WO2025148100A1 - 一种从头合成罗汉果甜苷的工程酵母的构建方法 - Google Patents

一种从头合成罗汉果甜苷的工程酵母的构建方法

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WO2025148100A1
WO2025148100A1 PCT/CN2024/073548 CN2024073548W WO2025148100A1 WO 2025148100 A1 WO2025148100 A1 WO 2025148100A1 CN 2024073548 W CN2024073548 W CN 2024073548W WO 2025148100 A1 WO2025148100 A1 WO 2025148100A1
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Prior art keywords
mogroside
yeast
engineered yeast
gene expression
expression frame
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English (en)
French (fr)
Inventor
刘龙
陈坚
宋云飞
谢永富
吕雪芹
韦锋锐
堵国成
曲冠颐
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Guilin Layn Synthetic Biotechnology Co Ltd
Jiangnan University
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Guilin Layn Synthetic Biotechnology Co Ltd
Jiangnan University
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Priority to EP24916164.7A priority Critical patent/EP4768591A1/en
Publication of WO2025148100A1 publication Critical patent/WO2025148100A1/zh
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Definitions

  • the invention relates to a method for constructing an engineered yeast, in particular to a method for constructing an engineered brewer's yeast for synthesizing mogroside from scratch.
  • Mogroside is a type of cucurbitane tetracyclic triterpenoid secondary metabolite derived from monk fruit. It uses mogroside alcohol as aglycone, and different numbers of sugar groups are connected by different glycosidic bonds at C-3 and C-24, including mogroside I A1, mogroside II E, mogroside III x , simenoside I, mogroside IV A, mogroside V and other structures. Among the many mogrosides, mogroside V is the component with high content and sweetness, and is also the main source of sweetness in monk fruit. Its chemical formula is C 60 H 102 O 29 and its relative molecular mass is 1287.43.
  • Mogroside V was certified as safe and non-toxic by FDA in 1995, allowing it to be used as a food additive. Due to its high sweetness, low calories, non-fermentability and other characteristics, it will not cause obesity or tooth decay, so mogroside V is an ideal natural sucrose substitute. Since these substances generally have anti-inflammatory effects and can regulate the body's sugar metabolism or lipid metabolism, other mogrosides are widely used in the production of medicines or cosmetics, even though their sweetness is not as high as that of mogroside V. It can be seen that mogrosides have broad application prospects.
  • the industrial production of mogroside is mainly extracted from monk fruit, but the average content of mogroside is less than 1% of the dry weight of monk fruit. Therefore, the direct extraction method has a large demand for monk fruit, but the planting volume of monk fruit is limited and is easily affected by soil conditions and climatic conditions. The extraction efficiency is low, which not only increases costs but also causes waste.
  • the rise of synthetic biology broke the limitation of specific species producing corresponding products, making the synthesis of mogroside by microorganisms a reality.
  • the synthesis of mogroside by microorganisms is not restricted by natural conditions, the production cycle is short, the product can be secreted outside the cell for easy separation, the product has little pollution, and is suitable for large-scale production.
  • the technical problem to be solved by the present invention is to provide a method for constructing engineered yeast for synthesizing mogroside from scratch.
  • the method is to integrate exogenous genes into the yeast genome and regulate and transform the yeast by means of metabolic engineering, so that the yeast can achieve efficient de novo synthesis of mogroside using glucose as a substrate by fermentation.
  • a method for constructing an engineered yeast for synthesizing mogroside from scratch comprises inserting the constructed cucurbitadienol synthase gene expression frame Ptef1 -cds- Tcyc1 , the epoxide hydrolase gene expression frame Ptef1 -eph3- Tcyc1 , the cytochrome P450 enzyme encoding gene CYP and the cytochrome P450 enzyme reductase gene expression frame Ttdh3 -cyp87d18-Pgal1 /10- atcpr2- Tcyc1 and the glycosyltransferase encoding gene expression frame Tadh1- ugt74ac1-Pgal1 /10- ugtms1- Tcyc1 into the yeast genome to obtain an engineered yeast for producing mogroside (hereinafter referred to as: recombinant engineered yeast).
  • a truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene expression frame Ptef1 -tHMG1- Thmg1 was constructed and inserted into the gal80 site in the recombinant yeast genome, thereby relieving the rate-limiting step of the MVA pathway. At the same time, it was not induced by galactose, and the synthesis pathway of mogroside was only regulated by glucose concentration.
  • the copy number of the squalene epoxidase gene erg1 of the recombinant engineered yeast was increased, which promoted more of the intermediate squalene to flow to the target product mogroside.
  • the engineered yeast is Saccharomyces cerevisiae, and the expression frame of the cucurbitadienol synthase gene Ptef1 -cds- Tcyc1 is inserted into the 1622b site of the yeast genome; the epoxide hydrolase gene expression frame Ptef1 -eph3- Tcyc1 is inserted into the 911b site of the yeast genome; the cytochrome P450 enzyme encoding gene CYP and the cytochrome P450 enzyme reductase gene expression frame Ttdh3 -cyp87d18-Pgal1 /10 -atcpr2- Tcyc1 are inserted into the 106a site of the yeast genome; the glycosyltransferase encoding gene expression frame Tadh1- ugt74ac1-Pgal1 /10- ugtms1- Tcyc1 is inserted into the 308a site of the yeast genome; the trunc
  • the final saccharomyces cerevisiae strain Mogrol 09 provided by the present invention has a yield of mogroside V of 5.6 mg/L, which is 467 times that of the saccharomyces cerevisiae strain Mogrol 04 that can initially synthesize mogroside V.
  • the yields of other mogrosides such as mogroside IIE, mogroside IIIx, mogroside IVA, and simanoside of the final strain Mogrol 09 are also significantly higher than those of the strain Mogrol 04 that can initially synthesize mogroside V.
  • the fermentation broth was collected and centrifuged at 8000 r/min for 5 min. 1 mL of the supernatant was added with chromatography-grade formic acid to make the final concentration of formic acid reach 3% (v/v). The mixture was allowed to stand at 4°C for 4-8 h to remove protein. The mixture was centrifuged at 8000 r/min for 5 min. 0.5 mL of the supernatant was taken and filtered through a 0.22 ⁇ m water filter membrane before being added to a liquid phase detection bottle for yield detection.
  • the 20bp PAM sequence of CRISPR Cas9 was selected according to the gene sequence of the integration sites 1622b, 911b, 106a, and 308a of the Saccharomyces cerevisiae genome, and the upstream and downstream homologous arm amplification primers of the gene expression frame were designed on both sides of the PAM sequence.
  • the upstream and downstream homologous arms were amplified by PCR using the Saccharomyces cerevisiae genomic DNA as the template.
  • the primer conditions are shown in Table 3.
  • the PCR amplification conditions are shown in Table 2.
  • the gene expression frames include: cucurbitadienol synthase gene expression frame P tef1 -cds-T cyc1 , epoxide hydrolase gene expression frame P tef1 -eph3-T cyc1 , cytochrome P450 enzyme encoding gene CYP and cytochrome P450 enzyme reductase gene expression frame T tdh3 -cyp87d18-P gal1/10 -atcpr2-T cyc1 and glycosyltransferase encoding gene expression frame T adh1 -ugt74ac1-P gal1/10 -ugtms1-T cyc1 .
  • the promoter P tef1 is a constitutive promoter endogenous to Saccharomyces cerevisiae, and the nucleotide sequence is shown in NA SEQ ID NO.8.
  • the promoter P gal1/10 is a bidirectional promoter composed of promoters gal1 and gal10, and the nucleotide sequence of P gal1/10 is shown in NA SEQ ID NO.9.
  • the terminators T cyc1 , T adh1 , and T tdh3 are endogenous terminators of Saccharomyces cerevisiae, and the nucleotide sequences are shown in NA SEQ ID NO.10, NA SEQ ID NO.11, and NA SEQ ID NO.12, respectively. As shown in SEQ ID NO.12.
  • the sgRNA plasmid of CRISPR Cas9 was constructed, and plasmid pML104 was used as a template. Circular PCR was performed with primers that added a 20bp PAM sequence (as shown in NA SEQ ID NO.13). The plasmid pML104-PAM with the corresponding PAM sequence was obtained by sequencing.
  • the constructed pML104-PAM and gene expression cassette were transformed into competent cells of the host strain Saccharomyces cerevisiae CEN PK2-1C MATa; ura3-52; trp1-289; leu2-3,112; his3 ⁇ 1; MAL2-8C; SUC2, and the correct transformants obtained by colony PCR were picked for sequencing verification, finally obtaining the recombinant Saccharomyces cerevisiae strain Mogrol 04 with the mogroside synthesis pathway.
  • the synthesis of mogroside belongs to secondary metabolism, and the accumulation of precursors is small.
  • the N-terminal endoplasmic reticulum localization signal peptide of gene hmg1 was removed to obtain thmg1.
  • a truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression frame P tef1 -thmg1-T hmg1 was constructed and transformed into strain Mogrol 04, and inserted into the gal80 site in the genome of Saccharomyces cerevisiae, that is, 1 copy of thmg1 was added to the gal80 site, and the obtained strain was named Mogrol 05.
  • Strain Mogrol 05 relieves the rate-limiting step of the MVA pathway, and the strain is not induced by galactose, and the opening of the synthesis pathway of mogroside is only regulated by glucose concentration.
  • strain Mogrol 08 was used as the starting strain, and the multi-copy plasmid pESC-G418-T tdh3 -cyp87d18-P gal1/10 -atcpr2-T cyc1 was transformed into it to obtain strain Mogrol 09.

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Abstract

提供了一种从头合成罗汉果甜苷工程酵母的构建方法。该方法包括：将构建的葫芦二烯醇合成酶基因表达框P tef1-cds-T cyc1、环氧水解酶基因表达框P tef1-eph3-T cyc1、细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框T tdh3-cyp87d18-P gal1/10-atcpr2-T cyc1和糖基转移酶编码基因表达框T adh1-ugt74ac1-P gal1/10-ugtms1-T cyc1，插入酵母基因组中，获得罗汉果甜苷生产工程酵母。该工程酵母能够以葡萄糖为底物进行发酵而从头合成罗汉果甜苷。

Description

一种从头合成罗汉果甜苷的工程酵母的构建方法 技术领域
本发明涉及工程酵母的构建方法，尤其一种从头合成罗汉果甜苷的工程酿酒酵母的构建方法。
背景技术
罗汉果甜苷(Mogroside)是一类来源于罗汉果果实的葫芦烷型四环三萜类次级代谢产物，以罗汉果醇作为苷元，在C-3、C-24分别以不同的糖苷键连接不同数量的糖基，包含罗汉果甜苷I A1、罗汉果甜苷II E、罗汉果甜苷IIIx、赛门苷I、罗汉果甜苷IV A、罗汉果甜苷V等多种结构。众多罗汉果甜苷中，罗汉果甜苷V是含量以及甜度均较高的成分，也是罗汉果的主要甜味来源，其化学式为C60H102O29，相对分子质量为1287.43。罗汉果甜苷V于1995年被FDA认证为安全无毒，允许其被用作食品添加剂，因其甜度高、热量低、无发酵性等特征，不会引起肥胖或者龋齿，故罗汉果甜苷V是一种理想的天然蔗糖替代品。由于此类物质普遍具有抗炎、调节人体糖代谢或脂代谢等作用，即使甜度不及罗汉果甜苷V，其他罗汉果甜苷也被广泛应用于药物或化妆品的生产。可见，罗汉果甜苷具有广阔的应用前景。
目前，罗汉果甜苷的工业生产主要是从罗汉果果实中进行提取的，但是，罗汉果甜苷的平均含量不足罗汉果干重的1％，因此直接提取法对于罗汉果果实的需求量大，但罗汉果的种植量有限，且易受到土壤条件以及气候条件的影响，提取效率低，不仅提高成本而且造成浪费。本世纪初，合成生物学的崛起，打破了特定物种生产对应产物的限制，使微生物合成罗汉果甜苷成为现实。微生物合成罗汉果甜苷不受自然条件制约，生产周期较短，产物能够分泌至胞外易于分离，产物污染小，适合大规模生产。
酵母不仅能够用于酿酒、制作面包和馒头等食品，随着基因编辑技术的发展，也能够以酿酒酵母为底盘细胞实现天然产物的生产。酵母具有低致病性，高抗逆性，受噬菌体污染的概率较低等优点，故其在基因工程领域发挥着重要作用。然 而，酵母中没有罗汉果甜苷合成的代谢通路，目前，未有关于微生物发酵法以葡萄糖为底物从头合成罗汉果甜苷的相关报道。
发明内容
本发明所要解决的技术问题是提供一种从头合成罗汉果甜苷工程酵母的构建方法。该方法是将外源基因整合至酵母基因组并通过代谢工程的手段对酵母进行调控、改造，使得酵母实现罗汉果甜苷的高效以葡萄糖为底物发酵从头合成。
具体地，一种从头合成罗汉果甜苷工程酵母的构建方法，将构建的葫芦二烯醇合成酶基因表达框Ptef1-cds-Tcyc1、环氧水解酶基因表达框Ptef1-eph3-Tcyc1、细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1和糖基转移酶编码基因表达框Tadh1-ugt74ac1-Pgal1/10-ugtms1-Tcyc1，插入酵母基因组中，获得罗汉果甜苷生产工程酵母(以下简称为：重组工程酵母)。
进一步地，构建截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因表达框Ptef1-tHMG1-Thmg1，并插入重组工程酵母基因组中的gal80位点，解除MVA途径的限速步骤，同时不受半乳糖的诱导，罗汉果甜苷的合成途径开启只受到葡萄糖浓度的调控。
进一步地，替换重组工程酵母中的羟甲基戊二酰辅酶A合酶基因erg13以及角鲨烯合成酶erg9的内源启动子为强启动子Ptef1，强化了罗汉果甜苷前体物质的累积。
进一步地，增加重组工程酵母的角鲨烯环氧化酶基因erg1的拷贝数，促使中间体角鲨烯更多地流向目标产物罗汉果甜苷。
进一步地，敲除重组工程酵母的旁路羊毛甾醇途经的关键酶羊毛甾醇合成酶基因erg7，避免旁路途径罗汉果甜苷的前体物质的竞争。
进一步地，为实现cyp87d18的多拷贝表达，向重组工程酵母中转化多拷贝质粒pESC-G418-Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1，获得优化的工程酵母。
本发明，所述工程酵母包括但不限于酿酒酵母、巴斯德毕赤酵母、发酵毕赤 酵母、异常汉逊酵母、解脂耶氏酵母、南极假丝酵母、产朊假丝酵母、热带假丝酵母、粟酒裂殖酵母、黏红酵母。
一个实施例中，所述工程酵母为酿酒酵母，葫芦二烯醇合成酶基因Ptef1-cds-Tcyc1的表达框插入酵母基因组1622b位点；环氧水解酶基因表达框Ptef1-eph3-Tcyc1插入酵母基因组911b位点；细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1插入酵母基因组的106a位点；糖基转移酶编码基因表达框Tadh1-ugt74ac1-Pgal1/10-ugtms1-Tcyc1插入酵母基因组的308a位点；截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因表达框Ptef1-tHMG1-Thmg1插入酵母基因组的gal80位点。
本发明还提供了从头合成罗汉果甜苷工程酵母。
具体地，从头合成罗汉果甜苷工程酵母，酵母基因组中含有葫芦二烯醇合成酶基因表达框Ptef1-cds-Tcyc1、环氧水解酶基因表达框Ptef1-eph3-Tcyc1、细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1和糖基转移酶编码基因表达框Tadh1-ugt74ac1-Pgal1/10-ugtms1-Tcyc1。
所述酵母基因组中含有截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因表达框Ptef1-tHMG1-Thmg1。
所述酵母中的羟甲基戊二酰辅酶A合酶基因erg13以及角鲨烯合成酶erg9的内源启动子为强启动子Ptef1。
所述酵母中的角鲨烯环氧化酶基因(erg1)的拷贝数为3个以上。
所述酵母中，敲除了旁路羊毛甾醇途经的关键酶羊毛甾醇合成酶基因erg7。
所述酿酒酵母中还包含多拷贝质粒pESC-G418-Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1。
进一步地，所述酵母为酿酒酵母、巴斯德毕赤酵母、发酵毕赤酵母、异常汉逊酵母、解脂耶氏酵母、南极假丝酵母、产朊假丝酵母、热带假丝酵母、粟酒裂殖酵母、黏红酵母。
一个实施例中，所述工程酵母为酿酒酵母，其基因组中，1622b位点插入所述葫芦二烯醇合成酶基因表达框Ptef1-cds-Tcyc1；911b位点插入所述环氧水解酶基因表达框Ptef1-eph3-Tcyc1；106a位点插入所述细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1；308a位点插入所述糖基转移酶编码基因表达框Tadh1-ugt74ac1-Pgal1/10-ugtms1-Tcyc1；gal80位点插入所述截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因表达框Ptef1-tHMG1-Thmg1。
本发明还提供了从头合成罗汉果甜苷工程酵母的应用，具体涉及其在生物发酵生产罗汉果甜苷中的应用。
本发明中，所述工程酵母发酵生产罗汉果甜苷的方法为：取所述工程酵母菌株于液态YPD培养基，30℃，220r/min条件下摇床培养18h，得发酵种子液；将所述发酵种子液以3％的接种率转入发酵培养基，于30℃，220r/min条件下培养96h。
本发明的有益效果：
1.本发明提供的酿酒酵母可以实现以葡萄糖为底物发酵生产罗汉果甜苷，产物可分泌至胞外，为下游的分离纯化步骤奠定了良好的基础。酿酒酵母具有内源的MVA途径，能够合成罗汉果甜苷的重要前体物质角鲨烯，随后角鲨烯依次在外源角鲨烯环氧化酶、葫芦二烯醇合成酶、环氧水解酶、细胞色素P450酶(及其伴生蛋白细胞色素P450还原酶)、糖基转移酶的催化作用下合成罗汉果甜苷。
本发明提供的最终的酿酒酵母菌株Mogrol 09的罗汉果甜苷V的产量达5.6mg/L，是初步能够合成罗汉果甜苷V的酿酒酵母菌株Mogrol 04的467倍。最终的菌株Mogrol 09的罗汉果甜苷IIE、罗汉果甜苷IIIx、罗汉果甜苷IVA、赛门苷等其他罗汉果甜苷产量同样显著高于初步能够合成罗汉果甜苷V的菌株Mogrol 04。
2.本发明提供的酵母构建方法简单，便于使用，具有良好的前景。
本发明采用CRISPR Cas9的基因编辑技术将合成通路关键酶葫芦二烯醇合成酶CDS、环氧水解酶EPH、细胞色素P450酶、细胞色素P450酶还原酶、糖 基转移酶UGT74AC1、UGTMS1即罗汉果甜苷的合成代谢途径整合至酵母，使得酵母实现罗汉果甜苷的以葡萄糖为底物发酵从头合成。
4.本发明提供的酵母以启动子Ptef1、Pgal1/10控制罗汉果甜苷合成通路基因的表达，整合截短的3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1，并增加角鲨烯环氧化酶基因erg1的拷贝数，敲除旁路羊毛甾醇途经的关键酶羊毛甾醇合成酶基因erg7，避免旁路途径罗汉果甜苷的前体物质的竞争并转化多拷贝质粒pESC-G418-Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1，实现cyp87d18的多拷贝表达，使得酵母能够高效地以葡萄糖为底物发酵从头合成罗汉果甜苷。
附图说明
图1为酿酒酵母菌株Mogrol 09和酿酒酵母菌株Mogrol 04的罗汉果甜苷V的产量对比。
图2为酿酒酵母菌株Mogrol 09和酿酒酵母菌株Mogrol 04的其他罗汉果甜苷的产量。
具体实施方式
下面将更详细地描述本发明的具体实施例。提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
本发明实施例的样品处理方法：
收集发酵液，8000r/min进行离心5min，取1mL上清液向其中加入色谱级甲酸，使甲酸终浓度达3％(v/v)。于4℃静置4-8h去除蛋白，8000r/min进行离心5min，取0.5mL上清液，过0.22μm水系滤膜后加入液相检测瓶，待进行产量的检测。
本发明实施例的检测方法：
高效液相-质谱联用仪(LC-MS)检测法：Waters(MICROMASS QUATTRO MICRO)，BEH-C18柱子，流动相乙腈和0.1％甲酸水溶液，柱温45℃，进样量1μL。
实施例1罗汉果甜苷生产菌株的构建
根据NCBI上公布的或来源于参考文献的罗汉果甜苷合成关键酶氨基酸序列，包含葫芦二烯醇合成酶CDS(NCBI ID：AUC64917.1)，氨基酸序列如SEQ ID NO.1所示；环氧水解酶EPH，氨基酸序列如SEQ ID NO.2所示；细胞色素P450酶CYP87D18(NCBI ID：K7NBR2.1)，氨基酸序列如SEQ ID NO.3所示；细胞色素P450酶还原酶AtCPR2(NCBI ID：UTD45115.1)，氨基酸序列如AA SEQ ID NO.4所示；糖基转移酶UGT74AC1，氨基酸序列如SEQ ID NO.5；糖基转移酶UGTMS1，氨基酸序列如SEQ ID NO.6。
截短的3-羟基-3-甲基戊二酰辅酶A还原酶tHMG1是将酿酒酵母内源酶HMG1的N-端2-530位氨基酸组成的跨膜结构域去除，其氨基酸序列如SEQ ID NO.7所示。
按照酿酒酵母的密码子偏好性进行密码子优化及全基因合成。
根据酿酒酵母基因组1622b、911b、106a、308a整合位点的基因序列选择CRISPR Cas9的20bp的PAM序列，并在PAM序列的两侧设计基因表达框的上下游同源臂扩增引物，分别以酿酒酵母基因组DNA为模板，通过PCR对上下游同源臂进行扩增。
引物条件如表1所示。PCR条件如表2所示。
表1同源臂扩增所需引物

表2 PCR扩增条件
根据重叠衍生PCR引物设计方法，设计引物扩增罗汉果甜苷合成通路中的关键酶及所涉及的启动子、终止子。以全基因合成的cds、eph3、cyp87d18、atcpr2、ugtac1、ugtms1质粒为模板，扩增获得相应的基因片段。以酿酒酵母基因组DNA为模板，扩增获得启动子Ptef1、Pgal1/10，终止子Tcyc1、Tadh1、Ttdh3。通过重叠延伸PCR构建基因表达框。
引物条件如表3所示。PCR扩增条件如表2所示。
表3片段扩增所需引物


基因表达框包括：葫芦二烯醇合成酶基因表达框Ptef1-cds-Tcyc1、环氧水解酶基因表达框Ptef1-eph3-Tcyc1、细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1和糖基转移酶编码基因表达框Tadh1-ugt74ac1-Pgal1/10-ugtms1-Tcyc1。
启动子Ptef1是酿酒酵母内源的组成型启动子，核苷酸序列如NA SEQ ID NO.8所示。启动子Pgal1/10是由启动子gal1和gal10组成的双向启动子，Pgal1/10的核苷酸序列如NA SEQ ID NO.9所示。所述终止子Tcyc1、Tadh1、Ttdh3是酿酒酵母内源终止子，核苷酸序列分别如NA SEQ ID NO.10、NA SEQ ID NO.11、NA  SEQ ID NO.12所示。
构建CRISPR Cas9的sgRNA质粒，以质粒pML104为模板，用添加20bp PAM序列的引物(如NA SEQ ID NO.13所示)进行环状PCR，经测序获得具有相应PAM序列质粒pML104-PAM。
将构建好的pML104-PAM与基因表达框转化至酿酒酵母Saccharomyces cerevisiae CEN PK2-1C MATa；ura3-52；trp1-289；leu2-3,112；his3Δ1；MAL2-8C；SUC2宿主菌的感受态细胞，挑取菌落PCR正确的转化子进行测序验证，最终获得具有罗汉果甜苷合成通路的重组酿酒酵母菌株Mogrol 04。
实施例2强化关键中间产物的累积
罗汉果甜苷合成属于次级代谢，前体积累较少，为提高前体的合成，解除甲羟戊酸途径中限速酶羟基甲基戊二酰-CoA还原酶的限制，将基因hmg1的N端内质网定位信号肽切除，获得thmg1。构建截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因表达框Ptef1-thmg1-Thmg1，转化至菌株Mogrol 04，于酿酒酵母基因组中的gal80位点插入，即gal80位点增加1拷贝thmg1，获得菌株命名为Mogrol 05。菌株Mogrol 05解除MVA途径的限速步骤，同时所述菌株不受半乳糖的诱导，罗汉果甜苷的合成途径开启只受到葡萄糖浓度的调控。
在此基础上过表达甲羟戊酸途径中的关键酶，将酿酒酵母内源羟甲基戊二酰辅酶A合酶基因erg13以及角鲨烯合成酶erg9的内源启动子替换为强启动子Ptef1，获得菌株Mogrol 06。
实施例3罗汉果甜苷合成通路的强化
为了促进中间体角鲨烯更多地流向罗汉果甜苷，以菌株Mogrol 06为出发菌株，增加角鲨烯环氧化酶基因erg1的拷贝数，具体增加3个拷贝，获得菌株Mogrol07。为避免角鲨烯流向旁路途径从而与罗汉果甜苷合成途径形成前体物质的竞争，以Mogrol 07为出发菌株，敲除旁路羊毛甾醇途经的关键酶羊毛甾醇合成酶基因erg7，获得菌株Mogrol 08。由于细胞色素P450的表达量较低，为提高cyp87d18的表达量，以菌株Mogrol 08为出发菌株，向其中转化多拷贝质粒pESC-G418-Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1，获得菌株Mogrol 09。
实施例4重组酿酒酵母发酵生产罗汉果甜苷
将具有罗汉果甜苷合成通路的酿酒酵母重组菌株划线与SD平板(添加尿嘧啶50mg/L)，30℃下培养直至长出大量菌落。
接种一环单菌落至种子培养基，30℃下220r/min培养18-20h至细胞生长对数前期。
按起始3％的接种量将种子培养液接种至发酵培养基中，30℃220r/min培养96h。每隔12h取一次菌液，测量OD600与葡萄糖剩余量。96h停止培养，将菌液转至50mL离心管，常温8000r/min进行离心5min，取1mL上清液向其中加入色谱级甲酸，使甲酸终浓度达3％(v/v)。于4℃静置4-8h去除蛋白，8000r/min进行离心5min，取0.5mL上清液，过水系滤膜后加入液相检测瓶，进行产量的检测。
种子培养基为液态YPD培养基。培养基成分为：葡萄糖20g/L，蛋白胨20g/L，酵母粉10g/L。
发酵培养基为调整葡萄糖浓度的液态YPD培养基。培养基成分为：葡萄糖30g/L，蛋白胨20g/L，酵母粉10g/L。
罗汉果甜苷V及其他罗汉果甜苷产量分别如图1、图2所示。重组菌株Mogrol09的罗汉果甜苷V产量为5.6mg/L，是最初构建的能够合成罗汉果甜苷V的菌株Mogrol 04的产量的467倍。酿酒酵母Mogrol 09的其他罗汉果甜苷产量同样比Mogrol 04显著提高，罗汉果甜苷IIE、罗汉果甜苷IIIx、罗汉果甜苷IVA、赛门苷I的产量分别可达150.2μg/L、212.3μg/L、183.5μg/L、110.4μg/L。

Claims (18)

  1. 一种从头合成罗汉果甜苷的工程酵母的构建方法，其特征是，将构建的葫芦二烯醇合成酶基因表达框Ptef1-cds-Tcyc1、环氧水解酶基因表达框Ptef1-eph3-Tcyc1、细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1和糖基转移酶编码基因表达框Tadh1-ugt74ac1-Pgal1/10-ugtms1-Tcyc1，插入酵母基因组中，获得罗汉果甜苷生产工程酵母。
  2. 根据权利要求1所述从头合成罗汉果甜苷的工程酵母的构建方法，其特征是，构建截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因表达框Ptef1-tHMG1-Thmg1，并插入所述罗汉果甜苷生产工程酵母基因组中的gal80位点。
  3. 根据权利要求2所述从头合成罗汉果甜苷的工程酵母的构建方法，其特征是，替换所述所述罗汉果甜苷生产工程酵母中的羟甲基戊二酰辅酶A合酶基因erg13以及角鲨烯合成酶erg9的内源启动子为强启动子Ptef1。
  4. 根据权利要求3所述从头合成罗汉果甜苷的工程酵母的构建方法，其特征是，增加所述罗汉果甜苷生产工程酵母的角鲨烯环氧化酶基因erg1的拷贝数，所述拷贝数为3个以上。
  5. 根据权利要求4所述从头合成罗汉果甜苷的工程酵母的构建方法，其特征是，敲除所述罗汉果甜苷生产工程酵母的旁路羊毛甾醇途经的关键酶羊毛甾醇合成酶基因erg7。
  6. 根据权利要求5所述从头合成罗汉果甜苷的工程酵母的构建方法，其特征是，向所述罗汉果甜苷生产工程酵母中转化多拷贝质粒pESC-G418-Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1。
  7. 根据权利要求1-6任一项所述从头合成罗汉果甜苷的工程酵母的构建方法，其特征是，其特征是，所述工程酵母包括但不限于酿酒酵母、巴斯德毕赤酵母、发酵毕赤酵母、异常汉逊酵母、解脂耶氏酵母、南极假丝酵母、产朊假丝酵母、热带假丝酵母、粟酒裂殖酵母、黏红酵母。
  8. 根据权利要求2-6任一项所述从头合成罗汉果甜苷的工程酵母的构建方法，其特征是，所述工程酵母为酿酒酵母，所述葫芦二烯醇合成酶基因Ptef1-cds-Tcyc1的表达框插入酵母基因组1622b位点；所述环氧水解酶基因表达框Ptef1-eph3-Tcyc1插入酵母基因组 911b位点；所述细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1插入酵母基因组的106a位点；所述糖基转移酶编码基因表达框Tadh1-ugt74ac1-Pgal1/10-ugtms1-Tcyc1插入酵母基因组的308a位点；所述截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因表达框Ptef1-tHMG1-Thmg1插入酵母基因组的gal80位点。
  9. 从头合成罗汉果甜苷的工程酵母，其特征是，酵母基因组中含有葫芦二烯醇合成酶基因表达框Ptef1-cds-Tcyc1、环氧水解酶基因表达框Ptef1-eph3-Tcyc1、细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1和糖基转移酶编码基因表达框Tadh1-ugt74ac1-Pgal1/10-ugtms1-Tcyc1。
  10. 根据权利要求9所述的从头合成罗汉果甜苷的工程酵母，其特征是，所述酵母基因组中含有截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因表达框Ptef1-tHMG1-Thmg1。
  11. 根据权利要求10所述的从头合成罗汉果甜苷的工程酵母，其特征是，所述酵母中的羟甲基戊二酰辅酶A合酶基因erg13以及角鲨烯合成酶erg9的内源启动子为强启动子Ptef1。
  12. 根据权利要求11所述的从头合成罗汉果甜苷的工程酵母，其特征是，所述酵母中的角鲨烯环氧化酶基因(erg1)的拷贝数为3个以上。
  13. 根据权利要求12所述的从头合成罗汉果甜苷的工程酵母，其特征是，所述酵母中，敲除了旁路羊毛甾醇途经的关键酶羊毛甾醇合成酶基因erg7。
  14. 根据权利要求13所述的从头合成罗汉果甜苷的工程酵母，其特征是，所述酿酒酵母中还包含多拷贝质粒pESC-G418-Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1。
  15. 根据权利要求9-14任一项权利要求所述的从头合成罗汉果甜苷的工程酵母，其特征是，所述工程酵母包括但不限于酿酒酵母、巴斯德毕赤酵母、发酵毕赤酵母、异常汉逊酵母、解脂耶氏酵母、南极假丝酵母、产朊假丝酵母、热带假丝酵母、粟酒裂殖酵母、黏红酵母。
  16. 根据权利要求10-14任一项权利要求所述的从头合成罗汉果甜苷的工程酵母，其特征是，所述工程酵母为酿酒酵母，其基因组中，1622b位点插入所述葫芦二烯醇合成酶基因表达框Ptef1-cds-Tcyc1；911b位点插入所述环氧水解酶基因表达框Ptef1-eph3-Tcyc1； 106a位点插入所述细胞色素P450酶编码基因CYP与细胞色素P450酶还原酶基因表达框Ttdh3-cyp87d18-Pgal1/10-atcpr2-Tcyc1；308a位点插入所述糖基转移酶编码基因表达框Tadh1-ugt74ac1-Pgal1/10-ugtms1-Tcyc1；gal80位点插入所述截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因表达框Ptef1-tHMG1-Thmg1。
  17. 权利要求9-16任一项权利要求所述的从头合成罗汉果甜苷的工程酵母在生物发酵生产罗汉果甜苷中的应用。
  18. 权利要求14所述的从头合成罗汉果甜苷的工程酵母发酵生产罗汉果甜苷的方法，其特征是，取所述工程酵母菌株于液态YPD培养基，30℃，220r/min条件下摇床培养18h，得发酵种子液；将所述发酵种子液以3％的接种率转入发酵培养基，于30℃，220r/min条件下培养96h。
PCT/CN2024/073548 2024-01-11 2024-01-23 一种从头合成罗汉果甜苷的工程酵母的构建方法 Pending WO2025148100A1 (zh)

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