WO2019237824A1 - 一种生产酪醇及羟基酪醇的酵母及构建方法 - Google Patents

一种生产酪醇及羟基酪醇的酵母及构建方法 Download PDF

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WO2019237824A1
WO2019237824A1 PCT/CN2019/082883 CN2019082883W WO2019237824A1 WO 2019237824 A1 WO2019237824 A1 WO 2019237824A1 CN 2019082883 W CN2019082883 W CN 2019082883W WO 2019237824 A1 WO2019237824 A1 WO 2019237824A1
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yeast
tyrosol
pcaas
adh
tyra
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方诩
郭伟
韩丽娟
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山东恒鲁生物科技有限公司
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Priority to EP19820261.6A priority Critical patent/EP3808836A4/en
Priority to JP2021506025A priority patent/JP7130284B2/ja
Priority to US17/053,983 priority patent/US11634720B2/en
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01025—Tyrosine decarboxylase (4.1.1.25)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)

Definitions

  • the invention belongs to the technical field of bioengineering, and relates to a gene recombinant yeast with high production of tyrosol, a Saccharomyces cerevisiae that heterologously synthesizes hydroxytyrosol, and a method for constructing the yeast.
  • Tyrosol is a natural antioxidant derived from olive oil and a derivative of phenethyl alcohol. Also known as Rhodiolaside, it is the main medicinal active ingredient of Rhodiola and is the precursor of salidroside and hydroxytyrosol. It can protect cells from oxidative damage. It is a phenolic compound with important industrial value. Tyrosol and its derivatives are synthetic precursors of many organic compounds. Tyrosol can be used as a pharmaceutical agent. Hydroxytyrosol, a derivative of tyrosol, has a strong antioxidant effect and a variety of physiological and medical functions. Hydroxytyrosol has stronger oxidation resistance than tyrosol, and can synthesize many polymers without known toxicity. It is widely used in biomedicine, functional food and other industries, and has the ability to prevent cardiovascular and bone diseases. At present, hydroxytyrosol is mainly obtained from olive leaves and from plants, which is costly and takes up a lot of arable land.
  • a phenylethanol synthesis method is used, and most of them first use a protected hydroxyl group, and then nitrate, reduce, diazotize, and hydrolyze to obtain p-hydroxyphenylethanol with a yield of 70%.
  • Phenylethanol is high in price and tight in supply. It is synthesized using nitrotoluene, which is cheap but has long steps and low yield. The yield of p-hydroxystyrene is 96%, the purity is 99%, and the yield and purity are high. Value, but higher raw material costs. Chemical preparation of tyrosol is costly and environmentally unfriendly, which directly restricts the industrial production of tyrosol. Therefore, biosynthesis of tyrosol and hydroxytyrosol 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 501-194-0, and the structural formula is
  • Patent name "A method and application of biosynthesis of tyrosol in E. coli” (application number 201310133238.7);
  • Patent name "A strain of E. coli expressing high production of tyrosol and / or salidroside and icariin D2 and its application” (Application No. 201410115011.4);
  • Patent name "Recombinant E. coli using glucose to produce hydroxytyrosol, and method and application thereof" (Application No. 201510242626.8);
  • Patent name "A recombinant strain producing tyrosol and its construction method” (application number 201710091999.9);
  • Patent name "Method for Production of Tyrosol and Hydroxytyrosol by Heterologous Metabolic Pathway” (Application No. 201711054680.5).
  • Endotoxin is a component of the cell wall of Gram-negative bacteria, also known as lipopolysaccharide. LPS can be toxic to the human body.
  • Methods for improvement" of "products” and “production” in “microorganism” and “additional” of “electron acceptors” discloses a method of recombinant yeast, which uses enzyme conversion to regulate metabolic pathways and produce glycerol.
  • one object of the present invention is to provide a yeast for producing tyrosol.
  • Develop a recombinant yeast construct a tyrosol biosynthetic pathway in yeast BY4741, knock out the pdc1 gene fragment in the yeast BY4741 gene template, weaken the pathway for synthesizing ethanol in yeast, and increase the production of tyrosol.
  • the gene for producing tyrosol was further transformed into the hydroxylase gene of hydroxytyrosol, and a yeast strain for heterologous synthesis of hydroxytyrosol was constructed.
  • Another object of the present invention is to provide a yeast for producing hydroxytyrosol.
  • a third object of the present invention is to provide a method for constructing a yeast for producing tyrosol.
  • a fourth object of the present invention is to provide a method for constructing a yeast for producing hydroxytyrosol.
  • the fifth object of the present invention is to provide the application of the yeast or the method for constructing tyrosol in the production of tyrosol.
  • a sixth object of the present invention is to provide the application of the yeast or the construction method for producing hydroxytyrosol in the production of hydroxytyrosol.
  • the technical solution of the present invention is:
  • a tyrosol-producing yeast is a tyrosine decarboxylase PcAAS (TryDC, Tyrosine decarboxylase) derived from Petroselinum crispum, and an alcohol dehydrogenase (ADH, Alcohol dehydrogenase) derived from Enterobacteriaceae
  • PcAAS TeryDC, Tyrosine decarboxylase
  • ADH Alcohol dehydrogenase
  • the amino acid sequence of tyrosine decarboxylase is SEQ ID NO.1; the DNA sequence of tyrosine decarboxylase is SEQ ID NO.2, which can express the amino acid sequence shown in SEQ ID NO.1.
  • the amino acid sequence of the alcohol dehydrogenase is SEQ ID No. 3; the DNA sequence of the alcohol dehydrogenase is SEQ ID NO. 4, and the amino acid sequence shown in SEQ ID NO. 3 can be expressed.
  • a pdc1 gene knockout cassette is introduced into the PcAAS-ADH recombinant yeast to obtain a PcAAS-ADH- ⁇ pdc1 recombinant yeast.
  • the method for constructing the pdc1 gene knockout box comprises the steps of using the yeast BY4741 genome as a template, using primers to amplify the 500bp homologous arms of the upstream and downstream of the pdc1 fragment, and using primers to amplify the G418 resistance gene. Fragment, the upstream and downstream 500bp homology arms and the G418 resistance gene fragment are fused to obtain a pdc1 knockout box.
  • a tyrA expression cassette is introduced into the PcAAS-ADH- ⁇ pdc1 recombinant yeast to obtain a PcAAS-ADH- ⁇ pdc1-tyrA recombinant yeast.
  • the method for constructing the tyrA expression cassette comprises the steps of using the yeast BY4741 genome as a template, using primers to amplify an upstream homologous arm of 500 bp, using the pdc1 knockout cassette as a template, and using primers to amplify A tyrA fragment, a 500bp upstream homology arm and a tyrA fragment were fused to construct a tyrA expression cassette.
  • a hydroxytyrosol-producing yeast a DNA sequence gene cluster of 4-hydroxyphenylacetic hydroxylase (HpaBC) derived from Escherichia coli is introduced into the tyrosol-producing PcAAS-ADH - ⁇ pdc1-tyrA recombinant yeast, PcAAS-ADH-HpaBC- ⁇ pdc1-tyrA recombinant yeast was produced.
  • HpaBC 4-hydroxyphenylacetic hydroxylase
  • the amino acid sequence gene cluster of the 4-hydroxyphenylacetate hydroxylase includes the amino acid sequence of 4-hydroxyphenylacetate hydroxylase (HpaB) as SEQ ID NO. 5,4-hydroxyphenylacetate hydroxylase (HpaC) ) 'S amino acid sequence is SEQ ID NO.7.
  • the DNA sequence of 4-hydroxyphenylacetic acid hydroxylase is SEQ ID No. 6, capable of expressing the amino acid sequence shown in SEQ ID No. 5;
  • DNA sequence of 4-hydroxyphenylacetic acid hydroxylase (HpaC) It is SEQ ID No. 8 and can express the amino acid sequence shown in SEQ ID No. 7.
  • a method for constructing tyrosol-producing yeast the specific steps are:
  • the primers were used to amplify the 500bp homologous arms upstream and downstream of the pdc1 fragment, the primer was used to amplify the G418 resistance gene fragment, and the resulting fragment was fused to obtain a pdc1 knockout box;
  • the expression vectors in step 1) are pJFE3, pUC19, p ⁇ BLE2.0, pGK series vectors, pXP318.
  • the vector is pJFE3.
  • a method for constructing hydroxytyrosol-producing yeast the specific steps are:
  • the HpaBC expression cassette is introduced into the recombinant PcAAS-ADH- ⁇ pdc1-tyrA yeast to obtain the recombinant PcAAS-ADH- ⁇ pdc1-tyrA-HpaBC yeast.
  • the specific mode of the application is to obtain tyrosol by fermenting and culturing the tyrosol-producing yeast;
  • the fermentation culture medium is one of glucose, fructose, sucrose, glucose and tyrosine mixture, or Their mixture.
  • the specific mode of the application is to ferment culture the hydroxytyrosol-producing yeast to obtain hydroxytyrosol;
  • the culture medium of the fermentation culture is one of a mixture of glucose, fructose, sucrose, glucose, and tyrosine. Species or mixtures thereof.
  • a tyrA expression cassette is introduced into the PcAAS-ADH- ⁇ pdc1 recombinant yeast, so that the metabolic steps for converting PREP to 4HPP are changed from two-way to one-way, and the yield of tyrosol is increased;
  • the present application develops a yeast, constructs a tyrosol biosynthetic pathway, improves the production of tyrosol, and introduces a hydroxylase gene capable of further converting tyrosol into hydroxytyrosol to construct a yeast that heterologously synthesizes hydroxytyrosol.
  • Strains Compared with E. coli, Saccharomyces cerevisiae has the advantages of good safety, stable yield, and strong anti-staining ability, which is suitable for large-scale industrial production;
  • the present invention provides a new and environmentally friendly technology for the preparation of tyrosol and hydroxytyrosol, which lays the foundation for the large-scale industrial production of tyrosol and hydroxytyrosol, and has important economic and social benefits.
  • FIG. 1 is a schematic diagram of a pathway for synthesizing tyrosol and hydroxytyrosol using glucose or tyrosine as a substrate.
  • the amino acid sequences of SEQ ID NO.1 and SEQ ID NO.2 are codon optimized according to the codon preference of the host Saccharomyces cerevisiae, and the optimized nucleotide sequences corresponding to SEQ ID NO.1 and SEQ ID NO.2 are obtained.
  • Perform gene synthesis The primers were selected and the target genes were obtained by amplification with KOD and FXDNA polymerase from TOYOBO. Agarose gel electrophoresis After verifying the correct size of the band, the band was excised and the gene fragment was recovered using the OMEGA gel extraction kit.
  • the primers for PCR amplification are as follows:
  • sequence of PcAAS-F is SEQ ID NO. 9; the sequence of PcAAS-R is SEQ ID NO. 10; the sequence of adh-F is SEQ ID NO. 11; the sequence of adh-R is SEQ ID NO. 12;
  • E.coli strain containing GBdir + pSC101-BAD-ETgA-tet plasmid was picked on a plate containing tetracycline resistance to an EP containing 1 mL of LB liquid medium (sterilized, containing 4 ug / mL tetracycline) Incubate at 30 ° C, 200 rpm in a tube overnight.
  • Use high-fidelity KODFX DNA polymerase to amplify the target gene fragment, and ensure that each fragment has a 50bp homologous sequence with adjacent fragments.
  • the PCR products are recovered by gel electrophoresis using a DNA fragment gel recovery kit, and the DNA is determined concentration. Fusion fragments were then used for RED / ET plasmid recombination:
  • T4 DNA polymerase was used to ligate the fragments.
  • the reaction system is shown in Table 2:
  • primers PDC1UF / PDC1UR and PDC1DF / PDC1DR were used to amplify the 500 bp homologous arm of the pdc1 fragment gene, and primers G418F / G418R were used to amplify the G418 resistance gene fragment.
  • the fusion PCR method was used to construct pdc1 Knock-out sequencing verification.
  • the sequence of PDC1UF is SEQ ID NO.13; the sequence of PDC1UR is SEQ ID NO.14; the sequence of G418F is SEQ ID NO.15; the sequence of G418R is SEQ ID NO.16; the sequence of PDC1DF is SEQ ID NO.17; The sequence of PDC1DR is SEQ ID NO.18.
  • the primers PDC1F-YZ / PDC1UF1 and a 500 bp upstream homology arm were used, and the pdc1 knockout cassette constructed in Example 2 was used as a template.
  • the primers G418F1 / PDCIR-YZ were used to amplify tyrA and 500 bp downstream of pdc1.
  • the fragment uses the E. coli BL-21 (DE3) genome as a template, and the primers tyrAF1 / tyrAR1 were used to amplify the gene tyrA fragment.
  • a fusion PCR method was used to construct a tyrA expression cassette and verified by sequencing.
  • the sequence of PDC1F-YZ is SEQ ID NO. 19; the sequence of PDC1UF1 is SEQ ID NO. 20; the sequence of TYRAF1 is SEQ ID NO. 21; the sequence of TYRAR1 is SEQ ID NO. 22; the sequence of G418F1 is SEQ ID NO. 23; The sequence of PDCIR-YZ is SEQ ID NO.24.
  • the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 7 were codon optimized according to the codon preference of the host Saccharomyces cerevisiae, and the optimized nucleotide sequences SEQ ID ID corresponding to SEQ ID NO. 5 and SEQ ID NO. 7 were obtained. After NO.6 and SEQ ID No.8, gene synthesis was performed.
  • the HpaBC expression cassette was constructed according to the method of Example 1.
  • Example 1 The strain obtained in Example 1 was inoculated into a liquid LB-containing medium, cultured at 37 ° C, 200 rpm for 14 hours, and the OMEGA plasmid extraction kit D6943-01 was used to obtain the pJFE3-PcAAS-ADH recombinant expression plasmid. Saccharomyces cerevisiae BY4741 was transformed by the PEG / LiAc method. Monoclonal selection was performed using URA selection medium, plasmids were extracted, and primers YZ1F and YZ1R were used for PCR verification to obtain PcAAS-ADH strains. The pdc1 knockout cassette of Example 2 and the tyrA expression cassette of Example 3 were introduced into the PcAAS-ADH strain, respectively, to obtain
  • PcAAS-ADH- ⁇ pdc1-tyrA strain Based on the PcAAS-ADH- ⁇ pdc1-tyrA strain, the HpaBC expression cassette constructed in Example 4 was introduced into the dpdc1-tyrA strain using the PEG / LiAc method to obtain the PcAAS-ADH- ⁇ pdc1-tyrA-HpaBC strain.
  • sequence of YZ1F is SEQ ID NO. 25; the sequence of YZ1R is SEQ ID NO. 26.
  • Monoclonals were picked from plates of tyrosol and hydroxytyrosol-producing strains PcAAS-ADH and PcAAS-ADH- ⁇ pdc1-tyrA, and inoculated into 5 mL of yeast selective auxotrophic medium at 30-32 ° C, 200 rpm After 24 hours of culture, transfer to 50 mL of yeast selective auxotrophic culture. The initial inoculation OD600 is 0.2. After incubating for 12 hours at 30 ° C and 200 rpm, transfer to 100 mL of yeast selective auxotrophic culture. The initial inoculation OD600 is 0.2. A 72-hour fermentation experiment was performed using glucose, fructose, sucrose, glucose and tyrosine. The HPLC method reported in the literature (Satoh, Tajima et al. 2012) was used to detect the concentration of tyrosol in the fermentation broth. Table 4 shows the yield of tyrosol under different carbon source culture conditions.
  • Monoclonals were picked on a plate of the hydroxytyrosol-producing strain PcAAS-ADH- ⁇ pdc1-tyrA-HpaBC, inoculated into a 5 mL yeast selective auxotrophic medium, cultured at 30-32 ° C, 200 rpm for 24 hours, transferred In a 50 mL yeast selective auxotrophic culture, the initial inoculation OD600 is 0.2, and after 30 hours of incubation at 200 rpm for 12 h, transfer to a 100 mL yeast selective auxotrophic culture, the initial inoculation OD600 is 0.2, and fermentation experiments are performed.
  • the HPLC method reported in the literature Satoh, Tajima et al. 2012
  • the yield of hydroxytyrosol was 978 mg / L after 72 hours of fermentation.

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Abstract

本发明涉及一种生产酪醇及羟基酪醇的酵母及构建方法，属于有机化合物制备技术领域，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。

Description

一种生产酪醇及羟基酪醇的酵母及构建方法 技术领域
本发明属于生物工程技术领域，涉及一种高产酪醇的基因重组酵母及一种异源合成羟基酪醇的酿酒酵母，以及所述酵母的构建方法。
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，生物法合成酪醇和羟基酪醇已经成为研究热点。
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 PCTCN2019082883-appb-000001
多项专利成功构建了产酪醇的基因重组大肠杆菌。
专利名：“一种在大肠杆菌中生物合成酪醇的方法及应用”(申请号201310133238.7)；
专利名：“一种高产酪醇和/或红景天苷和淫羊藿次苷D2的大肠杆菌表达菌株及其应用”(申请号201410115011.4)；
专利名：“利用葡萄糖生产羟基酪醇的重组大肠杆菌及重组方法及应用”(申请号201510242626.8)；
专利名：“一种产酪醇的重组菌株及其构建方法”(申请号201710091999.9)；
专利名：“异源代谢途径生产酪醇及羟基酪醇的方法”(申请号201711054680.5)。
但是大肠杆菌内毒素的去除是大规模工业生产中的一个重大挑战。内毒素是革兰氏阴性细菌细胞壁中的一种成分，也称为脂多糖。脂多糖对人体能够产生毒性。
“Methods for the improvement of product yield and production in a microorganism  thtough the addition of alternate electron acceptors”中公开了一种重组酵母，利用酶转化调节代谢途径，生产甘油的方法。
“Breeding of a high tyrosol-producing sake yeast by isolation of an ethanol-resistant mutant from a trp3mutant”公开了从酿酒酵母的色氨酸营养突变体中分离出一种耐乙醇的突变体，研制出了一种新型的高酪蛋白酵母菌育种策略。
同时，尚未检索到利用酵母，特别是酿酒酵母，生产酪醇和羟基酪醇的生物合成技术。
发明内容
针对上述现有技术中存在的问题，本发明的目的之一是提供一种生产酪醇的酵母。开发一种重组酵母，在酵母BY4741中构建酪醇生物合成途径，敲除酵母BY4741基因模板中的pdc1的基因片段，减弱酵母菌中合成乙醇的途径，提高酪醇的产量，并通过导入能够将生产酪醇的基因进一步转化成羟基酪醇的羟化酶基因，构建异源合成羟基酪醇的酵母菌株。
本发明的目的之二是提供一种生产羟基酪醇的酵母。
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一种生产羟基酪醇的酵母的构建方法。
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酪醇中的应用。
本发明的目的之六是提供所述一种生产羟基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，本发明的技术方案为：
一种生产酪醇的酵母，将来源于香芹(Petroselinum crispum)的酪氨酸脱羧酶PcAAS(TryDC,Tyrosine decarboxylase)和来源于肠杆菌属(Enterobacteriaceae)的醇脱氢酶(ADH，Alcohol dehydrogenase)的DNA序列导入酵母BY4741，得到PcAAS-ADH重组酵母。
优选的，酪氨酸脱羧酶的氨基酸序列为SEQ ID NO.1；酪氨酸脱羧酶的DNA序列为SEQ ID NO.2，能够表达SEQ ID NO.1所示氨基酸序列。
优选的，醇脱氢酶的氨基酸序列为SEQ ID NO.3；醇脱氢酶的DNA序列为SEQ ID NO.4，能够表达SEQ ID NO.3所示氨基酸序列。
优选的，在所述PcAAS-ADH重组酵母中导入pdc1基因敲除盒，得到PcAAS-ADH-Δpdc1重组酵母。
进一步优选的，所述pdc1基因敲除盒的构建方法，具体步骤为：以酵母BY4741的基因组为模板，利用引物分别扩增pdc1片段的上下游500bp同源臂，利用引物扩增G418抗性基因片段，将所述上下游500bp同源臂和G418抗性基因片段融合得到pdc1敲除盒。
优选的，在所述PcAAS-ADH-Δpdc1重组酵母中导入tyrA表达盒，得到PcAAS-ADH-Δpdc1-tyrA重组酵母。
进一步优选的，所述tyrA表达盒的构建方法，具体步骤为：以酵母BY4741的基因组为模板，利用引物扩增上游500bp的同源臂，以所述pdc1敲除盒为模板，利用引物扩增tyrA片段，将上游500bp的同源臂和tyrA片段融合构建tyrA表达盒。
一种生产羟基酪醇的酵母，将来源于大肠杆菌(Escherichia coli)的4-羟基苯乙酸羟化 酶(HpaBC，4-hydroxyphenylacetic hydroxylase)的DNA序列基因簇导入所述生产酪醇的PcAAS-ADH-Δpdc1-tyrA重组酵母中，得到生产羟基酪醇的PcAAS-ADH-HpaBC-Δpdc1-tyrA重组酵母。
优选的，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.5，4-羟基苯乙酸羟化酶(HpaC)的氨基酸序列为SEQ ID NO.7。
优选的，4-羟基苯乙酸羟化酶(HpaB)的DNA序列为SEQ ID NO.6，能够表达SEQ ID NO.5所示氨基酸序列；4-羟基苯乙酸羟化酶(HpaC)的DNA序列为SEQ ID NO.8，能够表达SEQ ID NO.7所示氨基酸序列。
一种生产酪醇的酵母的构建方法，具体步骤为：
1)将PcAAS和ADH的DNA序列插入表达载体，构建载体-PcAAS-ADH重组表达质粒；
2)以酵母BY4741的基因组为模板，利用引物分别扩增pdc1片段的上下游500bp同源臂，利用引物扩增G418抗性基因片段，将所得片段融合得到pdc1敲除盒；
3)以酵母BY4741的基因组为模板，利用引物扩增上游500bp的同源臂，以pdc1敲除盒为模板，利用引物扩增tyrA片段，将所得片段融合构建tyrA表达盒；
4)将步骤2)得到的pdc1敲除盒和步骤3)得到的tyrA表达盒插入到步骤1)得到的载体-PcAAS-ADH质粒，得到PcAAS-ADH-Δpdc1-tyrA质粒，导入到酵母BY4741中，得到重组PcAAS-ADH-Δpdc1-tyrA酵母菌株；
优选的，步骤1)中的表达载体为pJFE3、pUC19、pδBLE2.0、pGK系列载体、pXP318。
进一步优选的，载体为pJFE3。
一种生产羟基酪醇的酵母的构建方法，具体步骤为：
1)将HpaB和HpaC基因合成构建HpaBC表达盒；
2)将HpaBC表达盒导入重组PcAAS-ADH-Δpdc1-tyrA酵母中，得到重组PcAAS-ADH-Δpdc1-tyrA-HpaBC酵母。
所述生产酪醇的酵母和生产酪醇的酵母的构建方法在生产酪醇中的应用。
优选的，所述应用的具体方式为将所述生产酪醇的酵母进行发酵培养得到酪醇；所述发酵培养的培养液为葡萄糖、果糖、蔗糖、葡萄糖和酪氨酸混合物中的一种或者它们的混合物。
所述生产酪醇的酵母和生产羟基酪醇的酵母的构建方法在生产羟基酪醇中的应用。
优选的，所述应用的具体方式为将所述生产羟基酪醇的酵母进行发酵培养得到羟基酪醇；所述发酵培养的培养液为葡萄糖、果糖、蔗糖、葡萄糖和酪氨酸混合物中的一种或者它们的混合物。
本发明的有益效果：
1)本申请中在PcAAS-ADH重组酵母中导入pdc1敲除盒，切断了酵母中合成乙醇的途径；经过PEP合成酪醇的代谢途径，提高酪醇的产量；
2)本申请中在PcAAS-ADH-Δpdc1重组酵母中导入tyrA表达盒，使PREP转化为4HPP的代谢步骤由双向变为单向，提高了酪醇的产量；
3)本申请开发一种酵母，构建酪醇生物合成途径，提高酪醇的产量，并通过导入能够将酪醇进一步转化成羟基酪醇的羟化酶基因，构建异源合成羟基酪醇的酵母菌株；和大肠杆菌相比，酿酒酵母具有安全性好，产量稳定，抗染菌能力强等优点，适合大规模的工业生产；
4)本发明提供了一个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
构成本申请的一部分的说明书附图用来提供对本申请的进一步理解，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。
图1为以葡萄糖或酪氨酸为底物合成酪醇及羟基酪醇途径示意图。
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为简写与英文的对照
简写 英文全称 中文
  Glucose 葡萄糖
PEP Phosphoenolpyruvate 磷酸烯醇丙酮酸
PREP Prephenate 预苯酸
2HxThPP 2-(alpha-Hydroxyethyl)thiaminediphosphate 硫胺素二磷酸
4HPP 4-Hydroxyphenylpyruvate 对羟苯基丙酮酸双氧化酶
Tyr L-Tyrosine 酪氨酸
4HPAA 4-Hydroxyphenylacetaldehyde 4-羟基苯乙醛
TYR-OL 4-Hydroxyphenylethanol 酪醇
实施例1
pJFE3-PcAAS-ADH重组表达质粒的构建
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后，进行基因合成。选用引物，用TOYOBO公司的KOD FXDNA聚合酶扩增获得目标基因。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：
PcAAS-F的序列为SEQ ID NO.9；PcAAS-R的序列为SEQ ID NO.10；adh-F的序列为SEQ ID NO.11；adh-R的序列为SEQ ID NO.12；
将含有GBdir+pSC101-BAD-ETgA-tet质粒的E.coli菌株，在含有四环素抗性的平板上挑取单菌落至装有1mL LB液体培养基(灭菌，含4ug/mL四环素)的EP管中，30℃，200rpm培养过夜。使用高保真性KOD FX 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。随后使用融合片段进行RED/ET质粒重组：
(1)在培养过夜的菌液中吸取40μL至1mL新鲜的含4μg/mL四环素的LB培养基中，于30℃，200rpm摇菌2h。
(2)使用T4DNA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 PCTCN2019082883-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 PCTCN2019082883-appb-000003
(3)在步骤1所述菌液中加入40μL 10％L-阿拉伯糖溶液，37℃，200rpm摇菌40min；
(4)采用VSWP01300MF-Millipore白色MCE亲水0.025um光面13mm表面滤膜对步骤2所得反应后体系除盐40min；
(5)将步骤3菌液9000rpm离心1min，弃上清液，加入500μL无菌ddH 2O，重悬菌液，9000rpm离心1min，弃上清液，重复2次后弃上清液，加入50μL无菌ddH 2O重悬菌液并置于冰上；
(6)将除盐后的步骤4中的溶液与步骤5处理后的菌液混匀，冰上放置1min；
(7)吸取全部混合物进行电转(电转参数：1350V,200Ω,25mA,1uF)；
(8)加入1mL新鲜的LB液体培养基(灭菌，无抗性)至电转杯中，吹打均匀后吸出至EP管中。37℃，200rpm培养1h；
(9)取50μL菌液涂布至含有50-100ug/mlAmp的LB平板上，37℃培养过夜；
(10)挑单克隆进行PCR验证和测序验证。
实施例2
构建pdc1敲除盒
以酿酒酵母基因组为模板，使用引物PDC1UF/PDC1UR和PDC1DF/PDC1DR扩增pdc1片段基因的上下游500bp的同源臂，使用引物G418F/G418R扩增G418抗性基因片段采用融合PCR的方法，构建pdc1敲除盒测序验证。
引物序列：
PDC1UF的序列为SEQ ID NO.13；PDC1UR的序列为SEQ ID NO.14；G418F的序列为SEQ ID NO.15；G418R的序列为SEQ ID NO.16；PDC1DF的序列为SEQ ID NO.17；PDC1DR的序列为SEQ ID NO.18。
实施例3
构建tyrA表达盒
以酿酒酵母基因组为模板，使用引物PDC1F-YZ/PDC1UF1和上游500bp的同源臂，以实例2中构建的pdc1敲除盒为模板，以引物G418F1/PDCIR-YZ扩增tyrA和pdc1下游500bp的片段，以大肠杆菌BL-21(DE3)基因组为模板，使用引物tyrAF1/tyrAR1扩增基因tyrA片段采用融合PCR的方法，构建tyrA表达盒，测序验证。
引物序列：
PDC1F-YZ的序列为SEQ ID NO.19；PDC1UF1的序列为SEQ ID NO.20；TYRAF1的序列为SEQ ID NO.21；TYRAR1的序列为SEQ ID NO.22；G418F1的序列为SEQ ID NO.23；PDCIR-YZ的序列为SEQ ID NO.24。
实施例4
构建HpaBC表达盒
将SEQ ID NO.5和SEQ ID NO.7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.5和SEQ ID NO.7对应的优化后核苷酸序列SEQ ID NO.6和SEQ ID NO.8后，进行基因合成。根据实施例1的方法构建HpaBC表达盒。
实施例5
微生物异源合成酪醇菌株的构建，以酿酒酵母为例：
将实施例1获得的菌株接种于含有液体LB培养基中，在37℃，200rpm条件下培养14h，采用OMEGA质粒提取试剂盒D6943-01，获得pJFE3-PcAAS-ADH重组表达质粒。采用PEG/LiAc法转化酿酒酵母BY4741，利用URA选择培养基进行筛选挑取单克隆，提取质粒，利用引物YZ1F和YZ1R进行PCR验证，获得PcAAS-ADH菌株。并分别将实施例2的pdc1敲除盒和实施例3的tyrA表达盒导入PcAAS-ADH菌株，获得
PcAAS-ADH-Δpdc1-tyrA菌株。基于PcAAS-ADH-Δpdc1-tyrA菌株，采用PEG/LiAc法将实 施例4中构建的HpaBC表达盒导入dpdc1-tyrA菌株中，获得PcAAS-ADH-Δpdc1-tyrA-HpaBC菌株。
YZ1F的序列为SEQ ID NO.25；YZ1R的序列为SEQ ID NO.26。
实施例6
合成酪醇微生物的发酵，以酿酒酵母为例：
在产酪醇及羟基酪醇的菌株PcAAS-ADH和PcAAS-ADH-Δpdc1-tyrA的平板上挑取单克隆，接种到5mL酵母选择营养缺陷型培养基中，在30-32℃，200rpm条件下培养24h，转接到50mL酵母选择营养缺陷型培养中，初始接种OD600为0.2，30℃，200rpm条件下培养12h后，转接到100mL酵母选择营养缺陷型培养中，初始接种OD600为0.2，分别采用添加葡萄糖、果糖、蔗糖、葡萄糖和酪氨酸等进行72小时发酵实验。采用文献(Satoh,Tajima et al.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 PCTCN2019082883-appb-000004
实施例7
合成羟基酪醇微生物的发酵，以酿酒酵母为例：
在产羟基酪醇的菌株PcAAS-ADH-Δpdc1-tyrA-HpaBC的平板上挑取单克隆，接种到5mL酵母选择营养缺陷型培养基中，在30-32℃，200rpm条件下培养24h，转接到50mL酵母选择营养缺陷型培养中，初始接种OD600为0.2，30℃，200rpm条件下培养12h后，转接到100mL酵母选择营养缺陷型培养中，初始接种OD600为0.2，进行发酵实验。采用文献(Satoh,Tajima et al.2012)报道的HPLC法检测发酵液中羟基酪醇的浓度，发酵72小时后获得羟基酪醇的产量为978mg/L。
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。
Figure PCTCN2019082883-appb-000005
Figure PCTCN2019082883-appb-000006
Figure PCTCN2019082883-appb-000007
Figure PCTCN2019082883-appb-000008
Figure PCTCN2019082883-appb-000009
Figure PCTCN2019082883-appb-000010
Figure PCTCN2019082883-appb-000011
Figure PCTCN2019082883-appb-000012
Figure PCTCN2019082883-appb-000013
Figure PCTCN2019082883-appb-000014
Figure PCTCN2019082883-appb-000015
Figure PCTCN2019082883-appb-000016

Claims (10)

  1. 一种生产酪醇的酵母，其特征在于：具体步骤为：将来源于香芹的酪氨酸脱羧酶和来源于肠杆菌属的醇脱氢酶的DNA序列导入酵母BY4741，得到PcAAS-ADH重组酵母。
  2. 根据权利要求1所述生产酪醇的酵母，其特征在于：酪氨酸脱羧酶的氨基酸序列为SEQ ID NO.1；酪氨酸脱羧酶的DNA序列为SEQ ID NO.2，能够表达SEQ ID NO.1所示氨基酸序列；醇脱氢酶的氨基酸序列为SEQ ID NO.3；醇脱氢酶的DNA序列为SEQ ID NO.4，能够表达SEQ ID NO.3所示氨基酸序列。
  3. 根据权利要求1所述生产酪醇的酵母，其特征在于：在权利要求1所述PcAAS-ADH重组酵母中导入pdc1基因敲除盒，得到PcAAS-ADH-pdc1重组酵母；所述pdc1基因敲除盒的构建方法，具体步骤为：以酵母BY4741的基因组为模板，利用引物分别扩增pdc1片段的上下游500bp同源臂，利用引物扩增G418抗性基因片段，将所述上下游500bp同源臂和G418抗性基因片段融合得到pdc1敲除盒。
  4. 根据权利要求3所述的生产酪醇的酵母，其特征在于：在权利要求3所述PcAAS-ADH-Δpdc1重组酵母中导入tyrA表达盒，得到PcAAS-ADH-Δpdc1-tyrA重组酵母；所述tyrA表达盒的构建方法，具体步骤为：以酵母BY4741的基因组为模板，利用引物扩增上游500bp的同源臂，以权利要求4所述pdc1敲除盒为模板，利用引物扩增tyrA片段，将上游500bp的同源臂和tyrA片段融合构建tyrA表达盒。
  5. 一种生产羟基酪醇的酵母，其特征在于：将来源于大肠杆菌的4-羟基苯乙酸羟化酶的DNA序列基因簇导入权利要求5所述PcAAS-ADH-Δpdc1-tyrA重组酵母中，得到生产羟基酪醇的PcAAS-ADH-HpaBC-Δpdc1-tyrA重组酵母。
  6. 根据权利要求7所述的生产羟基酪醇的酵母，其特征在于：所述4-羟基苯乙酸羟化酶的氨基酸序列基因簇包括SEQ ID NO.5和SEQ ID NO.7；4-羟基苯乙酸羟化酶的DNA序列基因簇为SEQ ID NO.6和SEQ ID NO.8，分别能够表达SEQ ID NO.5所示氨基酸序列和SEQ ID NO.7所示氨基酸序列。
  7. 一种生产酪醇的酵母的构建方法，其特征在于：具体步骤为：
    1)将PcAAS和ADH的DNA序列插入表达载体，构建载体-PcAAS-ADH重组表达质粒；
    2)构建权利要求4所述pdc1敲除盒；
    3)构建权利要求6所述tyrA表达盒；
    4)将步骤2)得到的pdc1敲除盒和步骤3)得到的tyrA表达盒插入到步骤1)得到的载体-PcAAS-ADH质粒，得到PcAAS-ADH-Δpdc1-tyrA质粒，导入到酵母BY4741中，得到重组PcAAS-ADH-Δpdc1-tyrA酵母菌株；
    优选的，步骤1)中的表达载体为pJFE3、pUC19、pδBLE2.0、pGK系列载体、pXP318；
    进一步优选的，载体为pJFE3。
  8. 一种生产羟基酪醇的酵母的构建方法，其特征在于：具体步骤为：
    1)将HpaB和HpaC基因合成构建HpaBC表达盒；
    2)将HpaBC表达盒导入重组PcAAS-ADH-Δpdc1-tyrA酵母中，得到重组PcAAS-ADH-Δpdc1-tyrA-HpaBC酵母。
  9. 权利要求1-4所述生产酪醇的酵母和权利要求7所述的生产酪醇的酵母的构建方法在生产酪醇中的应用；
    所述应用的具体方式为将所述生产酪醇的酵母进行发酵培养得到酪醇；所述发酵培养的培养液为葡萄糖、果糖、蔗糖、葡萄糖和酪氨酸混合物中的一种或者它们的混合物。
  10. 权利要求5-6所述生产酪醇的酵母和权利要求8所述的生产羟基酪醇的酵母的构建方法在生产羟基酪醇中的应用；
    所述应用的具体方式为将所述生产酪醇的酵母进行发酵培养得到酪醇；所述发酵培养的培养液为葡萄糖、果糖、蔗糖、葡萄糖和酪氨酸混合物中的一种或者它们的混合物。
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