WO2023143136A1 - 一种发酵生产α-檀香烯的酵母工程菌及其应用 - Google Patents

一种发酵生产α-檀香烯的酵母工程菌及其应用 Download PDF

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WO2023143136A1
WO2023143136A1 PCT/CN2023/072128 CN2023072128W WO2023143136A1 WO 2023143136 A1 WO2023143136 A1 WO 2023143136A1 CN 2023072128 W CN2023072128 W CN 2023072128W WO 2023143136 A1 WO2023143136 A1 WO 2023143136A1
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santalene
synthase
gene
strain
sas
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连佳长
左一萌
程锦涛
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浙江大学杭州国际科创中心
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Definitions

  • the invention relates to the field of bioengineering, in particular to a yeast engineering bacterium for fermenting and producing ⁇ -santalene and its application.
  • Santalene, molecular formula C 15 H 24 belongs to bicyclic sesquiterpenes. There are two isomers of ⁇ -santalene and ⁇ -santalene.
  • ⁇ -Santalene has a boiling point of 252°C (100.39kPa), and exists in the volatile oil of sandalwood.
  • ⁇ -santalene has a long-lasting aroma and is widely used in the flavor and fragrance industries. It also has pharmacological activities such as antibacterial, antioxidative and antitumor, and can be used as a valuable health medicine.
  • Natural sandalwood oil mainly comes from the heartwood of sandalwood, and natural sandalwood essential oil is prepared by extraction methods such as organic reagent extraction and distillation, mainly by steam distillation, ether impregnation, solvent reflux, etc. The quantity is also different. Due to the different places of origin and different varieties of sandalwood, the composition of sandalwood essential oil is quite different, and the aroma effect is also inconsistent, so the standard of sandalwood essential oil cannot be controlled. In addition, due to the special growth characteristics of sandalwood and the long maturation period of heartwood, the natural sandalwood resources are becoming increasingly exhausted.
  • sandalwood With the continuous improvement of human living standards, the demand for sandalwood products is also increasing.
  • the biotransformation method is not limited by many conditions and is green and environmentally friendly.
  • synthetic biology technology if microbial cell factories are used to biosynthesize santalene heterologously, it can improve efficiency, reduce costs, and break through natural conditions. Effectively alleviate the contradiction between supply and demand of sandalwood essential oil.
  • Pichia pastoris As a recognized safe strain, Pichia pastoris has gained a good understanding of its genetic background, metabolic pathways, and regulatory mechanisms through years of research. At the same time, Pichia pastoris is a eukaryotic organism with fast growth rate, high environmental tolerance, and simple fermentation conditions. Now it can be widely used as an ideal host bacteria to express heterologous proteins and produce bio-based compounds.
  • the present invention provides a yeast engineered strain for fermenting and producing ⁇ -santalene and its application.
  • Yeast engineering strains firstly used gene editing technology to highly express santalene synthase SAS, and secondly, through high expression of 3-hydroxy-3-methylglutaryl-CoA reductase 1tHMG1 to release the rate-limiting step of mevalonate pathway, Then through the high expression pathway rate-limiting enzyme isopentenyl pyrophosphate isomerase IDI1 and farnesyl pyrophosphate synthase ERG20 to improve metabolic flux, then high expression of acetyl-CoA synthase ACS, and finally increase the copy number of SAS.
  • the present invention provides a yeast engineered bacterium for fermenting and producing ⁇ -santalene.
  • Yeast is used as a starting strain, and the yeast engineered bacterium is constructed by introducing genes, and the introduced genes include any one of the following groups:
  • one or more copies of the gene encoding santalene synthase SAS are introduced into the gene.
  • the gene encoding santalene synthase SAS among the introduced genes is 1-3 copies.
  • the starting strain is Pichia pastoris, Saccharomyces cerevisiae or Yarrowia lipolytica.
  • the starting strain is Pichia pastoris, such as Pichia pastoris GS115 strain.
  • the coding gene of the santalene synthase SAS is shown in SEQ ID NO.1, and the coding gene of 3-hydroxy-3-methylglutaryl-CoA reductase 1tHMG1 is shown in SEQ ID NO.2.
  • the coding gene of alkenyl pyrophosphate isomerase IDI1 is shown in SEQ ID NO.3, the coding gene of farnesyl pyrophosphate synthase ERG20 is shown in SEQ ID NO.4, and the coding gene of acetyl-CoA synthase ACS is shown in Shown in SEQ ID NO.5.
  • the present invention also provides a method for constructing yeast engineering bacteria for fermentatively producing ⁇ -santalene, and the introduced genes include the coding gene of santalene synthase SAS, 3-hydroxy-3-methylglutaryl-CoA reductase 1tHMG1
  • the coding gene of isopentenyl pyrophosphate isomerase IDI1 the coding gene of farnesyl pyrophosphate synthase ERG20 and the coding gene of acetyl-CoA synthase ACS, the construction method comprises the following steps:
  • strain Pa-3 is the yeast engineered strain for fermentatively producing ⁇ -santalene.
  • the promoter used in the construction process is pTEF or pGAP
  • the nucleotide sequence of the promoter pTEF is shown in SEQ ID NO.6
  • the nucleotide sequence of the promoter pGAP is shown in SEQ ID NO.7
  • the terminator It is tAOX1
  • the nucleotide sequence is shown in SEQ ID NO.8.
  • the present invention also provides the application of the above-mentioned yeast engineered bacteria for fermenting and producing ⁇ -santalene in the preparation of ⁇ -santalene.
  • the present invention also provides a method for preparing ⁇ -santalene, which involves fermenting and cultivating the above-mentioned yeast engineering bacteria for fermenting and producing ⁇ -santalene, and extracting to obtain ⁇ -santalene.
  • the specific fermentation culture method is as follows: in volume percentage, inoculate the yeast engineered bacterium into 50mL YPD medium according to the inoculation amount of 1-5%, ferment for 5 days at 30°C and 250rpm, and add 2 %glucose.
  • the present invention has the following advantages:
  • the yeast engineered bacteria of the present invention can efficiently produce ⁇ -santalene, and its shake flask fermentation yield is nearly 3.0 g/L, and the fermentation yield of a 1 L fermenter tank can reach up to 15 g/L.
  • the present invention improves the metabolic flux in the ⁇ -santalene biosynthetic pathway through high expression of key rate-limiting enzymes in the metabolic pathway, enhanced precursor supply and multi-copy strategy.
  • the present invention uses Pichia pastoris to efficiently generate ⁇ -santalene, which has the characteristics of short cycle, environmental protection and resource saving.
  • the yeast engineering bacteria with high ⁇ -santalene production of the present invention has broad application prospects.
  • Figure 1 is a metabolic flow diagram for the biosynthesis of santalene.
  • Figure 2 is a schematic diagram of the key enzyme expression cassette for pathway modification.
  • Figure 3 is a map of plasmid Int-pTEF-BamHI-tAOX1.
  • Figure 4 is a map of plasmid Int-pGAP-AatII-tAOX1.
  • Figure 5 is a map of plasmid HZP-sgRNA.
  • Figure 6 is a map of plasmid HHP-sgRNA.
  • Figure 7 is a map of plasmid HGP-sgRNA.
  • Fig. 8 is a GC-MS detection mass spectrum ion response value diagram of santalene.
  • Fig. 9 is a comparison chart of yields of shake flask cultures of different bacterial strains in Example 5.
  • Figure 10 is a GC-MS detection mass spectrum of santalene.
  • Embodiment 1 Construction of Pichia santalene recombinant strain Pa-1
  • the Int refers to the insertion site of the Pichia pastoris GS115 genome, and the used auxiliary plasmid Int-pTEF-tAOX1, Int-pGAP-tAOX1 consists of a general framework (ampicillin resistance gene expression cassette plus prokaryotic gene plasmid origin of replication ori), 500bp homology arms upstream and downstream of the site, and pTEF-BamHI-tAOX1 or pGAP-AatII-tAOX1 (see Figure 3 and Figure 4 for the plasmid map); helper plasmids HZP-sgRNA, HHP-sgRNA and HGP-sgRNA are composed of a general framework, resistance gene expression cassette, sgRNA corresponding to the site, and BsaI restriction site. Z, H, and G represent bleomycin resistance gene, hygromycin resistance gene and G418 respectively. Resistance gene (see Figure 5, Figure 6, and Figure 7 for the plasmid map); P is plasmid (plasmi
  • the gene encoding santalene synthase SAS was synthesized by KingScript Biotechnology Co., Ltd. Using the santalene synthase gene SAS as a template, the primers SAS-F/SAS-R were designed to amplify the target gene SAS, and the seamless cloning method was used It was assembled with the digested vector Int2-pTEF-tAOX1, and transformed into Escherichia coli DH5 ⁇ strain to obtain the recombinant vector Int2-pTEF-SAS-tAOX1.
  • the primer sequences used are as follows:
  • SAS-F atacattttagttattcgccaacGatgtctactcaacaagtttcttctg;
  • SAS-R CAAATGGCATTCTGACATCCTCTTGAGttagtcgtccaacttaactggg.
  • design primers Int2-sgRNA-F and Int2-sgRNA-R use the restriction site BsaI in the carrier HZP-sgRNA to introduce Int2-sgRNA, and connect it with T4 enzyme Recombinant vector HZP-sgRNA-Int2.
  • the primer sequences used are as follows:
  • Int2-sgRNA-F acgctcacggattcaggaaatacg;
  • Int2-sgRNA-R AAACcgtatttcctgaatccgtga.
  • design primers Int2-donor-F and Int2-donor-R perform PCR to obtain the corresponding linearized fragment Int2-donor, and combine it with the corresponding recombinant guide plasmid HZP-sgRNA -Int2 was simultaneously transformed into Pichia pastoris cells GS115-Cas9 to obtain recombinant strain Pa-1. Passage several times at the same time to discard the resistant plasmid.
  • the primer sequences used are as follows:
  • Int2-donor-F agaaggcaaagaatcttctgac
  • Int2-donor-R taggctaaaccaagtgatttttc.
  • Embodiment 2 Construction of Pichia santalene recombinant strain Pa-2
  • the primer sequences used are as follows:
  • tHMG1-F aatcaattgaacaactatcaaaacacaGATGGCTGCAGACCAATTGGTG;
  • tHMG1-R aggcaaatggcattctgacatCCTCTTGAGTTAGGATTTAATGCAGGTG.
  • design primers Int12-sgRNA-F and Int12-sgRNA-R use the restriction site BsaI in the carrier HZP-sgRNA to introduce Int12-sgRNA, and connect it with T4 enzyme Recombinant vector HZP-sgRNA-Int12.
  • the primer sequences used are as follows:
  • Int12-sgRNA-F acgcggggtttgaataacagacac
  • Int12-sgRNA-R AAACgtgtctgttattcaaaccccc.
  • design primers Int12-donor-F and Int12-donor-R perform PCR to obtain the corresponding linearized fragment Int12-donor, and combine it with the corresponding recombinant guide plasmid HZP-sgRNA -Int12 was simultaneously transformed into Pichia pastoris cell Pa-1 to obtain recombinant strain Pa-1-1. Passage several times at the same time to discard the resistant plasmid.
  • the primer sequences used are as follows:
  • Int12-donor-F CTGGGcagtagtgaattggttg; Int12-donor-R: acattgttcgtgaggctaatcc.
  • Design primers IDI1-F/IDI1-R to amplify the target gene IDI1 (as shown in SEQ ID NO.3) from the Pichia pastoris GS115 genome, and use the seamless cloning method to combine it with the vector Int1-pTEF digested by BamHI -tAOX1 (Int1: left gene PAS_FragB_0066, right gene PAS_FragB_0067) was assembled and transformed into Escherichia coli DH5 ⁇ strain to obtain the recombinant vector Int1-pTEF-IDI1-tAOX1.
  • the primer sequences used are as follows:
  • IDI1-F catacattttagttattcgccaacGatgactgccgacaacaatagtatg;
  • IDI1-R AAATGGCATTCTGACATCCTCTTGAGttatagcattctatgaatttgcc.
  • design primers Int1-sgRNA-F and Int1-sgRNA-R use the enzyme cutting site BsaI in the vector HHP-sgRNA to introduce Int1-sgRNA, and obtain by T4 enzyme ligation Recombinant vector HHP-sgRNA-Int1.
  • the primer sequences used are as follows:
  • Int1-sgRNA-F acgctatctgaagtattactggg;
  • Int1-sgRNA-R AAACcccagtaaatacttcagata.
  • the primer sequences used are as follows:
  • Int1-donor-F CTGGGcagtagtgaattggttg; Int1-donor-R: acattgttcgtgaggctaatcc.
  • Design primers ERG20-F/ERG20-R to amplify the target gene ERG20 (as shown in SEQ ID NO.4), and use the seamless cloning method to combine it with the vector Int20-pGAP-tAOX1 (Int20: left
  • the gene PAS_chr4_0465, the right gene PAS_chr4_0467) were assembled and transformed into Escherichia coli DH5 ⁇ strain to obtain the recombinant vector Int20-pGAP-ERG20-tAOX1.
  • the primer sequences used are as follows:
  • ERG20-F caattgaacaactatcaaacacacaGatggcttcagaaaaattagg;
  • ERG20-R GCAAATGGCATTCTGACATCCTCTTGAGctatttgcttctcttgtaac.
  • design primers Int20-sgRNA-F and Int20-sgRNA-R use the enzyme cutting site BsaI in the vector HGP-sgRNA to introduce Int20-sgRNA, and obtain by T4 enzyme ligation Recombinant vector HGP-sgRNA-Int20.
  • the primer sequences used are as follows:
  • Int20-sgRNA-F acgcagaagaaaatgcgaaacagg;
  • Int20-sgRNA-R AAACcctgtttcgcattttcttct.
  • design primers Int20-donor-F and Int20-donor-R perform PCR to obtain the corresponding linearized fragment Int20-donor, and combine it with the corresponding recombinant guide plasmid HGP-sgRNA -Int20 was simultaneously transformed into Pichia pastoris cells Pa-1-2 to obtain recombinant strain Pa-2. Passage several times at the same time to discard the resistant plasmid.
  • the primer sequences used are as follows:
  • Int20-donor-F tccgatcattgcatagatacc
  • Int20-donor-R ttggtcagaattacttcacac.
  • Embodiment 3 Construction of Pichia santalene recombinant strain Pa-3
  • the primer sequences used are as follows:
  • design primers Int16-sgRNA-F and Int16-sgRNA-R use the restriction site BsaI in the vector HZP-sgRNA to introduce Int16-sgRNA, and connect the recombinant vector HZP-sgRNA-Int16 by T4 enzyme .
  • the primer sequences used are as follows:
  • Int16-sgRNA-F acgcgatatgaagagcaaacacag
  • Int16-sgRNA-R AAACctgtgtttgctcttcatatc.
  • design primers Int16-donor-F and Int16-donor-R perform PCR to obtain the corresponding linearized fragment Int16-donor, and combine it with the corresponding recombinant guide plasmid HZP-sgRNA -Int16 was simultaneously transformed into Pichia pastoris cell Pa-2 to obtain recombinant strain Pa-3. Passage several times at the same time to discard the resistant plasmid.
  • the primer sequences used are as follows:
  • Int16-donor-F acaccaccacaaatacctaccaatg
  • Int16-donor-R acctgggagaatgttttcaatctgg.
  • Embodiment 4 Construction of multi-copy Pichia santalene recombinant strain Pa-4
  • the coding gene of santalene synthase SAS in Example 1 was combined with the vector Int33-pTEF-tAOX1 (Int33: left gene PAS_chr1-1_0053, right gene PAS_chr1- 1_0054) and Int6-pTEF-tAOX1 (Int6: left gene PAS_chr2-1_0010, right gene PAS_chr2-1_0011) were assembled respectively, and recombinant vectors Int33-pTEF-SAS-tAOX1 and Int6-pTEF-SAS were obtained after transformation of E. coli DH5 ⁇ -tAOX1.
  • the primer sequences used are the same as in Example 1.
  • Int33-sgRNA was introduced into Int33-sgRNA by using the restriction site BsaI in the vector HGP-sgRNA, and the recombinant vector HGP-sgRNA-Int33 was obtained by ligation with T4 enzyme;
  • primers Int6-sgRNA-F and Int6-sgRNA-R were designed , Int6-sgRNA was introduced into Int6-sgRNA by using restriction site BsaI in vector HHP-sgRNA, and recombinant vector HHP-sgRNA-Int6 was obtained by T4 enzyme ligation.
  • the primer sequences used are as follows:
  • Int33-sgRNA-F acgcccgtcactatgaggacaaag;
  • Int33-sgRNA-R AAACctttgtcctcatagtgacgg;
  • Int6-sgRNA-F gacgctatctgtgtaaagcgacgag;
  • Int6-sgRNA-R AAACctcgtcgctttacacagata.
  • design primers Int33-donor-F and Int33-donor-R perform PCR to obtain the corresponding linearized fragment Int33-donor, and combine it with the corresponding recombinant guide plasmid HGP-sgRNA -Int33 was simultaneously transformed into Pichia pastoris cell Pa-3 to obtain recombinant strain Pa-3-1.
  • design primers Int6-donor-F and Int6-donor-R perform PCR to obtain the corresponding linearized fragment Int6-donor, and combine it with the corresponding recombinant guide plasmid HHP-sgRNA -Int6 was simultaneously transformed into Pichia pastoris cell Pa-3-1 to obtain recombinant strain Pa-4. Passaging several times at the same time discarded the resistant plasmids of two consecutive rounds of transformation.
  • the primer sequences used are as follows:
  • Int33-donor-F aaagagagagtctaaaagtggtg
  • Int33-donor-R attataattagcacggtgttgc
  • Int6-donor-F agtaagggcagtgtaccatag
  • Int6-donor-R acattaagaaccaagatcatgca.
  • Example 5 Efficient production of santalene by engineering strains of Pichia pastoris
  • the recombinant strains in the above examples 1-4 were activated by solid YPD plates, picked a single colony and cultured in a 5mL test tube, then transferred to a 50mL shake flask with 1% inoculum size and cultured for 5 days, then processed the sample for product analysis. Fermentation tank fed-batch culture was carried out with Pa-4 to obtain high-concentration fermentation products.
  • the specific implementation is as follows:
  • Sample processing Take 1mL of the upper fermentation broth, centrifuge at 12000rpm for 3min, and take the upper organic phase for 0.22 ⁇ m nylon filter membrane, the sample was diluted 10 times with ethyl acetate for GC-MS detection.
  • GC-MS detection conditions chromatographic conditions: DB-5MS capillary column; mass spectrometry conditions: EI+ ionization source; electron energy 70eV; ion source temperature 250°C; inlet temperature 250°C; solvent delay 10min; 500m/z. Testing procedure: Initial temperature is 40°C, keep for 3 minutes; heat up to 130°C at a rate of 10°C/min, then rise to 180°C at a rate of 3°C/min, and finally rise to 300°C at a rate of 50°C/min, and hold for 10 minutes. Split injection with a split ratio of 20:1 and an injection volume of 1 ⁇ L.
  • glucose mass fraction in the fermenter drops to 1%, it is 40% (v/v) glucose solution to feed the mass fraction, control the stirring speed to 200-800rpm, keep dissolved oxygen above 20%, and feed the volume fraction in the later stage of fermentation Adjust the pH value between 5.5 and 6.0 with 25% ammonia water and 18% acetic acid, and finish the fermentation after 96 hours.

Abstract

公开了一种发酵生产α-檀香烯的酵母工程菌及其应用。以酵母作为出发菌株，通过导入基因构建获得可以高效生产α-檀香烯的酵母工程菌，其摇瓶发酵产量近3.0 g/L，1L发酵罐发酵产量最高可达 15 g/L。通过高表达代谢途径中关键限速酶、增强前体供应以及多拷贝策略，提高α-檀香烯生物合成途径中的代谢通量，利用毕赤酵母高效生成α-檀香烯，具有周期短、环保和节约资源的特点。

Description

一种发酵生产α-檀香烯的酵母工程菌及其应用 技术领域
本发明涉及生物工程领域，具体涉及一种发酵生产α-檀香烯的酵母工程菌及其应用。
背景技术
檀香烯，分子式为C15H24，属于双环倍半萜类化合物。有α-檀香烯和β-檀香烯两种异构体。α-檀香烯沸点252℃(100.39kPa)，存在于檀香木挥发油中。α-檀香烯作为檀香精油的主要成分之一，芳香味持久，被广泛应用于香精、香料行业，还具有抗菌、抗氧化和抗肿瘤等药理活性，又可用作名贵的保健药品。
天然的檀香油主要来自于檀香心材部分，通过有机试剂萃取，蒸馏等提取手段制备天然的檀香精油，主要用水蒸气蒸馏法，乙醚浸渍法，溶剂回流法等，不同的提取方法对精油提取量也不相同。由于檀香木的产地不同，品种不同等原因，檀香精油的成分差异较大，且结香效果也不一致，因此无法控制檀香精油的标准。除此之外，檀香由于其特殊的生长特性以及心材成熟周期较长的问题，天然的檀香木资源日渐枯竭。
随着人类生活水平的不断提高，对檀香产品的需求量也不断增加，早期天然的檀香资源由于受到人类不合理的砍伐利用，消耗殆尽，而檀香天然林的更新时间长达30年甚至更长，这些因素使得檀香这一珍贵树种的价格节节攀升，无法满足日益增长的市场需求。
生物转化法没有诸多条件的限制且绿色环保，近年来，随着合成生物学技术的发展，如果采用微生物细胞工厂异源生物合成檀香烯，则能提高效率、降低成本、打破自然条件限制而有效缓解檀香精油的供需矛盾。
目前已经有文献报道了檀香类植物合成α-檀香烯的关键基因，这为异源合成α-檀香烯奠定了良好的基础。随着合成生物学的发展，许多的天然产物能够在毕赤酵母体内表达，像萜类、黄酮类等物质。毕赤酵母作为公认的安全菌株，多年来的研究已经对其遗传背景，代谢通路，调控机理有了很好的认识。同时，毕赤酵母是真核生物，且生长速度快，环境耐受性高，发酵条件简单等，现在可以广泛作为理想的宿主菌来表达异源蛋白及生产生物基化合物。
尽管檀香烯有众多的应用前景，但是目前其产量低限制了其在不同领域的应 用。通过微生物合成植物源天然产物是一种非常环保而且高效的方式，目前暂未有利用毕赤酵母细胞工厂生产檀香烯的相关报道。
发明内容
为了解决现有技术中的不足，本发明提供了一种发酵生产α-檀香烯的酵母工程菌及其应用。
酵母工程菌株首先利用基因编辑技术高表达檀香烯合酶SAS，其次，通过高表达3-羟基-3-甲基戊二酰辅酶A还原酶1tHMG1以解除甲羟戊酸途径的限速步骤，再通过高表达途径限速酶异戊烯基焦磷酸异构酶IDI1和法尼基焦磷酸合成酶ERG20提高代谢通量，然后高表达乙酰辅酶A合酶ACS，最后增加SAS拷贝数获得。
本发明提供了一种发酵生产α-檀香烯的酵母工程菌，以酵母作为出发菌株，通过导入基因构建获得所述酵母工程菌，所述导入基因包括以下任意一组：
(1)檀香烯合酶SAS的编码基因、3-羟基-3-甲基戊二酰辅酶A还原酶1tHMG1的编码基因、异戊烯基焦磷酸异构酶IDI1的编码基因、法尼基焦磷酸合成酶ERG20的编码基因和乙酰辅酶A合酶ACS的编码基因；
(2)檀香烯合酶SAS的编码基因；
(3)檀香烯合酶SAS的编码基因、3-羟基-3-甲基戊二酰辅酶A还原酶1tHMG1的编码基因、异戊烯基焦磷酸异构酶IDI1的编码基因和法尼基焦磷酸合成酶ERG20的编码基因。
优选的，导入基因中檀香烯合酶SAS的编码基因为1个或多个拷贝。
更为优选的，导入基因中檀香烯合酶SAS的编码基因为1～3个拷贝。
优选的，所述出发菌株为毕赤酵母、酿酒酵母或解脂耶氏酵母。
更为优选的，所述出发菌株为毕赤酵母，例如毕赤酵母GS115菌株。
所述檀香烯合酶SAS的编码基因如SEQ ID NO.1所示，3-羟基-3-甲基戊二酰辅酶A还原酶1tHMG1的编码基因如SEQ ID NO.2所示，异戊烯基焦磷酸异构酶IDI1的编码基因如SEQ ID NO.3所示，法尼基焦磷酸合成酶ERG20的编码基因如SEQ ID NO.4所示，乙酰辅酶A合酶ACS的编码基因如SEQ ID NO.5所示。
本发明还提供了上述发酵生产α-檀香烯的酵母工程菌的构建方法，导入基因包括檀香烯合酶SAS的编码基因、3-羟基-3-甲基戊二酰辅酶A还原酶1tHMG1的编码基因、异戊烯基焦磷酸异构酶IDI1的编码基因、法尼基焦磷酸合成酶ERG20的编码基因和乙酰辅酶A合酶ACS的编码基因，所述构建方法包括以下步骤：
(1)将檀香烯合酶SAS的编码基因序列整合到毕赤酵母GS115-Cas9(由毕赤 酵母GS115菌株基因组引入Cas9蛋白编码基因得到)的基因组中，获得菌株Pa-1；
(2)将3-羟基-3-甲基戊二酰辅酶A还原酶1tHMG1的编码基因序列、异戊烯基焦磷酸异构酶IDI1的编码基因序列以及法尼基焦磷酸合成酶ERG20的编码基因序列整合到菌株Pa-1中，获得菌株Pa-2；
(3)将乙酰辅酶A合酶ACS的编码基因序列整合到菌株Pa-2中，获得菌株Pa-3；
(4)将2个单拷贝的檀香烯合酶SAS的编码基因序列整合到菌株Pa-3中，获得菌株Pa-4，即所述发酵生产α-檀香烯的酵母工程菌。
构建过程中使用的启动子为pTEF或pGAP，所述启动子pTEF的核苷酸序列如SEQ ID NO.6所示，启动子pGAP的核苷酸序列如SEQ ID NO.7所示；终止子为tAOX1，核苷酸序列如SEQ ID NO.8所示。
本发明还提供了上述发酵生产α-檀香烯的酵母工程菌在制备α-檀香烯中的应用。
本发明还提供了一种制备α-檀香烯的方法，发酵培养上述的发酵生产α-檀香烯的酵母工程菌，并提取获得α-檀香烯。
具体的发酵培养方法为：以体积百分比计，将所述酵母工程菌按1-5％接种量接种于50mL YPD培养基，在30℃、250rpm条件下，发酵5天，每24h补加一次2％葡萄糖。
本发明相对于现有技术具有如下优点：
(1)本发明的酵母工程菌可以高效生产α-檀香烯，其摇瓶发酵产量近3.0g/L，1L发酵罐发酵产量最高可达15g/L。
(2)本发明通过高表达代谢途径中关键限速酶、增强前体供应以及多拷贝策略，提高α-檀香烯生物合成途径中的代谢通量。
(3)本发明利用毕赤酵母高效生成α-檀香烯，具有周期短、环保和节约资源的特点。本发明高产α-檀香烯的酵母工程菌具有广阔的应用前景。
附图说明
图1为生物合成檀香烯的代谢流程图。
图2为途径改造关键酶表达盒示意图。
图3为质粒Int-pTEF-BamHI-tAOX1的图谱。
图4为质粒Int-pGAP-AatⅡ-tAOX1的图谱。
图5为质粒HZP-sgRNA的图谱。
图6为质粒HHP-sgRNA的图谱。
图7为质粒HGP-sgRNA的图谱。
图8为檀香烯的GC-MS检测质谱离子响应值图。
图9为实施例5中不同菌株摇瓶培养的产量对比图。
图10为檀香烯的GC-MS检测质谱图。
具体实施方式
生物合成檀香烯的代谢流程图如图1所示，途径改造关键酶表达盒如图2所示，详细合成途径详见实施例。
实施例1：檀香烯毕赤酵母重组菌株Pa-1的构建
所述Int均指的是毕赤酵母GS115基因组插入位点，所使用到的辅助质粒Int-pTEF-tAOX1，Int-pGAP-tAOX1由通用骨架(氨苄霉素抗性基因表达盒加原核生物基因质粒的复制起始位点ori)、位点上下游500bp同源臂以及pTEF-BamHI-tAOX1或pGAP-AatⅡ-tAOX1组成(质粒图谱见图3，图4)；辅助质粒HZP-sgRNA、HHP-sgRNA和HGP-sgRNA由通用骨架、抗性基因表达盒、位点对应的sgRNA以及BsaI酶切位点组成，Z、H、G分别代表博来霉素抗性基因、潮霉素抗性基因和G418抗性基因(质粒图谱见图5，图6，图7)；P为plasmid(质粒)。
以辅助质粒Int2-pTEF-tAOX1出发(实验室保存，Int2左侧基因PAS_chr1-3_0003，右侧基因PAS_chr1-3_0004)，利用载体中酶切位点BamHI引入檀香烯合酶编码基因SAS(如SEQ ID NO.1所示)得到重组载体Int2-pTEF-SAS-tAOX1；以辅助质粒HZP-sgRNA为出发(实验室保存)，利用载体中酶切位点BsaI引入sgRNA-Int2得到重组载体HZP-sgRNA-Int2。然后以重组载体Int2-pTEF-SAS-tAOX1为模板设计donor引物，进行PCR得到对应的Int2-donor，将其和对应的重组guide质粒HZP-sgRNA-Int2转化进入毕赤酵母细胞GS115-Cas9(由毕赤酵母GS115菌株基因组引入Cas9蛋白编码基因得到)获得重组菌株Pa-1，具体实施如下：
(1)檀香烯合酶重组表达载体的构建
檀香烯合酶SAS的编码基因由金斯瑞生物科技有限公司合成，以檀香烯合酶基因SAS为模板，设计引物SAS-F/SAS-R扩增目的基因SAS，利用无缝克隆方法将其与酶切后的载体Int2-pTEF-tAOX1进行组装，转化到大肠杆菌DH5α菌株从而获得重组载体Int2-pTEF-SAS-tAOX1。
所用引物序列如下：
SAS-F：atacattttagttattcgccaacGatgtctactcaacaagtttcttctg；
SAS-R：CAAATGGCATTCTGACATCCTCTTGAGttagtcgtccaacttaactggg。
(2)向导RNA重组表达载体HZP-sgRNA-Int2的构建
以质粒HZP-sgRNA为模板(质粒图谱见图5)，设计引物Int2-sgRNA-F和Int2-sgRNA-R，利用载体HZP-sgRNA中酶切位点BsaI引入Int2-sgRNA，通过T4酶连接得到重组载体HZP-sgRNA-Int2。
所用引物序列如下：
Int2-sgRNA-F：acgctcacggattcaggaaatacg；
Int2-sgRNA-R：AAACcgtatttcctgaatccgtga。
(3)檀香烯毕赤酵母重组菌株Pa-1的转化
以重组载体Int2-pTEF-SAS-tAOX1为模板设计引物Int2-donor-F和Int2-donor-R，进行PCR得到对应的线性化的片段Int2-donor，将其和对应的重组guide质粒HZP-sgRNA-Int2同时转化进入毕赤酵母细胞GS115-Cas9获得重组菌株Pa-1。同时传代几次丢掉抗性质粒。
所用引物序列如下：
Int2-donor-F：agaaggcaaagaatcttctgac；Int2-donor-R：taggctaaaccaagtgatttttc。
实施例2：檀香烯毕赤酵母重组菌株Pa-2的构建
分别构建高表达基因tHMG1、IDI1、ERG20的表达盒以及对应的guide质粒，并将其依次转入到上述重组菌株Pa-1中，经过逐轮转化分别得到毕赤酵母重组菌株Pa-1-1，Pa-1-2和Pa-2，具体实施如下：
(1)tHMG1重组表达载体的构建
以酿酒酵母基因组为模板，设计引物tHMG1-F/tHMG1-R扩增目的基因tHMG1(如SEQ ID NO.2所示)，利用无缝克隆方法将其与经AatⅡ酶切后的载体Int12-pGAP-tAOX1(Int12：左侧基因PAS_chr3_1202，右侧基因PAS_chr3_0618)进行组装，转化大肠杆菌DH5α菌株从而获得重组载体Int12-pGAP-tHMG1-tAOX1。
所用引物序列如下：
tHMG1-F：aatcaattgaacaactatcaaaacacaGATGGCTGCAGACCAATTGGTG；
tHMG1-R：aggcaaatggcattctgacatCCTCTTGAGTTAGGATTTAATGCAGGTG。
(2)向导RNA重组表达载体HZP-sgRNA-Int12的构建
以辅助质粒HZP-sgRNA(质粒图谱见图5)出发，设计引物Int12-sgRNA-F和Int12-sgRNA-R，利用载体HZP-sgRNA中酶切位点BsaI引入Int12-sgRNA，通过T4酶连接得到重组载体HZP-sgRNA-Int12。
所用引物序列如下：
Int12-sgRNA-F：acgcggggtttgaataacagacac；
Int12-sgRNA-R：AAACgtgtctgttattcaaacccc。
(3)檀香烯毕赤酵母重组菌株Pa-1-1的构建
以重组载体Int12-pGAP-tHMG1-tAOX1为模板设计引物Int12-donor-F和Int12-donor-R，进行PCR得到对应的线性化的片段Int12-donor，将其和对应的重组guide质粒HZP-sgRNA-Int12同时转化进入毕赤酵母细胞Pa-1获得重组菌株Pa-1-1。同时传代几次丢掉抗性质粒。
所用引物序列如下：
Int12-donor-F：CTGGGcagtagtgaattggttg；Int12-donor-R：acattgttcgtgaggctaatcc。
(4)异戊烯基焦磷酸异构酶IDI1的编码基因重组表达载体的构建
设计引物IDI1-F/IDI1-R，从毕赤酵母GS115基因组扩增目的基因IDI1(如SEQ ID NO.3所示)，利用无缝克隆方法将其与经BamHI酶切后的载体Int1-pTEF-tAOX1(Int1：左侧基因PAS_FragB_0066，右侧基因PAS_FragB_0067)进行组装，转化大肠杆菌DH5α菌株从而获得重组载体Int1-pTEF-IDI1-tAOX1。
所用引物序列如下：
IDI1-F：catacattttagttattcgccaacGatgactgccgacaacaatagtatg；
IDI1-R：AAATGGCATTCTGACATCCTCTTGAGttatagcattctatgaatttgcc。
(5)向导RNA重组表达载体HHP-sgRNA-Int1的构建
以辅助质粒HHP-sgRNA(质粒图谱见图6)出发，设计引物Int1-sgRNA-F和Int1-sgRNA-R，利用载体HHP-sgRNA中酶切位点BsaI引入Int1-sgRNA，通过T4酶连接得到重组载体HHP-sgRNA-Int1。
所用引物序列如下：
Int1-sgRNA-F：acgctatctgaagtatttactggg；
Int1-sgRNA-R：AAACcccagtaaatacttcagata。
(6)檀香烯毕赤酵母重组菌株Pa-1-2的构建
以重组载体Int1-pTEF-IDI1-tAOX1为模板设计引物Int1-donor-F和Int1-donor-R，进行PCR得到对应的线性化的片段Int1-donor，将其和对应的重组guide质粒HHP-sgRNA-Int1同时转化进入毕赤酵母细胞Pa-1-1获得重组菌株Pa-1-2。同时传代几次丢掉抗性质粒。
所用引物序列如下：
Int1-donor-F：CTGGGcagtagtgaattggttg；Int1-donor-R：acattgttcgtgaggctaatcc。
(7)法尼基焦磷酸合成酶ERG20的编码基因重组表达载体的构建
设计引物ERG20-F/ERG20-R扩增目的基因ERG20(如SEQ ID NO.4所示)，利用无缝克隆方法将其与经AatⅡ酶切后的载体Int20-pGAP-tAOX1(Int20：左侧基因PAS_chr4_0465，右侧基因PAS_chr4_0467)进行组装，转化大肠杆菌DH5α菌株从而获得重组载体Int20-pGAP-ERG20-tAOX1。
所用引物序列如下：
ERG20-F：caattgaacaactatcaaaacacaGatggcttcagaaaaagaaattagg；
ERG20-R：GCAAATGGCATTCTGACATCCTCTTGAGctatttgcttctcttgtaaac。
(8)向导RNA重组表达载体HGP-sgRNA-Int20的构建
以辅助质粒HGP-sgRNA(质粒图谱见图7)出发，设计引物Int20-sgRNA-F和Int20-sgRNA-R，利用载体HGP-sgRNA中酶切位点BsaI引入Int20-sgRNA，通过T4酶连接得到重组载体HGP-sgRNA-Int20。
所用引物序列如下：
Int20-sgRNA-F：acgcagaagaaaatgcgaaacagg；
Int20-sgRNA-R：AAACcctgtttcgcattttcttct。
(9)檀香烯毕赤酵母重组菌株Pa-2的构建
以重组载体Int20-pGAP-ERG20-tAOX1为模板设计引物Int20-donor-F和Int20-donor-R，进行PCR得到对应的线性化的片段Int20-donor，将其和对应的重组guide质粒HGP-sgRNA-Int20同时转化进入毕赤酵母细胞Pa-1-2获得重组菌株Pa-2。同时传代几次丢掉抗性质粒。
所用引物序列如下：
Int20-donor-F：tccgatcattgcatagatacc；Int20-donor-R：ttggtcagaattacttcacac。
实施例3：檀香烯毕赤酵母重组菌株Pa-3的构建
构建高表达乙酰辅酶A合酶ACS的编码基因的表达盒以及对应的guide质粒，将其电转化到上述重组菌株Pa-2中，最终得到毕赤酵母重组菌株Pa-3，具体实施如下：
(1)乙酰辅酶A合酶ACS的编码基因重组表达载体的构建
以实验室保存的乙酰辅酶A合酶ACS质粒为模板，设计引物ACS-F/ACS-R扩增目的基因ACS(如SEQ ID NO.5所示)，利用无缝克隆方法将其与经BamHI酶切后的载体Int16-pTEF-tAOX1(Int16：左侧基因PAS_chr3_1065，右侧基因 PAS_chr3_1067)进行组装，转化大肠杆菌DH5α菌株从而获得重组载体Int16-pTEF-ACS-tAOX1。
所用引物序列如下：
ACS-F：acatacattttagttattcgccaacGatgtcacaaacacacaaacatgc；
ACS-R：AATGGCATTCTGACATCCTCTTGAGtcatgatggcatagcaatagcttg。
(2)向导RNA重组表达载体HZP-sgRNA-Int16的构建
以辅助质粒HZP-sgRNA出发，设计引物Int16-sgRNA-F和Int16-sgRNA-R，利用载体HZP-sgRNA中酶切位点BsaI引入Int16-sgRNA，通过T4酶连接得到重组载体HZP-sgRNA-Int16。
所用引物序列如下：
Int16-sgRNA-F：acgcgatatgaagagcaaacacag；
Int16-sgRNA-R：AAACctgtgtttgctcttcatatc。
(3)檀香烯毕赤酵母重组菌株Pa-3的构建
以重组载体Int16-pTEF-ACS-tAOX1为模板设计引物Int16-donor-F和Int16-donor-R，进行PCR得到对应的线性化的片段Int16-donor，将其和对应的重组guide质粒HZP-sgRNA-Int16同时转化进入毕赤酵母细胞Pa-2获得重组菌株Pa-3。同时传代几次丢掉抗性质粒。
所用引物序列如下：
Int16-donor-F：acaccaccacaaatacctaccaatg；
Int16-donor-R：acctgggagaatgttttcaatctgg。
实施例4：多拷贝檀香烯毕赤酵母重组菌株Pa-4的构建
构建檀香烯合酶SAS的编码基因的表达盒以及对应的guide质粒，将其依次转入到上述重组菌株Pa-3中，经过转化最终得到重组菌株Pa-4，具体实施如下：
(1)SAS重组表达载体的构建
将实施例1中的檀香烯合酶SAS的编码基因通过无缝克隆方法将其与经BamHI酶切后的载体Int33-pTEF-tAOX1(Int33：左侧基因PAS_chr1-1_0053，右侧基因PAS_chr1-1_0054)与Int6-pTEF-tAOX1(Int6：左侧基因PAS_chr2-1_0010，右侧基因PAS_chr2-1_0011)分别组装，转化大肠杆菌DH5α后分别获得重组载体Int33-pTEF-SAS-tAOX1与Int6-pTEF-SAS-tAOX1。所用引物序列同实施例1。
(2)向导RNA重组表达载体HGP-sgRNA-Int33与HHP-sgRNA-Int6的构建
以辅助质粒HGP-sgRNA出发，设计引物Int33-sgRNA-F和Int33-sgRNA-R， 利用载体HGP-sgRNA中酶切位点BsaI引入Int33-sgRNA，通过T4酶连接得到重组载体HGP-sgRNA-Int33；以辅助质粒HHP-sgRNA出发，设计引物Int6-sgRNA-F和Int6-sgRNA-R，利用载体HHP-sgRNA中酶切位点BsaI引入Int6-sgRNA，通过T4酶连接得到重组载体HHP-sgRNA-Int6。
所用引物序列如下：
Int33-sgRNA-F：acgcccgtcactatgaggacaaag；
Int33-sgRNA-R：AAACctttgtcctcatagtgacgg；
Int6-sgRNA-F：gacgctatctgtgtaaagcgacgag；
Int6-sgRNA-R：AAACctcgtcgctttacacagata。
(3)檀香烯毕赤酵母重组菌株Pa-4的构建
以重组载体Int33-pTEF-SAS-tAOX1为模板设计引物Int33-donor-F和Int33-donor-R，进行PCR得到对应的线性化的片段Int33-donor，将其和对应的重组guide质粒HGP-sgRNA-Int33同时转化进入毕赤酵母细胞Pa-3获得重组菌株Pa-3-1。以重组载体Int6-pTEF-SAS-tAOX1为模板设计引物Int6-donor-F和Int6-donor-R，进行PCR得到对应的线性化的片段Int6-donor，将其和对应的重组guide质粒HHP-sgRNA-Int6同时转化进入毕赤酵母细胞Pa-3-1获得重组菌株Pa-4。同时传代几次丢掉两轮连续转化的抗性质粒。所用引物序列如下：
Int33-donor-F：aaagagagagtctaaaagtggtg；Int33-donor-R：attataattagcacggtgttgc；
Int6-donor-F：agtaagggcagtgtaccatag；Int6-donor-R：acattaagaaccaagatcatgca。
实施例5：毕赤酵母工程菌株高效生产檀香烯
将上述实施例1-4中重组菌株，经过固体YPD平板活化后，挑取单菌落到5mL试管中培养，再以1％接种量转接到50mL摇瓶中培5天后处理样品进行产物分析。以Pa-4进行发酵罐补料分批培养，得到高浓度发酵产物。具体实施如下：
(1)重组菌株的培养
从平板上挑取GS115-Cas9、Pa-1、Pa-2、Pa-3、Pa-4单菌落，分别接种于5mL YPD(葡萄糖20g/L，蛋白胨20g/L，酵母提取物10g/L)试管中培养，30℃，250rpm培养12h，然后以1％接种量分别接种到50mL的YPD摇瓶培养基中，每个菌株三个平行，培养基上层加入10％的正十二烷覆盖，30℃，250rpm培养5天，每24h补加2％的葡萄糖。
(2)檀香烯的检测方法
样品处理：取上层发酵液1mL，12000rpm离心3min，取上层有机相过0.22 μm尼龙滤膜，样品用乙酸乙酯稀释10倍后进行GC-MS检测。
GC-MS检测条件：色谱条件：DB-5MS毛细管色谱柱；质谱条件：EI+电离源；电子能量70eV；离子源温度250℃；进样口温度250℃；溶剂延迟10min；全质谱扫描，40-500m/z。检测程序：初始温度40℃，保持3min；以10℃/min速率升温至130℃，再以3℃/min速率升温至180℃，最后以50℃/min升温至300℃，保持10min。分流进样，分流比20:1，进样量为1μL。
(3)重组菌株Pa-4发酵罐培养
以毕赤酵母重组菌株Pa-4出发，首先将在YPD平板上活化的Pa-4单菌落接种于5mL YPD试管中培养12h，再以1％接种量转接到50mL YPD液体培养基，在30℃、摇床转速250rpm条件下培养12h作为种子液，以10％的接种量转接至1L发酵罐中(装液量600mL)，培养温度30℃，校正溶氧DO达100％，罐压为0.8个大气压，发酵24h后加入10％(v/v)无菌过滤的正十二烷。当发酵罐中的葡萄糖质量分数降至1％后流加质量分数为40％(v/v)的葡萄糖溶液，控制搅拌转速200-800rpm，保持溶氧在20％以上，发酵后期流加体积分数为25％的氨水、18％的醋酸调节pH值在5.5～6.0之间，96h后结束发酵。
(4)重组菌株檀香烯产量比较
表1
按照(1)中所述培养方法培养毕赤酵母工程菌株，采用(2)中所述检测方法测定檀香烯产量，检测质谱图如图8和图10所示，摇瓶中产量如表1和图9所示。按照(3)中所示方法对毕赤酵母工程菌株Pa-4进行分批补料发酵，96h后在1L发酵罐中檀香烯的最高产量大约为15g/L，为目前报道的最高产量，为α-檀香烯及其高附加值衍生物的工业化生产和应用奠定基础，也能够为其他萜烯类天然产物的高效绿色生物制造提供借鉴。

Claims (10)

  1. 一种发酵生产α-檀香烯的酵母工程菌，其特征在于，以酵母作为出发菌株，通过导入基因构建获得所述酵母工程菌，导入基因包括以下任意一组：
    (1)檀香烯合酶SAS的编码基因、3-羟基-3-甲基戊二酰辅酶A还原酶1 tHMG1的编码基因、异戊烯基焦磷酸异构酶IDI1的编码基因、法尼基焦磷酸合成酶ERG20的编码基因和乙酰辅酶A合酶ACS的编码基因；
    (2)檀香烯合酶SAS的编码基因；
    (3)檀香烯合酶SAS的编码基因、3-羟基-3-甲基戊二酰辅酶A还原酶1 tHMG1的编码基因、异戊烯基焦磷酸异构酶IDI1的编码基因和法尼基焦磷酸合成酶ERG20的编码基因。
  2. 如权利要求1所述发酵生产α-檀香烯的酵母工程菌，其特征在于，导入基因中檀香烯合酶SAS的编码基因为1个或多个拷贝。
  3. 如权利要求1所述发酵生产α-檀香烯的酵母工程菌，其特征在于，导入基因中檀香烯合酶SAS的编码基因为1～3个拷贝。
  4. 如权利要求1所述发酵生产α-檀香烯的酵母工程菌，其特征在于，所述出发菌株为毕赤酵母、酿酒酵母或解脂耶氏酵母。
  5. 如权利要求1所述发酵生产α-檀香烯的酵母工程菌，其特征在于，所述檀香烯合酶SAS的编码基因如SEQ ID NO.1所示，3-羟基-3-甲基戊二酰辅酶A还原酶1tHMG1的编码基因如SEQ ID NO.2所示，异戊烯基焦磷酸异构酶IDI1的编码基因如SEQ ID NO.3所示，法尼基焦磷酸合成酶ERG20的编码基因如SEQ ID NO.4所示，乙酰辅酶A合酶ACS的编码基因如SEQ ID NO.5所示。
  6. 如权利要求1-5任一项所述发酵生产α-檀香烯的酵母工程菌的构建方法，其特征在于，导入基因包括檀香烯合酶SAS的编码基因、3-羟基-3-甲基戊二酰辅酶A还原酶1 tHMG1的编码基因、异戊烯基焦磷酸异构酶IDI1的编码基因、法尼基焦磷酸合成酶ERG20的编码基因和乙酰辅酶A合酶ACS的编码基因，所述构建方法包括以下步骤：
    (1)将檀香烯合酶SAS的编码基因序列整合到毕赤酵母GS115-Cas9的基因组中，获得菌株Pa-1；
    (2)将3-羟基-3-甲基戊二酰辅酶A还原酶1 tHMG1的编码基因序列、异戊烯基焦磷酸异构酶IDI1的编码基因序列以及法尼基焦磷酸合成酶ERG20的编码基因序 列整合到菌株Pa-1中，获得菌株Pa-2；
    (3)将乙酰辅酶A合酶ACS的编码基因序列整合到菌株Pa-2中，获得菌株Pa-3；
    (4)将2个单拷贝的檀香烯合酶SAS的编码基因序列整合到菌株Pa-3中，获得菌株Pa-4，即所述发酵生产α-檀香烯的酵母工程菌。
  7. 如权利要求6所述的构建方法，其特征在于，构建过程中使用的启动子为pTEF或pGAP；终止子为tAOX1。
  8. 如权利要求1-5任一项所述发酵生产α-檀香烯的酵母工程菌在制备α-檀香烯中的应用。
  9. 一种制备α-檀香烯的方法，其特征在于，发酵培养权利要求1-5任一项所述的发酵生产α-檀香烯的酵母工程菌，并提取获得α-檀香烯。
  10. 如权利要求9所述制备α-檀香烯的方法，其特征在于，发酵培养方法为：以体积百分比计，将所述酵母工程菌按1-5％接种量接种于50mL YPD培养基，在30℃、250rpm条件下，发酵5天，每24h补加一次2％葡萄糖。
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