WO2021259384A1 - 一种酿酒酵母类胡萝卜素合成的培养方式 - Google Patents

一种酿酒酵母类胡萝卜素合成的培养方式 Download PDF

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WO2021259384A1
WO2021259384A1 PCT/CN2021/108449 CN2021108449W WO2021259384A1 WO 2021259384 A1 WO2021259384 A1 WO 2021259384A1 CN 2021108449 W CN2021108449 W CN 2021108449W WO 2021259384 A1 WO2021259384 A1 WO 2021259384A1
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saccharomyces cerevisiae
fermentation medium
yeast extract
carotenoid
solvent
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French (fr)
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朱红惠
苏卜利
王东东
李安章
邓名荣
冯广达
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广东省科学院微生物研究所(广东省微生物分析检测中心)
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    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes

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  • the invention belongs to the field of biotechnology, and specifically relates to a fermentation medium that is beneficial to the synthesis of carotenoids by Saccharomyces cerevisiae.
  • Carotenoids are a large group of colored substances that are naturally present in plants, algae, fungi and bacteria. This type of substance has strong antioxidant properties, has a protective effect on human health, and can be used in food, nutrition, cosmetics, etc.
  • chemical synthesis and natural extraction are the main methods for preparing carotenoids.
  • the purpose of the present invention is to provide a fermentation medium that is conducive to the synthesis of carotenoids by Saccharomyces cerevisiae in view of the phenomena found during the experiment.
  • the fermentation medium for the carotenoid synthesis of Saccharomyces cerevisiae of the present invention includes a basic medium for Saccharomyces cerevisiae and copper ions.
  • the copper ion concentration is 40-160 ⁇ M.
  • the copper ion concentration is 80 ⁇ M.
  • it also contains zinc ions, the concentration of zinc ions is 40 ⁇ M, and the concentration of copper ions is 40 ⁇ M.
  • the basal culture medium of Saccharomyces cerevisiae is: according to mass fraction, it includes 5-15 g/L of yeast extract, 15-25 g/L of peptone, 15-25 g/L of glucose, and water as the solvent.
  • the fermentation medium includes 5-15 g/L yeast extract, 15-25 g/L peptone, 15-25 g/L glucose, 5-15 g/L potassium dihydrogen phosphate, and heptahydrate based on mass fraction.
  • the fermentation medium includes yeast extract 10g/L, peptone 20g/L, glucose 20g/L, potassium dihydrogen phosphate 10g/L, and magnesium sulfate heptahydrate 5g/L in terms of mass fraction.
  • yeast extract 10g/L peptone 20g/L, glucose 20g/L, potassium dihydrogen phosphate 10g/L, and magnesium sulfate heptahydrate 5g/L in terms of mass fraction.
  • Potassium sulfate 3.5g/L, sodium phosphate 2.5g/L, TMS solution 1ml/L, solvent is water;
  • the TMS solution formula is: magnesium chloride hexahydrate 250mg/L, calcium chloride dihydrate 104.5mg/L, copper sulfate pentahydrate 0.4mg/L, sodium iodide 0.08mg/L, manganese chloride tetrahydrate 0.1mg/L , Sodium molybdate dihydrate 0.5mg/L, boric acid 1mg/L, cobalt chloride hexahydrate 0.3mg/L, zinc sulfate heptahydrate 6.25mg/L, ferrous sulfate heptahydrate 3.5mg/L, solvent is water
  • the second object of the present invention is to provide the application of the above-mentioned fermentation medium for promoting the fermentation of Saccharomyces cerevisiae to produce carotenoids.
  • the third object of the present invention is to provide the application of overexpression of transcription factor ACE1 in Saccharomyces cerevisiae in promoting carotenoid production by Saccharomyces cerevisiae.
  • the fourth objective of the present invention is to provide a method for promoting carotenoid production by Saccharomyces cerevisiae, which is to overexpress the transcription factor ACE1 in Saccharomyces cerevisiae.
  • the present invention has one of the following advantages and beneficial effects:
  • the present invention adds copper ions or a yeast extract containing copper ions to the fermentation medium of Saccharomyces cerevisiae, which can effectively promote the growth of microbial cells and the accumulation of carotenoids.
  • adding copper ions at a suitable concentration to the culture medium can significantly increase the accumulation of carotenoids in Saccharomyces cerevisiae, prevent the instability of fermentation raw materials and lead to unstable fermentation effects, and effectively improve the success rate of production.
  • Figure 1 shows the effect of different batches of yeast extract on carotenoid synthesis.
  • the accumulation of carotenoids in different companies and different batches of yeast extract medium, YPD1, contains the lot number LOT:2665431-02 OxIOD company YPD medium of yeast extract; YPD2, YPD medium containing yeast extract from OXIOD company with lot number LOT: 2315359-02; YPD3, YPD medium containing yeast extract from Angel Yeast Co., Ltd. with lot number LOT: 2018082210C9; Angel Yeast Co., Ltd. yeast extract YPM medium; YPM1, YPM2, YPM3;
  • B cell growth and production in YPM or YPD2 medium (three repetitions).
  • Figure 2 is a comparative transcriptome analysis, (A) gene differential expression; (B) GO enrichment; (C) KEGG enrichment; (D) significant up-regulation of metabolic pathways.
  • Figure 3 is the effect of different additives on the synthesis of carotenoids (A) YPD2, YPD medium containing lot number LOT: 2315359-02 OXIOD company yeast extract; YPD2+salt, containing lot number LOT: 2315359-02 OXIOD company Salt is added to the YPD medium of yeast extract; YPD2+Zn 2+ contains LOT: 2315359-02 OXIOD’s YPD medium contains Zn 2+ ; YPD2+Cu 2+ contains LOT: 2315359-02 The YPD medium of OXIOD's yeast extract is supplemented with Cu 2+ ; YPD2+TMS, containing lot number LOT: 2315359-02 The YPD medium of OXIOD's yeast extract is supplemented with TMS; YPD2+Cu 2+ +Zn 2+ , add Zn 2+ and Cu 2+ to YPD medium containing the yeast extract of OXIOD company with lot number LOT:2315359
  • Figure 4 is the reverse metabolic engineering
  • BL03-D-4 is Saccharomyces cerevisiae BL03, Cit1-tHMG1, ⁇ Ald6 strain
  • MO1 is knockout ADY2
  • MO2 is overexpression of HES1
  • MO3 is overexpression of ACE1.
  • the invention discloses a fermentation medium of Saccharomyces cerevisiae which is conducive to the accumulation of carotenoids.
  • the reagents involved in the embodiments of the present invention are all commercially available products, and all can be purchased through commercial channels.
  • Example 1 The effect of different batches of yeast extract on the synthesis of carotenoids
  • the fermentation medium of Saccharomyces cerevisiae in this embodiment is: YPD medium: yeast extract 10g/L (OXOID company or Angel Yeast Co., Ltd.), peptone 20g/L, glucose 20g/L, the solvent is water, and its preparation method It is to mix the ingredients uniformly according to their content and sterilize them for later use; or YPM medium: yeast extract 10g/L (Ange Yeast Co., Ltd.), peptone 20g/L, glucose 20g/L, salt (potassium dihydrogen phosphate) 10g/L, magnesium sulfate heptahydrate 5g/L, potassium sulfate 3.5g/L, sodium phosphate 2.5g/L), TMS solution 1ml/L, and the solvent is water. Sterilize for later use.
  • yeast extract 10g/L OXOID company or Angel Yeast Co., Ltd.
  • peptone 20g/L glucose 20g/L
  • the solvent is water, and its preparation method It
  • the TMS solution formula is: magnesium chloride hexahydrate 250mg/L, calcium chloride dihydrate 104.5mg/L, copper sulfate pentahydrate 0.4mg/L, sodium iodide 0.08mg/L, manganese chloride tetrahydrate 0.1mg/L , Sodium molybdate dihydrate 0.5mg/L, boric acid 1mg/L, cobalt chloride hexahydrate 0.3mg/L, zinc sulfate heptahydrate 6.25mg/L, ferrous sulfate heptahydrate 3.5mg/L, the solvent is water, its The preparation method is to mix the ingredients evenly according to their contents and sterilize them for later use.
  • Saccharomyces cerevisiae (application number: 2019103999462 Saccharomyces cerevisiae BL03, Cit1-tHMG1, ⁇ Ald6 strain) was streaked into fresh YPD solid medium, 48 hours later, a single colony was picked and inoculated into 5ml YPD liquid medium. After overnight incubation, they were respectively inoculated into 50 ml of the above-mentioned different YPD and YPM liquid media according to the volume ratio of 2% inoculum. Incubate at 30°C and 200 rpm for 96 hours. At the end of the culture, the carotenoid content and growth curve under each culture condition were determined according to the standard determination method.
  • yeast extracts from different companies have a significant impact on carotenoid accumulation, and different batches of OXOID company's yeast extracts also have a significant impact.
  • the yeast extract from Angel Yeast Company can not only promote the accumulation of carotenoids, but also promote cell growth, and the final content can reach 18.8mg/g dry cell weight. It is 10 times the content of other batches (1.85mg/g). This result reveals that certain substances contained in Angel Yeast's yeast extract can promote the accumulation of carotenoids.
  • transcriptome sequencing of Saccharomyces cerevisiae strains containing Angel Yeast's yeast extract culture conditions and ordinary YPD2 culture conditions for comparative transcriptome analysis. It can be seen from Figure 2 that a total of 464 genes have undergone significant changes (more than 2 times), of which 203 genes are significantly up-regulated and 261 genes are significantly down-regulated.
  • KEGG and GO enrichment analysis found that the glyoxylate pathway, glycolysis pathway, steroid synthesis pathway, fatty acid degradation pathway and pentose phosphate pathway were significantly up-regulated, especially most genes in the ergosterol synthesis pathway were significantly up-regulated.
  • the HES1 gene was up-regulated by 11 times
  • the ADY2 gene was down-regulated by 8 times
  • the CUP1-2 gene was up-regulated by 94 times.
  • the YPD2 liquid culture medium of this example is: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, solvent is water, pH is 5.0-7.0, and its preparation method is to mix the ingredients evenly according to their content and adjust pH value, sterilized for later use.
  • yeast extract comes from OXOID company, LOT: 2315359-02.
  • YPD2 solid medium is agar added to YPD2 liquid medium.
  • Saccharomyces cerevisiae BL03, Cit1-tHMG1, ⁇ Ald6 strains were streaked into fresh YPD2 solid medium (the yeast extract comes from OXOID company), 48 hours later, a single colony was picked and inoculated into 5ml YPD2 liquid medium, overnight After incubation, inoculate 50ml of salt (potassium dihydrogen phosphate 10g/L, magnesium sulfate heptahydrate 5g/L, potassium sulfate 3.5g/L, sodium phosphate 2.5g/L) in a volume ratio of 2% inoculum to 50ml respectively-corresponding diagram YPD2+Salt, TMS in 3A (magnesium chloride hexahydrate 250mg/L, calcium chloride dihydrate 104.5mg/L, copper sulfate pentahydrate 0.4mg/L, sodium iodide 0.08mg/L, manganese chloride tetrahydrate 0.1mg
  • the invention uses strains and plasmids

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Abstract

提供了一种利于酿酒酵母类胡萝卜素合成的发酵培养基，包括酿酒酵母基础培养基和铜离子，所述发酵培养基提升了重组酿酒酵母类胡萝卜素的积累。转录组学揭示了培养基中锌离子和铜离子之间存在协同增强作用，添加二者可以显著提升类胡萝卜素合成。

Description

一种酿酒酵母类胡萝卜素合成的培养方式 技术领域
本发明属于生物技术领域，具体涉及一种利于酿酒酵母类胡萝卜素合成的发酵培养基。
背景技术
类胡萝卜素是一大类有颜色的物质，天然存在于植物、藻类、真菌和细菌中。这类物质具有较强的抗氧化性，对人类健康有保护作用，可以用于食品、营养品、化妆品等。当前，化学合成和天然提取是制备类胡萝卜素的主要方式。但是，由于结构的复杂性，化学合成过程比较困难；而原料供应不稳定、有机溶剂污染、得率低等导致天然提取成本较高，无法规模化生产。所以，利用微生物合成特定类胡萝卜素越来越受到人们关注。
随着代谢工程和合成生物学的快速发展，构建微生物细胞工厂合成类胡萝卜素逐渐成为一种可持续生产方式。所以，当前有众多研究集中于优化内源代谢途径、引入外源途径增加代谢流量、辅因子平衡等。在酿酒酵母中，通过过表达tHMG1基因增加MVA途径代谢流、下调ERG9基因或者过表达ALD6和ACS增加前体物质乙酰辅酶A、增加TCA或PPP途径代谢流量等众多策略用于提高类胡萝卜素的产量。但是，通过优化培养基成分来提高类胡萝卜素产量的报道并不多见。
发明内容
有鉴于此，本发明的目的在于针对实验过程中发现的现象，提供一种利于酿酒酵母类胡萝卜素合成的发酵培养基。
在研究中我们发现，不同公司及不同批次的酵母提取物会对类胡萝卜素含量造成显著影响。为了找出这个现象背后隐藏的原因，我们进行了比较转录组分析并结合反向代谢工 程，确定了培养基中铜离子起了决定性作用。
本发明的利于酿酒酵母类胡萝卜素合成的发酵培养基，包括酿酒酵母基础培养基和铜离子。
优选，所述的铜离子浓度为：40～160μM。
进一步优选，所述的铜离子浓度为：80μM。
优选，还含有锌离子，所述的锌离子浓度为40μM，铜离子浓度为40μM。
所述的酿酒酵母基础培养基为：按质量分数计，包括酵母提取物5～15g/L，蛋白胨15～25g/L，葡萄糖15～25g/L，溶剂为水。
进一步优选，所述的发酵培养基按质量分数计，包括酵母提取物5～15g/L，蛋白胨15～25g/L，葡萄糖15～25g/L，磷酸二氢钾5～15g/L，七水硫酸镁5～15g/L，硫酸钾3.0～8.0g/L，磷酸钠0.5～3g/L，TMS溶液1ml/L，溶剂为水，pH为5.0～7.0。
更进一步优选为：所述的发酵培养基按质量分数计，包括酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，磷酸二氢钾10g/L，七水硫酸镁5g/L，硫酸钾3.5g/L，磷酸钠2.5g/L，TMS溶液1ml/L，溶剂为水；
所述TMS溶液配方为：六水氯化镁250mg/L，二水氯化钙104.5mg/L，五水合硫酸铜0.4mg/L，碘化钠0.08mg/L，四水合氯化锰0.1mg/L，二水合钼酸钠0.5mg/L，硼酸1mg/L，六水合氯化钴0.3mg/L，七水硫酸锌6.25mg/L，七水合硫酸亚铁3.5mg/L，溶剂为水
本发明的第二个目的是提供上述发酵培养基在用于促进酿酒酵母发酵产类胡萝卜素中的应用。
本发明的第三个目的是提供在酿酒酵母中过表达转录因子ACE1在促进酿酒酵母产类 胡萝卜素中的应用。
本发明的第四个目的是提供一种促进酿酒酵母产类胡萝卜素的方法，其是将在酿酒酵母中过表达转录因子ACE1。
相对于现有技术本发明具有以下优点和有益效果之一：
(1)本发明在酿酒酵母的发酵培养基中添加铜离子或含有铜离子的酵母提取物，可以有效促进微生物细胞的生长和类胡萝卜素的积累。
(2)本发明在培养基中添加合适浓度的铜离子，可以显著增加酿酒酵母类胡萝卜素的积累，防止发酵原料的不稳定而导致发酵效果的不稳定，有效提高生产的成功率。
附图说明
图1是不同批次酵母提取物对类胡萝卜素合成的影响，(A)不同公司及不同批次酵母提取物培养基中类胡萝卜素积累情况，YPD1,含有批号为LOT:2665431-02 OXIOD公司酵母提取物的YPD培养基；YPD2,含有批号为LOT:2315359-02 OXIOD公司酵母提取物的YPD培养基；YPD3,含有批号为LOT:2018082210C9安琪酵母股份公司酵母提取物的YPD培养基；含安琪酵母股份公司酵母提取物的YPM培养基；YPM1，YPM2，YPM3；(B)在YPM或YPD2培养基中细胞生长情况及生产情况(三次重复)。
图2是比较转录组分析，(A)基因差异表达情况；(B)GO富集情况；(C)KEGG富集情况；(D)显著上调代谢途径。
图3是不同添加物对类胡萝卜素合成的影响(A)YPD2,含有批号为LOT:2315359-02 OXIOD公司酵母提取物的YPD培养基；YPD2+salt,含有批号为LOT:2315359-02 OXIOD公司酵母提取物的YPD培养基中添加salt；YPD2+Zn 2+含有批号为LOT:2315359-02 OXIOD 公司酵母提取物的YPD培养基中添加Zn 2+；YPD2+Cu 2+,含有批号为LOT:2315359-02 OXIOD公司酵母提取物的YPD培养基中添加Cu 2+；YPD2+TMS,含有批号为LOT:2315359-02 OXIOD公司酵母提取物的YPD培养基中添加TMS；YPD2+Cu 2++Zn 2+,含有批号为LOT:2315359-02 OXIOD公司酵母提取物的YPD培养基中添加Zn 2+和Cu 2+；在YPD2培养基中分别添加0.04mM,0.8mM,0.16mM的铜离子，对应图中的0.04mM、0.8mM、1.6mM。不同酵母提取物中铜离子浓度含量测定(B)YPD1,含有批号为LOT:2665431-02 OXIOD公司酵母提取物的YPD培养基；YPD2,含有批号为LOT:2315359-02 OXIOD公司酵母提取物的YPD培养基；YPD3,含有批号为LOT:2018082210C9安琪酵母股份公司酵母提取物的YPD培养基；含不同铜离子浓度YPD2条件下细胞生长曲线(C)。
图4是反向代谢工程改造，BL03-D-4是酿酒酵母BL03，Cit1-tHMG1，ΔAld6菌株；MO1是敲除ADY2；MO2是过表达HES1；MO3是过表达ACE1。
具体实施方式
本发明公开了一种利于类胡萝卜素积累的酿酒酵母的发酵培养基。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及产品已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或适当变更与组合，来实现和应用本发明技术。
为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获 得的所有其他实施例，都属于本发明保护的范围。
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。
实施例1 不同批次酵母提取物对类胡萝卜素合成的影响
本实施例的酿酒酵母发酵培养基为：YPD培养基：酵母提取物10g/L(OXOID公司或安琪酵母股份有限公司)，蛋白胨20g/L，葡萄糖20g/L，溶剂为水，其配制方法是将各成分按其含量混合均匀，灭菌备用；或YPM培养基：酵母提取物10g/L(安琪酵母股份有限公司)，蛋白胨20g/L，葡萄糖20g/L，salt(磷酸二氢钾10g/L，七水硫酸镁5g/L，硫酸钾3.5g/L，磷酸钠2.5g/L)，TMS溶液1ml/L，溶剂为水，其配制方法是将各成分按其含量混合均匀，灭菌备用。
所述TMS溶液配方为：六水氯化镁250mg/L，二水氯化钙104.5mg/L，五水合硫酸铜0.4mg/L，碘化钠0.08mg/L，四水合氯化锰0.1mg/L，二水合钼酸钠0.5mg/L，硼酸1mg/L，六水合氯化钴0.3mg/L，七水硫酸锌6.25mg/L，七水合硫酸亚铁3.5mg/L，溶剂为水，其配制方法是将各成分按其含量混合均匀，灭菌备用。
在长期的实验过程中，我们发现使用不同批次的酵母提取物，类胡萝卜素的含量会出现波动情况，导致实验结果的不稳定。为了弄清楚这里面的原因，我们首先对不同批次酵母提取物类胡萝卜素含量进行表征。
将酿酒酵母(申请号为：2019103999462中酿酒酵母BL03，Cit1-tHMG1，ΔAld6菌株)将菌株划线于新鲜YPD固体培养基中，48小时后，挑取单菌落接种于5ml YPD液体培养基中，过夜培养后，按体积比2％接种量分别接种到50ml上述不同的YPD和YPM液体培 养基中。于30℃，200rpm条件下培养96小时。培养结束，按标准的测定方法，分别测定各培养条件下类胡萝卜素含量和生长曲线。
从图1可以看出，不同公司的酵母提取物对类胡萝卜素积累有显著影响，并且不同批次OXOID公司的酵母提取物同样会有显著影响。其中安琪酵母公司来源的酵母提取物不仅可以促进类胡萝卜素的积累，还可以促进细胞生长，最后含量可以达到18.8mg/g细胞干重。是其它批次含量的10倍(1.85mg/g)。此结果揭示了，在安琪酵母公司的酵母提取物中含有某种物质可以促进类胡萝卜素的积累。
实施例2 比较转录组揭示潜在机制
为了搞清楚培养基中何种物质促进了类胡萝卜素积累，我们将含有安琪酵母公司的酵母提取物培养条件和普通YPD2培养条件下的酿酒酵母菌株进行转录组测序，进行比较转录组分析。从图2可以看出，总计有464个基因发生了显著变化(大于2倍)，其中203个基因显著上调，261基因显著下调。KEGG和GO富集分析发现，乙醛酸途径、糖酵解途径、类固醇合成途径、脂肪酸降解途径和磷酸戊糖途径显著上调，特别是麦角固醇合成途径中的大部分基因都显著上调。其中，HES1基因上调了11倍，ADY2基因下调了8倍，CUP1-2基因上调94倍，这些显著变化的基因有可能与类胡萝卜素积累有关。
实施例3 铜离子对类胡萝卜素合成的影响
本实施例YPD2液体培养基为：酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，溶剂为水，pH为5.0～7.0，其配制方法是将各成分按其含量混合均匀，调pH值，灭菌备用。其中酵母提取物有来至OXOID公司,LOT:2315359-02。YPD2固体培养基是在YPD2液体培养基中加入琼脂。
从比较转录组获得启示，安琪酵母公司的酵母提取物对类胡萝卜素积累的促进作用可能与锌离子和铜离子有关。所以我们尝试在培养基中添加多种营养成分，考察其对类胡萝卜素合成的影响。
将酿酒酵母BL03，Cit1-tHMG1，ΔAld6菌株划线于新鲜YPD2固体培养基(其中酵母提取物有来至OXOID公司)中，48小时后，挑取单菌落接种于5ml YPD2液体培养基中，过夜培养后，按体积比2％接种量分别接种到50ml含有salt(磷酸二氢钾10g/L，七水硫酸镁5g/L，硫酸钾3.5g/L，磷酸钠2.5g/L)-对应图3A中的YPD2+Salt,TMS(六水氯化镁250mg/L，二水氯化钙104.5mg/L，五水合硫酸铜0.4mg/L，碘化钠0.08mg/L，四水合氯化锰0.1mg/L，二水合钼酸钠0.5mg/L，硼酸1mg/L，六水合氯化钴0.3mg/L，七水硫酸锌6.25mg/L，七水合硫酸亚铁3.5mg/L)-对应图3A中的YPD2+TMS,Zn 2+(80μM)-对应图3A中的YPD2+Zn 2+,Cu 2+(80μM)-对应图3A中的YPD2+Cu 2+，Zn 2+(40μM)+Cu 2+(40μM)-对应图3A中的YPD2+Cu2+Zn 2+，Cu 2+(0.04M)-对应图3A中的0.04mM，Cu 2+(0.8M)-对应图3A中的0.08mM，Cu 2+(1.6mM)-对应图3A中的1.6mM等不同成分的YPD2液体培养基中。于30℃，200rpm条件下培养96小时。培养结束，按标准的测定方法，分别测定各培养条件下类胡萝卜素含量和生长曲线。
从图3可以看出，80μM锌离子和80μM铜离子的添加都可以促进类胡萝卜素合成，其中铜离子起主导作用。锌离子和铜离子同时添加有协同作用，可以达到YPM培养基的效果，最高产量可以达到19.8mg/g细胞干重(图3A)。我们还发现铜离子浓度对类胡萝卜素积累也有影响，浓度高于160μM会对细胞造成抑制作用，浓度低于40μM促进作用有限(图3A)。
随后，我们对出发菌株进行反向代谢工程研究，参考文献(Chen,Y.；Xiao,W.；Wang,Y.；Liu,H.；Li,X.；Yuan,Y.,Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering.Microb Cell Fact.2016,15,(1),113.)一步法基因失活方法，利用组氨基酸(HIS3)缺陷对ADY2(Gene ID:850368)基因进行敲除，对HES1(Gene ID:854412)和ACE1(Gene ID:852710)基因进行过表达，获得ADY2基因敲除的突变菌株MO1；HES1基因过表达的突变菌株MO2；ACE1基因过表达的突变菌株MO3。发现ADY2基因敲除和HES1基因过表达对类胡萝卜素积累无促进作用，而与铜离子有关的转录因子ACE1的过表达，可以使类胡萝卜素含量提高6.4倍到11.9mg/g细胞干重(图4)。
本发明使用菌株和质粒
Figure PCTCN2021108449-appb-000001
Figure PCTCN2021108449-appb-000002
本发明所使用引物
Figure PCTCN2021108449-appb-000003
Figure PCTCN2021108449-appb-000004

Claims (10)

  1. 一种利于酿酒酵母类胡萝卜素合成的发酵培养基，其特征在于，包括酿酒酵母基础培养基和铜离子。
  2. 根据权利要求1所述的发酵培养基，其特征在于，所述的铜离子浓度为：40～160μM。
  3. 根据权利要求2所述的发酵培养基，其特征在于，所述的铜离子浓度为：80μM。
  4. 根据权利要求1、2或3所述的发酵培养基，其特征在于，还含有锌离子，所述的锌离子浓度为40μM，铜离子浓度为40μM。
  5. 根据权利要求1所述的发酵培养基，其特征在于，所述的酿酒酵母基础培养基为：按质量分数计，包括酵母提取物5～15g/L，蛋白胨15～25g/L，葡萄糖15～25g/L，溶剂为水。
  6. 根据权利要求5所述的发酵培养基，其特征在于，所述的发酵培养基按质量分数计，包括酵母提取物5～15g/L，蛋白胨15～25g/L，葡萄糖15～25g/L，磷酸二氢钾5～15g/L，七水硫酸镁5～15g/L，硫酸钾3.0～8.0g/L，磷酸钠0.5～3g/L，TMS溶液1ml/L，溶剂为水，pH为5.0～7.0。
  7. 根据权利要求6所述的发酵培养基，其特征在于，所述的发酵培养基按质量分数计，包括酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，磷酸二氢钾10g/L，七水硫酸镁5g/L，硫酸钾3.5g/L，磷酸钠2.5g/L，TMS溶液1ml/L，溶剂为水；
    所述TMS溶液配方为：六水氯化镁250mg/L，二水氯化钙104.5mg/L，五水合硫酸铜0.4mg/L，碘化钠0.08mg/L，四水合氯化锰0.1mg/L，二水合钼酸钠0.5mg/L，硼酸1mg/L，六水合氯化钴0.3mg/L，七水硫酸锌6.25mg/L，七水合硫酸亚铁3.5mg/L，溶剂为水。
  8. 权利要求1、2、3、4、5、6或7所述的发酵培养基在用于促进酿酒酵母发酵产类胡萝卜素中的应用。
  9. 在酿酒酵母中过表达转录因子ACE1在促进酿酒酵母产类胡萝卜素中的应用。
  10. 一种促进酿酒酵母产类胡萝卜素的方法，其特征在于，是将在酿酒酵母中过表达转录因子ACE1。
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