WO2022166648A1 - 利用区室化提高酵母菌中7-脱氢胆固醇产量的方法 - Google Patents

利用区室化提高酵母菌中7-脱氢胆固醇产量的方法 Download PDF

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WO2022166648A1
WO2022166648A1 PCT/CN2022/073487 CN2022073487W WO2022166648A1 WO 2022166648 A1 WO2022166648 A1 WO 2022166648A1 CN 2022073487 W CN2022073487 W CN 2022073487W WO 2022166648 A1 WO2022166648 A1 WO 2022166648A1
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sterol
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reductase
yeast
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刘龙
陈坚
吕雪芹
堵国成
李江华
刘延峰
修翔
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Jiangnan University
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Definitions

  • the invention belongs to the technical field of metabolic engineering, and in particular relates to a method for improving the yield of 7-dehydrocholesterol in yeast by utilizing compartmentalization.
  • 7-Dehydrocholesterol is a kind of sterol with high added value.
  • the 7-dehydrocholesterol (7-DHC) synthesized in the human body is directly converted into vitamin D3 after being irradiated by sunlight in the skin tissue.
  • Vitamin D3 is not only a fat-soluble vitamin necessary for the growth and development of human skeletal muscles, but also reduces the risk of immune system disorders and various cancers, and also has a preventive effect on cardiovascular diseases. Vitamin D deficiency has been recognized as a global problem. Health concerns, which also increase the annual global demand for vitamin D3 or its immediate precursor 7-DHC.
  • 7-DHC is an important precursor for the synthesis of sterol drugs such as androstenedione and 9 ⁇ -hydroxyandrost-4-ene-3,17-dione. 7-DHC not only has important applications in medicine and clinic, but also is an important raw material for the preparation of cholesteric liquid crystal materials, and is also widely used in display manufacturing.
  • the traditional preparation method of 7-DHC adopts chemical synthesis method.
  • the first one uses cholesterol acetate as raw material, and 7-DHC is synthesized through 7-position bromination, elimination and hydrolysis reaction.
  • the second one uses cholesterol acetate as raw material, and synthesizes 7-DHC through oxidation, hydrazone, elimination and hydrolysis.
  • the chemical synthesis method has the problem of the selectivity of the reaction substrate, high energy consumption, harsh reaction conditions and serious pollution, which is not conducive to sustainable development.
  • the method of microbial fermentation to produce 7-DHC is environmentally friendly and sustainable.
  • the microbial fermentation method of 7-DHC mainly focuses on the screening and molecular modification of strains.
  • the screening of strains is time-consuming and labor-intensive, and large-scale screening is required. Even if the strains producing 7-DHC are screened, the initial product concentration is extremely low, and It is not conducive to the later transformation and industrial production.
  • Molecular modification mainly focuses on the elimination of branch paths and the supply of precursor substances, but it is very easy to cause the accumulation of toxic intermediates, which is not conducive to the growth and production of strains.
  • Chinese patent CN107075551B US patent US2007/0204059A1, Chinese patent CN109154015A; Chinese patent CN109154015A produces sterol mixture in yeast, in order to achieve the effect of preferential production of 7-DHC, it is necessary to inactivate ERG5 and ERG6, reduce or eliminate ARE2 and ARE1 It is a heterologous enzyme that expresses sterol ⁇ 24-reductase activity, but inactivates key genes in the ergosterol synthesis pathway, which will definitely affect the growth of bacteria and cause the accumulation and metabolism of intermediates in the cytoplasm of yeast cells. imbalance.
  • the present invention uses the acetyl-CoA in the peroxisome and mitochondria to pass the enzymes in the 7-DHC synthesis path through peroxidase
  • Body and mitochondrial localization protein tags are located in mitochondria and peroxisomes, respectively, so that yeast can not only synthesize important 7-DHC in the cytoplasm, but also in peroxisomes and mitochondria, so as to improve metabolism.
  • the conversion efficiency of the substrate in the pathway increases the storage space of the final product and reduces the feedback inhibition in the metabolic pathway.
  • the yield of 7-DHC in yeast is 52.31 mg/L by this method.
  • the first object of the present invention is to provide a method for improving the production of 7-dehydrocholesterol in yeast by using compartmentalization, including expressing heterologous sterol ⁇ 24- in the peroxisome and mitochondria of the yeast host, respectively.
  • Reductase and delta-cholesterolase, and localized enzymes in the 7-dehydrocholesterol synthesis pathway were expressed in peroxisomes and mitochondria using peroxisome-localized tags and mitochondria-localized tags, respectively.
  • amino acid sequence of the sterol ⁇ 24-reductase is shown in SEQ ID NO.1.
  • amino acid sequence of the ⁇ -cholesterolase is shown in SEQ ID NO.2.
  • the enzymes in the 7-dehydrocholesterol synthesis pathway are hydroxymethylglutaryl-CoA synthase ERG13, mevalonate kinase ERG12, hydroxymethylglutaryl-CoA reductase HMG1, phosphate mevalonate Kinase ERG8, meglutarate diphosphate decarboxylase MVD1, isopentenyl diphosphate delta-isomerase IDI1, diphosphate farnesyltransferase ERG9, squalene monooxygenase ERG1, lanosterol synthase ERG7 , sterol 14 ⁇ -demethylase ERG11, ⁇ 14-sterol reductase ERG24, methylsterol monooxygenase ERG25, sterol 4 ⁇ -carboxylate 3-dehydrogenase ERG26, 3-ketosteroid reductase ERG27, ⁇ 7-sterol 5-desaturase ERG3, sterol ⁇ 24
  • the NCBI number of hydroxymethylglutaryl-CoA synthase ERG13 is XM_033912304.1
  • the NCBI number of mevalonate kinase ERG12 is XM_033912620.1
  • the NCBI number of hydroxymethylglutaryl-CoA reductase HMG1 It is XM_033912352.1
  • the NCBI number of phosphomevalonate kinase ERG8 is NM_001182727.1
  • the NCBI number of diphosphate meglutarate decarboxylase MVD1 is NM_001183220.1
  • the NCBI number is NM_001183931.1
  • the NCBI number for diphosphate farnesyltransferase ERG9 is NM_001179321.1
  • the NCBI number for squalene monooxygenase ERG1 is NM_001181304.1
  • the NCBI number of sterol 14 ⁇ -demethylase ERG11 is NM_001179137.1
  • the NCBI number of ⁇ 14-sterol reductase ERG24 is NM_001183118.1
  • the NCBI number of methylsterol monooxygenase ERG25 is NM_001181189 .3.
  • the NCBI number of sterol 4 ⁇ -carboxylate 3-dehydrogenase ERG26 is NM_001180866.1
  • the NCBI number of 3-ketosteroid reductase ERG27 is NM_001181987.1
  • the NCBI number of ⁇ 7-sterol 5-desaturase ERG3 The NCBI number is NM_001181943.1
  • the NCBI number for sterol ⁇ 24-reductase DHCR24 is NM_001031288.1
  • the NCBI number for ⁇ -cholestenolase EBP is KR709569.1.
  • the peroxisome localization tag is the peroxisome localization signal peptide PTS1, and its nucleotide sequence is shown in SEQ ID NO.3.
  • mitochondrial localization tag is the mitochondrial localization signal peptide MMF1, and its nucleotide sequence is shown in SEQ ID NO.4.
  • yeast host is Saccharomyces cerevisiae, Pichia or Candida tropicalis.
  • yeast host is Saccharomyces cerevisiae S288C.
  • the second object of the present invention is to provide a yeast engineering bacterium that expresses heterologous sterol ⁇ 24-reductase and ⁇ -cholestene in the peroxisome and mitochondria of the yeast host, respectively alcoholase, and express enzymes in the 7-dehydrocholesterol synthesis pathway in peroxisomes and mitochondria using peroxisome-localized tags and mitochondria-localized tags, respectively.
  • the present invention has at least the following advantages:
  • yeast is used as the starting strain, and heterologous sterol ⁇ 24-reductase and ⁇ -cholestenolase are simultaneously expressed in peroxisome and mitochondria respectively, and the output of 7-DHC in Saccharomyces cerevisiae is detected as follows: 10.15mg/L, through our invention, the partial enzymes in the 7-DHC synthesis pathway are localized in each compartment in Saccharomyces cerevisiae using oxisome and mitochondrial localization tags, forming a relatively independent 7-DHC synthesis pathway, and also The storage space of precursor substances required for 7-DHC synthesis is increased, and the feedback effect is also reduced. In the same compartment, the conversion efficiency between enzymes is improved, the loss of the action substrate is reduced, and the final 7-DHC production is realized. It increased by 4 times, reaching 53.31mg/L.
  • Figure 1 is a schematic diagram of yeast compartmentalized production of 7-DHC
  • Fig. 2 is the liquid phase diagram of 7-DHC standard substance
  • Figure 3 is a graph showing the results of the liquid phase fermentation of Saccharomyces cerevisiae strain CDHC-17.
  • the 7-DHC in Saccharomyces cerevisiae was detected by high performance liquid chromatography.
  • the fermented Saccharomyces cerevisiae liquid for 60h was centrifuged, resuspended with 10ml sterile water, added with 0.5mm glass beads, and crushed with a high-speed homogenizer for 10min. Take out the crushed mixed solution, add 1g ascorbic acid and 0.5g HBT, mix well, add 20ml of absolute ethanol and 10ml of 1.5mol/L potassium hydroxide methanol solution in turn, saponify in water at 80°C for 2 hours, and the saponification is over.
  • step (b) The four fragments P TEF1 -DHCR24-T cyc1 -P GAP -DIC-T ADH1 , 208F-F, 208-R and locxT gene fragments obtained in step (a) were subjected to overlap extension PCR, 1% agar After verification by glycogel electrophoresis, the fragment was recovered by cutting the gel to obtain the fusion gene fragment 208-F-loxT-P TEF1 -DHCR24-T cyc1 -P GAP -DIC-T ADH1-208 -R gene fragment.
  • step (c) The gene fragment obtained in step (b) was transformed into the competent state of Saccharomyces cerevisiae S288C strain, spread on SD Trp plate, cultivated at 30°C for 3 days, using primers YZ-24-DIC-F, YZ- 24-DIC-R was verified by colony PCR to obtain strain CDHC-1
  • step (d) The strain CDHC-1 obtained in step (c) is made competent, and the plasmid PY26-Cre is transformed into Saccharomyces cerevisiae. After SD Ura has screened a single colony on the solid plate, it is inserted into YPD medium Cultured for 12 h in the medium and on the YPD solid plate containing 5-FOA, cultured at 30 °C for 3 days, and then transferred the grown single colonies to the YPD solid plate and SD Ura screening solid plate for comparison and verification. The single colony that cannot grow on the SD Ura screening plate is the correct genetically engineered bacteria, and is named CDHC-2.
  • loxT-F ccatacacacgtccgctaaacaacaggaaacagctatgaccatgattacg
  • loxT-R acctttagtacgggtaattaacgataaaacgacggccagtgccaaggtcg
  • YZ-24-DIC-R caatgaaacagctggttccattcgaaactaagttctggtgttttaaactaa.
  • Fragment GAL80F1 use primers GAL80R1-F, GAL80R1-R to amplify the gene fragment to obtain GAL80R1, use CYC1-S-F, CYC1-S-R to amplify the gene fragment to obtain CYC1, use primers ADH1-S-F, ADH1-S-R to amplify the gene fragment to obtain ADH1, Using MMF1-F, MMF1-R, MMF2-F and MMF2-R as primers to amplify the fragments to obtain MMF1-1 and MMF2-2.
  • step (c) The single colony verified by colony PCR in step (d) was inserted into SD trp liquid medium and cultivated for 16 hours, then streaked on the YPD solid plate containing 5-FOA, and cultivated at 30°C for 3 days, after which the growth The single colony was transferred to the YPD solid plate and the SD his screening solid plate to compare and verify that the single colony that grew normally on the YPD plate but could not grow on the SD his screening plate was the correct genetically engineered bacteria, and was named CDHC-3.
  • ERG12-F1 tcaaggagaaaaaactataatgtctcagaacgtttacattgtat
  • ERG12-MMF1-R aacggaatttcttaaaaacattatcttttcaatgacaatagaggaa
  • ERG13-MMF1-R ttttgaaaattcaatataaatgaaactctcaactaaactttgttg
  • GAL-F1 agttgagagtttcatttatattgaattttcaaaaattcttacttttttttttgg
  • GAL-R1 taaacgttctgagacattatagttttttctccttgacgttaaagt
  • loxT-M-F cttattgaccacacctctaccggcaggaaacagctatgaccatgattacg
  • LoxT-M-R aatatcagagcatccataaaacgacggccagtgccaaggtcga
  • GAL80F2-F acaagttcgtacttttccaggataaatgc
  • GAL80F2-R ggctttaatttgcggccaccttgcattacaaattgtgagg
  • CYC1-M-F tgtaatgcaaggtggccgcaaattaaagccttcgagcgtc
  • CYC1-M-R atctctgagaaggccgaataatcatgtaattagttatgtcac
  • ADH1-M-F ctctgagaaggccgaagcgaatttcttatgatttatgatttttttatt
  • ADH1-M-R tcatggtcatagctgtttcctgccggtagaggtgtggtcaataag
  • GAL80R2-F cactggccgtcgttttatggatgctctgatattacacaggttaat
  • GAL80R2-R accgggtgataggtttgctcaaccat
  • YZM-10-13-F ggaaaagctgcataaccactttaact
  • YZM-10-13-R ctgagaaagcaacctgacctacagg
  • MMF1-F tacatgattattcggccttctcagagatagaaccttgaac
  • MMF1-R atcttacgatgttaaaaaaatgtttttaagaaattccgtt
  • MMF2-F attgtcattgaaagataatgttttttaagaaattccgttttgagaacag
  • MMF2-R aaatcataagaaattcgcttcggccttctcagagatagaa.
  • Fragment GAL80F3 use primers GAL80R3-F, GAL80R3-R to amplify the gene fragment to obtain GAL80R3, use primers CYC1-F, CYC1-13-R to amplify the gene fragment to obtain CYC1, use primers ADH1-10-F, ADH1-R to amplify The gene fragment yields ADH1.
  • step (c) The single colony verified by colony PCR in step (d) was inserted into SD trp liquid medium and cultivated for 16 hours, then streaked on the YPD solid plate containing 5-FOA, and cultivated at 30°C for 3 days, after which the growth The single colonies were transferred to YPD solid plates and SD hiss screening solid plates for comparison and verification. The single colonies that grew normally on YPD plates but could not grow on SD his screening plates were the correct genetically engineered bacteria, and were named CDHC-7.
  • ERG12-F tcaaggagaaaaaactataatgtctcagaacgtttacattgtat
  • ERG12-PTS1-R ttgggaagaggtagaagatccaaattgtatcttttcaatgacaatagaggaagca
  • ERG13-F acaatttggatcttctacctcttcccaattttttaacatcgtaag
  • ERG13-PTS1-R ttttgaaaattcaatataaatgaaactctcaactaaactttgttg
  • GAL-F agttgagagtttcatttatattgaattttcaaaaattcttacttttttttttgg
  • GAL-R taaacgttctgagacattatagttttttctccttgacgttaaagt
  • LoxT-pts-R gcattactcaattttagactaaaaacgacggccagtgccaaggtcg
  • GAL80F3-F ccaatgctaatccggtcactgccactgc
  • GAL80F3-R aggctttaatttgcggcctggcaatagaagtctcaatttt
  • CYC1-F gagacttctattgccaggccgcaaattaaagccttcgagc
  • CYC1-13-R gtagaagatccaaattgtaatcatgtaattagttatgtcacgctt
  • ADH1-10-F aatttggatcttctacctcttcccaagcgaatttcttatgattta
  • ADH1-R tcatggtcatagctgtttcctgccggtagaggtgtggtcaataag
  • GAL80R3-F gccgtcgttttagtctaaaattgagtaatgccactgcttttccca
  • GAL80R3-R atgtattgtaaatatcgattgtgt
  • YZ-10-13-F ggaaaagctgcataaccactttaact
  • YZ-10-13-R ctgagaaagcaacctgacctacagg.
  • Saccharomyces cerevisiae CDHC-10 single colony and CDHC-17 single colony on solid YPD plate were inoculated into 2ml YPD medium, cultured at 30°C and 220rpm for 16-20h, respectively inoculated with 25mL YPD liquid culture at 1% of the inoculum amount.
  • a 250mL round-bottomed shaker flask cultured at 30°C and 220rpm for 60h. When the fermentation was up to 16h, ethanol was added to supplement the carbon source.
  • the 7-DHC yield of the successfully constructed mitochondrial compartmentalized strain CDHC-10 was 29.35 mg/L.
  • the successfully constructed peroxisome compartmentalized strain CDHC-17 had a -DHC yield was 53.31 mg/L.

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Abstract

本发明涉及一种利用区室化提高酵母菌中7-脱氢胆固醇产量的方法，本发明以酵母菌为出发菌株，表达异源的甾醇△24-还原酶和△-胆甾烯醇酶，在酿酒酵母S288C中经过检测7-DHC的产量为10.15mg/L，通过我们的发明，将7-DHC合成路径中的部分酶使用氧化酶体和线粒体定位标签定位在酿酒酵母中各区室化中，形成相对独立的7-DHC合成途径，同时也增加了7-DHC合成所需前体物质的储存空间，也减少了反馈作用，在同一区室中，酶与酶之间的转化效率提高，减少作用底物的损失，最终实现7-DHC产量提升4倍，达到53.31mg/L。

Description

利用区室化提高酵母菌中7-脱氢胆固醇产量的方法 技术领域
本发明属于代谢工程技术领域，尤其涉及一种利用区室化提高酵母菌中7-脱氢胆固醇产量的方法。
背景技术
7-脱氢胆固醇是一种具有高附加值的在甾醇，人体中合成的7-脱氢胆固醇(7-DHC)在皮肤组织中被阳光照射后直接转化为维生素D3。而维生素D3不仅是人体骨骼肌肉生长发育所必须脂溶性维生素，还能降低免疫系统紊乱和多种癌症的风险，对心血管疾病也有预防作用，维生素D的缺乏已经被公认为是一个全球性的健康问题，这也增加每年全球对维生素D3或其直接前体7-DHC的需求量。7-DHC是雄甾烯二酮和9α-羟基雄甾-4-烯-3,17-二酮等甾醇药物合成的重要前体物质。7-DHC不光在医药和临床有重要应用，它也是制备胆甾相液晶材料的重要原料，在显示制造等方面也有广发应用。
传统的7-DHC制备方法采用化学合成法，主流的化学合成法主要有两种，第一种是以胆固醇醋酸酯为原料，经过7-位的溴化，消除和水解反应合成7-DHC。第二种是以胆固醇醋酸酯为原料，经过氧化，腙化、消除和水解反应合成7-DHC。化学合成的方法中存在反应底物的选择性问题，能耗高，反应条件苛刻且污染严重，不利于可持续发展。微生物发酵生产7-DHC的方法绿色环保可持续。
7-DHC的微生物发酵方法主要集中于菌株的筛选和分子改造方面，菌株筛选费时费力，需要进行大规模的筛选，即使筛选到生产7-DHC的菌株，其初始产物浓度也是极低的，并且不利于后期的改造和工业化生产。分子改造方面主要集中于分支路径的消除，前体物质的供应，但极容易造成有毒中间产物的积累，不利于菌株的生长和生产。如中国专利CN107075551B、美国专利US2007/0204059A1、中国专利CN109154015A；其中中国专利CN109154015A在酵母中生产甾醇混合物，为了达到优先生产7-DHC的效果，需要将ERG5和ERG6失活，降低或者消除ARE2和ARE1的活性，表达甾醇△24-还原酶活性的异源酶，但是失活麦角固醇合成路径中的关键基因，必定会影响菌体生长，并造成酵母细胞中胞质中的中间产物积累及代谢失衡。
现有生产7-DHC的技术主要集中在增强胞质的甲羟戊酸途径，敲除麦角甾醇合成路径中的ERG5和ERG6，实现酿酒酵母中7-DHC的积累，但酿酒酵母中7-DHC合成路径长， 途径中关键的酶分布在不同的细胞器中，在整个合成过程中会造成酶到酶的转化效率低，底物在反应之前扩散损失，中间产物的积累对前期合成路径反馈抑制等突出性问题，使在酿酒酵母菌中生产7-DHC的产量一直难以实现大的提升。
发明内容
为了解决目前酵母菌胞质中合成7-DHC存在的乙酰辅酶A不足的问题，本发明通过利用过氧化酶体和线粒体中的乙酰辅酶A，将7-DHC合成路径中的酶通过过氧化酶体和线粒体定位蛋白标签分别定位于线粒体和过氧化酶体中，这样使酵母菌不光能在胞质能合成重要的7-DHC，还能在过氧化酶体和线粒体中合成，达到了提高代谢途径中底物的转化效率，增加最终产物的储存空间，减少代谢途径中的反馈抑制，最终通过这种方法实现了在酵母菌中7-DHC的产量为52.31mg/L。
本发明的第一个目的是提供一种利用区室化提高酵母菌中7-脱氢胆固醇产量的方法，包括分别在酵母菌宿主的过氧化酶体和线粒体中，表达异源甾醇△24-还原酶和△-胆甾烯醇酶，并分别使用过氧化酶体定位标签和线粒体定位标签将7-脱氢胆固醇合成路径中的酶定位在过氧化酶体和线粒体中表达。
进一步地，所述的甾醇△24-还原酶的氨基酸序列如SEQ ID NO.1所示。
进一步地，所述的△-胆甾烯醇酶的氨基酸序列如SEQ ID NO.2所示。
进一步地，7-脱氢胆固醇合成路径中的酶为羟甲基戊二酰辅酶A合酶ERG13、甲羟戊酸激酶ERG12、羟甲基戊二酰辅酶A还原酶HMG1、磷酸甲羟戊酸激酶ERG8、二磷酸甲戊二酸酯脱羧酶MVD1、异戊烯基二磷酸δ-异构酶IDI1、二磷酸法尼基转移酶ERG9、角鲨烯单加氧酶ERG1、羊毛甾醇合酶ERG7、固醇14α-脱甲基酶ERG11、△14-固醇还原酶ERG24、甲基固醇单加氧酶ERG25、固醇4α-羧酸酯3-脱氢酶ERG26、3-酮类固醇还原酶ERG27、△7-甾醇5-去饱和酶ERG3、甾醇△24-还原酶DHCR24和△-胆甾烯醇酶EBP。
进一步地，羟甲基戊二酰辅酶A合酶ERG13的NCBI编号为XM_033912304.1、甲羟戊酸激酶ERG12的NCBI编号为XM_033912620.1、羟甲基戊二酰辅酶A还原酶HMG1的NCBI编号为XM_033912352.1、磷酸甲羟戊酸激酶ERG8的NCBI编号为NM_001182727.1、二磷酸甲戊二酸酯脱羧酶MVD1的NCBI编号为NM_001183220.1、异戊烯基二磷酸δ-异构酶IDI1的NCBI编号为NM_001183931.1、二磷酸法尼基转移酶ERG9的NCBI编号为NM_001179321.1、角鲨烯单加氧酶ERG1的NCBI编号为NM_001181304.1、羊毛甾醇合酶ERG7的NCBI编号为NM_001179202.2、固醇14α-脱甲基酶ERG11的NCBI编号为NM_001179137.1、△14-固醇还原酶ERG24的NCBI编号为NM_001183118.1、甲基固醇单 加氧酶ERG25的NCBI编号为NM_001181189.3、固醇4α-羧酸酯3-脱氢酶ERG26的NCBI编号为NM_001180866.1、3-酮类固醇还原酶ERG27的NCBI编号为NM_001181987.1、△7-甾醇5-去饱和酶ERG3的NCBI编号为NM_001181943.1、甾醇△24-还原酶DHCR24的NCBI编号为NM_001031288.1和△-胆甾烯醇酶EBP的NCBI编号为KR709569.1。
进一步地，所述的过氧化酶体定位标签为过氧化酶体定位信号肽PTS1，其核苷酸序列如SEQ ID NO.3所示。
进一步地，所述的线粒体定位标签为线粒体定位信号肽MMF1，其核苷酸序列如SEQ ID NO.4所示。
进一步地，所述的酵母菌宿主为酿酒酵母、毕赤酵母或热带假丝酵母。
进一步地，所述的酵母菌宿主为酿酒酵母S288C。
本发明的第二个目的是提供一种酵母工程菌，所述的酵母工程菌分别在酵母菌宿主的过氧化酶体和线粒体中，表达异源甾醇△24-还原酶和△-胆甾烯醇酶，并分别使用过氧化酶体定位标签和线粒体定位标签在过氧化酶体和线粒体中表达7-脱氢胆固醇合成路径中的酶。
借由上述方案，本发明至少具有以下优点：
本发明以酵母菌为出发菌株，分别在过氧化酶体和线粒体中同时表达异源的甾醇△24-还原酶和△-胆甾烯醇酶，在酿酒酵母中经过检测7-DHC的产量为10.15mg/L，通过我们的发明，将7-DHC合成路径中的部分酶使用氧化酶体和线粒体定位标签定位在酿酒酵母中各区室化中，形成相对独立的7-DHC合成途径，同时也增加了7-DHC合成所需前体物质的储存空间，也减少了反馈作用，在同一区室中，酶与酶之间的转化效率提高，减少作用底物的损失，最终实现7-DHC产量提升4倍，达到53.31mg/L。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细说明如后。
附图说明
图1为酵母区室化生产7-DHC概论图；
图2为7-DHC标品液相图；
图3为酿酒酵母菌株CDHC-17发酵液相结果图。
具体实施方式
涉及的检测方法：
通过高效液相色谱对酿酒酵母中的7-DHC进行检测，将发酵60h的酿酒酵母菌液离心，使用10ml无菌水重悬，加入0.5mm的玻璃珠，使用高速匀浆破碎仪破碎10min，取出破碎 后的混合液，加入1g抗坏血酸和0.5gHBT，混匀，依次加入20ml的无水乙醇，10ml的1.5mol/L的氢氧化钾甲醇溶液，在80℃的水域中皂化2小时，皂化结束后使用萃取液(异丙醇：正己烷＝1:3)进行超声震荡30分钟，使用分液漏斗出去下层杂质后，将萃取混合液进行冷冻干燥处理，处理结束后使用甲醇或乙腈或甲醇和乙腈的混合物复溶，0.55μm过滤后进行高效液相色谱分析。流动相使用甲醇和水，检测器使用紫外检测器，检测波长为265nm。
实施例1：构建酿酒酵母菌CDHC-2
(a)人工合成基因片段P TEF1-DHCR24-T cyc1-P GAP-DIC-T ADH1，以酿酒酵母S228C基因组为模板，使用引物208F-F、208F-R扩增得到基因片段208-F，使用引物208R-F、208R-R扩增得到基因片段208-R，以pMHyLp-trp质粒为模板，使用引物loxT-F、loxT-R扩增得到基因片断loxT片段。
(b)将步骤(a)中获得的四个片段P TEF1-DHCR24-T cyc1-P GAP-DIC-T ADH1、208F-F、208-R和locxT基因片段进行重叠延伸PCR，1％的琼脂糖凝胶电泳验证正确后，切胶回收片段，得到融合基因片段208-F-loxT-P TEF1-DHCR24-T cyc1-P GAP-DIC-T ADH1-208-R基因片段。
(c)将步骤(b)得到的基因片段，转化进入酿酒酵母S288C菌株的感受态中，涂布在SD Trp平板上，30℃培养3天，使用引物YZ-24-DIC-F、YZ-24-DIC-R进行菌落PCR验证得到菌株CDHC-1
(d)将步骤(c)得到的菌株CDHC-1制成感受态，并将PY26-Cre这个质粒转化进入酿酒酵母中，待SD Ura筛选固体平板上长出单菌落后，接入YPD培养基中培养12h，于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD Ura筛选固体平板上对比验证，YPD平板上正常生长但SD Ura筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为CDHC-2。
引物序列：
208F-F:ccaggttaatgtgttctctgaaattcgc
208F-R:gtaatcatggtcatagctgtttcctgttgtttagcggacgtgtgtatggt
208R-F:cagcttttgttccctttagtgtttgacgatgtcagtgaatcccgg
208R-R:cttgtagtccaccattgccatttttca
loxT-F:ccatacacacgtccgctaaacaacaggaaacagctatgaccatgattacg
loxT-R:acctttagtacgggtaattaacgataaaacgacggccagtgccaaggtcg
YZ-24-DIC-F:atggatttcgccaatcaaacccat
YZ-24-DIC-R:caatgaaacagctggttccattcgaaactaagttctggtgttttaaaactaa。
实施例2：构建酿酒酵母线粒体区室化
(a)以酿酒酵母工程菌S228C基因组为模板，选取线粒体定位信号肽MMF1。采用引物ERG12-F，ERG12-MMF1-R扩增得到基因片段ERG12-MMF1，采用引物ERG13-F1，ERG13-MMF1-R1扩增得到基因片段ERG13-MMF1，采用引物GAL-F1、GAL-R1扩增得到GAL1启动子和GAL10启动子基因片段GAL1-10，采用引物loxT-GAL-F、LoxT-GAL-R扩增得到基因片段Lox-Trp，采用引物GAL80F1-F、GAL80F1-R扩增得到基因片段GAL80F1，采用引物GAL80R1-F、GAL80R1-R扩增基因片段得到GAL80R1，采用CYC1-S-F、CYC1-S-R扩增基因片段得到CYC1，采用引物ADH1-S-F、ADH1-S-R扩增基因片段得到ADH1，采用MMF1-F、MMF1-R、MMF2-F、MMF2-R为引物扩增片段得到MMF1-1、MMF2-2。
(b)将实例1中得到的CDHC-2菌株制成酵母感受态细胞，将步骤(a)中得到的10个基因片段转化进入CDHC-2中，使用引物YZS-12-13-F、YZS-12-13-R进行菌落PCR验证。
(c)将步骤(d)中菌落PCR验证正确的单菌落接入SD trp液体培养基内培养16h，之后划线于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD his筛选固体平板上对比验证，YPD平板上正常生长但SD his筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为CDHC-3。
(d)按照上述步骤将HMG1、ERG8、MVD1、IDI1、ERG9、ERG1、ERG7、ERG11、ERG24、ERG25、ERG26、ERG27、ERG3、DHCR24、EBP基因固定于线粒体中，最终得到菌株的CDHC-10
引物序列：
ERG12-F1：tcaaggagaaaaaactataatgtctcagaacgtttacattgtat
ERG12-MMF1-R：aacggaatttcttaaaaacattatcttttcaatgacaatagaggaa
ERG13-F1：tcttaaaaacatttttttaacatcgtaagatcttctaaat
ERG13-MMF1-R：ttttgaaaattcaatataaatgaaactctcaactaaactttgttg
GAL-F1：agttgagagtttcatttatattgaattttcaaaaattcttactttttttttgg
GAL-R1：taaacgttctgagacattatagttttttctccttgacgttaaagt
loxT-M-F：cttattgaccacacctctaccggcaggaaacagctatgaccatgattacg
LoxT-M-R：aatatcagagcatccataaaacgacggccagtgccaaggtcga
GAL80F2-F：acaagttcgtacttttccaggataaatgc
GAL80F2-R：ggctttaatttgcggccaccttgcattacaaattgtgagg
CYC1-M-F:tgtaatgcaaggtggccgcaaattaaagccttcgagcgtc
CYC1-M-R:atctctgagaaggccgaataatcatgtaattagttatgtcac
ADH1-M-F:ctctgagaaggccgaagcgaatttcttatgatttatgatttttatt
ADH1-M-R:tcatggtcatagctgtttcctgccggtagaggtgtggtcaataag
GAL80R2-F：cactggccgtcgttttatggatgctctgatattacacaggttaat
GAL80R2-R：accgggtgataggtttgctcaaccat
YZM-10-13-F：ggaaaagctgcataaccactttaact
YZM-10-13-R：ctgagaaagcaacctgacctacagg
MMF1-F:tacatgattattcggccttctcagagatagaaccttgaac
MMF1-R:atcttacgatgttaaaaaaatgtttttaagaaattccgtt
MMF2-F:attgtcattgaaaagataatgtttttaagaaattccgttttgagaacag
MMF2-R:aaatcataagaaattcgcttcggccttctcagagatagaa。
实施例3：构建酿酒酵母过氧化酶体区室化
(a)以酿酒酵母工程菌S228C基因组为模板，选取过氧化酶体定位信号肽PTS1。采用引物ERG12-F，ERG12-PTS1-R扩增得到基因片段ERG12-PTS1，采用引物ERG13-F，ERG13-PTS1-R扩增得到基因片段ERG13-PTS1，采用引物GAL-F、GAL-R扩增得到GAL1启动子和GAL10启动子基因片段GAL1-10，采用引物loxT-GAL-F、LoxT-GAL-R扩增得到基因片段Lox-Trp，采用引物GAL80F3-F、GAL80F3-R扩增得到基因片段GAL80F3，采用引物GAL80R3-F、GAL80R3-R扩增基因片段得到GAL80R3，采用引物CYC1-F、CYC1-13-R扩增基因片段得到CYC1，采用引物ADH1-10-F、ADH1-R扩增基因片段得到ADH1。
(b)将实例2中得到的CDHC-10菌株制成酵母感受态细胞，将步骤(a)中得到的8个基因片段转化进入CDHC-6中，使用引物YZ-10-13-F、YZ-10-13-R进行菌落PCR验证。
(c)将步骤(d)中菌落PCR验证正确的单菌落接入SD trp液体培养基内培养16h，之后划线于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD his筛选固体平板上对比验证，YPD平板上正常生长但SD his筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为CDHC-7。
(d)按照上述步骤将HMG1、ERG8、MVD1、IDI1、ERG9、ERG1、ERG7、ERG11、ERG24、ERG25、ERG26、ERG27、ERG3、DHCR24、EBP基因固定于线粒体中，最终得到菌株的CDHC-17。
引物序列：
ERG12-F：tcaaggagaaaaaactataatgtctcagaacgtttacattgtat
ERG12-PTS1-R：ttgggaagaggtagaagatccaaattgtatcttttcaatgacaatagaggaagca
ERG13-F：acaatttggatcttctacctcttcccaattttttaacatcgtaag
ERG13-PTS1-R：ttttgaaaattcaatataaatgaaactctcaactaaactttgttg
GAL-F：agttgagagtttcatttatattgaattttcaaaaattcttactttttttttgg
GAL-R：taaacgttctgagacattatagttttttctccttgacgttaaagt
loxT-pts-F：cttattgaccacacctctaccggcaggaaacagctatgaccatgattacg
LoxT-pts-R：gcattactcaattttagactaaaacgacggccagtgccaaggtcg
GAL80F3-F：ccaatgctaatccggtcactgccactgc
GAL80F3-R：aggctttaatttgcggcctggcaatagaagtctcaatttt
CYC1-F:gagacttctattgccaggccgcaaattaaagccttcgagc
CYC1-13-R:gtagaagatccaaattgtaatcatgtaattagttatgtcacgctt
ADH1-10-F:aatttggatcttctacctcttcccaagcgaatttcttatgattta
ADH1-R:tcatggtcatagctgtttcctgccggtagaggtgtggtcaataag
GAL80R3-F：gccgtcgttttagtctaaaattgagtaatgccactgcttttccca
GAL80R3-R：atgtattgtaaaatatcgattgtgt
YZ-10-13-F：ggaaaagctgcataaccactttaact
YZ-10-13-R：ctgagaaagcaacctgacctacagg。
实施例4：构建成功的酿酒酵母菌进行发酵培养
在2ml YPD培养基中接种固体YPD平板上的酿酒酵母菌CDHC-10单菌落和CDHC-17单菌落，30℃，220rpm培养16-20h后，分别按接种量1％接入含有25mL YPD液体培养基的250mL圆底摇瓶内，30℃，220rpm培养60h。发酵至16h时，加入乙醇进行碳源的补充。发酵结束108h后，离心取沉淀，除去上清，使用10ml无菌水重悬，加入0.5mm的玻璃珠，使用高速匀浆破碎仪破碎10min，取出破碎后的混合液，加入1g抗坏血酸和0.5gHBT，混匀，依次加入20ml的无水乙醇，10ml的1.5mol/L的氢氧化钾甲醇溶液，在80℃的水域中皂化2小时，皂化结束后使用萃取液(异丙醇：正己烷＝1:3)进行超声震荡30分钟，使用分液漏斗出去下层杂质后，将萃取混合液进行冷冻干燥处理，处理结束后使用甲醇或乙腈或甲醇和乙腈的混合物复溶，0.55μm过滤后进行高效液相色谱分析。流动相使用甲醇和水，检测器使用紫外检测器，检测波长为265nm。
经过液相分析，构建成功的线粒体区室化菌株CDHC-10的7-DHC产量为29.35mg/L，以CDHC-10基础上，构建成功的过氧化酶体区室化菌株CDHC-17的7-DHC产量为53.31mg/L。
以上仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Figure PCTCN2022073487-appb-000001
Figure PCTCN2022073487-appb-000002
Figure PCTCN2022073487-appb-000003
Figure PCTCN2022073487-appb-000004
Figure PCTCN2022073487-appb-000005

Claims (10)

  1. 一种利用区室化提高酵母菌中7-脱氢胆固醇产量的方法，其特征在于，包括分别在酵母菌宿主的过氧化酶体和线粒体中，表达异源甾醇△24-还原酶和△-胆甾烯醇酶，并分别使用过氧化酶体定位标签和线粒体定位标签将7-脱氢胆固醇合成路径中的酶定位在过氧化酶体和线粒体中表达。
  2. 根据权利要求1所述的方法，其特征在于，所述的甾醇△24-还原酶的氨基酸序列如SEQ ID NO.1所示。
  3. 根据权利要求1所述的方法，其特征在于，所述的△-胆甾烯醇酶的氨基酸序列如SEQ ID NO.2所示。
  4. 根据权利要求1所述的方法，其特征在于，7-脱氢胆固醇合成路径中的酶为羟甲基戊二酰辅酶A合酶ERG13、甲羟戊酸激酶ERG12、羟甲基戊二酰辅酶A还原酶HMG1、磷酸甲羟戊酸激酶ERG8、二磷酸甲戊二酸酯脱羧酶MVD1、异戊烯基二磷酸δ-异构酶IDI1、二磷酸法尼基转移酶ERG9、角鲨烯单加氧酶ERG1、羊毛甾醇合酶ERG7、固醇14α-脱甲基酶ERG11、△14-固醇还原酶ERG24、甲基固醇单加氧酶ERG25、固醇4α-羧酸酯3-脱氢酶ERG26、3-酮类固醇还原酶ERG27、△7-甾醇5-去饱和酶ERG3、甾醇△24-还原酶DHCR24和△-胆甾烯醇酶EBP。
  5. 根据权利要求4所述的方法，其特征在于，羟甲基戊二酰辅酶A合酶ERG13的NCBI编号为XM_033912304.1、甲羟戊酸激酶ERG12的NCBI编号为XM_033912620.1、羟甲基戊二酰辅酶A还原酶HMG1的NCBI编号为XM_033912352.1、磷酸甲羟戊酸激酶ERG8的NCBI编号为NM_001182727.1、二磷酸甲戊二酸酯脱羧酶MVD1的NCBI编号为NM_001183220.1、异戊烯基二磷酸δ-异构酶IDI1的NCBI编号为NM_001183931.1、二磷酸法尼基转移酶ERG9的NCBI编号为NM_001179321.1、角鲨烯单加氧酶ERG1的NCBI编号为NM_001181304.1、羊毛甾醇合酶ERG7的NCBI编号为NM_001179202.2、固醇14α-脱甲基酶ERG11的NCBI编号为NM_001179137.1、△14-固醇还原酶ERG24的NCBI编号为NM_001183118.1、甲基固醇单加氧酶ERG25的NCBI编号为NM_001181189.3、固醇4α-羧酸酯3-脱氢酶ERG26的NCBI编号为NM_001180866.1、3-酮类固醇还原酶ERG27的NCBI编号为NM_001181987.1、△7-甾醇5-去饱和酶ERG3的NCBI编号为NM_001181943.1、甾醇△24-还原酶DHCR24的NCBI编号为NM_001031288.1和△-胆甾烯醇酶EBP的NCBI编号为KR709569.1。
  6. 根据权利要求1所述的方法，其特征在于，所述的过氧化酶体定位标签为过氧化酶体定位信号肽PTS1，其核苷酸序列如SEQ ID NO.3所示。
  7. 根据权利要求1所述的方法，其特征在于，所述的线粒体定位标签为线粒体定位信 号肽MMF1，其核苷酸序列如SEQ ID NO.4所示。
  8. 根据权利要求1所述的方法，其特征在于，所述的酵母菌宿主为酿酒酵母、毕赤酵母或热带假丝酵母。
  9. 根据权利要求8所述的方法，其特征在于，所述的酵母菌宿主为酿酒酵母S288C。
  10. 一种酵母工程菌，其特征在于，所述的酵母工程菌分别在酵母菌宿主的过氧化酶体和线粒体中，表达异源甾醇△24-还原酶和△-胆甾烯醇酶，并分别使用过氧化酶体定位标签和线粒体定位标签在过氧化酶体和线粒体中表达7-脱氢胆固醇合成路径中的酶。
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