WO2020199952A1 - 重组酵母、构建方法和其在制备酪醇及衍生物中的应用 - Google Patents

重组酵母、构建方法和其在制备酪醇及衍生物中的应用 Download PDF

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WO2020199952A1
WO2020199952A1 PCT/CN2020/080627 CN2020080627W WO2020199952A1 WO 2020199952 A1 WO2020199952 A1 WO 2020199952A1 CN 2020080627 W CN2020080627 W CN 2020080627W WO 2020199952 A1 WO2020199952 A1 WO 2020199952A1
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bifidobacterium
yeast
tyrosol
candida
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French (fr)
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方诩
郭伟
侯少莉
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烟台华康荣赞生物科技有限公司
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Priority to EP20783979.6A priority Critical patent/EP3835411B9/en
Priority to ES20783979T priority patent/ES2954682T3/es
Priority to JP2021502824A priority patent/JP7083959B2/ja
Priority to US17/264,889 priority patent/US20220213512A1/en
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  • the present invention relates to recombinant yeast, a construction method and its application in the preparation of tyrosol and its derivatives, in particular to a strain that passes exogenous fructose-6-phosphate phosphoketolase (Fructose-6-phosphate phosphoketolase, fxp)
  • the method for introducing expressed genes into yeast and using the strain to efficiently produce tyrosol and its derivatives belongs to the technical field of microbial genetic engineering.
  • Tyrosol is a natural antioxidant derived from olive oil and is a derivative of phenethyl alcohol. Also known as salidroside aglycone, it is the main medicinal active ingredient of rhodiola root and the precursor substance of salidroside and hydroxytyrosol. It can protect cells from oxidative damage and is a phenolic compound with important industrial value. Tyrosol and its derivatives are synthetic precursors of a variety of organic compounds. Tyrosol can be used as a pharmaceutical agent.
  • the derivative of tyrosol, hydroxytyrosol has a strong antioxidant effect and a variety of physiological and medical functions. The antioxidant capacity of hydroxytyrosol is stronger than that of tyrosol.
  • phenylethyl alcohol is used to synthesize, most of which firstly protect the hydroxyl group, then nitrify, reduce, diazotize, and hydrolyze to obtain p-hydroxyphenyl alcohol with a yield of 70%.
  • Phenylethanol is high in price and tight in supply. It is synthesized from nitrotoluene, and the price is low, but the steps are longer and the yield is low.
  • the synthetic yield of p-hydroxystyrene reaches 96%, the purity is 99%, and the yield and purity are both high. Value, but the raw material cost is higher.
  • the chemical method for preparing tyrosol has high raw material costs and is not environmentally friendly, which directly restricts the industrial production of tyrosol. Therefore, biosynthesis of tyrosol and its derivatives has become a research hotspot.
  • Tyrosol has the following characteristics: the chemical name is 4-(2-Hydroxyethyl)phenol, the molecular formula is C 8 H 10 O 2 , the molecular weight is 138.164, the CAS number is 501-94-0, and the structural formula is
  • Chinese patent document CN108753636A discloses a yeast producing tyrosol and hydroxytyrosol and a construction method.
  • PcAAS and ADH sequences are introduced into yeast BY4741 to obtain tyrosol-producing PcAAS-ADH recombinant yeast; in the PcAAS -Introduce the pdc1 gene knockout cassette and tyrA expression cassette into the ADH recombinant yeast to obtain the PcAAS-ADH- ⁇ pdc1-tyrA recombinant yeast producing tyrosol; introduce the DNA sequence of HpaBC into the PcAAS-ADH- ⁇ pdc1-tyrA recombinant yeast to obtain the hydroxyphenol production PcAAS-ADH-HpaBC- ⁇ pdc1-tyrA recombinant yeast.
  • the present invention provides recombinant yeast, a construction method and its application in the preparation of tyrosol and derivatives.
  • One of the objectives of the present invention is to express fructose-6-phosphate phosphoketolase from Bifidobacterium adolescentis (Bifidobacterium adolescentis) strain number ATCC 15703 on the basis of the invention patent application (application number 201810601213.8) (Fructose-6-phosphate phosphoketolase, EC 4.1.2.22) (The amino acid sequence is shown in GenBank: BAF39468.1, SEQ ID NO.1) or the fructose-6-phosphate phosphate ketone of Bifidobacterium breve BBRI4 Enzyme (Fructose-6-phosphate phosphoketolase, EC 4.1.2.22) (amino acid sequence is shown in GenBank: KND53308.1, SEQ ID NO.
  • the second objective of the present invention is to provide a method for producing hydroxytyrosol.
  • the third objective of the present invention is to provide a method for constructing tyrosol-producing yeast.
  • the fourth objective of the present invention is to provide the application of the tyrosol-producing yeast or construction method in tyrosol production.
  • the fifth object of the present invention is to provide the application of the tyrosol-producing yeast or the construction method in the production of hydroxytyrosol.
  • the recombinant yeast is constructed by introducing an exogenous fructose-6-phosphate phosphoketolase expression gene into a modified yeast cell, and the modified yeast cell is Yeast cells with metabolic pathways for synthesizing tyrosol via 4-phosphoerythrose and phosphoenolpyruvate.
  • the modified yeast cell is obtained by integrating aromatic aldehyde synthase and chorismate mutase/prephenate dehydratase; or the modified yeast cell is obtained by integrating aromatic aldehyde synthase. Obtained later
  • the aromatic aldehyde synthase is derived from parsley, and the system number of the enzyme is EC4.1.1.25; the chorismate mutase is derived from E. coli, and the system number of the enzyme is EC1 .3.1.12; The prephenate dehydratase is derived from Escherichia coli (E.coli), and the system number of the enzyme is EC1.3.1.12, EC 5.4.99.5.
  • the expression gene of fructose-6-phosphate phosphoketolase is derived from Bifidobacterium adolescentis, Bifidobacterium animalis, and Bifidobacterium breve ( Bifidobacterium bifidum, Aspergillus nidulans, Bifidobacterium breve, Bifidobacterium lactis, Clostridium acetobutylicum, Bifidobacterium longum, Bifidobacterium longum, Bifidobacterium dentium, Leuconostoc mesenteroides, Bifidobacterium mongoliense, Lactobacillus paraplantarum, Lactobacillus plantarum, False Bifidobacterium pseudolongum, Candida tropicalis, Cryptococcus neoformans, Cupriavidus necator, Gardnerella vaginalis, saprophytic yeast Rhodotorul
  • the amino acid sequence of the fructose-6-phosphate phosphoketolase is shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the nucleotide sequence of the expressed gene is shown in SEQ ID NO. 3 or SEQ ID NO. 4 shown;
  • the amino acid sequence of the fructose-6-phosphate phosphoketolase is shown in SEQ ID NO. 30, and the nucleotide sequence of the expressed gene is shown in SEQ ID NO. 31.
  • the yeast cells are preferably: Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces pombe, Kluyveromyces lactis, Max Kluyveromyces marxianus, Candida lipolytica, Torulopsis glabrata, saprophytic yeast Rhodotorula glutinis, Rhodotorula graminis, Barbados Saccharomyces pastorianus, Candida tropicalis, Zygosaccharomyces rouxii, Candida glabrata, Torulaspora delbrueckii, Hans de Pastoris ( Debaryomyces hansenii), Scheffersomyces stipites, Meyerozyma guilliermondii, Lodderomyces elongisporus, Candida albicans, Candida parapsilosis orthopsilosis), Candida metapsilosis (Candida metasilosis), Dublin yeast (Candida dubliniens
  • the yeast cell is Saccharomyces cerevisiae, the strain number is CICC1964; and the Kluyveromyces marxianus, the strain number is NBRC1777.
  • the modified yeast cells are those that integrate aromatic aldehyde synthase derived from Parsley (Petroselinum crispum) into the delta12 site of Saccharomyces cerevisiae CICC1964 and are derived from E. coli
  • the chorismate mutase/prephenate dehydratase was obtained by replacing the pdc1 gene of Saccharomyces cerevisiae CICC1964.
  • a method for constructing recombinant yeast with high tyrosol production includes the following steps:
  • the modified yeast cell is a yeast cell that has a metabolic pathway for synthesizing tyrosol via erythrose 4-phosphate and phosphoenolpyruvate.
  • the modified yeast cells are those that integrate aromatic aldehyde synthase derived from Parsley (Petroselinum crispum) into the delta12 site of Saccharomyces cerevisiae CICC1964 and are derived from E. coli Replace the pdc1 gene of Saccharomyces cerevisiae CICC1964 with chorismate/prephenate dehydratase of
  • the modified yeast cell is an aromatic aldehyde synthase derived from Parsley (Petroselinum crispum) integrated into the delta12 site of Kluyveromyces marxianus NBRC1777, and is derived from Escherichia coli (E .coli) chorismate mutase/prephenate dehydratase was obtained by replacing the pdc1 gene of Kluyveromyces marxianus NBRC1777.
  • the expression gene of fructose-6-phosphate phosphoketolase in the step (1) is preferably derived from Bifidobacterium adolescentis, Bifidobacterium animalis, and Bifidobacterium bifidum.
  • Bifidobacterium breve (Bifidobacterium bifidum), Aspergillus nidulans (Aspergillus nidulans), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium lactis (Bifidobacterium lactis), Clostridium acetobutylicum (Clostridium acetobutylicum), Bifidobacterium longum ( Bifidobacterium longum), Bifidobacterium dentium, Leuconostoc mesenteroides, Bifidobacterium mongoliense, Lactobacillus paraplantarum, Lactobacillus paraplantarum ( Lactobacillus plantarum, Bifidobacterium pseudolongum, Candida tropicalis, Cryptococcus neoformans, Cupriavidus necator, Gardnerella vaginalis, saprophytic bacteria Sexual yeast Rhodotorul
  • the amino acid sequence of the fructose-6-phosphate phosphoketolase is shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the nucleotide sequence of the expressed gene is shown in SEQ ID NO. 3 or SEQ ID NO. .4 shown.
  • the amino acid sequence of the fructose-6-phosphate phosphoketolase is shown in SEQ ID NO. 30, and the nucleotide sequence of the expressed gene is shown in SEQ ID NO. 31.
  • the homology arm uses the genome of Saccharomyces cerevisiae strain CICC1964 or Kluyveromyces marxianus NBRC1777 as a template, and primers are used to amplify the upstream of prephenate dehydratase gene pha2 And the 500bp downstream gene fragment, the nucleotide sequences of the upstream homology arm amplification primers are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively; the nucleotide sequences of the downstream homology arm amplification primers are respectively As shown in SEQ ID NO.7 and SEQ ID NO.8;
  • the promoter in the step (1) is a promoter tpi1 amplified with primers using the genome of Saccharomyces cerevisiae strain CICC1964 or Kluyveromyces marxianus NBRC1777 as a template, and an amplification primer of the promoter tpi1
  • the nucleotide sequences of are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively;
  • the promoter in the step (1) is a terminator gpm1 amplified with primers using the genome of Saccharomyces cerevisiae strain CICC1964 or Kluyveromyces marxianus NBRC1777 as a template, and an amplification primer of the terminator gpm1
  • the nucleotide sequences of are shown in SEQ ID NO.9 and SEQ ID NO.10 respectively;
  • the modified yeast cell is obtained by integrating aromatic aldehyde synthase and chorismate mutase/prephenate dehydratase; or the modified yeast cell is Obtained after integration of aromatic aldehyde synthase;
  • the aromatic aldehyde synthase is derived from parsley (Petroselinum crispum), and the system number of the enzyme is EC4.1.1.25; the chorismate mutase/pre-phenic acid dehydration
  • the enzyme is derived from Escherichia coli (E. coli), the system number of the enzyme is EC1.3.1.12, EC 5.4.99.5.
  • the yeast cells in the step (2) are: Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces pombe, Kluyveromyces lactis Yeast (Kluyveromyces lactis), Kluyveromyces marxianus, Candida lipolytica, Torulopsis glabrata, saprophytic yeast Rhodotorula glutinis, Graminea red Yeast (Rhodotorula graminis), Pasteurys yeast (Saccharomyces pastorianus), Candida tropicalis (Candida tropicalis), Lu's yeast (Zygosaccharomyces rouxii), Candida glabrata (Candida glabrata), Deborer spore yeast (Torulaspora delbrueckii), Han Debaryomyces hansenii, Scheffersomyces stipites, Meyerozyma guilliermondii, Lodderomyces
  • the yeast cell is Saccharomyces cerevisiae, the strain number is CICC1964; the Kluyveromyces marxianus, the strain number is NBRC1777.
  • the modified yeast cell is obtained by integrating an aromatic aldehyde synthase derived from petroselinum crispum into the delta12 site of Saccharomyces cerevisiae CICC1964.
  • the modified yeast cell in the step (2), is to integrate aromatic aldehyde synthase derived from petroselinum crispum into the delta12 site of Saccharomyces cerevisiae CICC1964, and will be derived from the large intestine
  • the chorismate mutase/prephenate dehydratase of E. coli was obtained by replacing the pdc1 gene of Saccharomyces cerevisiae CICC1964.
  • the modified yeast cell is obtained by integrating an aromatic aldehyde synthase derived from petroselinum crispum into the delta12 site of Kluyveromyces marxianus NBRC1777.
  • the modified yeast cell integrates aromatic aldehyde synthase derived from petroselinum crispum into the delta12 site of Kluyveromyces marxianus NBRC1777,
  • the pdc1 gene of Kluyveromyces marxianus NBRC1777 was replaced by chorismate mutase/prephenate dehydratase derived from E. coli.
  • Recombinant yeast with high tyrosol production constructed by the above method.
  • the fermentation medium for fermentation contains at least one of glucose, fructose, sucrose or a combination of two or more and tyrosine.
  • hydroxytyrosol is obtained through hydroxylase reaction.
  • the tyrosol obtained by fermentation of the above-mentioned recombinant yeast with high tyrosol production is catalyzed by E. coli overexpressing 4-hydroxyphenylacetic acid hydroxylase to obtain hydroxytyrosol.
  • the fermentation medium for fermentation contains at least one or a combination of glucose, fructose, sucrose and tyrosine.
  • the present invention discloses for the first time that during expression in yeast, fructose-6-phosphate (Fructose-6-phosphate) synthesizes beta-D-Fructose 1,6-bisphosphate at the same time, It is catalyzed into Erythrose-4-phosphate and Acetyl-phosphate, and Xylulose-5-phosphate is catalyzed into Glyceraldehyde-3-phosphate ( Glyceraldehydes-3-phosphate) and acetyl phosphate, which change the carbon metabolism flux distribution in yeast, enhance the synthesis of erythrose-4-phosphate, which is an important intermediate in tyrosol biosynthesis, and optimize the metabolic pathways for synthesizing tyrosol.
  • the present invention introduces the expression gene of exogenous fructose-6-phosphate phosphoketolase into modified yeast cells to obtain a recombinant yeast, which can increase the yield of tyrosol and has used E. coli expressing 4-hydroxyphenylacetic acid hydroxylase catalyzes tyrosol to obtain hydroxytyrosol.
  • the present invention provides a new and environment-friendly tyrosol and hydroxytyrosol biological preparation technology, which lays the foundation for large-scale industrial production of tyrosol and hydroxytyrosol, and has important economic value and social benefits.
  • Figure 1 is a structural diagram of the recombinant plasmid pUG6 described in Example 1;
  • E. coli BL21 and expression vector pET-28a are commonly used commercially.
  • the experimental methods without specific conditions in the following examples are carried out according to conventional conditions, such as the conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions recommended by the manufacturers of the corresponding biological reagents.
  • the reaction procedure of the PCR amplification reaction may be a conventional PCR amplification reaction procedure.
  • Saccharomyces cerevisiae CICC1964 purchased from China Industrial Microbial Culture Collection Management Center, strain number CICC1964, is a known non-preserved strain;
  • the aromatic aldehyde synthase (AAS, aromatic aldehyde synthase, EC4.1.1.25) derived from petroselinum crispum was integrated into the delta12 site of Saccharomyces cerevisiae CICC1964, and Replaced chorismate mutase/prephenate dehydrogenase (TyrA, fused chorismate mutase T/prephenate dehydrogenase, EC1.3.1.12, EC 5.4.99.5) from E. coli for pdc1 of Saccharomyces cerevisiae CICC1964 Gene to obtain SC-1 strain.
  • AAS aromatic aldehyde synthase
  • the amino acid sequences of SEQ ID NO. 1 and SEQ ID NO. 2 were codon-optimized according to the codon preference of the host Saccharomyces cerevisiae, and the optimized nucleotide sequence SEQ ID corresponding to SEQ ID NO. 1 and SEQ ID NO. 2 was obtained. After NO.3 and SEQ ID NO.4, perform gene synthesis.
  • the primer pairs Bafxpk-F/Bafxpk-R and Bbfxpk-F/Bbfxpk-R were used to amplify the target gene fructose-6-phosphate phosphoketolase expression gene (Bafxpk) with PhantaMax High-Fidelity DNA polymerase from Vazyme.
  • the nucleotide sequence of the fragment is shown in SEQ ID NO. 3, and the nucleotide sequence of the Bbfxpk fragment is shown in SEQ ID NO. 4).
  • primer pairs UF/UR and DF/DR PCR were used to amplify the DNA fragments of the upstream and downstream homology arms, and primer pairs Ptpi1-F/Ptpi1-R and Tgpm1-F/Tgpm1-R were used for amplification. Increase the promoter tpi1 and terminator gpm1 fragments.
  • the sequence of the primer pair Bafxpk-F/Bafxpk-R is SEQ ID NO.13 and SEQ ID NO.14; the sequence of the primer pair Bbfxpk-F/Bbfxpk-R is SEQ ID NO.15 and SEQ ID NO.16; the primer pair The sequence of UF/UR is SEQ ID NO.5 and SEQ ID NO.6; the sequence of primer pair DF/DR is SEQ ID NO.7 and SEQ ID NO.8; the sequence of primer pair Ptpi1-F/Ptpi1-R is SEQ ID NO.9 and SEQ ID NO.10; the sequence of primer pair Tgpm1-F/Tgpm1-R is SEQ ID NO.11 and SEQ ID NO.12; the sequence of primer pair G418-F/G418-R is SEQ ID NO.17 and SEQ ID NO.18;
  • step 2 Use the PCR reaction product of step 1 as a PCR amplification template, select the primer pair Yzaw-F/Yzaw-R, and use Vazyme's PhantaMax High-Fidelity DNA polymerase to amplify the target fragment. After agarose gel electrophoresis to verify the correct size of the band, cut the band, and use the OMEGA gel extraction kit to recover the DNA fragments, namely the DNA fragments of Bafxpk and Bbfxpk expression cassettes.
  • the primers for PCR amplification are as follows:
  • sequence of Yzaw-F is SEQ ID NO.19; the sequence of Yzaw-R is SEQ ID NO.20;
  • the PEG/LiAc method was used to transform the Saccharomyces cerevisiae tyrosol synthesis strain CICC1964, the single clone was selected by adding G418 resistance to the medium, the genome was extracted, and the Yzaw-F/Yzaw-R was verified by PCR using primers to obtain SC- bafxpk and SC-bbfxpk strains.
  • the initial inoculation OD 600 is 0.2, and after culturing for 12 hours at 30°C and 200 rpm, it is transferred to 100 mL YPD liquid medium.
  • the initial inoculation OD 600 is 0.2, and the medium contains 2% glucose or 2% sucrose.
  • the DNA sequence gene cluster of 4-hydroxyphenylacetate hydroxylase (HpaBC, 4-hydroxyphenylacetate 3-hydroxylase, enzyme system number EC 1.5.1.37) derived from E. coli was overexpressed in E. coli using whole E. coli cells The tyrosol is catalyzed to hydroxytyrosol.
  • the amino acid sequence gene cluster of 4-hydroxyphenylacetate hydroxylase includes 4-hydroxyphenylacetate hydroxylase (HpaB).
  • the amino acid sequence of 4-hydroxyphenylacetate hydroxylase (HpaB) is SEQ ID NO. 21, and the corresponding nucleotide sequence is SEQ ID NO. 23;
  • the amino acid sequence of 4-hydroxyphenylacetic acid hydroxylase (HpaC) is SEQ ID NO. 22, and the corresponding nucleotide sequence is SEQ ID NO. 24.
  • the bacterial genome kit was used to extract the E. coli DE3 genome as a template, and the primer pairs hpaB-F/hpaB-R and hpaC-F/hpaC-R were used to amplify SEQ ID NO. 23 and SEQ ID NO. 24, and sequenced verification.
  • the sequence of hpaB-F is SEQ ID NO.25; the sequence of hpaB-R is SEQ ID NO.26; the sequence of hpaC-F is SEQ ID NO.27; the sequence of hpaC-R is SEQ ID NO.28.
  • pET-28a as an expression vector, culture E. coli containing an empty pET-28a vector, extract the pET-28a plasmid using a bacterial plasmid extraction kit, and use conventional molecular biology methods to construct an expression vector pEThpaBC (SEQ ID NO.29). Then pEThpaBC was transformed into E. coli expression vector BL21, and kanamycin was used as a selection marker to obtain monoclonal BL21-pEThpaBC.
  • BL21-pEThpaBC was cultured in shake flasks and 1mM IPTG was used to induce the expression of HPAB/C.
  • Table 5 The production of hydroxytyrosol after adding BL21-pEThpaBC bacteria to the culture solution shown in Table 4 and mixing for 3 hours
  • Kluyveromyces marxianus Kluyveromyces marxianus (Kluyveromyces marxianus), the strain number is NBRC1777, according to the invention patent application (application number 201810601213.8), the aromatic aldehyde synthase (AAS, aromatic aldehyde synthase) derived from petroselinum crispum, EC4.1.1.25) was integrated into the delta12 site of Kluyveromyces marxianus, and the chorismate mutase/prephenate dehydratase (TyrA, fused chorismate mutase T/prephenate) derived from E.
  • AAS aromatic aldehyde synthase
  • T/prephenate dehydratase TeyrA, fused chorismate mutase T/prephenate
  • coli dehydrogenase EC1.3.1.12, EC 5.4.99.5 replace the pdc1 gene of Kluyveromyces marxianus to obtain Kluyveromyces marxianus which has a metabolic pathway to synthesize tyrosol via 4-phosphate erythrose and phosphoenolpyruvate Strains.
  • the KM-bafxp and KM-bbfxp strains obtained by preparing fructose-6-phosphate phosphoketolase (EC 4.1.2.22) according to the method described in Example 1-2 of the present invention, the difference
  • the culturing temperature of Kluyveromyces marxianus cells is 42 to 49°C.
  • fructose-6-phosphate phosphoketolase (bdfxp, GenBank: BAQ26957.1) derived from Bifidobacterium dentium
  • the amino acid sequence is shown in SEQ ID NO. 30, according to Example 3 and Example 4.
  • the described method is carried out, the sequence of the primer pair Bdfxp-F/Bdfxp-R used is SEQ ID NO.32 and SEQ ID NO.33; the results are similar to those of Example 3 and Example 4, and tyrosol and hydroxytyrosol are also increased.
  • the output of alcohol The specific results are as follows:
  • Table 7 The production of hydroxytyrosol after adding BL21-pEThpaBC bacteria to the culture solution shown in Table 6 after mixing for 3 hours
  • the aromatic aldehyde synthase (AAS, aromatic aldehyde synthase, EC4.1.1.25) derived from parsley (Petroselinum crispum) was integrated into the delta12 site of Saccharomyces cerevisiae CICC1964, and the Fructose-6-phosphate phosphoketolase (EC 4.1.2.22) shown in SEQ ID NO. 4 was introduced into the above yeast to obtain the SC-bbfxpk-AAS strain. The result was similar to the tyrosol production of the SC-bbfxpk strain in Example 3.
  • AAS aromatic aldehyde synthase
  • Table 9 The production of hydroxytyrosol after adding BL21-pEThpaBC bacteria to the culture solution shown in Table 8 and mixing for 3 hours
  • fructose-6-phosphate phosphoketolase (baifxp, GenBank: WP_052826255.1) derived from Bifidobacterium animalis (Bifidobacterium animalis) is shown in SEQ ID NO.34.
  • the amino acid sequence of fructose-6-phosphate phosphoketolase (bbifxp, GenBank: WP_047289945.1) of Bifidobacterium breve (Bifidobacterium bifidum) is shown in SEQ ID NO.35
  • Bifidobacterium lactis Bifidobacterium lactis
  • the amino acid sequence of fructose-6-phosphate phosphoketolase (blafxp, GenBank: CAC29121.1) of lactis is shown in SEQ ID NO.
  • Bifidobacterium longum fructose-6-phosphate phosphoketolase The amino acid sequence of blofxp, GenBank: PWH09343.1) is shown in SEQ ID NO. 37, and the fructose-6-phosphate phosphoketolase of Bifidobacterium mongoliense (bmfxp, GenBank: CAC29121.1) ) The amino acid sequence is shown in SEQ ID NO.38, and the fructose-6-phosphate phosphoketolase (bpfxp, GenBank: WP_034883174.1) amino acid sequence of Bifidobacterium pseudolongum (Bifidobacterium pseudolongum) is shown in SEQ ID NO.39
  • the amino acid sequence of fructose-6-phosphate phosphoketolase (Anfxp, GenBank: CBF76492.1) of Aspergillus nidulans (Aspergillus nidulans) is shown in SEQ ID NO.40, and the
  • fructose-6-phosphate phosphoketolase (Lmfxp, GenBank: Leuconostoc mesenteroides)
  • the amino acid sequence of AAV66077.1) is shown in SEQ ID NO. 42
  • the amino acid sequence of fructose-6-phosphate phosphoketolase (Lprfxp, GenBank: ALO04878.1) of Lactobacillus paraplantarum (Lactobacillus paraplantarum) is shown in SEQ ID NO.
  • Table 9 The production of hydroxytyrosol after adding BL21-pEThpaBC bacteria to the culture solution shown in Table 8 and mixing for 3 hours
  • Example 7 Based on the results of Example 7, the inventors simultaneously transformed the above-mentioned gene fragments corresponding to fructose-6-phosphate phosphoketolase into the SC-1 strain obtained according to the invention patent application (application number 201810601213.8), namely:
  • the aromatic aldehyde synthase (AAS, aromatic aldehyde synthase, EC4.1.1.25) of celery (Petroselinum crispum) was integrated into the delta12 site of Saccharomyces cerevisiae CICC1964, and the chorismate mutase derived from E.
  • coli /Prephenate dehydratase (TyrA, fused chorismate mutase T/prephenate dehydrogenase, EC1.3.1.12, EC5.4.99.5) was obtained by replacing the pdc1 gene of Saccharomyces cerevisiae CICC1964; the results showed that baifxp, bbifxp, blafxp, blofxp, bmfxp, The results of bpfxp, Anfxp, Cafxp, Lmfxp, Lprfxp, Lplfxp are similar to the results of bbfxpk in Example 7, indicating whether there is chorismate mutase/prephenate dehydrogenase (TyrA, fused chorismate mutase T/prephenate dehydrogenase, EC1) in yeast .3.1.12, EC 5.4.99.5) The introduction of genes has little effect on the production of t

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Abstract

提供一种重组酵母、其构建方法和其在制备酪醇及衍生物中的应用。该重组酵母为将外源性的果糖-6-磷酸盐磷酸酮酶的表达基因导入经过改造的酵母细胞中构建获得，经过改造的酵母细胞为具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞。在该酵母中表达果糖-6-磷酸盐磷酸酮酶的过程中，果糖-6-磷酸在合成1，6-二磷酸果糖的同时，被催化为赤藓糖-4-磷酸和乙酰磷酸，木酮糖-5-磷酸被催化为甘油醛-3-磷酸和乙酰磷酸，这改变了酵母中的碳代谢流量分布，增强酪醇生物合成重要中间物质的赤藓糖-4-磷酸的合成，提高酪醇及羟基酪醇等衍生物的产率。

Description

重组酵母、构建方法和其在制备酪醇及衍生物中的应用 技术领域
本发明涉及重组酵母、构建方法和其在制备酪醇及衍生物中的应用，特别涉及一株通过外源性的果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase，fxp)的表达基因导入酵母，以及利用该菌株高效生产酪醇及其衍生物的方法，属于微生物基因工程技术领域。
背景技术
酪醇是天然的抗氧化剂，来源于橄榄油，是苯乙醇的一种衍生物。别名红景天苷元，是红景天的主要药用活性成分，是红景天苷、羟基酪醇的前体物质。可以保护细胞免受氧化伤害，是一种具有重要工业价值的酚类化合物，酪醇及其衍生物是多种有机化合物的合成前体，酪醇可被用于医药剂。酪醇的衍生物羟基酪醇是一种具有很强的抗氧化作用及多种生理医药功能，羟基酪醇的抗氧化性强于酪醇，同时可以合成很多聚合物，且没有已知毒性，在生物医药、功能食品等行业应用广泛，具有预防心血管、骨质缺乏等疾病的发生。目前，羟基酪醇的获得主要是从橄榄叶中提取，从植物中提取，成本高，占用大量的耕地。
化学法中利用苯乙醇合成法，大多先采用保护羟基，然后硝化、还原、重氮化、水解得到对羟基苯乙醇，收率为70％。苯乙醇价格高供应紧张，利用硝基甲苯合成，价格低廉但步骤较长，产率低，利用对羟基苯乙烯合成产率达到96％，纯度99％，产率和纯度都很高，具有一定的价值，但原料成本较高。化学法制备酪醇原料成本高且环境不友好，这些都直接制约了酪醇的工业化生产。因此，生物法合成酪醇及其衍生物已经成为研究热点。
酪醇(Tyrosol)具有以下特征：化学名称为4-(2-Hydroxyethyl)phenol，分子式为C 8H 10O 2，分子量为138.164，CAS号为501-94-0，结构式为
Figure PCTCN2020080627-appb-000001
中国专利文献CN108753636A(申请号201810601213.8)公开了一种生产酪醇及羟基酪醇的酵母及构建方法，将PcAAS和ADH序列导入酵母BY4741，得到生产酪醇的PcAAS-ADH重组酵母；在所述PcAAS-ADH重组酵母中导入pdc1基因敲除盒、tyrA表达盒得到生产酪醇的PcAAS-ADH-Δpdc1-tyrA重组酵母；将HpaBC的DNA序列导入PcAAS-ADH-Δpdc1-tyrA重组酵母，得到生产羟基酪醇的PcAAS-ADH-HpaBC-Δpdc1-tyrA重组酵母。在酵母BY4741中构建酪醇或羟基酪醇生物合成途径，提高酪醇或羟基酪醇的产量。该技 术虽然可以提高酵母中酪醇的产量，但酪醇产量仍然无法达到工业化生产的要求。受限于酵母中酪醇的合成受多种代谢途径的影响，且相关代谢途径并未完全研究清楚，因此，如何实现酪醇酵母发酵工业化仍然是目前亟需解决的技术难题。
发明内容
本发明针对现有技术的不足，提供重组酵母、构建方法和其在制备酪醇及衍生物中的应用。
本发明的目的之一是在发明专利申请(申请号201810601213.8)的基础上，在酵母中表达来源于青春双岐杆菌(Bifidobacterium adolescentis)菌种编号为ATCC 15703的果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase,EC 4.1.2.22)(氨基酸序列如GenBank:BAF39468.1所示，SEQ ID NO.1)或短双歧杆菌(Bifidobacterium breve)BBRI4的果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase,EC 4.1.2.22)(氨基酸序列如GenBank:KND53308.1所示，SEQ ID NO.2)的基因片段等，通过构建由果糖-6-磷酸催化生成酪醇生物合成重要前体物质赤藓糖-4-磷酸的新途径，提高酪醇的产率。
本发明的目的之二是提供一种生产羟基酪醇的方法。
本发明的目的之三是提供一种生产酪醇的酵母的构建方法。
本发明的目的之四是提供所述一种生产酪醇的酵母或构建方法在生产酪醇中的应用。
本发明的目的之五是提供所述一种生产酪醇的酵母或构建方法在生产羟基酪醇中的应用。
为了解决以上技术问题，本发明的技术方案如下：
重组酵母在生产酪醇中的应用，所述重组酵母为将外源性的果糖-6-磷酸盐磷酸酮酶的表达基因导入经过改造的酵母细胞中构建获得，所述经过改造的酵母细胞为具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞。
根据本发明优选的，所述经过改造的酵母细胞为将芳香醛合成酶和分支酸变位酶/预苯酸脱水酶整合后获得；或者所述经过改造的酵母细胞为将芳香醛合成酶整合后获得；
根据本发明进一步优选的，所述芳香醛合成酶来源于香芹，酶的系统编号EC4.1.1.25；所述分支酸变位酶来源于大肠杆菌(E.coli)，酶的系统编号EC1.3.1.12；所述预苯酸脱水酶来源于大肠杆菌(E.coli)，酶的系统编号EC1.3.1.12，EC 5.4.99.5。
根据本发明优选的，所述果糖-6-磷酸盐磷酸酮酶的表达基因来源于青春双岐杆菌(Bifidobacterium adolescentis)、动物双歧杆菌(Bifidobacterium animalis)、两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)、构巢曲霉(Aspergillus nidulans)、短双歧杆菌(Bifidobacterium breve)、乳酸双歧杆菌(Bifidobacterium lactis)、丙酮丁醇梭菌(Clostridium  acetobutylicum)、长双歧杆菌(Bifidobacterium longum)、齿双歧杆菌(Bifidobacterium dentium)、肠系膜明串珠菌(Leuconostoc mesenteroides)、摩恩格里艾恩斯双歧(Bifidobacterium mongoliense)、类植物乳杆菌(Lactobacillus paraplantarum)、胚牙乳杆菌(Lactobacillus plantarum)、假长双歧杆菌(Bifidobacterium pseudolongum)、热带假丝酵母(Candida tropicalis)、新生隐球菌(Cryptococcus neoformans)、钩虫贪铜菌(Cupriavidus necator)、加德纳菌(Gardnerella vaginalis)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)等。
更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示，表达基因核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；
更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.30所示，表达基因核苷酸序列如SEQ ID NO.31所示。
根据本发明优选的，所述酵母细胞为：酿酒酵母(Saccharomyces cerevisiae)、耶氏解脂酵母(Yarrowia lipolytica)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、解脂假丝酵母(Candida lipolytica)、光滑球拟酵母(Torulopsis glabrata)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)、热带假丝酵母(Candida tropicalis)、鲁氏酵母(Zygosaccharomyces rouxii)、光滑念珠菌(Candida glabrata)、德布尔有孢酵母(Torulaspora delbrueckii)、汉斯德巴氏酵母菌(Debaryomyces hansenii)、树干毕赤酵母(Scheffersomyces stipites)、季也蒙毕赤酵母(Meyerozyma guilliermondii)、长孢洛德酵母(Lodderomyces elongisporus)、白假丝酵母菌(Candida albicans)、拟平滑念珠菌(Candida orthopsilosis)、似平滑念珠菌(Candida metapsilosis)、都柏林酵母菌(Candida dubliniensis)、葡萄牙棒孢酵母(Clavispora lusitaniae)、耳念珠菌(Candida auris)。
进一步优选的，所述酵母细胞为酿酒酵母(Saccharomyces cerevisiae)，菌种编号为CICC1964；所述马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777。
根据本发明更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1基因，获得。
一种高产酪醇的重组酵母的构建方法，包括如下步骤：
(1)构建表达盒，表达盒由启动子、终止子、同源臂以及果糖-6-磷酸盐磷酸酮酶的表达基因经融合后获得；
(2)将步骤(1)构建的表达盒转化至经过改造的酵母细胞，制得高产酪醇的重组酵母；
所述经过改造的酵母细胞为具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞。
根据本发明更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1基因，获得；
根据本发明更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换马克斯克鲁维酵母NBRC1777的pdc1基因，获得。
根据本发明优选的，所述步骤(1)中果糖-6-磷酸盐磷酸酮酶的表达基因来源于青春双岐杆菌(Bifidobacterium adolescentis)、动物双歧杆菌(Bifidobacterium animalis)、两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)、构巢曲霉(Aspergillus nidulans)、短双歧杆菌(Bifidobacterium breve)、乳酸双歧杆菌(Bifidobacterium lactis)、丙酮丁醇梭菌(Clostridium acetobutylicum)、长双歧杆菌(Bifidobacterium longum)、齿双歧杆菌(Bifidobacterium dentium)、肠系膜明串珠菌(Leuconostoc mesenteroides)、摩恩格里艾恩斯双歧(Bifidobacterium mongoliense)、类植物乳杆菌(Lactobacillus paraplantarum)、胚牙乳杆菌(Lactobacillus plantarum)、假长双歧杆菌(Bifidobacterium pseudolongum)、热带假丝酵母(Candida tropicalis)、新生隐球菌(Cryptococcus neoformans)、钩虫贪铜菌(Cupriavidus necator)、加德纳菌(Gardnerella vaginalis)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)等。
更优的，所述果糖-6-磷酸盐磷酸酮酶的的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示，表达基因核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示。
更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.30所示，表达基因核苷酸序列如SEQ ID NO.31所示。
根据本发明优选的，所述步骤(1)中同源臂为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板，利用引物扩增预苯酸脱水酶(prephenate dehydratase)基因pha2上游及下游500bp的基因片段，所述上游同源臂扩增引物的核苷酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示；下游同源臂扩增引物的核苷酸序列分别如SEQ ID NO.7和SEQ ID NO.8所示；
根据本发明优选的，所述步骤(1)中启动子为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板利用引物扩增的启动子tpi1，所述启动子tpi1的扩增引物的核苷酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示；
根据本发明优选的，所述步骤(1)中启动子为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板利用引物扩增的终止子gpm1，所述终止子gpm1的扩增引物的核苷酸序列分别如SEQ ID NO.9和SEQ ID NO.10所示；
根据本发明优选的，所述步骤(2)中，经过改造的酵母细胞为将芳香醛合成酶和分支酸变位酶/预苯酸脱水酶整合后获得；或者所述经过改造的酵母细胞为将芳香醛合成酶整合后获得；
根据本发明进一步优选的，所述步骤(2)中，芳香醛合成酶来源于香芹(Petroselinum crispum)，酶的系统编号EC4.1.1.25；所述分支酸变位酶/预苯酸脱水酶来源于大肠杆菌(E.coli)，酶的系统编号EC1.3.1.12，EC 5.4.99.5。
根据本发明优选的，所述步骤(2)中所述酵母细胞为：酿酒酵母(Saccharomyces cerevisiae)、耶氏解脂酵母(Yarrowia lipolytica)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、解脂假丝酵母(Candida lipolytica)、光滑球拟酵母(Torulopsis glabrata)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)、热带假丝酵母(Candida tropicalis)、鲁氏酵母(Zygosaccharomyces rouxii)、光滑念珠菌(Candida glabrata)、德布尔有孢酵母(Torulaspora delbrueckii)、汉斯德巴氏酵母菌(Debaryomyces hansenii)、树干毕赤酵母(Scheffersomyces stipites)、季也蒙毕赤酵母(Meyerozyma guilliermondii)、长孢洛德酵母(Lodderomyces elongisporus)、白假丝酵母菌(Candida albicans)、拟平滑念珠菌(Candida orthopsilosis)、似平滑念珠菌(Candida metapsilosis)、都柏林酵母菌(Candida dubliniensis)、葡萄牙棒孢酵母(Clavispora lusitaniae)、耳念珠菌(Candida auris)等。
进一步优选的，所述酵母细胞为酿酒酵母，菌种编号为CICC1964；所述马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777。
进一步优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，获得。
根据本发明更优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1 基因，获得。
进一步优选的，所述步骤(2)中，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，获得。
根据本发明更优选的，所述步骤(2)中，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换马克斯克鲁维酵母NBRC1777的pdc1基因，获得。
上述方法构建的高产酪醇的重组酵母。
上述高产酪醇的重组酵母在发酵制备酪醇中的应用。
根据本发明优选的，所述发酵的发酵培养基中至少含有葡萄糖、果糖、蔗糖之一或二者以上的组合与酪氨酸。
上述高产酪醇的重组酵母在发酵制备羟基酪醇中的应用。
根据本发明优选的，将上述高产酪醇的重组酵母通过发酵制备酪醇后，经过羟化酶反应获得羟基酪醇。
根据本发明优选的，将上述高产酪醇的重组酵母经发酵所得酪醇，利用过表达4-羟基苯乙酸羟化酶的大肠杆菌催化，获得羟基酪醇。
根据本发明进一步优选的，所述的发酵的发酵培养基中至少含有葡萄糖、果糖、蔗糖之一或二者以上的组合与酪氨酸。
本发明的有益效果：
1、本发明首次公开了在酵母中表达过程中，果糖-6-磷酸(Fructose-6-phosphate)在合成1,6-二磷酸果糖(beta-D-Fructose 1,6-bisphosphate)的同时，被催化为赤藓糖-4-磷酸(Erythrose-4-phosphate)和乙酰磷酸(Acetyl-phosphate)，木酮糖-5-磷酸(Xylulose-5-phosphate)被催化为甘油醛-3-磷酸(Glyceraldehydes-3-phosphate)和乙酰磷酸，这改变了酵母中的碳代谢流量分布，增强酪醇生物合成重要中间物质的赤藓糖-4-磷酸的合成，优化了合成酪醇的代谢途径，提高酪醇及羟基酪醇等衍生物的产率；
2、本发明将外源性的果糖-6-磷酸盐磷酸酮酶的表达基因导入经过改造的酵母细胞中，获得了一种重组酵母，该重组酵母可以提高酪醇的产率，并利用过表达4-羟基苯乙酸羟化酶的大肠杆菌催化酪醇后，获得羟基酪醇。
3、本发明提供了一种新型且环境友好型酪醇及羟基酪醇生物制备技术，为酪醇及羟基酪醇的大规模工业生产奠定了基础，具有重要的经济价值和社会效益。
附图说明
图1为实施例1中所述的重组质粒pUG6的结构图；
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
下面结合实施例对本发明进一步说明：
表1中英文名称的对照
英文 中文
Glucose 葡萄糖
Glucose-6-phosphate 葡萄糖-6-磷酸
Fructose-6-phosphate 果糖-6-磷酸
Fructose-1,6-bisphosphate 果糖-1,6-二磷酸
Dihydroxyacetone phosphate 果糖1,6-双磷酸
Glyceraldehyde-3-phosphate 磷酸二羟丙酮
Phosphoenolpyruvate 磷酸烯醇丙酮酸
Pyruvate 丙酮酸
Tricarboxylic acid cycle 三羧酸循环
Ethanol 乙醇
6-Phospho-D-glucono-1,5-lactone 6-磷酸葡糖酸内酯
6-Phospho-D-gluconate 磷酸葡萄糖酸
Ribulose-5-phosphate 核酮糖-5-磷酸酯
Ribose-5-phosphate 核糖-5-磷酸
Xylulose-5-phosphate 木酮糖-5-磷酸
Sedoheptulose-7-phosphate 景天庚酮糖-7-磷酸
Erythrose-4-phosphate 赤藓糖-4-磷酸
2-Dehydro-3-deoxy-D-arabino-heptonate-7-phosphate 3-脱氧-D-阿糖基-庚酮糖
  酸7-磷酸
Chorismate 分支酸
4-hydroxyphenylpyruvate 4-羟基苯丙酮酸
tyrosine 酪氨酸
4-hydroxyphenylacetaldehyde 4-羟基苯乙醛
Tyrosol 酪醇
Hydroxytyrosol 羟基酪醇
以下实施例中，大肠杆菌BL21和表达载体pET-28a为市售常用。下列实施例中未注明具体条件的实验方法，按照常规条件进行，例如《分子克隆：实验室手册》中所述的条件，或按照相应生物学试剂的制造厂商所建议的条件。PCR扩增反应的反应程序可以为常规的PCR扩增反应程序。
酿酒酵母CICC1964，购自中国工业微生物菌种保藏管理中心，菌种编号CICC1964，为已知非保藏菌株；
按照发明专利申请(申请号201810601213.8)的方法，将来源于香芹(Petroselinum crispum)的芳香醛合成酶(AAS，aromatic aldehyde synthase，EC4.1.1.25)整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenate dehydrogenase，EC1.3.1.12，EC 5.4.99.5)替换酿酒酵母CICC1964的pdc1基因，获得SC-1菌株。
实施例1
Bafxpk和Bbfxpk表达盒的构建
将SEQ ID NO.1和SEQ ID NO.2的氨基酸序列根据宿主酿酒酵母密码子偏好性进行密码子优化，获得SEQ ID NO.1和SEQ ID NO.2对应的优化后核苷酸序列SEQ ID NO.3和SEQ ID NO.4后，进行基因合成。选用引物对Bafxpk-F/Bafxpk-R和Bbfxpk-F/Bbfxpk-R，用Vazyme公司的Phanta Max High-Fidelity DNA聚合酶扩增获得目标基因果糖-6-磷酸盐磷酸酮酶的表达基因(Bafxpk片段核苷酸序列如SEQ ID NO.3所示，Bbfxpk片段核苷酸序列如SEQ ID NO.4所示)。以酿酒酵母CICC1964的基因组为模板，以引物对U-F/U-R和D-F/D-R PCR扩增上下游同源臂的DNA片段，以引物对Ptpi1-F/Ptpi1-R和Tgpm1-F/Tgpm1-R扩增启动子tpi1和终止子gpm1片段。以带有遗传霉素(Geneticin)抗性基因KanMX4(核苷酸序列如SEQ ID NO.24所示)的重组质粒pUG6见附图1的DNA为模板，以引物对G418-F/G418-R 经PCR扩增抗性基因KanMX4的DNA片段。琼脂糖凝胶电泳验证条带大小正确后切取条带，用OMEGA凝胶提取试剂盒回收基因片段。PCR扩增引物如下：
引物对Bafxpk-F/Bafxpk-R的序列为SEQ ID NO.13和SEQ ID NO.14；引物对Bbfxpk-F/Bbfxpk-R的序列为SEQ ID NO.15和SEQ ID NO.16；引物对U-F/U-R的序列为SEQ ID NO.5和SEQ ID NO.6；引物对D-F/D-R的序列为SEQ ID NO.7和SEQ ID NO.8；引物对Ptpi1-F/Ptpi1-R的序列为SEQ ID NO.9和SEQ ID NO.10；引物对Tgpm1-F/Tgpm1-R的序列为SEQ ID NO.11和SEQ ID NO.12；引物对G418-F/G418-R的序列为SEQ ID NO.17和SEQ ID NO.18；
使用高保真性Phanta Max High-Fidelity DNA聚合酶扩增目的基因片段，并保证每条片段都与相邻片段存在50bp的同源序列，PCR产物经凝胶电泳后使用DNA片段胶回收试剂盒回收，并测定DNA浓度。随后将获得的纯化后的带有同源序列的DNA片段采用融合PCR的方法进行融合：
(1)使用Phanta Max High-Fidelity DNA聚合酶对片段进行连接。反应体系如表2所示：
表2 反应体系
Figure PCTCN2020080627-appb-000002
将上述试剂加入PCR管中，反应条件如表3所示：
表3 反应条件
Figure PCTCN2020080627-appb-000003
Figure PCTCN2020080627-appb-000004
(2)以步骤1的PCR反应产物作为PCR扩增模板，选用引物对Yzaw-F/Yzaw-R，用Vazyme公司的Phanta Max High-Fidelity DNA聚合酶扩增获得目标片段。琼脂糖凝胶电泳验证条带大小正确后切取条带，用OMEGA凝胶提取试剂盒回收DNA片段即Bafxpk和Bbfxpk表达盒的DNA片段。PCR扩增引物如下：
Yzaw-F的序列为SEQ ID NO.19；Yzaw-R的序列为SEQ ID NO.20；
(3)将步骤2所获得的Bafxpk和Bbfxpk表达盒的DNA片段，测序验证。
实施例2
Bafxpk和Bbfxpk异源表达菌株的构建，以酿酒酵母为例：
采用PEG/LiAc法转化酿酒酵母酪醇合成菌株CICC1964，通过在培养基中添加G418抗性进行筛选挑取单克隆，提取基因组，利用引物对Yzaw-F/Yzaw-R进行PCR验证，获得SC-bafxpk和SC-bbfxpk菌株。
实施例3
合成酪醇微生物的发酵，以酿酒酵母为例：
在产酪醇的菌株CICC1964和SC-bafxpk和SC-bbfxpk的平板上挑取单克隆，接种到5mL YPD液体培养基中，在30～32℃，200rpm条件下培养24h，转接到50mL YPD液体培养基中，初始接种OD 600为0.2，30℃，200rpm条件下培养12h后，转接到100mL YPD液体培养基中，初始接种OD 600为0.2，培养基中分别含有2％葡萄糖或者2％蔗糖或者2％葡萄糖和1％酪氨酸等碳源，培养24小时后，再次加入2％葡萄糖或者2％蔗糖或者2％葡萄糖和1％酪氨酸等碳源，共进行72小时发酵。采用文献(Satoh et al.,Journal of Agricultural and Food Chemistry,60,979-984,2012)报道的HPLC法检测发酵液中酪醇的浓度。不同碳源培养条件下酪醇产量如表4所示。
表4 不同碳源下发酵72小时后酪醇的产量
Figure PCTCN2020080627-appb-000005
Figure PCTCN2020080627-appb-000006
实施例4
基因HpaBC及大肠杆菌表达载体的获得方法
将来源于大肠杆菌的4-羟基苯乙酸羟化酶(HpaBC，4-hydroxyphenylacetate 3-hydroxylase，酶的系统编号EC 1.5.1.37)的DNA序列基因簇在大肠杆菌中过表达，利用大肠杆菌全细胞将酪醇催化为羟基酪醇。
所述4-羟基苯乙酸羟化酶的氨基酸序列基因簇包括4-羟基苯乙酸羟化酶(HpaB)的氨基酸序列为SEQ ID NO.21，对应的核苷酸序列为SEQ ID NO.23；4-羟基苯乙酸羟化酶(HpaC)的氨基酸序列为SEQ ID NO.22，对应的核苷酸序列为SEQ ID NO.24。
利用细菌基因组试剂盒提取大肠杆菌DE3基因组作为模板，分别使用引物对hpaB-F/hpaB-R和hpaC-F/hpaC-R来扩增SEQ ID NO.23和SEQ ID NO.24，测序验证。hpaB-F的序列为SEQ ID NO.25；hpaB-R的序列为SEQ ID NO.26；hpaC-F的序列为SEQ ID NO.27；hpaC-R的序列为SEQ ID NO.28。
以pET-28a作为表达载体，培养含有pET-28a空载体的大肠杆菌，利用细菌质粒提取试剂盒提取pET-28a质粒，采用常规分子生物学方法，构建表达载体pEThpaBC(SEQ ID NO.29)。然后将pEThpaBC转化大肠杆菌表达型载体BL21，以卡那霉素为筛选标记，获得单克隆BL21-pEThpaBC。
摇瓶培养BL21-pEThpaBC，使用1mM IPTG诱导表达HPAB/C的表达。
将获得的BL21-pEThpaBC菌培养液添加到实施例3的培养液中，反应3小时后，检测获得的羟基酪醇产量结果如表5所示：
表5 在表4所示的培养液中添加BL21-pEThpaBC菌后混合3小时后羟基酪醇的产量
Figure PCTCN2020080627-appb-000007
实施例5
采用马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777，按照发明专利申请(申请号201810601213.8)的方法，将来源于香芹(Petroselinum crispum)的芳香醛合成酶(AAS，aromatic aldehyde synthase，EC4.1.1.25)整合到马克斯克鲁维酵母的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenate dehydrogenase，EC1.3.1.12，EC 5.4.99.5)替换马克斯克鲁维酵母的pdc1基因，获得具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的马克斯克鲁维酵母菌株。
按照本发明实施例1-2所述方法，将果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase,EC 4.1.2.22)制备获得的KM-bafxp和KM-bbfxp菌株，不同之处在于，马克斯克鲁维酵母细胞的培养温度为42～49℃。
实施例6
采用来源于齿双歧杆菌(Bifidobacterium dentium)的果糖-6-磷酸盐磷酸酮酶(bdfxp，GenBank:BAQ26957.1)，氨基酸序列如SEQ ID NO.30所示，按照实施例3和实施例4所述的方法进行，所用引物对Bdfxp-F/Bdfxp-R的序列为SEQ ID NO.32和SEQ ID NO.33；结果与实施例3和实施例4的相似，也提高了酪醇和羟基酪醇的产量。具体结果如下：
表6 不同碳源下发酵72小时后酪醇的产量
Figure PCTCN2020080627-appb-000008
表7 在表6所示的培养液中添加BL21-pEThpaBC菌后混合3小时后羟基酪醇的产量
Figure PCTCN2020080627-appb-000009
实施例7
按照实施例1-2所述的方法，将来源于香芹(Petroselinum crispum)的芳香醛合成酶(AAS，aromatic aldehyde synthase，EC4.1.1.25)整合到酿酒酵母CICC1964的delta12位点，并且将如SEQ ID NO.4所示的果糖-6-磷酸盐磷酸酮酶(Fructose-6-phosphate phosphoketolase,EC  4.1.2.22)导入上述酵母中，获得SC-bbfxpk-AAS菌株。结果与实施例3中SC-bbfxpk菌株的酪醇产量相似。
将获得的BL21-pEThpaBC菌培养液添加到上述制得的含有酪醇的培养液中，反应3小时后，检测获得的羟基酪醇产量也和实施例4中所述SC-bbfxpk菌株的产量相似。说明酵母中没有分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenate dehydrogenase，EC1.3.1.12，EC 5.4.99.5)基因导入，也可以达到相近的酪醇和羟基酪醇产量；具体结果如表8和表9所示：
表8 不同碳源下发酵72小时后酪醇的产量
Figure PCTCN2020080627-appb-000010
表9 在表8所示的培养液中添加BL21-pEThpaBC菌后混合3小时后羟基酪醇的产量
Figure PCTCN2020080627-appb-000011
实施例8
按照实施例6所述的方法，分别将来源于动物双歧杆菌(Bifidobacterium animalis)的果糖-6-磷酸盐磷酸酮酶(baifxp，GenBank:WP_052826255.1)氨基酸序列如SEQ ID NO.34所示，两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)的果糖-6-磷酸盐磷酸酮酶(bbifxp，GenBank:WP_047289945.1)氨基酸序列如SEQ ID NO.35所示，乳酸双歧杆菌(Bifidobacterium lactis)的果糖-6-磷酸盐磷酸酮酶(blafxp，GenBank:CAC29121.1)氨基酸序列如SEQ ID NO.36所示，长双歧杆菌(Bifidobacterium longum)果糖-6-磷酸盐磷酸酮酶(blofxp，GenBank:PWH09343.1)氨基酸序列如SEQ ID NO.37所示，摩恩格里艾恩斯双歧(Bifidobacterium mongoliense)的果糖-6-磷酸盐磷酸酮酶(bmfxp，GenBank:CAC29121.1) 氨基酸序列如SEQ ID NO.38所示，假长双歧杆菌(Bifidobacterium pseudolongum)的果糖-6-磷酸盐磷酸酮酶(bpfxp，GenBank:WP_034883174.1)氨基酸序列如SEQ ID NO.39所示，构巢曲霉(Aspergillus nidulans)的果糖-6-磷酸盐磷酸酮酶(Anfxp，GenBank:CBF76492.1)氨基酸序列如SEQ ID NO.40所示，丙酮丁醇梭菌(Clostridium acetobutylicum)的果糖-6-磷酸盐磷酸酮酶(Cafxp，GenBank:KHD36088.1)氨基酸序列如SEQ ID NO.41所示，肠系膜明串珠菌(Leuconostoc mesenteroides)的果糖-6-磷酸盐磷酸酮酶(Lmfxp，GenBank:AAV66077.1)氨基酸序列如SEQ ID NO.42所示，类植物乳杆菌(Lactobacillus paraplantarum)的果糖-6-磷酸盐磷酸酮酶(Lprfxp，GenBank:ALO04878.1)氨基酸序列如SEQ ID NO.43所示，胚芽乳杆菌(Lactobacillus plantarum)的果糖-6-磷酸盐磷酸酮酶(Lplfxp，GenBank:KRU19755.1)氨基酸序列如SEQ ID NO.44所示对应的基因片段转化改造的酿酒酵母；分别获得改造菌株SC-baifxp，SC-bbifxp，SC-blafxp，SC-blofxp，SC-bmfxp，SC-bpfxp，SC-Anfxp，SC-Cafxp，SC-Lmfxp，SC-Lprfxp，SC-Lplfxp；所述改造的酿酒酵母为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点。结果与实施例3和实施例4的相似，也提高了酪醇和羟基酪醇的产量。具体结果如表8和表9所示：
表8 不同碳源下发酵72小时后酪醇的产量
Figure PCTCN2020080627-appb-000012
表9 在表8所示的培养液中添加BL21-pEThpaBC菌后混合3小时后羟基酪醇的产量
Figure PCTCN2020080627-appb-000013
从以上的结果来看，当酵母细胞中表达以上外源性的果糖-6-磷酸盐磷酸酮酶时，虽然产量有所不同，和实施例3、实施例4和实施例5的来源于青春双岐杆菌、短双歧杆菌以及齿双歧杆菌的果糖-6-磷酸盐磷酸酮酶相比，产量较低，但是和出发菌株相比，酪醇和羟基酪醇的产量均有显著提高。
基于实施例7的结果，发明人同时对以上果糖-6-磷酸盐磷酸酮酶对应的基因片段转化按照发明专利申请(申请号201810601213.8)的方法获得的SC-1菌株，即：将来源于香芹(Petroselinum crispum)的芳香醛合成酶(AAS，aromatic aldehyde synthase，EC4.1.1.25)整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenate dehydrogenase，EC1.3.1.12，EC5.4.99.5)替换酿酒酵母CICC1964的pdc1基因获得；结果显示baifxp，bbifxp，blafxp，blofxp，bmfxp，bpfxp，Anfxp，Cafxp，Lmfxp，Lprfxp，Lplfxp与实施例7中bbfxpk的结果类似，从而说明酵母中有没有分支酸变位酶/预苯酸脱水酶(TyrA，fused chorismate mutase T/prephenate dehydrogenase，EC1.3.1.12，EC 5.4.99.5)基因的导入，对酪醇和羟基酪醇产量影响不大具有普遍性。
结果分析
通过上述结果本领域技术人员可以发现，当酵母细胞中表达外源性的果糖-6-磷酸盐磷酸酮酶时，可以显著提高酪醇的产量，并且该现象并非仅限于一种来源的果糖-6-磷酸盐磷酸酮酶和特异的酵母细胞，本领域技术人员通过本发明的技术教导可以预期，具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞中通过表达果糖-6-磷酸盐磷酸酮酶后，由于代谢外源性的果糖-6-磷酸盐磷酸酮酶的表达影响了酵母细胞的该糖代谢途径，从而促进了酪醇产物的生成，因此均能够产生本申请所述的技术效果。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。

Claims (10)

  1. 重组酵母在生产酪醇中的应用，所述重组酵母为将外源性的果糖-6-磷酸盐磷酸酮酶的表达基因导入经过改造的酵母细胞中构建获得，所述经过改造的酵母细胞为具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞。
  2. 如权利要求1所述的应用，其特征在于，所述经过改造的酵母细胞为将芳香醛合成酶和分支酸变位酶/预苯酸脱水酶整合后获得；或者所述经过改造的酵母细胞为将芳香醛合成酶整合后获得；
    进一步优选的，所述芳香醛合成酶来源于香芹，酶的系统编号EC4.1.1.25；所述分支酸变位酶来源于大肠杆菌，酶的系统编号EC1.3.1.12和EC 5.4.99.5；所述预苯酸脱水酶来源于大肠杆菌，酶的系统编号5.4.99.5。
  3. 如权利要求1所述的应用，其特征在于，所述果糖-6-磷酸盐磷酸酮酶的表达基因来源于青春双岐杆菌(Bifidobacterium adolescentis)、动物双歧杆菌(Bifidobacterium animalis)、两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)、构巢曲霉(Aspergillus nidulans)、短双歧杆菌(Bifidobacterium breve)、乳酸双歧杆菌(Bifidobacterium lactis)、丙酮丁醇梭菌(Clostridium acetobutylicum)、长双歧杆菌(Bifidobacterium longum)、齿双歧杆菌(Bifidobacterium dentium)、肠系膜明串珠菌(Leuconostoc mesenteroides)、摩恩格里艾恩斯双歧(Bifidobacterium mongoliense)、类植物乳杆菌(Lactobacillus paraplantarum)、胚牙乳杆菌(Lactobacillus plantarum)、假长双歧杆菌(Bifidobacterium pseudolongum)、热带假丝酵母(Candida tropicalis)、新生隐球菌(Cryptococcus neoformans)、钩虫贪铜菌(Cupriavidus necator)、加德纳菌(Gardnerella vaginalis)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)等；
    更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示，表达基因核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；
    更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.30所示，表达基因核苷酸序列如SEQ ID NO.31所示。
  4. 如权利要求1所述的应用，其特征在于，所述酵母细胞为：酿酒酵母(Saccharomyces cerevisiae)、耶氏解脂酵母(Yarrowia lipolytica)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、解脂假丝酵母(Candida lipolytica)、光滑球拟酵母(Torulopsis glabrata)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)、热带假丝酵母(Candida tropicalis)、鲁氏酵母(Zygosaccharomyces rouxii)、 光滑念珠菌(Candida glabrata)、德布尔有孢酵母(Torulaspora delbrueckii)、汉斯德巴氏酵母菌(Debaryomyces hansenii)、树干毕赤酵母(Scheffersomyces stipites)、季也蒙毕赤酵母(Meyerozyma guilliermondii)、长孢洛德酵母(Lodderomyces elongisporus)、白假丝酵母菌(Candida albicans)、拟平滑念珠菌(Candida orthopsilosis)、似平滑念珠菌(Candida metapsilosis)、都柏林酵母菌(Candida dubliniensis)、葡萄牙棒孢酵母(Clavispora lusitaniae)、耳念珠菌(Candida auris)等；
    进一步优选的，所述酵母细胞为酿酒酵母(Saccharomyces cerevisiae)，菌种编号为CICC1964；所述马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777；
    进一步优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，获得。
    更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1基因，获得；
    进一步优选的，所述步骤(2)中，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，获得。
    更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换马克斯克鲁维酵母NBRC1777的pdc1基因，获得。
  5. 一种高产酪醇的重组酵母的构建方法，其特征在于，包括如下步骤：
    (1)构建表达盒，表达盒由启动子、终止子、同源臂以及果糖-6-磷酸盐磷酸酮酶的表达基因经融合后获得；
    (2)将步骤(1)构建的表达盒转化至经过改造的酵母细胞，制得高产酪醇的重组酵母；
    所述经过改造的酵母细胞为具有经4-磷酸赤藓糖和磷酸烯醇式丙酮酸合成酪醇代谢途径的酵母细胞。
  6. 如权利要求5所述的构建方法，其特征在于，所述步骤(1)中果糖-6-磷酸盐磷酸酮酶的表达基因来源于青春双岐杆菌(Bifidobacterium adolescentis)、动物双歧杆菌(Bifidobacterium animalis)、两歧双歧杆菌短双歧杆菌(Bifidobacterium bifidum)、构巢曲霉(Aspergillus nidulans)、短双歧杆菌(Bifidobacterium breve)、乳酸双歧杆菌(Bifidobacterium lactis)、丙酮丁醇梭菌(Clostridium acetobutylicum)、长双歧杆菌(Bifidobacterium longum)、齿双歧杆菌(Bifidobacterium dentium)、肠系膜明串珠菌(Leuconostoc mesenteroides)、 摩恩格里艾恩斯双歧(Bifidobacterium mongoliense)、类植物乳杆菌(Lactobacillus paraplantarum)、胚牙乳杆菌(Lactobacillus plantarum)、假长双歧杆菌(Bifidobacterium pseudolongum)、热带假丝酵母(Candida tropicalis)、新生隐球菌(Cryptococcus neoformans)、钩虫贪铜菌(Cupriavidus necator)、加德纳菌(Gardnerella vaginalis)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces pastorianus)等；
    更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示，表达基因核苷酸序列如SEQ ID NO.3或SEQ ID NO.4所示；
    更优的，所述果糖-6-磷酸盐磷酸酮酶的氨基酸序列如SEQ ID NO.30所示，表达基因核苷酸序列如SEQ ID NO.31所示；
    优选的，所述步骤(1)中同源臂为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板，利用引物扩增预苯酸脱水酶(prephenate dehydratase)基因pha2上游及下游500 bp的基因片段，所述上游同源臂扩增引物的核苷酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示；下游同源臂扩增引物的核苷酸序列分别如SEQ ID NO.7和SEQ ID NO.8所示；
    优选的，所述步骤(1)中启动子为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板利用引物扩增的启动子tpi1，所述启动子tpi1的扩增引物的核苷酸序列分别如SEQ ID NO.9和SEQ ID NO.10所示；
    优选的，所述步骤(1)中启动子为以酿酒酵母菌株CICC1964或马克斯克鲁维酵母NBRC1777的基因组为模板利用引物扩增的终止子gpm1，所述终止子gpm1的扩增引物的核苷酸序列分别如SEQ ID NO.11和SEQ ID NO.12所示；
    优选的，所述步骤(2)中，经过改造的酵母细胞为将芳香醛合成酶和分支酸变位酶/预苯酸脱水酶整合后获得；或者所述经过改造的酵母细胞为将芳香醛合成酶整合后获得；
    进一步优选的，所述步骤(2)中，芳香醛合成酶来源于香芹(Petroselinum crispum)，酶的系统编号EC4.1.1.25；所述分支酸变位酶/预苯酸脱水酶来源于大肠杆菌(E.coli)，酶的系统编号EC1.3.1.12，EC 5.4.99.5；
    优选的，所述步骤(2)中所述酵母细胞为：酿酒酵母(Saccharomyces cerevisiae)、耶氏解脂酵母(Yarrowia lipolytica)、粟酒裂殖酵母(Schizosaccharomyces pombe)、乳酸克鲁维酵母(Kluyveromyces lactis)、马克斯克鲁维酵母(Kluyveromyces marxianus)、解脂假丝酵母(Candida lipolytica)、光滑球拟酵母(Torulopsis glabrata)、腐生性酵母菌红酵母菌(Rhodotorula glutinis)、禾本红酵母(Rhodotorula graminis)、巴氏酵母(Saccharomyces  pastorianus)、热带假丝酵母(Candida tropicalis)、鲁氏酵母(Zygosaccharomyces rouxii)、光滑念珠菌(Candida glabrata)、德布尔有孢酵母(Torulaspora delbrueckii)、汉斯德巴氏酵母菌(Debaryomyces hansenii)、树干毕赤酵母(Scheffersomyces stipites)、季也蒙毕赤酵母(Meyerozyma guilliermondii)、长孢洛德酵母(Lodderomyces elongisporus)、白假丝酵母菌(Candida albicans)、拟平滑念珠菌(Candida orthopsilosis)、似平滑念珠菌(Candida metapsilosis)、都柏林酵母菌(Candida dubliniensis)、葡萄牙棒孢酵母(Clavispora lusitaniae)、耳念珠菌(Candida auris)等；
    进一步优选的，所述酵母细胞为酿酒酵母，菌种编号为CICC1964；所述马克斯克鲁维酵母(Kluyveromyces marxianus)，菌种编号为NBRC1777；
    进一步优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，获得。
    更优选的，所述步骤(2)中，经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到酿酒酵母CICC1964的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换酿酒酵母CICC1964的pdc1基因，获得；
    进一步优选的，所述步骤(2)中，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，获得。
    更优选的，所述经过改造的酵母细胞为将来源于香芹(Petroselinum crispum)的芳香醛合成酶整合到马克斯克鲁维酵母NBRC1777的delta12位点，并将来源于大肠杆菌(E.coli)的分支酸变位酶/预苯酸脱水酶替换马克斯克鲁维酵母NBRC1777的pdc1基因，获得。
  7. 权利要求5所述方法构建的高产酪醇的重组酵母。
  8. 权利要求7所述高产酪醇的重组酵母在发酵制备酪醇中的应用；
    优选的，所述发酵的发酵培养基中至少含有葡萄糖、果糖、蔗糖之一或二者以上的组合与酪氨酸。
  9. 权利要求7所述高产酪醇的重组酵母在发酵制备羟基酪醇中的应用。
  10. 如权利要求9所述的应用，其特征在于，将权利要求7所述高产酪醇的重组酵母通过发酵制备酪醇后，经过4-羟基苯乙酸羟化酶反应获得羟基酪醇；
    优选的，利用过表达4-羟基苯乙酸羟化酶大肠杆菌催化权利要求8所述制备的酪醇后，获得羟基酪醇；
    进一步优选的，所述的发酵的发酵培养基中至少含有葡萄糖、果糖、蔗糖之一或二者以上的组合与酪氨酸。
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