CN117343888A - 一种高效合成乳糖-n-二岩藻四糖的基因工程菌及生产方法 - Google Patents
一种高效合成乳糖-n-二岩藻四糖的基因工程菌及生产方法 Download PDFInfo
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Classifications
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Abstract
本发明公开了一种高效合成乳糖‑N‑二岩藻四糖的基因工程菌及生产方法,属于基因工程技术领域。本发明利用合成生物学手段,在宿主菌中组合调控了GDP‑L‑岩藻糖合成途径中的manB、manC、gmd和wcaG,并在此基础上异源表达了限速酶α‑1,2‑岩藻糖基转移酶突变体和α‑1,3‑岩藻糖基转移酶突变体。基于质粒拷贝数模块优化通路基因,通过限速酶的表达上调(酶源筛选、截短/延伸突变、定点突变、插入位点模式优化、染色体整合)获得了从头高效生产乳糖‑N‑二岩藻四糖的基因工程菌株,菌株生产成本低廉且产物生产水平较高,具有明显的工业化生产潜力。
Description
技术领域
本发明涉及一种高效合成乳糖-N-二岩藻四糖的基因工程菌及生产方法,属于基因工程技术领域。
背景技术
母乳低聚糖(Human Milk Oligosaccharides,HMOs)是母乳中天然存在且结构多样的非共轭聚糖家族,不易被婴儿消化吸收,可充当有效的抗粘附抗菌剂、益生元和炎症免疫细胞反应级联调节剂来改善新生儿健康。几种简单的HMOs包括2’-岩藻糖基乳糖(2’-FL)、3-岩藻糖基乳糖(3-FL)、乳糖-N-四糖(LNT)和乳糖-N-新四糖(LNnT)等已在工程微生物中得到普遍应用。乳糖-N-二岩藻四糖(LDFT)是岩藻糖基化HMOs的重要成分,其结构是由C2’和C3位置岩藻糖基化的核心乳糖单元组成。目前,LDFT已被美国食品药品管理局(FDA)批准为一般公认安全食品(GRAS),可作为新资源食品配料添加到食品中。相关研究表明:LDFT可有效预防新生儿空肠弯曲菌引起的腹泻并抑制血小板诱导的炎症。作为胃肠道和免疫调节剂,LDFT潜在的治疗用途有待进一步研究。为进一步阐明其作用机制,需制备大量结构均一的该类化合物,然而,从天然产物中分离提取的产量极低,且LDFT的高成本和有限的市场供应(1400美元/10mg,Biosynth Carbosynth)成为生物学研究的阻碍。因此,寻求更高效、环保且经济的生产方式获得LDFT是目前寇待解决的问题。
LDFT的合成方法主要包括三种:分别是化学合成、酶法合成和发酵法合成。LDFT的化学合成需要众多繁琐的保护和脱保护步骤。酶促合成由于供体底物核苷酸糖价格昂贵且产量低,使得合成LDFT的成本较高,不适合工业化生产。利用改造的工程菌生物合成LDFT,可以从廉价碳源(葡萄糖、甘油、乳糖)从头制备,该过程具有绿色、清洁、可持续等典型特征。目前为止,仅有Zhang和Lee等人利用补救合成途径在大肠杆菌中合成LDFT(Zhang等人,2021;Lee等人,2022),该方法以L-岩藻糖为底物,过程涉及ATP和GTP参与,生产成本较高且产量偏低,实现LDFT工业化生产仍存在一些困难。本发明旨在利用合成生物学手段,通过重构LDFT从头合成途径、弱化代谢分支途径、模块优化通路基因、限速酶的表达上调(酶源筛选、截短/延伸突变、定点突变、插入位点模式优化、染色体整合)等策略实现低成本无抗和有抗菌株的构建及LDFT的高效合成。该研究方法将丰富和发展微生物代谢调控的技术研究,为重构微生物代谢网络、提高碳原子经济性提供新方法和案例,并为理性设计和构建新一代微生物细胞工厂提供新思路,在理论和实际应用方面均具有重要价值。
发明内容
[技术问题]
现有技术生产乳糖-N-二岩藻四糖的成本高昂,且产量较低,不足以实现工业化生产,无法提供高效生产乳糖-N-二岩藻四糖的菌株,也不能提供成本低廉且绿色高效生产乳糖-N-二岩藻四糖的方法。
[技术方案]
本发明利用合成生物学技术,以廉价甘油为底物,通过几个连续的生物催化过程从头合成关键中间体GDP-L-岩藻糖,随后在α-1,2-岩藻糖基转移酶(α-1,2-FucT)和α-1,3-岩藻糖基转移酶(α-1,3-FucT)的双酶催化作用下对GDP-L-岩藻糖和乳糖进行两步岩藻糖基化,最终合成高附加值产物LDFT。该方法对LDFT技术瓶颈的解决具有借鉴意义,为LDFT的批量生产提供了有效途径,同时具有较强的理论研究价值和社会经济效益,市场开发前景广阔。本发明为了解决现有生物法合成的乳糖-N-二岩藻四糖产量较低的问题,提供了一种产乳糖-N-二岩藻四糖的基因工程菌及其构建方法。
本发明的第一个目的是提供一种底盘细胞I,以大肠杆菌BZWNDPAL为宿主,敲除了谷氨酰胺-果糖-6-磷酸氨基转移酶基因glmS。
本发明的第二个目的是提供一种底盘细胞II,以底盘细胞I为宿主,敲除了泛醌依赖性丙酮酸脱氢酶基因poxB和磷酸乙酰转移酶-乙酸激酶基因簇pta-ackA,整合表达两个拷贝的α-1,2-岩藻糖基转移酶HpfutC和两个拷贝的α-1,3-岩藻糖基转移酶HpM32。
在一种实施方式中,所述α-1,2-岩藻糖基转移酶HpfutC为(a)或(b):
(a)氨基酸序列如SEQ ID NO.15所示的HpfutC;
(b)在(a)的基础上进行突变的突变体:K102T、R105C、Y251F、K282E、K102T/R105C、Y251F/K282E、R105C/K282E、K102T/R105C/K282E或K102T/R105C/Y251F/K282E。
在一种实施方式中,(b)中,所述突变体的氨基酸序列如SEQ ID NO.16~SEQ IDNO.24所示。
在一种实施方式中,所述α-1,3-岩藻糖基转移酶HpM32为(a)~(c)中的任一:
(a)氨基酸序列如SEQ ID NO.1所示的HpM32;
(b)在(a)的基础上进行截短获得的截短体:截短HpM32的C末端7肽重复序列,分别截短49、35、14和7个氨基酸。
(c)在(a)的基础上进行延伸获得的延伸体:延伸HpM32的C末端7肽重复序列,分别延长7、14、21和28个氨基酸。
在一种实施方式中,(b)中,所述突变体的氨基酸序列分别如SEQ ID NO.7~SEQID NO.10所示。
在一种实施方式中,(c)中,所述突变体的氨基酸序列分别如SEQ ID NO.11~SEQID NO.14所示。
本发明的第三个目的是提供一种产乳糖-N-二岩藻四糖的工程菌,以底盘细胞I或底盘细胞II为宿主,游离表达磷酸甘露糖变位酶基因manB、甘露糖-1-磷酸鸟嘌呤基转移酶基因manC、GDP-甘露糖-6-脱氢酶基因gmd、GDP-岩藻糖合成酶基因wcaG、α-1,2-岩藻糖基转移酶HpfutC和α-1,3-岩藻糖基转移酶HpM32。
在一种实施方式中,所述α-1,2-岩藻糖基转移酶HpfutC为(a)或(b):
(a)氨基酸序列如SEQ ID NO.15所示的HpfutC;
(b)在(a)的基础上进行突变的突变体:K102T、R105C、Y251F、K282E、K102T/R105C、Y251F/K282E、R105C/K282E、K102T/R105C/K282E或K102T/R105C/Y251F/K282E。
在一种实施方式中,(b)中,所述突变体的氨基酸序列如SEQ ID NO.16~SEQ IDNO.24所示。
在一种实施方式中,所述α-1,3-岩藻糖基转移酶HpM32为(a)~(c)中的任一:
(a)氨基酸序列如SEQ ID NO.1所示的HpM32;
(b)在(a)的基础上进行截短获得的截短体:截短HpM32的C末端7肽重复序列,分别截短49、35、14和7个氨基酸。
(c)在(a)的基础上进行延伸获得的延伸体:延伸HpM32的C末端7肽重复序列,分别延长7、14、21和28个氨基酸。
在一种实施方式中,(b)中,所述突变体的氨基酸序列分别如SEQ ID NO.7~SEQID NO.10所示。
在一种实施方式中,(c)中,所述突变体的氨基酸序列分别如SEQ ID NO.11~SEQID NO.14所示。
在一种实施方式中,利用质粒pRSFDuet-1、pETDuet-1或pCDFDuet-1游离表达manB、manC、gmd和wcaG。
在一种实施方式中,利用质粒pRSFDuet-1、pETDuet-1或pCDFDuet-1游离表达HpM32和双串联的HpfutC。
在一种实施方式中,利用BBa_B0034替换质粒pRSFDuet-1、pETDuet-1或pCDFDuet-1的原始RBS。
在一种实施方式中,将双串联的HpfutC和HpM32分别插入质粒的MCS1区和MCS2区。
在一种实施方式中,在质粒的MCS1区串联连接双拷贝HpfutC,在MCS2区连接HpM32。
在一种实施方式中,敲除poxB位点之后,在poxB位点整合表达双串联的α-1,2-岩藻糖基转移酶HpfutC。
在一种实施方式中,敲除pta-ackA位点之后,在pta-ackA位点整合表达双串联的α-1,3-岩藻糖基转移酶HpM32。
在一种实施方式中,使用强启动子替换大肠杆菌基因组中manA、manC、manB和gmd-wcaG编码基因的启动子。
在一种实施方式中,使用强启动子起始HpfutC和HpM32的表达。
在一种实施方式中,所述强启动子包括T7启动子。
在一种实施方式中,所述大肠杆菌为BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlonΔglmS。
在一种实施方式中,所述谷氨酰胺-果糖-6-磷酸氨基转移酶基因glmS的Gene ID为948241,泛醌依赖性丙酮酸脱氢酶基因poxB的Gene ID为946132,磷酸乙酰转移酶基因pta的Gene ID为946778,乙酸激酶基因ackA的Gene ID为946775,甘露糖-6-磷酸异构酶基因manA的Gene ID为944840,磷酸甘露糖变位酶基因manB的Gene ID为946574,甘露糖-1-磷酸鸟嘌呤基转移酶基因manC的Gene ID为946580,GDP-甘露糖-6-脱氢酶基因gmd的Gene ID为946562,GDP-岩藻糖合成酶基因wcaG的Gene ID为946563。
本发明的第四个目的是提供一种生产2’-岩藻糖基乳糖或3-岩藻糖基乳糖的基因工程菌,以底盘细胞I或底盘细胞II为宿主,游离表达manB、manC、gmd、wcaG,游离表达HpfutC或HpM32。
在一种实施方式中,游离表达HpfutC的基因工程菌生产2’-岩藻糖基乳糖,游离表达HpM32的基因工程菌生产3-岩藻糖基乳糖。
在一种实施方式中,所述α-1,2-岩藻糖基转移酶HpfutC为(a)或(b):
(a)氨基酸序列如SEQ ID NO.15所示的HpfutC;
(b)在(a)的基础上进行突变的突变体:K102T、R105C、Y251F、K282E、K102T/R105C、Y251F/K282E、R105C/K282E、K102T/R105C/K282E或K102T/R105C/Y251F/K282E。
在一种实施方式中,(b)中,所述突变体的氨基酸序列如SEQ ID NO.16~SEQ IDNO.24所示。
在一种实施方式中,所述α-1,3-岩藻糖基转移酶HpM32为(a)~(c)中的任一:
(a)氨基酸序列如SEQ ID NO.1所示的HpM32;
(b)在(a)的基础上进行截短获得的截短体:截短HpM32的C末端7肽重复序列,分别截短49、35、14和7个氨基酸。
(c)在(a)的基础上进行延伸获得的延伸体:延伸HpM32的C末端7肽重复序列,分别延长7、14、21和28个氨基酸。
在一种实施方式中,(b)中,所述突变体的氨基酸序列分别如SEQ ID NO.7~SEQID NO.10所示。
在一种实施方式中,(c)中,所述突变体的氨基酸序列分别如SEQ ID NO.11~SEQID NO.14所示。
本发明的第五个目的是提供生产乳糖-N-二岩藻四糖的方法,所述方法为以所述产乳糖-N-二岩藻四糖的工程菌为发酵菌株发酵生产乳糖-N-二岩藻四糖。
在一种实施方式中,所述方法为将所述工程菌接种至在以甘油、葡萄糖为碳源的发酵体系中发酵生产乳糖-N-二岩藻四糖。
在一种实施方式中,将菌株在摇瓶发酵体系中培养至OD600=0.6±0.1;加入终浓度为0.2~0.5mM的IPTG,同时加入乳糖至终浓度为5~12g/L,20~30℃,150~220rpm的条件下诱导培养不少于60h。
在一种实施方式中,将菌株在发酵罐体系中培养至OD600=20±3;加入终浓度为0.2~0.5mM的IPTG,同时加入乳糖至终浓度为5~12g/L,20~30℃,控制发酵体系中pH为6.5~7.0;溶氧控制在30%±5%,发酵不少于70h。
在一种实施方式中,所述发酵培养基包括甘油20~40g/L,酪蛋白氨基酸1~5g/L,酵母提取物4~10g/L,磷酸二氢钾1~5g/L,磷酸氢二钠5~10g/L,氯化铵0.1~1.0g/L,氯化钠0.1~1.0g/L,七水硫酸镁1~1.5g/L,盐酸硫胺素5~20mg/L,微量金属溶液0.1~1.0mL/L。
在一种实施方式中,所述微量金属溶液包括六水氯化铁20~30g/L,二水氯化钙1~5g/L,氯化锌1~5g/L,二水钼酸钠1~5g/L,五水硫酸铜1~5g/L,一水硫酸锰1~5g/L,硼酸0.1~1.0g/L。
本发明的第六个目的是提供所述底盘细胞I或所述底盘细胞II或所述产乳糖-N-二岩藻四糖的工程菌或所述方法在制备乳糖-N-二岩藻四糖中的应用。
本发明还提供了所述底盘细胞I或所述底盘细胞II在制备母乳低聚糖中的应用。
在一种实施方式中,所述母乳低聚糖包括岩藻糖基乳糖。
在一种实施方式中,所述岩藻糖基乳糖为2’-岩藻糖基乳糖、3-岩藻糖基乳糖或乳糖-N-二岩藻四糖。
本发明的有益效果:
本发明在大肠杆菌BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlonΔglmS宿主菌中组合调控了GDP-L-岩藻糖合成途径中的磷酸甘露糖变位酶基因manB、甘露糖-1-磷酸鸟嘌呤基转移酶基因manC、GDP-甘露糖-6-脱氢酶基因gmd和GDP-岩藻糖合成酶基因wcaG,并在此基础上异源表达了α-1,2-岩藻糖基转移酶和α-1,3-岩藻糖基转移酶突变体。基于质粒拷贝数模块优化通路基因,通过限速酶的表达上调(酶源筛选、截短/延伸突变、定点突变、插入位点模式优化、染色体整合)获得了从头高效生产乳糖-N-二岩藻四糖的基因工程菌株。
通过摇瓶发酵培养,本申请构建的基因工程菌摇瓶生产乳糖-N-二岩藻四糖的能力由初始的2.18g/L提升至4.68g/L;在3L发酵罐中,乳糖-N-二岩藻四糖的产量达到了41.02g/L,是目前报道的最高产量,本发明为乳糖-N-二岩藻四糖的工业化生产奠定了基础。
附图说明
图1为乳糖-N-二岩藻四糖的代谢通路图。
图2为为乳糖-N-二岩藻四糖标准品及发酵样品的HPLC对比图。
图3α-1,3-岩藻糖基转移酶截短或延伸示意图及突变体对LDFT生成的影响。
图4为菌株BDFL53的3L发酵罐的补料分批发酵。
具体实施方式
以下结合实例与附图对本发明的具体实施作进一步的说明。
1、以下实例中所使用的质粒、PCR试剂、限制性内切酶、质粒抽提试剂盒、DNA胶回收试剂盒等采用商业产品,具体操作按照试剂盒说明书进行;本发明的实施方式不限于此,其他未注明的实验操作和工艺参数按照常规技术进行。
2、载体pRSFDuet-1、pETDuet-1和pCDFDuet-1购自Addgene。
3、DNA产物、质粒的测序工作交予天霖生物科技(无锡)有限公司完成。
4、大肠杆菌感受态的制备:上海生工生物工程公司试剂盒。
5、LB液体培养基:10g/L蛋白胨,5g/L酵母提取物,10g/L氯化钠。
6、LB固体培养基:10g/L蛋白胨,5g/L酵母提取物,10g/L氯化钠,18g/L琼脂粉。
7、发酵培养基:甘油30.0g/L,酪蛋白氨基酸2.0g/L,酵母提取物5.0g/L,磷酸二氢钾3.0g/L,磷酸氢二钠6.8g/L,氯化铵1.0g/L,氯化钠0.5g/L,七水硫酸镁1.4g/L,盐酸硫胺素10.0mg/L,微量金属溶液1.0mL/L(六水氯化铁25.0g/L,二水氯化钙2.3g/L,氯化锌2.6g/L,二水钼酸钠2.6g/L,五水硫酸铜2.0g/L,一水硫酸锰2.5g/L,硼酸0.7g/L),pH 6.8。
8、下述实施例中菌株的摇瓶发酵条件:工程菌单菌落接种于LB液体培养基,37℃,200rpm,摇瓶培养12h,得到种子液;将种子液以3%(v/v)的接种量接入50mL发酵培养基,37℃,200rpm,摇瓶培养至OD600为0.6;加入终浓度为0.3mM的异丙基-β-D-硫代吡喃半乳糖苷(IPTG),同时加入乳糖至终浓度为8g/L,25℃,200rpm的条件下诱导培养72h,获得发酵液,使用HPLC方法测定乳糖-N-二岩藻四糖的含量。2’-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵条件与乳糖-N-二岩藻四糖的摇瓶发酵条件相同。
9、乳糖-N-二岩藻四糖、2’-岩藻糖基乳糖和3-岩藻糖基乳糖的测定方法:使用HPLC测定:1mL发酵液于100℃煮沸10min,12000r/min离心5min,上清液经0.22μm膜过滤处理,利用HPLC检测乳糖-N-二岩藻四糖、2’-岩藻糖基乳糖和3-岩藻糖基乳糖的生成量以及乳糖和甘油的消耗量。HPLC检测条件:示差折光检测器;色谱柱为Rezex ROA-organic acid(Phenomenex,USA),柱温为50℃;流动相为0.005mol/L的H2SO4水溶液,流速为0.6mL/min;进样量为10μL。
实施例1:CRISPR-Cas9基因编辑技术敲除大肠杆菌染色体的glmS基因
分析乳糖-N-二岩藻四糖的从头合成途径,其关键中间体果糖-6-磷酸向UDP-乙酰葡糖胺(LNT、LNnT、3’-SL和6’-SL合成的关键中间体)转化的分支途径可能会阻碍LDFT的生物合成和积累(图1所示)。因此,本实施例以大肠杆菌BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlon(BZWNDPAL)为出发菌株(菌株BZWNAPAL的构建方法已公开于公开号为CN114480240A的专利文献中),利用CRISPR-Cas9基因敲除系统敲除了大肠杆菌BZWNDPAL基因组中的谷氨酰胺-果糖-6-磷酸氨基转移酶基因glmS,具体步骤如下(所涉及到的引物序列见表1):
(1)通过http://www.regenome.net/cas-offinder查找glmS基因的特异性靶点gRNA(20bp),使用glmS-gRNA_F/glmS-gRNA_R上下游引物,以pTargetF质粒(Addgene:#62226)为模板进行PCR扩增,扩增产物经限制性内切酶Dpn I酶切,以去除多余的环状质粒pTargetF。后将扩增产物转化E.coli DH5α感受态细胞,小提质粒,经公司测序鉴定,将构建成功的敲除质粒命名为pTargetF-glmS。
(2)以大肠杆菌BZWNDPAL基因组为模板,利用上游同源臂引物glmS-US_F/glmS-US_R和下游同源臂引物glmS-DS_F/glmS-DS_R分别扩增同源臂序列片段,产物纯化回收后采用SOE-PCR方法使用引物glmS-US_F/glmS-DS_R将上下游同源臂片段连接获得基因同源修复模板。
(3)取pCas9质粒(Addgene:#62225)电转至大肠杆菌BZWNDPAL电转感受态细胞,冰上放置5min后进行感受态融化,取10μL质粒加入100μL感受态细胞中,轻轻混匀。将质粒和电转感受态细胞转入预冷的电转杯中,2.5kV电击5ms,电击后迅速加入预冷的液体LB,轻轻吹打混匀后将混有质粒和感受态细胞的培养基转移到新的离心管中扩大培养1.5h。6000r/min离心2min,弃去上清液,菌体涂布于含有卡那抗性的LB平板上,放置于30℃培养箱过夜培养。
(4)挑取大肠杆菌BZWNDPAL/pCas9单菌落于LB培养基中,30℃培养1.0h,加入终浓度为30mM的L-阿拉伯糖以诱导λ-red系统表达。当OD600达到0.6-0.8时,制备大肠杆菌BZWNDPAL/pCas9感受态。
(5)将500ng步骤(1)构建的质粒pTargetF-glmS和1000ng步骤(2)构建的基因同源修复模板电转至步骤(4)制备的大肠杆菌BZWNDPAL/pCas9感受态细胞,涂布于LB平板(卡那霉素和壮观霉素),30℃培养16-24h,对平板上长出的单菌落进行菌落PCR验证,筛选阳性转化子和进行基因测序。
(6)对验证正确的单菌落进行pTargetF-glmS和pCas9质粒的消除,单菌落接种于LB液体培养基(卡那抗性)中,30℃、200r/min培养至对数生长期,加入终浓度为0.5mmol/L的IPTG过夜培养,诱导pTargetF-glmS质粒失活。菌液划线含有Kan的LB平板上,于30℃、200r/min培养12h。单菌落点板于卡那和壮观霉素的双抗性平板上,若无菌落生长,表明pTargetF-glmS质粒消除成功。
(7)pCas9质粒为温敏型质粒,将消除成功pTargetF-glmS质粒的单菌落转接至LB无抗性液体培养基中,42℃传代培养消除pCas9质粒。菌液划线无抗性的LB平板后37℃恒温培养,单菌落点板于含卡那抗性的LB培养基中,若单菌落不生长,则表明pCas9质粒消除成功,将构建好的无pTargetF-glmS质粒和pCas9质粒的基因缺失菌株在-80℃条件下保存备用。
(8)将构建成功的大肠杆菌底盘宿主命名为BZWNDPALS。
表1.glmS基因敲除引物
实施例2:从头合成乳糖-N-二岩藻四糖及模块优化基因表达
以实施例1中的BZWNDPAL和BZWNDPALS为底盘宿主,使用双质粒系统共表达通路基因。重组质粒和菌株的构建具体步骤如下(所涉及到的引物序列见表2):
(1)manB,manC,gmd和wcaG片段的获得:以大肠杆菌K12的基因组为模板,使用引物manCB_F/R和GW_F/R进行PCR扩增,胶回收DNA,获得manC-manB、gmd-wcaG基因片段。
(2)以pRSFDuet-1、pETDuet-1、pCDFDuet-1为模板,使用引物V1_F/R和V2_F/R扩增载体骨架序列。根据In-Fusion克隆技术,将片段manC-manB分别插入上述载体的Nco I/BamHI酶切位点之间,片段gmd-wcaG分别插入上述载体的Nde I/Xho I酶切位点之间,筛选阳性克隆并测序,最终得到重组质粒pRSF-CBGW、pET-CBGW和pCDF-CBGW。
(3)HpfutC和HpM32基因片段的获得:委托天霖生物科技(上海)有限公司合成来源于幽门螺杆菌ATCC 26695的HpfutC基因序列(SEQ ID NO.15)和来源于幽门螺杆菌NCTC11639的突变体HpM32(S45F/D127N/R128E/H131I/Y199N/E340D/V368A)基因序列(SEQ IDNO.1)。将合成后的HpfutC片段通过无缝克隆试剂盒(南京诺唯赞生命科技有限公司)连接到载体pETDuet-1的Nco I/BamHI酶切位点之间,获得质粒pET-HpfutC,再将HpM32基因片段连接到载体质粒pET-HpfutC的Nde I/Xho I酶切位点之间,获得的质粒为pET-HpfutC-HpM32。并且,质粒pET-HpfutC-HpM32的原始RBS(T7)的-35和-10区域之间替换为RBS(BBa_B0034)。RBS(BBa_B0034)序列及引物序列见表2,以pET-HpfutC、pET-HpM32、pET-HpfutC-HpM32质粒为模板,使用引物BBa_B0034_CF/R和BBa_B0034_MF/R扩增载体骨架序列,胶回收DNA,产物经限制性内切酶Dpn I酶切,去除多余的环状模板。使用无缝克隆试剂盒对线性片段进行重组反应,随后转化至E.coli DH5α并过夜培养,挑取平板上的单克隆至4mL的LB培养基,扩大培养后质粒抽提并测序,将构建成功的质粒分别命名为pET-R34-HpfutC、pET-R34-HpM32和pET-R34-HpfutC-R34-HpM32。
(4)以pRSFDuet-1和pCDFDuet-1为模板,使用引物V3_F/R扩增载体骨架片段;以pET-R34-HpfutC-R34-HpM32为模板,使用引物CM_F/R扩增R34-HpfutC-R34-HpM32基因片段,将R34-HpfutC-R34-HpM32和载体骨架片段无缝连接,最终获得重组质粒pRSF-R34-HpfutC-R34-HpM32和pCDF-R34-HpfutC-R34-HpM32。
表2.质粒构建引物
(5)上述通过质粒表达系统对途径基因进行组装,构建了不同拷贝数的表达载体以增加上游关键基因和下游糖基转移酶的表达水平。采用3种不同强度的表达载体pCDFDuet-1(拷贝数20~40)、pETDuet-1(拷贝数>40)和pRSFDuet-1(拷贝数>100)进行模块优化。将关键基因gmd,wcaG,manC和manB划分为模块一,将含有岩藻糖基转移酶的HpfutC和HpM32划分为模块二。在不同质粒和菌株组合下,构建了7种LDFT工程菌株(见表3),比较不同组合下LDFT菌株的生产性能。
表3.模块质粒组合工程菌的详细信息
(6)摇瓶发酵实验结果显示,以E.coli BL21(DE3)ΔlacZΔwcaJΔnudDΔpfkAΔlonΔglmS(BZWNDPALS)为宿主菌产LDFT的最佳质粒组合为pRSF-CBGW和pET-R34-HpfutC-R34-HpM32,即菌株BDFL2。在模块二固定表达下,产物积累随模块一表达强度的增加而呈上升趋势,固定模块一的表达后,模块二在略低于模块的表达强度一时,产物积累较多。最佳菌株BDFL2经60h发酵后产物LDFT的产量最高达到2.18g/L。
实施例3:不同来源α-1,3-岩藻糖基转移酶的筛选
(1)HpM32、Hp3/4FT、HpfutA、Hc3FT、Hh3FT和Bf3FT基因片段的获得:委托天霖生物科技(上海)有限公司合成来源于幽门螺杆菌NCTC 11639的突变体HpM32(S45F/D127N/R128E/H131I/Y199N/E340D/V368A)基因序列、来源于幽门螺杆菌UA948的Hp3/4FT基因序列、来源于幽门螺杆菌ATCC 26695的HpfutA基因序列、来源于胃螺杆菌的Hc3FT基因序列、来源于肝螺杆菌的Hh3FT基因序列和来源于脆弱拟杆菌的Bf3FT基因序列。将合成后的目的基因片段通过无缝克隆试剂盒(南京诺唯赞生命科技有限公司)连接到载体pET-R34-HpfutC的Nde I/Xho I酶切位点之间,分别获得质粒pET-R34-HpfutC-R34-HpM32、pET-R34-HpfutC-R34-Hp3/4FT、pET-R34-HpfutC-R34-HpfutA、pET-R34-HpfutC-R34-Hc3FT、pET-R34-HpfutC-R34-Hh3FT和pET-R34-HpfutC-R34-Bf3FT。
(2)将实施例2中的表达载体pRSF-CBGW和上述(1)中几种原核生物来源的α-1,3-岩藻糖基转移酶表达载体共转化至E.coli BZWNDPALS,所得菌株如表4所示。摇瓶发酵结果显示,通过测定LDFT的产量,来源于幽门螺杆菌NCTC 11639的突变体HpM32(S45F/D127N/R128E/H131I/Y199N/E340D/V368A)具有更高的LDFT生产能力。
表4.含不同来源α-1,3-岩藻糖基转移酶的工程菌
实施例4:α-1,3-岩藻糖基转移酶的截短或延伸突变体对乳糖-N-二岩藻四糖产量
的影响
α-1,2-岩藻糖基转移酶和α-1,3-岩藻糖基转移酶参与LDFT生物合成的最后两步,其催化GDP-L-岩藻糖中岩藻糖基团的转移,并通过α-1,2和α-1,3键连接乳糖的半乳糖基和葡萄糖基。根据以往的报道,岩藻糖基转移酶的溶解度差和催化活性低已成为微生物制备岩藻糖基乳糖或乳糖-N-二岩藻四糖的普遍问题。α-1,3-岩藻糖基转移酶的C末端由七肽重复序列和两亲性螺旋组成,分别负责酶的二聚化和外围膜锚定区域。在多数α-1,3-岩藻糖基转移酶中,催化结构域的C端有2-10个保守序列的七肽重复序列,七肽重复序列和/或两亲性螺旋区域的截短或延伸可能导致酶活性和可溶性表达量增加。
本实施例在幽门螺杆菌NCTC 11639来源的突变体HpM32(C末端有7个七肽重复序列)的基础上进行C末端七肽重复序列的截短或延伸优化,通过调控α-1,3-岩藻糖基转移酶的溶解度和表达强度,构建并筛选了高效生产LDFT的工程菌株。选取了8种不同长度的七肽重复序列,构建的质粒突变体见表4,工程菌株见表5,分别包含4个截短突变体和4个延伸突变体,具体构建流程如下(涉及的引物序列见表5)。
(1)以截短突变体H6和延伸突变体H8为例,使用引物H6_F/R和H8_F/R,以实施例2构建的pET-R34-HpfutC-R34-HpM32质粒为模板扩增载体骨架序列,胶回收DNA,产物经限制性内切酶Dpn I酶切,去除多余的环状模板。使用One Step Cloning Kit(Vazyme)试剂盒对线性片段进行重组反应,随后转化至E.coli DH5α并过夜培养,挑取平板上的单克隆至4mL的LB培养基,扩大培养后质粒抽提并测序,将构建成功的敲除质粒命名为pET-R34-HpfutC-R34-H6和pET-R34-HpfutC-R34-H8。采用相同的方法分别构建突变体H0、H2、H5、H9、H10、H11,其中,截短突变体H0、H2、H5的质粒模板为pET-R34-HpfutC-R34-HpM32,延伸突变体H9、H10和H11的质粒模板分别为pET-R34-HpfutC-R34-H8、pET-R34-HpfutC-R34-H9和pET-R34-HpfutC-R34-H10。
表5.截短或延伸突变体的引物序列
(2)将上述步骤中成功构建的质粒转入含有pRSF-CBGW的宿主大肠杆菌BZWNDPALS,获得的工程菌株如表6所示。
(3)将得到的工程菌通过摇瓶发酵生产LDFT,结果同时展示于图3,延长七肽重复序列的长度可以提高LDFT的产量和酶的可溶性表达,最佳菌株BDFL19获得LDFT的产量提升至3.03g/L。
表6.截短或延伸突变体工程菌的详细信息
实施例5:α-1,2-岩藻糖基转移酶的突变体对乳糖-N-二岩藻四糖产量的影响
据报道,多数α-1,2-岩藻糖基转移酶在微生物表达系统中均表现出低催化活力和可溶性表达差等问题,且未见有微生物来源的α-1,2-岩藻糖基转移酶的晶体结构报道。本实施例基于前期突变体的研究工作(已公开于公开号为CN116064455A的专利文献中),选用几种催化活力提高的α-1,2-岩藻糖基转移酶的单点(K102T、R105C、Y251F、K282E)和组合突变体(K102T/R105C,Y251F/K282E,R105C/K282E,K102T/R105C/K282E,K102T/R105C/Y251F/K282E),并将其应用于乳糖-N-二岩藻四糖的发酵生产。在质粒pET-R34-HpfutC-R34-H10的基础上进行突变体的构建工作,具体流程和所用引物如下(表7):
(1)以突变体K102T为例进行质粒构建,以pET-R34-HpfutC-R34-H10为模板,使用K102T_F/R为上下游引物,通过反向PCR线性化扩增表达质粒,随后使用Dpn I酶消化模板质粒,纯化回收线性化载体片段。使用One Step Cloning Kit(Vazyme)试剂盒对线性片段进行重组连接,重组反应体系在37℃下反应30min,随后转化至E.coli DH5α并过夜培养,挑取平板上的单克隆至4mL的LB培养基,扩大培养后质粒抽提并测序。将构建成功的质粒命名为pET-R34-K102T-R34-H10。其他几个单点突变体和V1的质粒模板均同K102T,组合突变体V7、V8、V9和V10的质粒模板则是在单点突变体的基础上进行叠加突变,相应的模板分别是Y251F、R105C、V1和V9。最终,获得的单点突变质粒分别为pET-R34-K102T-R34-H10、pET-R34-R105C-R34-H10、pET-R34-Y251F-R34-H10和pET-R34-K282E-R34-H10,获得的组合突变质粒分别为pET-R34-V1-R34-H10、pET-R34-V7-R34-H10、pET-R34-V8-R34-H10、pET-R34-V9-R34-H10和pET-R34-V10-R34-H10。
表7.α-1,2-岩藻糖基转移酶突变体的引物序列
(2)将上述步骤中成功构建的质粒转入含有pRSF-CBGW的宿主大肠杆菌BZWNDPALS,获得的工程菌株如表8所示。
(3)将得到的工程菌通过摇瓶发酵生产LDFT,产量结果展示于表7中。
表8.不同α-1,2-岩藻糖基转移酶突变体的工程菌的详细信息
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实施例6:限速酶的插入位点及模式对乳糖-N-二岩藻四糖产量的影响
有研究表明,途径基因在多克隆位点(MCS)的插入位置和模式可能影响目标化合物的合成。为了增加乳糖-N-二岩藻四糖的合成效率,本实施例比较了两个限速酶的插入顺序、位置及串联拷贝数。
为验证插入位点对限速酶基因表达和LDFT生产的影响,将α-1,2/3-岩藻糖基转移酶突变体V10/H10分别插入原始RBS(T7)替换为BBa_B0034的载体pETDuet-1的MCS1区和MCS2区,获得四个质粒分别为pET-R34-1H10、pET-R34-2H10、pET-R34-1V10和pET-R34-2V10。随后,为了验证不同通路配置和顺序对产物合成的影响,使用操纵子和假操纵子形式优化限速酶,获得四个质粒分别为pET-R34-1V10-R34-1H10、pET-R34-2V10-R34-2H10、pET-R34-1V10-R34-2H10和pET-R34-1H10-R34-2V10。最后,为了强化限速酶的表达,在质粒pET-R34-1V10-R34-2H10的MCS1区后的酶切位点NotI处串联插入α-1,2/3-岩藻糖基转移基因,获得质粒pET-R34-1V10-R34-2V10-R34-3H10和pET-R34-1V10-R34-2H10-R34-3H10;在质粒pET-R34-1V10-R34-2V10-R34-3H10的基础上,将α-1,3-岩藻糖基转移基因插入酶切位点BamHI,获得质粒pET-R34-1V10-R34-2V10-R34-3H10-R34-4H10。质粒的构建方法同实施例2,所用引物如下(表9)。其中,目的基因位于MCS1区的质粒pET-R34-1H10和pET-R34-1V10分别使用1H_F/R和1V_F/R为引物,载体骨架则共用引物V_1H/V_F/R;目的基因位于MCS2区的质粒pET-R34-2H10和pET-R34-2V10分别使用2H_F/R和2V_F/R为引物,载体骨架则共用引物V_2H/V_F/R。在此基础上,质粒pET-R34-1V10-R34-1H10和pET-R34-2V10-R34-2H10使用1V1H_F/R和2V2H_F/R扩增目的基因,使用V_1V1H-F/R和V_2V2H_F/R扩增载体骨架,质粒pET-R34-1H10-R34-2V10则是以pET-R34-1H10为模板,分别使用2V_F/R和V_2H/V_F/R扩增基因和载体片段。此外,基因串联质粒pET-R34-1V10-R34-2V10-R34-3H10和pET-R34-1V10-R34-2H10-R34-3H10所用目的基因扩增引物分别为1V2V3H_F/R和1V2H3H_F/R,载体扩增引物为V_1V2V/H3H_F/R;质粒pET-R34-1V10-R34-2V10-R34-3H10-R34-4H10使用1V2V2H3H_F/R扩增目的基因,使用V_1V2V2H3H_F/R扩增载体骨架。
将上述构建的表达载体转化至含有pRSF-CBGW的宿主大肠杆菌BZWNDPALS,获得的10种工程菌株如表10所示。将工程菌通过摇瓶发酵生产LDFT,其中含有重组质粒pRSF-CBGW和pET-R34-1V10-R34-2V10-R34-3H10的工程菌(即菌株BDFL37)获得了3.94g/L的最高产量。
表9.限速酶的插入位点及模式优化的质粒构建引物
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表10.工程菌的详细信息
实施例7:途径酶的染色体整合强化菌株生产LDFT
为了加强前体GDP-L-岩藻糖的合成,本实施例通过启动子工程策略适度调节GDP-L-岩藻糖模块中“关键酶”的表达水平,即将强启动子T7替换大肠杆菌基因组上manA,manB,manC和gmd本身的启动子。此外,为了弱化糖酵解途径副产物乙酸的积累,将下游限速酶中的α-1,2-岩藻糖基转移酶突变体V10-V10(双串联)整合至染色体的poxB位点,并将α-1,3-岩藻糖基转移酶突变体H10-H10(双串联)整合至染色体的ackA-pta位点。乳糖-N-二岩藻四糖合成通路中涉及的基因编辑步骤如下(所涉及到的引物序列见表11):
(1)以在poxB位点染色体整合V10-V10基因为例,通过http://www.regenome.net/cas-offinder查找poxB基因的特异性靶点gRNA(20bp),使用poxB-gRNA_F/poxB-gRNA_R上下游引物,以pTargetF质粒(Addgene:#62226)为模板进行PCR扩增,扩增产物经限制性内切酶Dpn I酶切,以去除多余的环状质粒pTargetF。后将扩增产物转化E.coli DH5α感受态细胞,小提质粒,经公司测序鉴定,将构建成功的敲除质粒命名为pTargetF-poxB。
(2)以大肠杆菌BZWNDPAL基因组为模板,利用上游同源臂引物poxB-US_F/poxB-US_R,中游同源臂引物V10-V10-MS_F/V10-V10-MS_R和下游同源臂引物poxB-DS_F/poxB-DS_R分别扩增三条序列片段,产物纯化回收后采用SOE-PCR方法使用引物poxB-US_F/poxB-DS_R将三片段连接获得基因同源修复模板。
(3)取pCas9质粒(Addgene:#62225)电转至大肠杆菌BZWNDPALS电转感受态细胞,冰上放置5min后进行感受态融化,取10μL质粒加入100μL感受态细胞中,轻轻混匀。将质粒和电转感受态细胞转入预冷的电转杯中,2.5kV电击5ms,电击后迅速加入预冷的液体LB,轻轻吹打混匀后将混有质粒和感受态细胞的培养基转移到新的离心管中扩大培养1.5h。6000r/min离心2min,弃去上清液,菌体涂布于含有卡那抗性的LB平板上,放置于30℃培养箱过夜培养。
(4)挑取大肠杆菌BZWNDPAL/pCas9单菌落于LB培养基中,30℃培养1.0h,加入终浓度为30mM的L-阿拉伯糖以诱导λ-red系统表达。当OD600达到0.6-0.8时,制备大肠杆菌BZWNDPAL/pCas9感受态。
(5)将500ng步骤(1)构建的pTargetF-poxB和1000ng步骤(2)构建的基因同源修复模板电转至步骤(4)制备的大肠杆菌BZWNDPAL/pCas9感受态细胞,涂布于LB平板(卡那霉素和壮观霉素),30℃培养16-24h,对平板上长出的单菌落进行菌落PCR验证,筛选阳性转化子和进行基因测序。
(6)对验证正确的单菌落进行pTargetF-poxB和pCas9质粒的消除,单菌落接种于LB液体培养基(卡那抗性)中,30℃、200r/min培养至对数生长期,加入终浓度为0.5mmol/L的IPTG过夜培养,诱导pTargetF-poxB质粒失活。菌液划线含有Kan的LB平板上,于30℃、200r/min培养12h。单菌落点板于卡那和壮观霉素的双抗性平板上,若无菌落生长,表明pTargetF-poxB质粒消除成功。
(7)pCas9质粒为温敏型质粒,将消除成功pTargetF-poxB质粒的单菌落转接至LB无抗性液体培养基中,42℃传代培养消除pCas9质粒。菌液划线无抗性的LB平板后37℃恒温培养,单菌落点板于含卡那抗性的LB培养基中,若单菌落不生长,则表明pCas9质粒消除成功,将构建好的无pTargetF-poxB质粒和pCas9质粒的基因缺失菌株在-80℃条件下保存备用。
(8)染色体上“关键酶”的启动子替换和突变体H10的整合操作参照上述步骤。
表11.启动子替换和限速酶整合引物
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本实施例中构建的工程菌株如表12所示,检测工程菌株的LDFT的合成能力、菌体生物量以及副产物积累情况。在删除poxB和ackA-pta基因后,重组菌株在发酵期间未检测到乙酸的积累,最佳无抗菌株BDFL46合成LDFT的产量达到1.68g/L,最佳有抗菌株BDFL53将LDFT的产量提升至4.68g/L,比初始BDFL1菌株产量提升了114%。
表12.基因组整合乳糖-N-二岩藻四糖代谢通路的工程菌的详细信息
实施例8:新型底盘宿主菌株在2’-岩藻糖基乳糖和3-岩藻糖基乳糖生产中的应用
2’-FL、3-FL和LDFT是母乳中分泌最丰富的岩藻糖基化母乳寡糖。2’-FL和3-FL是LDFT合成的前体,其在胞内的可用性是微生物高效合成LDFT的关键。为了验证本发明中构建的新型底盘菌株在前体合成中的有益效果,将2’-FL/3-FL生产所需的表达质粒pRSF-CBGW和含有α-1,2/3-岩藻糖基转移酶的突变质粒共转化至宿主菌株。选用的新型底盘菌株分别为大肠杆菌BZWNDPAL、BZWNDPALS和BDFL46,选用的α-1,2-岩藻糖基转移酶质粒分别为pET-R34-HpfutC、pET-R34-V10和pET-R34-1V10-R34-2V10,选用的α-1,3-岩藻糖基转移酶质粒分别为pET-R34-HpM32、pET-R34-H10和pET-R34-1H10-R34-2H10。其中,质粒pET-R34-HpfutC和pET-R34-HpM32来源于实施例2,其他质粒的构建引物如下表13。以质粒pET-R34-HpfutC和pET-R34-HpM32为载体模板,使用引物V10_F/R和H10_F/R进行目的片段V10和H10的线性扩增,将目的片段分别替换至载体的HpfutC和HpM32位置,最终得到质粒pET-R34-V10和pET-R34-H10。在质粒pET-R34-V10和pET-R34-H10的基础上,使用引物R34-V10_F/R和R34-H10_F/R扩增R34-2V10和R34-2H10线性片段,将其插入载体pET-R34-V10和pET-R34-H10的MCS2区,得到双串联α-1,2/3-岩藻糖基转移酶质粒pET-R34-1V10-R34-2V10和pET-R34-1H10-R34-2H10。
本实施例中构建的2’-FL和3-FL工程菌株如表14和15所示,比较各工程菌株的摇瓶生产性能,摇瓶发酵的条件与摇瓶发酵生产乳糖-N-二岩藻四糖的条件相同。发酵结果显示,改造菌株BZWNDPALS生产2’-FL和3-FL的能力高于BZWNDPAL,α-1,2/3-岩藻糖基转移酶突变体质粒表现出明显的产物合成优势。特别地,当质粒上串联二拷贝突变基因时,宿主菌BZWNDPALS中2’-FL和3-FL的产量比对照组(B2FL1和B3FL1)提高了60%和93%,而在改造菌株BDFL46中2’-FL和3-FL的产量比对照组(B2FL1和B3FL1)进一步提高了80%和114%,上述结果表明glmS基因的删除、关键限速酶的正向突变以及底盘菌株中通路酶的染色体整合可显著增强2’-FL/3-FL代谢通路的碳通量,促进了限速酶的正确折叠和功能表达,从而实现了2’-FL/3-FL的高效生物制备。
表13.α-1,2/3-岩藻糖基转移酶突变体的引物序列
表14.2’-FL工程菌的详细信息
表15.3-FL工程菌的详细信息
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实施例9:3L发酵罐补料分批发酵生产乳糖-N-二岩藻四糖
为了制备高产量的LDFT,使用重组菌BDFL53分别在3L发酵罐进行高密度的补料分批发酵。
发酵条件:将-80℃冰箱保藏的甘油菌在冰上解冻,取接种环沾取少量菌液“Z”字划线于固体LB平板(氨苄青霉素100μg/mL,硫酸卡那霉素50μg/mL),置于37℃恒温培养箱过夜培养。挑取平板上的单菌落接种于50mL的LB液体培养基,于37℃,200rpm条件下培养12h,即为种子液。3-L发酵罐中含有1L发酵培养基(甘油的初始浓度为30g/L),按照5%(v/v)的接种量进行菌株培养。诱导前培养温度设为37℃,待OD600达到20,加入IPTG诱导蛋白表达使得其在发酵体系中的浓度为0.2mmol/L,初始乳糖浓度为10g/L,诱导发酵温度为25℃。发酵全程使用NH4OH控制罐体pH恒定为6.80。为了维持菌体生长以及岩藻糖基乳糖的合,待初始甘油消耗完后流加800g/L的甘油(含20g/L的MgSO4·7H2O)以补充碳源,通过pH反馈调节(设置流速为20mL/h)使甘油在发酵体系中的浓度维持在较低浓度水平(甘油用于菌体生长和代谢,浓度约为0g/L,即维持在刚好被菌株消耗完的情况)至发酵结束,待初始乳糖消耗完后补加300g/L的乳糖并使其在发酵体系中的终浓度维持在10±0.5g/L左右,发酵过程中若乳糖消耗至较低浓度时继续补加乳糖至发酵结束。发酵过程中系统级联控制,通过调节转速,通气量和氧气使罐内溶氧为30%±5%。
发酵全程定时取样并测定菌体OD600,1mL发酵液煮沸15min使细胞破碎完全,12000r/min离心10min,上清液经0.22μm膜过滤处理,发酵过程中利用HPLC检测LDFT的生成量(图2)以及乳糖和甘油的消耗量。结果显示,发酵结束后(共发酵80小时),产物LDFT的浓度可达41.02g/L(图4)。
在发酵64、72、80小时LDFT的浓度分别为32.46、38.88、41.02g/L,转化率分别达到0.49、0.52、0.53mol LDFT/mol乳糖。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一种底盘细胞,其特征在于,以大肠杆菌为宿主,敲除了β-半乳糖苷酶基因lacZ、UDP-葡萄糖脂质载体转移酶基因wcaJ、GDP-甘露糖甘露糖基水解酶基因nudD、6-磷酸果糖激酶-1基因pfkA、蛋白酶基因lon、谷氨酰胺-果糖-6-磷酸氨基转移酶基因glmS。
2.根据权利要求1所述的底盘细胞,其特征在于,还敲除了泛醌依赖性丙酮酸脱氢酶基因poxB和磷酸乙酰转移酶-乙酸激酶基因簇pta-ackA,整合表达两个拷贝的α-1,2-岩藻糖基转移酶HpfutC和两个拷贝的α-1,3-岩藻糖基转移酶HpM32;
所述α-1,2-岩藻糖基转移酶HpfutC为(a)或(b):
(a)氨基酸序列如SEQ ID NO.15所示的HpfutC;
(b)在(a)的基础上进行突变的突变体:K102T、R105C、Y251F、K282E、K102T/R105C、Y251F/K282E、R105C/K282E、K102T/R105C/K282E或K102T/R105C/Y251F/K282E;
所述α-1,3-岩藻糖基转移酶HpM32为(a)~(c)中的任一:
(a)氨基酸序列如SEQ ID NO.1所示的HpM32;
(b)在(a)的基础上进行截短获得的截短体:截短HpM32的C末端7肽重复序列,分别截短49、35、14和7个氨基酸;
(c)在(a)的基础上进行延伸获得的延伸体:延伸HpM32的C末端7肽重复序列,分别延长7、14、21和28个氨基酸。
3.一种产乳糖-N-二岩藻四糖的工程菌,其特征在于,以权利要求1或2的底盘细胞为宿主,游离表达磷酸甘露糖变位酶基因manB、甘露糖-1-磷酸鸟嘌呤基转移酶基因manC、GDP-甘露糖-6-脱氢酶基因gmd、GDP-岩藻糖合成酶基因wcaG、α-1,2-岩藻糖基转移酶HpfutC和/或α-1,3-岩藻糖基转移酶HpM32;
所述α-1,2-岩藻糖基转移酶HpfutC为(a)或(b):
(a)氨基酸序列如SEQ ID NO.15所示的HpfutC;
(b)在(a)的基础上进行突变的突变体:K102T、R105C、Y251F、K282E、K102T/R105C、Y251F/K282E、R105C/K282E、K102T/R105C/K282E或K102T/R105C/Y251F/K282E;
所述α-1,3-岩藻糖基转移酶HpM32为(a)~(c)中的任一:
(a)氨基酸序列如SEQ ID NO.1所示的HpM32;
(b)在(a)的基础上进行截短获得的截短体:截短HpM32的C末端7肽重复序列,分别截短49、35、14和7个氨基酸;
(c)在(a)的基础上进行延伸获得的延伸体:延伸HpM32的C末端7肽重复序列,分别延长7、14、21和28个氨基酸。
4.根据权利要求3所述的工程菌,其特征在于,利用质粒pRSFDuet-1、pETDuet-1或pCDFDuet-1游离表达manB、manC、gmd和wcaG,利用质粒pRSFDuet-1、pETDuet-1或pCDFDuet-1游离表达HpM32和双串联的HpfutC。
5.根据权利要求3所述的工程菌,其特征在于,利用BBa_B0034替换质粒pRSFDuet-1、pETDuet-1或pCDFDuet-1的原始RBS。
6.根据权利要求4或5所述的工程菌,其特征在于,将双串联的HpfutC和HpM32分别插入质粒的MCS1区和MCS2区。
7.根据权利要求3所述的工程菌,其特征在于,敲除poxB位点之后,在poxB位点整合表达双串联的α-1,2-岩藻糖基转移酶HpfutC;敲除pta-ackA位点之后,在pta-ackA位点整合表达双串联的α-1,3-岩藻糖基转移酶HpM32。
8.根据权利要求3~7任一所述的工程菌,其特征在于,使用强启动子替换大肠杆菌基因组中manA、manC、manB和gmd-wcaG编码基因的启动子,使用强启动子起始HpfutC和HpM32的表达。
9.一种产2’-岩藻糖基乳糖或3-岩藻糖基乳糖的工程菌,其特征在于,以权利要求1或2的底盘细胞为宿主,游离表达manB、manC、gmd、wcaG,游离表达HpfutC或HpM32;游离表达HpfutC的基因工程菌生产2’-岩藻糖基乳糖,游离表达HpM32的基因工程菌生产3-岩藻糖基乳糖;
所述α-1,2-岩藻糖基转移酶HpfutC为(a)或(b):
(a)氨基酸序列如SEQ ID NO.15所示的HpfutC;
(b)在(a)的基础上进行突变的突变体:K102T、R105C、Y251F、K282E、K102T/R105C、Y251F/K282E、R105C/K282E、K102T/R105C/K282E或K102T/R105C/Y251F/K282E;
所述α-1,3-岩藻糖基转移酶HpM32为(a)~(c)中的任一:
(a)氨基酸序列如SEQ ID NO.1所示的HpM32;
(b)在(a)的基础上进行截短获得的截短体:截短HpM32的C末端7肽重复序列,分别截短49、35、14和7个氨基酸;
(c)在(a)的基础上进行延伸获得的延伸体:延伸HpM32的C末端7肽重复序列,分别延长7、14、21和28个氨基酸。
10.生产乳糖-N-二岩藻四糖的方法,其特征在于,所述方法为以权利要求3~8任一所述工程菌为发酵菌株发酵生产乳糖-N-二岩藻四糖。
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