Construction of recombinant plasmid pUC-HUH-IntA-Pc4CL1

[0050] The promoter P of the Pc4CL1 gene expression cassette was amplified using primers IntA-Pc4CL1-1-F / IntA-Pc4CL1-1-R and IntA-Pc4CL1-3-F / IntA-Pc4CL1-3-R, respectively, and the Yarrowia lipolytica Po1f genome as a template. TEF and terminator T xpr2t Using primers IntA-Pc4CL1-2-F / IntA-Pc4CL1-2-R as primers and pUC57-Pc4CL1 plasmid as template, amplify the two ends of the TEF and T xpr2t Homology arms of the Pc4CL1 gene.

[0051] The IntA site integration plasmid was digested with restriction endonuclease Kpn I from NEB, and the linearized IntA site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntA site integration plasmid and the various elements of the constructed Pc4CL1 gene expression cassette (promoter P TEF , Pc4CL1 gene, terminator T xpr2t ) One-step cloning was achieved using ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The Pc4CL1 gene expression cassette was inserted between the downstream homology arm of the IntA site integration plasmid and the hisG tag encoding gene to obtain a circular recombinant vector. The circular recombinant vector was transformed into Escherichia coli Trans1-T1 competent cells, and the positive recombinant plasmid pUC-HUH-IntA-Pc4CL1 was obtained by plate screening for ampicillin resistance, combined with colony PCR and sequencing verification.

[0052] (2) Construction of recombinant plasmid pUC-HUH-IntA-Pc4CL1-PtSTS

[0053] The promoter region of the PtSTS gene expression cassette was amplified using primers IntA-PtSTS-1-F / IntA-PtSTS-1-R and IntA-PtSTS-3-F / IntA-PtSTS-3-R, respectively, with the Yarrowia lipolytica Po1f genome as a template. TEF and terminator T lip2t Using primers IntA-PtSTS-2-F / IntA-PtSTS-2-R and pUC57-PtSTS plasmid as template, we amplified the fragments with P at both ends. TEF and T lip2t Homology arms of the PtSTS gene.

[0054] The pUC-HUH-IntA-Pc4CL1 integration plasmid was digested with the restriction endonuclease Not I from NEB, and the linearized pUC-HUH-IntA-Pc4CL1 integration plasmid was recovered by agarose gel electrophoresis. Then, the linearized pUC-HUH-IntA-Pc4CL1 site integration plasmid and the elements of the constructed PtSTS gene expression cassette (promoter P TEF , PtSTS gene, terminator T lip2t ) One-step cloning was achieved using ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The PtSTS gene expression cassette was inserted between the downstream homology arm of the IntA site integration plasmid and the Pc4CL1 gene expression cassette to obtain a circular recombinant vector. The circular recombinant vector was transformed into Escherichia coli Trans1-T1 competent cells, and the positive recombinant plasmid pUC-HUH-IntA-Pc4CL1-PtSTS was obtained by plate screening for ampicillin resistance, combined with colony PCR and sequencing verification.

[0055] (3) Construction of recombinant plasmid pUC-HUH-IntA-PtrPAL3-Pc4CL1-PtSTS

[0056] The promoter region of the PtrPAL3 gene expression cassette was amplified using primers IntA-PtrPAL3-1-F / IntA-PtrPAL3-1-R and IntA-PtrPAL3-3-F / IntA-PtrPAL3-3-R, respectively, and the Yarrowia lipolytica Po1f genome as a template. TEF and terminator T xpr2t Using primers IntA-PtrPAL3-2-F / IntA-PtrPAL3-2-R as primers and pUC57-PtrPAL3 plasmid as template, amplify the two ends of the TEF and T xpr2t Homology arms of the PtrPAL3 gene.

[0057] The pUC-HUH-IntA-Pc4CL1-PtSTS integration plasmid was digested with restriction endonuclease Pac I from NEB, and the linearized pUC-HUH-IntA-Pc4CL1-PtSTS integration plasmid was recovered by agarose gel electrophoresis. Then, the linearized pUC-HUH-IntA-Pc4CL1-PtSTS integration plasmid and the elements in the constructed PtrPAL3 gene expression cassette (promoter P TEF , PtrPAL3 gene, terminator T xpr2t) One-step cloning was achieved using ClonExpress MultiSOne Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The gene expression cassette was inserted between the upstream homology arm of the IntA site integration plasmid and the Pc4CL1 gene expression cassette to obtain a circular recombinant vector. The circular recombinant vector was transformed into Escherichia coli Trans1-T1 competent cells, and the positive recombinant plasmid pUC-HUH-IntA-PtrPAL3-Pc4CL1-PtSTS was obtained by plate screening for ampicillin resistance, combined with colony PCR and sequencing verification.

[0058] 2. Construction of multi-copy integration expression plasmid pUC-HUH-IntD-PtSTS03

[0059] The integration expression plasmid pUC-HUH-IntD-PtSTS03 is based on the plasmid pUC57-hisG-Ura-hisG, which is inserted into the homology arm IntD-up upstream of the start codon and the homology arm IntD-down downstream of the stop codon of the IntD site on chromosome D in the Po1f genome of Yarrowia lipolytica. Three copies of the PtSTS gene expression cassette (named PtSTS01 gene expression cassette, PtSTS02 gene expression cassette, and PtSTS03 gene expression cassette, respectively, are inserted between the downstream homology arm and the hisG tag encoding gene. TEF -PtSTS-T lip2t Composition), Ura expression cassette (promoter TEFin , 3 orotidine-5'-phosphate decarboxylase encoding gene and terminator T xpr2t The specific structure of the integration expression plasmid pUC-HUH-IntD-PtSTS03 is shown in Figure 3 As shown, the specific construction process is as follows:

[0060] The promoter region of the PtSTS01 gene expression cassette was amplified using the primers IntD-PtSTS01-1-F / IntD-PtSTS01-1-R and IntD-PtSTS01-3-F / IntD-PtSTS01-3-R, respectively, and the Yarrowia lipolytica Po1f genome as a template. TEF and terminator T xpr2t Using primers IntD-PtSTS01-2-F / IntD-PtSTS01-2-R as primers and pUC57-PtSTS plasmid as template, the amplified fragments were labeled with P TEF and T lip2t Homology arms of the PtSTS01 gene.

[0061] The IntD site integration plasmid was digested with restriction endonuclease Pac I from NEB, and the linearized IntD site integration plasmid was recovered by agarose gel electrophoresis. The linearized IntD site integration plasmid and the constructed PtSTS01 gene expression cassette were cloned in one step using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The PtSTS01 gene expression cassette was inserted between the downstream homology arm and the hisG tag encoding gene of the IntD site integration plasmid to obtain a circular recombinant vector. The circular recombinant vector was transformed into Escherichia coli Trans1-T1 competent cells, and the positive recombinant plasmid pUC-HUH-IntD-PtSTS01 was obtained by plate screening for ampicillin resistance, combined with colony PCR and sequencing verification.

[0062] Based on the recombinant plasmid pUC-HUH-IntD-PtSTS01, the above construction process was repeated using corresponding primers (IntD-PtSTS02-1-F / IntD-PtSTS02-1-R, IntD-PtSTS02-3-F / IntD-PtSTS02-3-R, IntD-PtSTS02-2-F / IntD-PtSTS02-2-R), and the PtSTS02 gene expression cassette was inserted between the downstream homologous arm of the pUC-HUH-IntD-PtSTS01 site integration plasmid and the PtSTS01 expression cassette gene to construct the positive recombinant plasmid pUC-HUH-IntD-PtSTS02.

[0063] Based on the recombinant plasmid pUC-HUH-IntD-PtSTS02, the above construction process was repeated using corresponding primers (IntD-PtSTS03-1-F / IntD-PtSTS03-1-R, IntD-PtSTS03-3-F / IntD-PtSTS03-3-R, IntD-PtSTS03-2-F / IntD-PtSTS03-2-R), and the PtSTS03 gene expression cassette was inserted between the downstream homologous arm of the pUC-HUH-IntD-PtSTS01 site integration plasmid and the PtSTS02 expression cassette gene, and finally the positive recombinant plasmid pUC-HUH-IntD-PtSTS03 was constructed.

[0064] 3. Construction of integration expression plasmid pUC-HUH-IntF-ARO3

[0065] The promoter region of the ARO3 gene expression cassette was amplified using primers IntF-ARO3-1-F / IntF-ARO3-1-R and IntF-ARO3-3-F / IntF-ARO3-3-R, respectively, and the Yarrowia lipolytica Po1f genome as a template. TEF and terminator T mig1t Using primers IntF-ARO3-2-F / IntF-ARO3-2-R, the Yarrowia lipolytica Po1f genome was used as a template to amplify the fragment with P TEF and T lip2t ARO3 gene of homology arm. Wherein, the nucleotide sequence of the ARO3 gene is shown as SEQ ID NO.4.

[0066] The IntF site integration plasmid was digested with restriction endonuclease Pac I from NEB, and the linearized IntF site integration plasmid was recovered by agarose gel electrophoresis. Then, the linearized IntF site integration plasmid and the elements in the constructed ARO3 gene expression cassette (promoter P TEF , AR03 gene, terminator T mig1t ) One-step cloning was achieved using ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The ARO3 gene expression cassette was inserted between the downstream homology arm of the IntF site integration plasmid and the hisG tag encoding gene to obtain a circular recombinant vector. The circular recombinant vector was transformed into Escherichia coli Trans1-T1 competent cells, and the positive recombinant plasmid pUC-HUH-IntF-ARO3 was obtained by plate screening for ampicillin resistance, combined with colony PCR and sequencing verification. The specific structure is as follows Figure 4 shown.

[0067] 4. Construction of integration expression plasmid pUC-HUH-IntC-ACC1

[0068] The promoter P of the ACC1 gene expression cassette was amplified using primers IntC-ACC1-1-F / IntC-ACC1-1-R and IntC-ACC1-3-F / IntC-ACC1-3-R, plasmid pUC57-HP4D and Yarrowia lipolytica Po1f genome as templates. HP4D and terminator T mig1t Primers IntC-ACC1-2-F / IntC-ACC1-2-R were used as primers and the Yarrowia lipolytica genome was used as a template to amplify the fragments with P HP4D and Tlip2t The ACC1 gene of the homology arm. The nucleotide sequence of the ACC1 gene is shown in SEQ ID NO.5.

[0069] The IntC site integration plasmid was digested with restriction endonuclease Pac I from NEB, and the linearized IntC site integration plasmid was recovered by agarose gel electrophoresis. Then, the linearized IntC site integration plasmid and the elements in the constructed ACC1 gene expression cassette (promoter P HP4D , ACC1 gene, terminator T mig1t ) One-step cloning was achieved using ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. The ACC1 gene expression cassette was inserted between the downstream homology arm of the IntC site integration plasmid and the hisG tag encoding gene to obtain a circular recombinant vector. The circular recombinant vector was transformed into Escherichia coli Trans1-T1 competent cells, and the positive recombinant plasmid pUC-HUH-IntC-ACC1 was obtained by plate screening for ampicillin resistance, combined with colony PCR and sequencing verification. The specific structure is as follows Figure 5 shown.

[0070] 2. Construction of knockout plasmid

[0071] 1. Construction of knockout plasmid pUC-HUH-HPD

[0072] The knockout plasmid pUC-HUH-HPD is based on the plasmid pUC57-hisG-Ura-his and the Yarrowia lipolytica Po1f genome template. The upstream homology arm (HPD-up) and downstream homology arm (HPD-down) of the HPD gene are amplified by PCR. The ClonExpress MultiS One Step Cloning Kit of Nanjing Novizan Biotechnology Co., Ltd. is used to achieve one-step cloning. The two homology arms are inserted into the plasmid pUC57-hisG-Ura-his respectively. The two hisG tag encoding genes are between the upstream and downstream homology arms of the HPD gene. The hisG tag encoding gene Ura expression cassette (promoter TEFin , 3 orotidine-5'-phosphate decarboxylase encoding gene and terminator T xpr2t The composition is also between the upper and lower homologous arms. The specific structure is as follows Figure 6 Wherein, the nucleotide sequence of the HPD gene is shown in SEQ ID NO.6.

[0073] The construction process is described as follows:

[0074] HPD-up-F / HPD-up-R were used as primers and the Po1f genome of Yarrowia lipolytica was used as a template to amplify the upstream homology arm HPD-up of the HPD gene. After digestion with the restriction endonuclease EcoR I from NEB, the linearized pUC57-hisG-Ura-his plasmid was recovered by agarose gel electrophoresis. The linearized pUC57-hisG-Ura-his plasmid and the upstream homology arm HPD-up were cloned in one step using the ClonExpress MultiS OneStep Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. to obtain a circular recombinant vector. The circular recombinant vector was then transformed into Escherichia coli Trans1-T1 competent cells, screened on a plate for ampicillin resistance, and combined with colony PCR and sequencing verification to obtain the positive recombinant plasmid pUC-HUH-HPD-up.

[0075] HPD-down-F / HPD-down-R were used as primers and the Po1f genome of Yarrowia lipolytica was used as a template to amplify the downstream homology arm HPD-down of the HPD gene. After digestion with the restriction endonuclease Hind III from NEB, the linearized pUC-HUH-HPD-up plasmid was recovered by agarose gel electrophoresis. The linearized pUC-HUH-HPD-up plasmid and the downstream homology arm HPD-down were cloned in one step using the ClonExpress MultiS OneStep Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. to obtain a circular recombinant vector. The circular recombinant vector was then transformed into Escherichia coli Trans1-T1 competent cells, screened on a plate for ampicillin resistance, and combined with colony PCR and sequencing verification to obtain the positive recombinant plasmid pUC-HUH-HPD.

[0076] 2. Construction of knockout plasmid pUC-HUH-ARO10

[0077] The corresponding primers were used to obtain the upstream homology arm ARO10-up and the downstream homology arm ARO10-down of the ARO10 gene. The construction process of the knockout plasmid pUC-HUH-HPD was referred to and the positive recombinant plasmid pUC-HUH-ARO10 was finally obtained. The specific structure is as follows Figure 7 Wherein, the nucleotide sequence of the ARO10 gene is shown in SEQ ID NO.7.

[0078] The primers used in the construction of the above-mentioned integration expression plasmid and knockout plasmid are shown in Table 1.

[0079] Table 1 Primer list

[0080]

[0081]

[0082] Example 2: Construction of recombinant Yarrowia

[0083] The pUC-HUH-IntA-PtrPAL3-Pc4CL1-PtSTS plasmid, pUC-HUH-IntD-PtSTS03 plasmid, pUC-HUH-IntF-ARO3 plasmid, pUC-HUH-IntC-ACC1 plasmid, pUC-HUH-HPD plasmid, and pUC-HUH-ARO10 plasmid obtained in Example 1 were digested with restriction endonucleases EcoR I, Not I, Not I, EcoR I, Hind III, and Hind IIII from NEB, respectively, and the corresponding linearized plasmids were recovered by agarose gel electrophoresis.

[0084] Then, the purified fragments of each linearized plasmid were sequentially introduced into Yarrowia lipolytica Po1fΔku70 using the ZymogenFrozen EZ Yeast Transformation Kit II of Zymo Research Corporation to obtain the corresponding recombinant Yarrowia lipolytica. The specific process is as follows:

[0085] 1. Preparation of competent cells: Yarrowia lipolytica Po1fΔku70 was cultured in YPD20 liquid medium (formula: 20 g / L peptone, 10 g / L yeast extract and 20 g / L glucose) at 30°C, 200 rpm overnight to prepare competent cells.

[0086] 2. Chemical transformation and screening: The linearized plasmid pUC-HUH-IntA-PtrPAL3-Pc4CL1-PtSTS was transferred into Yarrowia lipolytica Po1fΔku70 competent cells using Zymogen Frozen EZYeast Transformation Kit II from Zymo Research Corporation for homologous recombination. Positive clones were screened using the screening medium SD-Ura purchased from Point Biotech, and recombinant Yarrowia lipolytica TAN1 was obtained by PCR identification.

[0087] 3. Remove the screening marker: Dissolve 1g of 5-fluoroorotic acid powder in 10mL of dimethyl sulfoxide to obtain a 5-fluoroorotic acid solution with a concentration of 100g / L. Add the 5-fluoroorotic acid solution to the YPD20 medium at an addition amount of 1% (v / v) to prepare a YPD20 plate containing 5-fluoroorotic acid. Spread the recombinant Yarrowia lipolytica TAN1 on a YPD plate containing 5-fluoroorotic acid, and after PCR identification, successfully lose a hisG tag and Ura screening marker under the pressure of 5-fluoroorotic acid, and obtain the strain Q-TAN1 with the correct removal of the screening marker.

[0088] 4. The linearized recombinant plasmid pUC-HUH-IntD-PtSTS03 was introduced into the competent cells of the strain Q-TAN1 with the selection marker removed using the Zymogen Frozen EZ Yeast Transformation Kit II kit for homologous recombination. The positive clones were screened using the screening medium SD-Ura purchased from Puint Biotech, and the recombinant Yarrowia lipolytica TAN2 was obtained by PCR identification. The recombinant Yarrowia lipolytica TAN2 was coated on a YPD20 plate containing 5-fluoroorotic acid. After PCR identification, a hisG tag and Ura selection marker were successfully lost under the pressure of 5-fluoroorotic acid, and the strain Q-TAN2 with the correct selection marker removal was obtained.

[0089] 5. The linearized recombinant plasmids pUC-HUH-IntF-ARO3 and pUC-HUH-IntC-ACC1, and the knockout plasmids pUC-HUH-HPD and pUC-HUH-ARO10 were introduced into competent cells of the strain Q-TAN2 in which the selection marker was correctly removed using the Zymogen Frozen EZ Yeast Transformation Kit II kit for homologous recombination. The positive clones were screened using the screening medium SD-Ura purchased from Puint Biotech, and the recombinant strain TAN3 of Yarrowia lipolytica was obtained by PCR identification.

[0090] Example 3: Fermentation of recombinant Yarrowia lipolytica and determination of sutsugamotolin concentration

[0091] 1. Shake flask culture of Yarrowia lipolytica and product extraction

[0092] The initial Yarrowia lipolytica and the recombinant Yarrowia lipolytica TAN1-3 in Example 2 were used to produce sutsugamotolin. The specific method is as follows:

[0093] The activated initial Yarrowia lipolytica and recombinant Yarrowia lipolytica TAN1-3 were inoculated into YPD20 liquid culture medium, shaken and cultured at 30°C and 200rpm for 24h to obtain seed liquid respectively. The seed liquid was inoculated into a shake flask containing 20mL fermentation medium (i.e., YPD20 liquid culture medium) at an inoculum amount of 1% v / v, and shaken and cultured at 30°C and 200rpm for 5 days. After the fermentation was completed, 0.5mL of fermentation broth was taken into a 1.5mL centrifuge tube and 0.5mL of ethyl acetate was added. Vortex for 3min, centrifuged at 1200rpm for 1min, the supernatant was transferred to a new 1.5mL centrifuge tube, and evaporated at 65°C for 1h. The residue was redissolved in methanol and filtered through an organic nylon filter membrane (0.22μm), and then HPLC was used to quantitatively analyze the red pinocytogenin.

[0094] 2. Quantitative analysis of pine tin

[0095] (1) Reagents and solutions:

[0096] Acetonitrile: chromatography grade; acetic acid: chromatography grade; distilled water: freshly distilled twice; redistilled pine alkali standard; mobile phase: 1.5% acetic acid and 100% acetonitrile in a volume ratio of 60:40.

[0097] (2) Instrument: High performance liquid chromatography with adjustable wavelength UV detector; Chromatographic column: Agilentr Eclipse XDB-C18 reverse phase column (250 mm×4.6 mm, 5 μm).

[0098] (3) HPLC operating conditions

[0099] The detection wavelength was 294 nm; the mobile phase A was 100% acetonitrile and B was 1.5% acetic acid, and 40% of the mobile phase A was used for isocratic elution. The flow rate was 1 mL / min and the column temperature was 30°C. Under these conditions, the peak times of cinnamic acid (t-ca) and pinus erythrin were 6.175 and 11.238 min, respectively. The results are shown in Figure 8 The calibration curves of cinnamic acid (t-ca) and pinus erythritol are shown in Figure 8 As shown in B and C.

[0100] (4) Preparation of standard solution

[0101] Dissolve 2 mg of pine sapogenin in 10 mL of methanol to prepare a 200 mg / L pine sapogenin mother solution. Then dilute the mother solution to obtain 20, 100, 120, 160, and 200 mg / L pine sapogenin solutions. Take 100 μL of the 20, 100, 120, 160, and 200 mg / L solutions for HPLC analysis and draw a standard curve.

[0102] (3) Detection of pine cone concentration in fermentation broth

[0103] The concentration of sutchondroitin in the fermentation broth of the initial Yarrowia lipolytica and the recombinant Yarrowia lipolytica TAN1-3 was determined according to the above method. The specific results are shown in Table 2. The results showed that after 3 days of fermentation, no accumulation of sutchondroitin was detected in the initial Yarrowia lipolytica; 60 mg / L of sutchondroitin was detected in the recombinant Yarrowia lipolytica TAN1; 120 mg / L of sutchondroitin was detected in the recombinant Yarrowia lipolytica TAN2; and 230 mg / L of sutchondroitin was detected in the recombinant Yarrowia lipolytica TAN3.

[0104] 3. Fermentation of recombinant Yarrowia lipolytica TAN3 in a 5L fermenter

[0105] The recombinant Yarrowia lipolytica TAN3 was inoculated into 5 mL of YPD20 liquid culture medium, and cultured at 30°C and 200 rpm for 24 h to obtain a seed solution; the seed solution was expanded to 100 mL of YPD20 liquid culture medium at an inoculum volume of 1% v / v, and cultured at 30°C and 200 rpm for 24 h to obtain a fermentation tank seed solution; the fermentation tank seed solution was inoculated into 100 mL of YPD20 liquid culture medium at an initial OD 600 =0.5 was connected to a 5L fermenter (containing 2L fermentation medium, i.e., YPD20 liquid medium: 20g / L peptone, 10g / L yeast extract and 60g / L glucose) for fermentation culture. When the residual sugar concentration of glucose was lower than 5g / L, feed medium (formula: 800g / L glucose) was added to control the glucose concentration to 5g / L, and the fermentation was carried out for 84h. During the fermentation period, the temperature was controlled to be 30°C, the dissolved oxygen was 30-40%, the pH value was constant to be 5.0, the stirring speed was 300-1000rpm, and the ventilation volume was 2-3vvm.

[0106] After 84 h of fermentation, the results are shown in Table 2. It was determined that the yield of kaempferol by the recombinant Yarrowia lipolytica TAN3 reached 1200 mg / L.

[0107] Table 2 The yield of pine tin in the fermentation broth of each strain

[0108] Fermentation method strain Pinus erythritol yield (mg / L) Shake bottle Yarrowia lipolytica 0 Shake bottle Recombinant Yarrowia lipolytica TAN1 60 Shake bottle Recombinant Yarrowia lipolytica TAN2 120 Shake bottle Recombinant Yarrowia lipolytica TAN3 230 Fermentation tank Recombinant Yarrowia lipolytica TAN3 1200

[0109] The present invention provides a recombinant Yarrowia lipolytica with high yield of styrax tin and its construction method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A recombinant Yarrowia lipolytica for high yield of kaempferol, characterized in that: The recombinant Yarrowia lipolytica is constructed by using Yarrowia lipolytica as a chassis cell, inserting the phenylalanine ammonia lyase gene PtrPAL3, the p-coumaroyl-CoA ligase gene Pc4CL1 and the stilbene synthase gene PtSTS into the genome, overexpressing the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene ARO3 and the acetyl-CoA carboxylase gene ACC1, and knocking out the 4-hydroxyphenylpyruvate dioxygenase gene HPD and the phenylpyruvate decarboxylase gene ARO10.

2. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The phenylalanine ammonia lyase gene PtrPAL3, the p-coumaroyl-CoA ligase gene Pc4CL1 and the stilbene synthase gene PtSTS are derived from Populus trichocarpa, Petroselinum crispum and Pinus thunbergii respectively.

3. The recombinant Yarrowia lipolytica according to claim 2, characterized in that The nucleotide sequence of the phenylalanine ammonia lyase gene PtrPAL3 is the sequence shown in SEQ ID NO.1, or a nucleotide sequence with a homology greater than 90% and having an equivalent function for the synthesis of pinostom; the nucleotide sequence of the p-coumaroyl-CoA ligase gene Pc4CL1 is the sequence shown in SEQ ID NO.2, or a nucleotide sequence with a homology greater than 90% and having an equivalent function for the synthesis of pinostom; the nucleotide sequence of the stilbene synthase gene PtSTS is the sequence shown in SEQ ID NO.3, or a nucleotide sequence with a homology greater than 90% and having an equivalent function for the synthesis of pinostom.

4. The recombinant Yarrowia lipolytica according to claim 1, characterized in that The 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene ARO3, the acetyl-CoA carboxylase gene ACC1, the 4-hydroxyphenylpyruvate dioxygenase gene HPD and the phenylpyruvate decarboxylase gene ARO10 are all derived from Yarrowia lipolytica; wherein the nucleotide sequence of the 3-deoxy-D-arabinoheptulose-7-phosphate synthase gene ARO3 is shown in SEQ ID NO.4, the nucleotide sequence of the acetyl-CoA carboxylase gene ACC1 is shown in SEQ ID NO.5, the nucleotide sequence of the 4-hydroxyphenylpyruvate dioxygenase gene HPD is shown in SEQ ID NO.6, and the nucleotide sequence of the phenylpyruvate decarboxylase gene ARO10 is shown in SEQ ID NO.

7.

5. The method for constructing the recombinant Yarrowia lipolytica according to claim 1, characterized in that: The integration expression plasmids carrying PtrPAL3, Pc4CL1 and PtSTS genes, the integration expression plasmid carrying PtSTS gene, the integration expression plasmid carrying ARO3 gene, the integration expression plasmid carrying ACC1 gene, the HPD gene knockout plasmid and the ARO10 gene knockout plasmid are linearized and sequentially introduced into chassis cells to construct the recombinant Yarrowia lipolytica.

6. The construction method according to claim 5, characterized in that: The integration expression plasmid carrying the PtSTS gene is a multi-copy integration expression plasmid, and the copy number is ≥2.

7. The construction method according to claim 5, characterized in that: The integration expression plasmids are all inserted into corresponding integration sites in the form of expression cassettes; wherein the integration sites are the IntA site, IntD site, IntC site and IntF site of the chassis cells.

8. The construction method according to claim 7, characterized in that: The expression cassette contains a promoter and a terminator; wherein the promoter is promoter P HP4D , promoter P TEF , promoter P TEFin Any one of the above, wherein the terminator is terminator T xpr2t , terminator T lip2t , terminator T mig1t Any one of .

9. Use of the recombinant Yarrowia lipolytica according to any one of claims 1 to 8 in the biosynthesis of pinospermum officinale.

10. The use according to claim 9, characterized in that: The formula of the fermentation medium for biosynthesis is: 10-40 g / L peptone, 5-20 g / L yeast extract and 20-120 g / L glucose; the fermentation conditions for biosynthesis are: fermentation temperature of 28-30°C, dissolved oxygen of 30-40%, pH 5.0-6.0, stirring speed of 200-1000 rpm, ventilation volume of 2-3 vvm, fermentation time of 72-200 h, and glucose concentration is controlled at 5-10 g / L during fermentation.

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