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Recombinant engineering bacterium for producing 2 '-fucosyllactose as well as construction method and application of recombinant engineering bacterium

A technology of fucosyllactose and recombinant engineering bacteria, applied in the field of genetic engineering, can solve the problems of cumbersome synthetic steps of chemical synthesis method, high cost of enzyme-catalyzed synthesis method, poor enzyme stability, etc. Efficient expression effect

Active Publication Date: 2022-06-17
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The extraction method needs human milk donation, but there are few human milk donors, so the extraction is difficult. The synthesis steps of the chemical synthesis method are cumbersome and require the use of toxic reagents. In contrast, the biosynthe

Method used

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  • Recombinant engineering bacterium for producing 2 '-fucosyllactose as well as construction method and application of recombinant engineering bacterium
  • Recombinant engineering bacterium for producing 2 '-fucosyllactose as well as construction method and application of recombinant engineering bacterium
  • Recombinant engineering bacterium for producing 2 '-fucosyllactose as well as construction method and application of recombinant engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0044]实施例1 Lac4缺陷型的马克斯克鲁维酵母的筛选

[0045]本实施例中，Lac4缺陷型的马克斯克鲁维酵母的构建，以野生型马克斯克鲁维（K.marxianus） KM-0（为现有技术中已公开的菌株，可从中国微生物菌种保藏管理委员会普通微生物中心购买得到，保藏编号为CGMCC NO.21978）为出发菌株，用HPT基因完全取代野生型马克斯克鲁维（K. marxianus）KM-0基因组中所有的Lca4基因，以此实现Lca4基因的敲除。

[0046]根据Lac4的启动子、终止子及HPT基因来源PMT015质粒序列信息，设计扩增引物，如表1所示（F-HPTcycle1, R-HPTcycle1, F-HPTcycle2, R-HPTcycle2）；然后分别以PMT015质粒和野生型马克斯克鲁维（K. marxianus）KM-0基因组为模板，PCR扩增得到HPT基因片段、Lac4的部分启动子和Lac4的部分终止子，扩增条件为：预变性95℃ 5 min；变性95℃ 20 s，30个循环；退火55℃ 20 s，30个循环；延伸72℃ 1 kbp / 60 s，30个循环；最终延伸72℃ 10 min。所述HPT的核苷酸序列如SEQ ID NO.1所示，其表达的潮霉素B的氨基酸序列如SEQ ID NO.2所示；所述Lac4的部分启动子的核苷酸序列如SEQ ID NO.3所示；所述Lac4的部分终止子的核苷酸序列如SEQ ID NO.4所示。

[0047]常规方法构建得到重组基因片段：Lac4的部分启动子-HPT-Lac4的部分终止子，称之为HPTT。将该重组基因片段转入野生型马克斯克鲁维（K. marxianus）KM-0的感受态细胞中，转化条件为：电压1.5 KV，脉冲时间5 ms，电阻200 Ω，电容25 μF。在含有潮霉素（100 μg / ml）的YPD培养基上培养，筛选得到阳性的转化子。本实施例筛选得到24个转化子。

[0048]电泳检测：用HPT取代Lca4后，基因片段长度差别明显，因此可以根据核酸电泳条带的大小判断是否完全取代。设计Lac4缺陷型转化子核酸电泳验证引物，如表1所示（F-Lac4check，R-Lac4check），提取各转化子基因组为模板，进行PCR扩增，扩增条件为：预变性95℃ 5 min；变性95℃ 20 s，30个循环；退火55℃ 20 s...

Example Embodiment

[0057]实施例2 产2′-岩藻糖基乳糖的重组工程菌的设计

[0058]基于PGASO策略，组装5个基因盒（命名及顺序为：SOG418t，SOGmdt，SOWcaGt，SOFucTt，SOCDT2t），构建产2′-岩藻糖基乳糖的重组工程菌。为使各基因在宿主细胞内充分表达，每个基因都匹配了一对强启动子、终止子。

[0059]在第1个基因盒中，G418抗性筛选基因通过同源重组，与从野生型马克斯克鲁维K.marxianus KM-0基因组体外克隆的ADH2启动子和TPI1终止子首尾相接。

[0060]在第2个基因盒中，Gmd基因通过同源重组，与PGK启动子和INU1终止子首尾相接。

[0061]在第3个基因盒中，WcaG基因通过同源重组，与FKP1的启动子和终止子首尾连接。

[0062]在第4个基因盒中，FucT基因通过同源重组，与TEF启动子和TPS1终止子首尾连接。

[0063]在第5个基因盒中，CDT2基因通过同源重组，与GAPDH的启动子和终止子首尾连接。

[0064]这5个基因盒的插入的多克隆位点两端分别命名为rDNA1和rDNA2，基因盒的组装顺序和连接如图3所示，5个基因及启动子、终止子的扩增引物如表2所示，启动子、终止子的具体来源如下：乙醇脱氢酶基因ADH2（GenBank: AF225206.1）启动子，磷酸丙糖异构酶基因TPI1（GenBank: AJ577476.2）终止子；磷酸甘油酸激酶基因PGK（GenBank: XM_022822209.1）启动子，菊粉酶基因INU1（GenBank: XM_022822162.1）终止子；6-磷酸果糖激酶基因FKP1（GenBank: XM_022820285.1）启动子和终止子；翻译延伸因子基因TEF（GenBank: XP_022678358.1）启动子、海藻糖-6-磷酸合成酶基因TPS1（GenBank: XP_022678366.1）终止子；甘油醛-3-磷酸脱氢酶基因GAPDH（GenBank: XP_022678116.1）启动子和终止子。

[0065]表2

[0066]引物名称碱基序列（5’ to 3’）F-G418ATGGGTAAGGAAAAGACTR-G418TTAATTTAGAAAAACTCATCGAGCF-GmdATGTCTAAGGTTGCTTTGR-GmdTTAGGATTCCAATGCGATAGF...

Example Embodiment

[0072]实施例3 产2′-岩藻糖基乳糖的重组工程菌的验证

[0073]设计验证引物，如表4所示，以转化子KMdeLac24-FL15为模板，利用表4所示的引物进行扩增，扩增产物进行电泳检测，结果如图5所示，结果表明，核酸电泳扩增出5条与5个基因盒实际大小一致的单一条带，证明通过PGASO策略已成功按照预定顺序转入了SOG418T等5个基因盒。

[0074]表4

[0075]引物名称碱基序列（5’ to 3’）F-rDNA1GCGGCCGCCTGGTTGATCCTGCCAGTR-checkSOG418TAGCATGTAGCAAGCGAGGGCCATGF-checkSOGmdGCAGTTTCATTTGATGCTCGATGAGR-checkSOGmdCTAAACACTAAACTATGCACCAGCTCF-checkSOWcaGCATGTGCCGGTCGCTTTCCCTTTCR-checkSOWcaGCACTGGGGCAGAAGCCAGGTCTGGGF-checkSOFucTGATGAGATATACTGACACACAAAGGCR-checkSOFucTGTGGATGAGGGCCTCTCGAGTGF-checkSOCDT2GAATGCATATCGAAATGTCTGCACR-rDNA2GCGGCCGCAATGATCCTTCCGCAG

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Abstract

The invention discloses a recombinant engineering bacterium for producing 2 '-fucosyllactose as well as a construction method and application of the recombinant engineering bacterium, and belongs to the technical field of genetic engineering. The invention discloses a recombinant engineering bacterium for producing 2 '-fucosyllactose, the strain name of the recombinant engineering bacterium is KMdeLac24-FL15, the recombinant engineering bacterium is preserved in the China General Microbiological Culture Collection Center on March 21, 2022, the preservation number is CGMCC NO.24549, and the recombinant engineering bacterium is classified and named as Kluyveromyces marxianus. The invention further discloses a preparation method of the recombinant engineering bacterium for producing 2'-fucosyllactose. The strain can efficiently express the 2 '-fucosyllactose and is applied to preparation of the 2'-fucosyllactose. The invention also discloses a recombinant engineering bacterium for producing the 2 '-fucosyllactose and a construction method of the recombinant engineering bacterium, and also discloses application of the recombinant engineering bacterium in preparation of the 2'-fucosyllactose.

Description

Technical field [0001] The invention relates to a recombinant engineering bacterium that produces 2'-fucosyllactose and its construction method and application, and belongs to the technical field of genetic engineering. Background technique [0002] 2′-Fucosyllactose (2′-FL) is a non-reducing trisaccharide composed of lactose and L-fucose. It is a kind of human milk oligosaccharide. The molecular formula is C 18 H 32 O 15 , its reducing end is connected to a lactose, and the fucose at the non-reducing end is connected to the galactose in the lactose structure through α-1,2-bonds. It has many functions such as anti-infection, regulating intestinal flora and immune response, and promoting brain development. functional activity. 2′-Fucosyllactose can not only be used as a food ingredient added to infant formula, but can also be used as dietary supplements and medical foods, which has broad market potential. Although 2′-fucosyllactose has been industrially produced and its p...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/65C12N15/60C12N15/53C12N15/54C12N15/31C12N15/56C12P19/18C12P19/00C12R1/645
CPCC12N15/52C12N9/88C12N9/0006C12N9/1051C07K14/37C12N9/2471C12N15/81C12N15/65C12P19/18C12P19/00C12Y402/01047C12Y302/01023C12Y101/01271
Inventor 毛相朝姜宏周文婷全永奕
Owner OCEAN UNIV OF CHINA
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