CN111235048B - 一株木糖利用酵母及其用途 - Google Patents
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Abstract
本发明公开了一株木糖利用酵母及其用途。该酵母为整合型工程菌，所述的整合型工程菌含有经过突变的木糖还原酶基因XYL1m和木糖醇脱氢酶基因XLY2，木糖还原酶的氨基酸序列含有1个点突变，所述突变为第271位氨基酸由K变为N，还含有磷酸转酮酶基因xPK和磷酸转乙酰酶基因PTA，并且Pho13基因被敲除，还含有经过突变的己糖转运蛋白Gal2，所述突变为Gal2第376位氨基酸由N变为F，并且Gal2基因上游的启动子替换为SSA1，木酮糖激酶XK基因启动子替换为强启动子HSP82。本发明构建一套利用木糖合成类胡萝卜素的酿酒酵母平台，为更加经济地利用生物发酵法制备类胡萝卜素提供重要支撑。
Description
技术领域
本发明属于基因工程和生物技术领域，涉及一株木糖利用酵母及其用途，具体涉及一株可利用木糖酿酒酵母及其在类胡萝卜素合成中的应用。
背景技术
木质纤维素生物质资源是地球上最丰富、最廉价的可再生资源；我国是农业大国，农业废弃物等生物质资源异常丰富。目前，这些资源大部分还没有得到充分利用，而且常常还会造成不同程度的污染；随着化石燃料等不可再生资源的日益枯竭及其产生的环境污染、气候变化等问题，可再生资源的开发与利用逐渐受到人们的重视，利用生物质资源生产生物基化学品和生物燃料是当前研究的热点。木质纤维素包括纤维素(34-50％)、半纤维素(19-35％)和木质素(11-30％)，这些组分会随着植物生长条件和植物种类的不同而有所变化。纤维素是木质纤维素的主要成分，其由葡萄糖单体通过β-1,4糖苷键形成的线性同聚多糖；半纤维素是木质纤维素的第二大组成成分，其成分较为复杂，单体分子包括多种糖类如木糖、阿拉伯糖、葡萄糖、半乳糖和甘露糖等；木质素是木质纤维素的第三大组成成分，其由复杂的聚合物组成，构成细胞的细胞壁，抵御外在微生物的侵袭。如何利用这三大组分生产生物基产品有过大量研究，其中利用纤维素生产燃料乙醇的研究已经很成熟并有工业化生产实例，但目前受限于成本因素，所售价格往往低于生产成本，需政府补贴才能维持生存，企业开发热情不高。木质素由于成分较为复杂，研究较为缓慢，并没有取得重大进展。而半纤维素结构相对简单，有关半纤维素的研究正引起人们的广泛关注。半纤维素的主要成分是木聚糖，其经过简单的酸解就能获得以木糖为主要成分的糖分；利用木糖生产高附加值化学品是一条经济可行的途径；对降低生产成本，增加产品竞争力都具有很重要意义。
所以，构建高效细胞工厂利用木糖进行目标产品的合成是一条切实可行的路径。类胡萝卜素是一类具有超强抗氧化性能的物质，在饲料添加剂、医药、食品、化妆品等领域有着重要的应用。目前，通过生物发酵法生产类胡萝卜素主要是以葡萄糖为碳源，生产成本较高，无法大规模生产。通过酿酒酵母细胞，利用木糖合成类胡萝卜素的研究还未见报道。
发明内容
本发明的目的是提供一株可利用木糖酿酒酵母及其在类胡萝卜素合成中的应用。通过构建酿酒酵母利用木糖合成类胡萝卜素平台，为进一步利用可再生生物质资源合成类胡萝卜素打下基础。
一株可利用木糖合成类胡萝卜素的整合型工程菌，包括酿酒酵母和搭建的木糖到番茄红素的合成途径，所述的整合型工程菌含有经过突变的来源于Scheffersomycesstipitis的木糖还原酶基因XYL1m和木糖醇脱氢酶基因XLY2。该工程菌可以转化木糖合成类胡萝卜素。
优选，所述的整合型工程菌中，所述的木糖还原酶的氨基酸序列含有1个点突变，所述突变为第271位氨基酸由K变为N，辅酶专一性变为NADH。
优选，所述的整合型工程菌含有来源于Leuconostoc mesenteroides的磷酸转酮酶基因xPK和来源于Clostridium butyricum的磷酸转乙酰酶基因PTA，此途径显著缩短了木糖到乙酰辅酶A的途径。
优选，所述的整合型工程菌的Pho13基因被敲除。
优选，所述的整合型工程菌含有经过突变的己糖转运蛋白Gal2，所述突变为己糖转运蛋白氨基酸序列第376位氨基酸由N变为F，其转运木糖活性不受葡萄糖抑制。
优选，所述的整合型工程菌中，所述的己糖转运蛋白基因Gal2上游的启动子替换为SSA1。
优选，所述的整合型工程菌中，所述的酿酒酵母基因组中木酮糖激酶XK基因启动子替换为强启动子HSP82。
本发明还提供上述的整合型工程菌在合成类胡萝卜素中的应用。
与现有技术相比，本发明具有以下有益效果：
(1)本发明首先搭建了木糖利用途径，使工程菌可以利用生物质资源中的木糖进行高附加值化学品的生物合成，此工艺可以降低生产成本。
(2)本发明引入磷酸戊糖酮解(PK)途径，使木糖尽可能多的流入类胡萝卜素合成途径，减少木糖的浪费。
(3)本发明基因工程菌中己糖转运蛋白Gal2经过突变，消除葡萄糖的抑制作用，并经过启动子替换进行过表达，使重组菌可以利用木糖-葡萄糖混合糖，进一步降低生产成本。
附图说明
图1为构建木糖代谢途径合成番茄红素示意图。
图2为不同改造菌株在添加木糖和葡萄糖混合物的YPD培养基中进行摇瓶发酵的菌体生长(圆形)及木糖消耗曲线(正形)。A：菌株BL03，Cit1-tHMG1，ΔAld6，B：菌株SC101，C：菌株SC102，D：菌株SC103，E：菌株SC104，和F：菌株SC105。
图3为不同改造菌株在添加木糖和葡萄糖混合物(斜线图)，或添加葡萄糖(横线图)的YPD培养基中进行摇瓶发酵的番茄红素产量及得率。A：各菌株番茄红素产量(mg/L)；B：各菌株番茄红素得率(mg/g DCW)；其中，ΔAld6为菌株BL03，Cit1-tHMG1，ΔAld6。
图4为添加不同糖比例下SC105菌株进行摇瓶发酵的番茄红素产量及得率。
具体实施方式
下面结合具体实施例对本发明做进一步详细的说明，但本发明并不限于以下实施例。
本发明是在申请号为CN201910399946.2、名为“一种重组酵母菌株及其在类胡萝卜素合成中的应用”发明专利申请的基础上做的进一步研究。
上述发明专利申请中公开了一种可以合成番茄红素的酿酒酵母。
下列实施例中，质粒pHCas9-gRNA,菌株BL03，Cit1-tHMG1，ΔAld6均来自申请号为201910399946.2、名为“一种重组酵母菌株及其在类胡萝卜素合成中的应用”发明专利申请文献中。
本发明构建木糖代谢途径合成番茄红素示意图见图1所示。
下属实施例中标准番茄红素的测定方法的参考文献为：Xie W,Ye L,Lv X,Xu H,Yu H:Sequential control of biosynthetic pathways for balanced utilization ofmetabolic intermediates in Saccharomyces cerevisiae.Metab Eng 2015,28:8-18。
实施例1酿酒酵母中木糖利用代谢途径构建
由于酿酒酵母本身不能代谢木糖，为了能够利用木糖合成类胡萝卜素，本实例引入来源于Scheffersomyces stipitis的木糖还原酶基因XYL1和木糖醇脱氢酶基因XLY2。以质粒pHCas9-gRNA为模板(pHCas9-gRNA含有引导序列N20)，利用引物gRNA-XK-F/gRNA-R扩增功能模块，构建基因组整合质粒pHCas9-XK。以酿酒酵母基因组为模板，用引物EFT1-F-2、EFT1-F/EFT1-R，PGK-F/PGK-R，HSP82-F/HSP82-R分别扩增EFT1、PGK1、HSP82启动子；以Scheffersomyces stipitis基因组为模板，用引物XYL1-F/XYL1-R、XYL1-R-2，XYL2-F/XYL2-R、XYL2-R-2分别扩增XYL1(Gene ID:4839234)，XYL2(Gene ID:4852013)基因。利用重叠PCR，搭建EFT1-XYL1-PGK1-XYL2-HSP82模块，与pHCas9-XK一起导入菌株BL03，Cit1-tHMG1，ΔAld6，并用鉴定引物XK-check-F/XK-check-R进行鉴定，获得重组菌SC101。同时以pACYC-Duet(购自Novagen)为模板，用引物pACYC-XK-F/pACYC-XK-R扩增质粒骨架，与EFT1-XYL1-PGK1-XYL2-HSP82模块构建重组质粒pACYC-XK(pACYC-EFT1-XYL1-PGK1-XYL2-HSP82-XK)，方便后续扩增。以pACYC-XK质粒为模板，用引物EFT1-XYL1-F/EFT1-XYL1-R，PGK-XYL2-F/PGK-XYL2-R扩增模块，对XYL1进行突变(木糖还原酶的氨基酸序列第271位氨基酸由K突变为N，辅酶专一性变为NADH)，用突变后的模块与pHCas9-XK一起导入菌株BL03，Cit1-tHMG1，ΔAld6，并用鉴定引物XK-check-F/XK-check-R进行鉴定，获得重组菌SC102。
将获得的重组菌SC101和SC102进行摇瓶发酵制备番茄红素，具体的摇瓶发酵方法如下：250mL中装载50mL改良YPD培养基(10g/L酵母浸粉，20g/L蛋白胨，10g/L KH2PO4，2.5g/L MgSO4，3.5g/L K2SO4，0.25g/L Na2SO4，溶剂为水；配制：将各成分溶于水，搅拌溶解，灭菌，即得)，往YPD培养基中添加30g/L木糖和10g/L葡萄糖混合物或添加40g/L葡萄糖，得到摇瓶培养基。从新鲜平板上挑取各菌株单菌落，接种于含有5mL YPD培养基的试管中，30℃培养16小时后，接种于上述摇瓶培养基中，30℃培养96小时或者120小时，定点取样测定OD600，木糖含量，菌株SC101和SC102的生长及木糖消耗曲线见图2B和图2C。发酵结束后按标准的番茄红素测定方法分别测定菌株SC101和SC102在添加木糖和葡萄糖混合物，或添加葡萄糖的YPD培养基中进行摇瓶发酵的番茄红素产量及得率，结果见图3。从图2和图3可以看出，SC101和SC102都可以利用木糖合成番茄红素。
引物如下，加粗黑体为N20识别位点，斜体为合成终止子，大写字母为突变位点，下划线代表同源臂。
PCR反应体系：PCR反应体系为50μL，其中PrimerSTAR Max DNA Polymerase 25μL，上下游引物(10μM)各1.5μL，模板(50ng/ml)1μL，ddH2O 21μL；
PCR反应程序为：95℃预变性2min；98℃变性10s，Tm退火15s，72℃延伸5s/kb，30个循环；72℃延伸5min，4℃保温。
实施例2磷酸戊糖酮解(PK)途径构建
在上述实施例1中，在酿酒酵母中引入木糖代谢途径后，虽然工程菌已经可以利用木糖，但目标产物含量还比较低。为了提高产物产量，本实施例中引入磷酸戊糖酮解(PK)途径。以质粒pHCas9-gRNA为模板(pHCas9-gRNA含有引导序列N20)，利用引物gRNA-720a-F/gRNA-R扩增功能模块，构建基因组整合质粒pHCas9-720a。以Leuconostoc mesenteroides基因组为模板，用引物xpk-F/xpk-R、xpk-R-2扩增xPK基因(GenBank:TJY30451.1)；以Clostridium butyricum基因组为模板，用引物pta-F/pta-R、pta-R-2、720-R扩增PTA基因(GenBank:EEP53689.1)。以酿酒酵母基因组为模板，用引物720-F、HSP104-F/HSP104-R，SSA1-F0220/SSA1-R0220分别扩增HSP104、SSA1启动子。利用重叠PCR，搭建HSP104-xPK-SSA1-PTA模块，与pHCas9-720a一起导入实施例1构建的菌株SC102，并用鉴定引物720-check-F/720-check-R进行鉴定，获得重组菌SC103。将获得的重组菌SC103进行摇瓶发酵制备番茄红素(培养方法同上)，其生长及木糖消耗曲线见图2D。发酵结束后按标准的番茄红素测定方法分别测定菌株SC103在添加木糖和葡萄糖混合物，或添加葡萄糖的YPD培养基中进行摇瓶发酵的番茄红素产量及得率，结果见图3。从图3可以看出，导入PK途径后，工程菌番茄红素得率有一定提升。
引物如下，斜体为合成终止子，加粗黑体为N20识别位点,下划线代表同源臂。
PCR反应体系：PCR反应体系为50μL，其中PrimerSTAR Max DNA Polymerase 25μL，上下游引物(10μM)各1.5μL，模板(50ng/ml)1μL，ddH2O 21μL；
PCR反应程序为：95℃预变性2min；98℃变性10s，Tm退火15s，72℃延伸5s/kb，30个循环；72℃延伸5min，4℃保温。
实施例3Pho13基因敲除
经过上述实施例2后，工程菌的木糖利用速率还是相对较慢，为了提高木糖利用效率，本实施例对Pho13基因(Gene ID:851362)进行敲除。以质粒pHCas9-gRNA为模板，利用引物gRNA-Pho13-F/gRNA-R扩增功能模块，构建基因组整合质粒pHCas9-Pho13。以酿酒酵母基因组为模板，用引物Pho13-UP-F/Pho13-UP-R，Pho13-DOWN-F/Pho13-DOWN-R分别扩增Pho13基因上下游同源臂，利用重叠PCR构建Pho13基因敲除模块，与pHCas9-Pho13一起导入实施例2构建的菌株SC103，利用鉴定引物Pho13-check-F/Pho13-check-R进行鉴定，获得工程菌SC104。将获得的重组菌SC104进行摇瓶发酵制备番茄红素(培养方法同上)，其生长及木糖消耗曲线见图2E。发酵结束后按标准的番茄红素测定方法分别测定菌株SC104在添加木糖和葡萄糖混合物，或添加葡萄糖的YPD培养基中进行摇瓶发酵的番茄红素产量及得率，结果见图3。从图3可以看出，Pho13基因的敲除对番茄红素的得率有一定的促进作用。
引物如下，加粗黑体为N20识别位点，下划线代表同源臂。
PCR反应体系：PCR反应体系为50μL，其中PrimerSTAR Max DNA Polymerase 25μL，上下游引物(10μM)各1.5μL，模板(50ng/ml)1μL，ddH2O 21μL；
PCR反应程序为：95℃预变性2min；98℃变性10s，Tm退火15s，72℃延伸5s/kb，30个循环；72℃延伸5min，4℃保温。
实施例4改造酿酒酵母己糖转运蛋白Gal2
为了进一步提高木糖利用效率，本实施例对己糖转运蛋白Gal2进行突变并对其启动子进行替换。以质粒pHCas9-gRNA为模板，利用引物gRNA-Gal2-P1/gRNA-R扩增功能模块，构建基因组整合质粒pHCas9-Gal2。以酿酒酵母基因组为模板，用引物SSA1-F0523-2、SSA1-F0523/SSA1-R，Gal2-F/Gal2-R、Gal2-R-2分别扩增SSA1启动子和Gal2突变基因(Gene ID:850770)，实现对Gal2进行突变(己糖转运蛋白Gal2氨基酸序列第376位氨基酸由N变为F，其转运木糖活性不受葡萄糖抑制)。利用重叠PCR构建SSA1-Gal2m模块，与pHCas9-Gal2一起导入实施例3构建的SC104，利用鉴定引物Gal2-check-F/Gal2-check-R进行鉴定，获得工程菌SC105。将获得的重组菌SC105进行摇瓶发酵制备番茄红素(培养方法同上)，其生长及木糖消耗曲线见图2F。发酵结束后按标准的番茄红素测定方法分别测定菌株SC105在添加木糖和葡萄糖混合物，或添加葡萄糖的YPD培养基中进行摇瓶发酵的番茄红素产量及得率，结果见图3。从图2，3可以看出，Gal2的突变可以显著提高木糖的利用及番茄红素的产量。
通过调整摇瓶发酵过程中往YPD培养基添加的木糖和葡萄糖混合物比例(仅含木糖、木糖：葡萄糖＝2:1、木糖：葡萄糖＝1:1)，评价不同比例的木糖和葡萄糖对菌株SC105发酵生产番茄红素产量及得率的影响，结果见图4。图4表明，不同比例的木糖和葡萄糖对产物的产量并没有显著影响，这样就更利于木糖的利用，进一步降低生产成本。
引物如下，加粗黑体为N20识别位点，大写字母为突变位点，下划线代表同源臂。
PCR反应体系：PCR反应体系为50μL，其中PrimerSTAR Max DNA Polymerase 25μL，上下游引物(10μM)各1.5μL，模板(50ng/ml)1μL，ddH2O 21μL；
PCR反应程序为：95℃预变性2min；98℃变性10s，Tm退火15s，72℃延伸5s/kb，30个循环；72℃延伸5min，4℃保温。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一株可利用木糖合成番茄红素的整合型工程菌，其特征在于，所述的整合型工程菌为在酿酒酵母工程菌株BL03，Cit1- tHMG1，ΔAld6的基础上，含有经过突变的来源于Scheffersomyces stipitis的木糖还原酶基因XYL1m和木糖醇脱氢酶基因XYL2，Gene ID:4852013；
所述的木糖还原酶基因XYL1m，其编码的氨基酸序列是在Gene ID: 4839234编码的氨基酸序列上含有1个突变位点，所述的突变位点为第270位氨基酸由K变为N，辅酶专一性变为NADH；
所述的整合型工程菌含有来源于Leuconostoc mesenteroides的磷酸转酮酶基因xPK，GenBank: TJY30451.1和来源于Clostridium butyricum的磷酸转乙酰酶基因PTA，GenBank: EEP53689.1；
所述的整合型工程菌的Pho13基因被敲除，所述的Pho13基因，Gene ID: 851362；
所述的整合型工程菌含有经过突变的己糖转运蛋白Gal2，所述突变为Gene ID:850770编码的己糖转运蛋白的氨基酸序列第376位氨基酸由N变为F，其转运木糖活性不受葡萄糖抑制；
所述的己糖转运蛋白Gal2的编码基因Gal2上游的启动子替换为SSA1。
2.根据权利要求1所述的整合型工程菌，其特征在于，将木酮糖激酶XK基因启动子替换为强启动子HSP82，所述的XK基因，Gene ID: 853108。
3.权利要求1-2任一所述的整合型工程菌在合成番茄红素中的应用。
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