CN116536298A - 一种蛋白质序列n端修饰的木糖异构酶及其应用 - Google Patents
一种蛋白质序列n端修饰的木糖异构酶及其应用 Download PDFInfo
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Abstract
本发明公开了一种蛋白质序列N端修饰的木糖异构酶及其应用,该木糖异构酶具有IDNO.1~SEQ ID NO.5中任一氨基酸序列,它们的单独表达或组合表达能够赋予酵母细胞转化木糖为木酮糖的能力,进而赋予宿主细胞将木糖转化为其他产物的能力。这些木糖异构酶是由原来在酿酒酵母中无活性的木糖异构酶通过N端修饰后获得。当该木糖异构酶在酿酒酵母等酵母细胞中被表达时,能够使原来不具备转化木糖为木酮糖能力的宿主获得该转化能力,并赋予宿主细胞利用木糖或木质纤维素水解液等富含木糖的原料生产乙醇等化学品的能力。
Description
技术领域
本发明属于生物技术领域,尤其涉及一种蛋白质序列N端修饰的木糖异构酶及其应用,这种木糖异构酶是由原来在酿酒酵母中无活性的木糖异构酶通过蛋白质N端修饰后获得,其应用为该木糖异构酶赋予宿主细胞利用木糖或木质纤维素水解液生产多种发酵产品的应用。
背景技术
表达外源活力的木糖异构酶(xylose isomerase,XI)可以使酵母能够获得利用木糖的能力,并且木糖异构酶催化木糖异构化的过程不需要辅因子,不会生成副产物木糖醇,是目前最受关注的木糖代谢途径的关键酶之一(Annals of Microbiology,2020,70(1):50;Biotechnology for biofuels,2014,7(1):122)。研究者们在酵母中表达了大量木糖异构酶,但因为蛋白质错误折叠、翻译后修饰、二硫键形成和不适的胞内pH等原因,只有少数木糖异构酶在酵母中表现出活性(Applied and environmental microbiology,2009,75(8):2304-2311;Biofuels,2007:179-204;Scientific reports,2021,11(1):4766;Letters in Applied Microbiology,2022,74(6):941-948)。推测原因可能是蛋白的错误折叠、翻译后修饰、二硫键形成。前期有研究对在酿酒酵母中能够活性表达的木糖异构酶进行氨基酸序列分析,发现了一些底物结合和金属离子结合的保守位点,但也不是在酵母中活性表达的充分条件。目前并不能够解析出木糖异构酶在酵母中活性表达的关键因素,因而通过蛋白质工程改造对目前不能够在酵母中表达的木糖异构酶进行赋能,赋予其能够在酵母中活性表达的能力。
发明内容
针对现有技术的不足,本发明提供了一种蛋白质序列N端修饰的木糖异构酶及其应用。
本发明的目的是通过以下技术方案实现的:
一种蛋白质序列N端修饰的木糖异构酶,其氨基酸序列为以下氨基酸序列之一:
(1)SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5所示的氨基酸序列;
(2)SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5所示的氨基酸序列添加、缺失、取代或插入了1个或多个氨基酸的氨基酸序列;
(3)具有与SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5的任一者所示的氨基酸序列具有70%以上的同一性的氨基酸序列。
进一步地,其核苷酸序列为以下核苷酸序列之一:
(1)SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示的核苷酸序列;
(2)SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示的核苷酸序列添加、缺失、取代或插入了1个或多个核苷酸的核苷酸序列;
(3)具有与SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10中任一者所示的核苷酸序列具有70%以上的同一性的核苷酸序列;
(4)由于遗传密码子的简并性区别于SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示核苷酸序列的核苷酸序列。
进一步地,所述木糖异构酶由对来自Anaeromyces robustus、Neocallimastixcaliforniae、Rhizoclosmatium globosum的XI进行人工改造获得。
进一步地,所述木糖异构酶的表达均能够赋予宿主细胞转化木糖为木酮糖能力,从而赋予宿主细胞同化木糖的能力,所述的宿主细胞为酿酒酵母细胞Saccharomyces、耶氏酵母Yarrowia、假丝酵母Candida、毕赤酵母Pichia、裂殖酵母Schizosaccharomyces、汉逊酵母Hansenula或克鲁维酵母Kluyveromyces。
进一步地,所述宿主细胞为酿酒酵母细胞。
进一步地,所述木糖异构酶在宿主的表达方式为以下方式之一:
(1)木糖异构酶基因连接到宿主的游离质粒上,在宿主中进行游离表达;
(2)木糖异构酶基因整合到宿主细胞的染色体上,在宿主中进行整合表达;
(3)木糖异构酶基因在宿主中同时进行游离表达和整合表达。
进一步地,所述木糖异构酶单独在宿主菌株中表达或共同在宿主细胞中表达。
进一步地,所述酵母细胞是野生菌株或是进行了一个或多个遗传修饰的酵母细胞。
一种上述木糖异构酶的应用,该应用具体为:所述木糖异构酶赋予宿主细胞利用木糖或木质纤维素水解液生产多种发酵产品,包括木酮糖、果糖、乙醇、丁醇、微生物油脂、游离脂肪酸、糠醛、乳酸、琥珀酸、柠檬酸、丙酸、3-羟基丙酸、己二酸、木酮糖-5-磷酸、异戊二烯、聚羟基脂肪酸酯、赖氨酸、谷氨酸、苯丙氨酸、丙氨酸、香草酸、香草醛。
本发明的有益效果是,本发明公开了四种新的可以在酵母细胞中高活性表达的木糖异构酶的氨基酸序列和核苷酸序列。这四种木糖异构酶均来自人工构建,它们的单独表达或组合表达能够赋予酵母细胞转化木糖为木酮糖的能力,进而赋予宿主细胞将木糖转化为其他产物的能力。本发明还涉及此四种木糖异构酶在酵母利用木糖为底物生产乙醇等化学品上的应用。当该木糖异构酶在酿酒酵母等酵母细胞中被表达时,能够使原来不具备转化木糖为木酮糖能力的宿主获得该转化能力,并赋予宿主细胞利用木糖或木质纤维素水解液等富含木糖的原料生产乙醇等化学品的能力。
附图说明
图1来自不同菌株的木糖异构酶的序列比对示意图。
图2是来自于Neocallimastix californiae、Anaeromyces robustus和Rhizoclosmatium globosum的原始XI(NeoXI、AnaXI和RhiXI)和进行了N-端改造的XI(NeoXI-1、NeoXI-2、AnaXI-1、AnaXI-2和RhiXI)在酿酒酵母中游离表达时,重组酵母以初始40g/L木糖为碳源发酵96小时后的发酵液成分柱状图。
具体实施方式
以下实施例中所举的质粒、菌株只是用于对本发明作进一步详细说明,并不对本发明的实质内容加以限制。实际上,用本发明发现的核苷酸序列,本领域技术人员可以得到其它多种具有将木糖转化为木酮糖能力的遗传工程菌株,其均不能脱离本发明的精神和思路。除特别指出以外,实施例中的百分比为质量百分比。
对目前已经报道的能够在酵母中活性表达的木糖异构酶和已经报道的不能够在酵母中活性表达的木糖异构酶的氨基酸序列进行比对。结果发现许多非活力木糖异构酶相比于有活力的木糖异构酶,其N端氨基酸序列有明显不同。例如,相对于在酵母中有活性的木糖异构酶,RhiXI(来自Rhizoclosmatium globosum的木糖异构酶)、AnaXI(来自Anaeromyces robustus的木糖异构酶)分别缺失了约37和22个N端氨基酸,而NeoXI(来自Neocallimastix californiae的木糖异构酶)则多出来11个N端氨基酸(图1)。总所周知,蛋白质的合成从N端开始,蛋白质N端的序列组成影响蛋白质的整体生物学功能(BiochemicalJournal,2018,475(20):3201-3219;Proteomics,2015,15(14):2385-2401;Scientificreports,2017,7(1):1-13.)。例如,N端序列影响蛋白质的半衰期,并与蛋白质亚细胞器的定位有关。由此,可以推测假如能够对非活力的木糖异构酶的N端进行改造,则很有可能获得具有活性的木糖异构酶。
本申请中,利用组合合成生物学的理念,将来自于活性木糖异构酶的N端氨基酸序列对无活性木糖异构酶RhiXI和AnaXI的N端进行拼接,组合后的重组木糖异构酶获得了在酵母中进行活力表达的能力。此外,针对木糖异构酶NeoXI相比于已报道的活性氨基酸的N端多出11个氨基酸,通过对这些氨基酸进行删除,获得的N端截短的木糖异构酶同样在酵母中能够展示出较高的活力。
因此,本申请提供一种蛋白质序列N端修饰的木糖异构酶,其氨基酸序列为以下氨基酸序列之一:
(1)SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5所示的氨基酸序列;
(2)SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5所示的氨基酸序列添加、缺失、取代或插入了1个或多个氨基酸的氨基酸序列;
(3)具有与SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5的任一者所示的氨基酸序列具有70%以上的同一性的氨基酸序列。
上述的木糖异构酶的核苷酸序列为以下核苷酸序列之一:
(1)SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示的核苷酸序列;
(2)SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示的核苷酸序列添加、缺失、取代或插入了1个或多个核苷酸的核苷酸序列;
(3)具有与SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10中任一者所示的核苷酸序列具有70%以上的同一性的核苷酸序列;
(4)由于遗传密码子的简并性区别于SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示核苷酸序列的核苷酸序列。
具体地,所述木糖异构酶由对来自Anaeromyces robustus、Neocallimastixcaliforniae、Rhizoclosmatium globosum的XI进行人工改造获得。
具体地,所述木糖异构酶的表达均能够赋予宿主细胞转化木糖为木酮糖能力,从而赋予宿主细胞同化木糖的能力。所述的宿主细胞为酿酒酵母细胞(Saccharomyces)、耶氏酵母(Yarrowia)、假丝酵母(Candida)、毕赤酵母(Pichia)、裂殖酵母(Schizosaccharomyces)、汉逊酵母(Hansenula)、克鲁维酵母(Kluyveromyces)。
具体地,所述宿主细胞优选为酿酒酵母细胞。
具体地,所述木糖异构酶在宿主的表达方式为以下方式之一:
(1)木糖异构酶基因连接到宿主的游离质粒上,在宿主中进行游离表达;
(2)木糖异构酶基因整合到宿主细胞的染色体上,在宿主中进行整合表达;
(3)木糖异构酶基因在宿主中同时进行游离表达和整合表达。
具体地,所述木糖异构酶可以单独在宿主菌株中表达,也可以共同在宿主细胞中表达。
具体地,所述酵母细胞可以是野生菌株,也可以是进行了一个或多个遗传修饰的酵母细胞。
具体地,该应用具体为:所述木糖异构酶赋予宿主细胞利用木糖或木质纤维素水解液生产多种发酵产品,包括木酮糖、果糖、乙醇、丁醇、微生物油脂、游离脂肪酸、糠醛、乳酸、琥珀酸、柠檬酸、丙酸、3-羟基丙酸、己二酸、木酮糖-5-磷酸、异戊二烯、聚羟基脂肪酸酯、赖氨酸、谷氨酸、苯丙氨酸、丙氨酸、香草酸、香草醛。
实施例1:木糖异构酶的获得及表达
1.1、XI的获得
为了挖掘在酿酒酵母中具有活性的新型XI,我们从NCBI数据库中选择3个未被表征的XI,其分别来自Anaeromyces robustus(AnaXI)、Neocallimastix californiae(NeoXI)、Rhizoclosmatium globosum(RhiXI)。委托擎科生物科技股份有限公司对异构酶核苷酸序列分别进行合成。之后将合成的三条核苷酸大分子分别插入酿酒酵母游离表达载体,具体步骤为:将G418抗性基因插入至酿酒酵母游离表达载体pESC-URA的SmaI-SalI位点,获得G418_pESC-URA质粒;然后将酿酒酵母启动子TDH3序列插入至G418_pESC-URA质粒的KpnI-NheI位点,获得TDH3_G418_pESC-URA质粒;最后分别将合成的SEQ ID NO.5、SEQ IDNO.6、SEQ ID NO.7、SEQ ID NO.8对应的大分子核苷酸片段插入TDH3_G418_pESC-URA质粒的NheI位点,获得木糖异构酶游离表达载体pESC-Ana、pESC-Neo、pESC-Rhi。在获得这些酿酒酵母游离表达载体中木糖异构酶基因5’侧为TDH3启动子,3’侧为CYC1终止子1.2、XI的表达
将具有木糖异构酶基因的质粒pESC-Ana、pESC-Neo、pESC-Rhi转化至双倍体酿酒酵母CRD3(ATCC 26603,MATa/α,ΔGre3,
pho13::TPI1p-XKS1-ADH1t-FBA1p-TKL1-FBA1t-PGK1p-RKI1-GAL2t,pyk2::TEF1p-GAL2N376F-TEF1t-TDH3p-TAL1-PGI1t),转化子在YPD平板(400μg/mLG418)筛选,未转化的细胞不能在这些平板上生长。以平板上的单菌落为模板,PCR扩增相应的木糖异构酶基因并测序,鉴定含有相应木糖异构酶基因质粒的转化子,分别命名为CRD3Ana、CRD3Neo、CRD3Rhi。将其转接至YPX40培养基进行培养。
结果1:
如图2所示,YPX培养基初始木糖浓度为40g/L,当酿酒酵母CRD3Ana、CRD3Neo、CRD3Rhi在其中培养96h后,菌株没有利用木糖进行生长,说明这3种木糖异构酶没有在酿酒酵母中转化木糖的能力。
实施例2:木糖异构酶基因的突变及表达
2.1、木糖异构酶基因的突变
对AnaXI、NeoXI、RhiXI进行氨基酸位点改造,具体方法见表1。改造后的氨基酸及核苷酸序列见SEQ ID NO.1-5、SEQ ID NO.6-10。
表1对AnaXI、NeoXI、RhiXI的突变方式
2.2、游离表达载体的转化及转化子的筛选
将具有木糖异构酶基因的质粒pESC-Ana1、pESC-Ana2、pESC-Neo1、pESC-Neo2、pESC-Rhi转化至双倍体酿酒酵母CRD3(ATCC 26603,MATa/α,ΔGre3,pho13::TPI1p-XKS1-ADH1t-FBA1p-TKL1-FBA1t-PGK1p-RKI1-GAL2t,pyk2::TEF1p-GAL2N376F-TEF1t-TDH3p-TAL1-PGI1t),转化子在YPD平板(400μg/mLG418)筛选,未转化的细胞不能在这些平板上生长。以平板上的单菌落为模板,PCR扩增相应的木糖异构酶基因并测序,鉴定含有相应木糖异构酶基因质粒的转化子,分别命名为CRD3Ana1、CRD3Ana2、CRD3Neo1、CRD3Neo2、CRD3Rhi1。
2.3、重组菌株利用木糖能力测定
将酵母CRD3Ana-1、CRD3Ana-2、CRD3Neo-1、CRD3Neo-2、CRD3Rhi-1于YPD(2%蛋白胨、1%酵母提取物、2%葡萄糖)培养基过夜培养,然后以初始OD600为1.0转接到YPX(2%蛋白胨、1%酵母提取物、4%木糖)培养基,30℃,150rpm进行厌氧培养。高效液相色谱(HPLC)测定培养基中的木糖和乙醇浓度。使用紫外分光光度计在600nm波长下测量OD600来监测酵母生长。
结果2:
如图2所示,YPX培养基初始木糖浓度为40g/L,当酿酒酵母CRD3Ana-1、CRD3Ana-2、CRD3Neo-1、CRD3Neo-2、CRD3Rhi-1在其中培养96h后,消耗的木糖量分别为11.55、10.63、18.61、17.26、7.32g/L木糖,并且伴随着菌体的生长和乙醇的生成。该结果表明,本实验室中涉及的五种木糖异构酶在酿酒酵母中表达后,均赋予了酿酒酵母转化木糖为木酮糖的能力,使其可以利用木糖生长,并生成乙醇。
XI只有在形成二聚体或四聚体时才能正常发挥功能,而N端序列对于亚基A和D、亚基B和C形成的对角线二聚体的稳定有着重要作用。以AnaXI和AnaXI-1的亚基B、C为例,两个亚基间相互作用的氨基酸为棍棒形式,主要来自于XI的N端和C端。AnaXI的N端序列较短,只有His4、Tyr5和相邻亚基间进行作用。而延长N-端序列的AnaXI-1中,Lys17、Asp18、Lys20、Asn21、Pro22、Leu23、His26、Tyr27均和相邻亚基之间有作用力,并且有多个氨基酸和相邻亚基的氨基酸形成氢键,这有助于提升四聚体结构的稳定性。对RhiXI添加N端序列后,RhiXI-1获得在酿酒酵母中的活性,应该也是基于这个原因。而NeoXI冗余的N端结构可能对四聚体的空间结构有影响,导致无法转化木糖。据我们所知,这是第一次通过修饰氨基酸序列将非活性XI改变为活性XI。
Claims (9)
1.一种蛋白质序列N端修饰的木糖异构酶,其特征在于,其氨基酸序列为以下氨基酸序列之一:
(1)SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5所示的氨基酸序列;
(2)SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5所示的氨基酸序列添加、缺失、取代或插入了1个或多个氨基酸的氨基酸序列;
(3)具有与SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5的任一者所示的氨基酸序列具有70%以上的同一性的氨基酸序列。
2.根据权利要求1所述木糖异构酶,其特征在于,其核苷酸序列为以下核苷酸序列之一:(1)SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示的核苷酸序列;
(2)SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10所示的核苷酸序列添加、缺失、取代或插入了1个或多个核苷酸的核苷酸序列;
(3)具有与SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10中任一者所示的核苷酸序列具有70%以上的同一性的核苷酸序列;
(4)由于遗传密码子的简并性区别于SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQIDNO.9、SEQ ID NO.10所示核苷酸序列的核苷酸序列。
3.根据权利要求1或2所述的木糖异构酶,其特征在于,所述木糖异构酶由对来自Anaeromyces robustus、Neocallimastix californiae、Rhizoclosmatium globosum的XI进行蛋白质N端的改造获得。
4.根据权利要求3所述的木糖异构酶,其特征在于,所述木糖异构酶的表达均能够赋予宿主细胞转化木糖为木酮糖能力,从而赋予宿主细胞同化木糖的能力,所述的宿主细胞为酿酒酵母细胞Saccharomyces、耶氏酵母Yarrowia、假丝酵母Candida、毕赤酵母Pichia、裂殖酵母Schizosaccharomyces、汉逊酵母Hansenula或克鲁维酵母Kluyveromyces。
5.根据权利要求4所述的木糖异构酶,其特征在于,所述宿主细胞为酿酒酵母细胞。
6.根据权利要求3所述的木糖异构酶,其特征在于,所述木糖异构酶在宿主的表达方式为以下方式之一:
(1)木糖异构酶基因连接到宿主的游离质粒上,在宿主中进行游离表达;
(2)木糖异构酶基因整合到宿主细胞的染色体上,在宿主中进行整合表达;
(3)木糖异构酶基因在宿主中同时进行游离表达和整合表达。
7.根据权利要求3所述的木糖异构酶,其特征在于,所述木糖异构酶单独在宿主菌株中表达或共同在宿主细胞中表达。
8.根据权利要求3所述的木糖异构酶,其特征在于,所述酵母细胞是野生菌株或是进行了一个或多个遗传修饰的酵母细胞。
9.一种权利要求1-8中任一项所述的木糖异构酶的应用,其特征在于,该应用具体为:所述木糖异构酶赋予宿主细胞利用木糖或木质纤维素水解液生产多种发酵产品,包括木酮糖、果糖、乙醇、丁醇、微生物油脂、游离脂肪酸、糠醛、乳酸、琥珀酸、柠檬酸、丙酸、3-羟基丙酸、己二酸、木酮糖-5-磷酸、异戊二烯、聚羟基脂肪酸酯、赖氨酸、谷氨酸、苯丙氨酸、丙氨酸、香草酸、香草醛。
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