CN114891774A - 在酵母细胞中高活性表达的木糖异构酶及应用 - Google Patents

在酵母细胞中高活性表达的木糖异构酶及应用 Download PDF

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CN114891774A
CN114891774A CN202210481456.9A CN202210481456A CN114891774A CN 114891774 A CN114891774 A CN 114891774A CN 202210481456 A CN202210481456 A CN 202210481456A CN 114891774 A CN114891774 A CN 114891774A
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金明杰
陈思同
许召贤
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Nanjing University of Science and Technology
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Abstract

本发明公开了一种在酵母细胞中高活性表达的木糖异构酶，具有ID NO.1～SEQ ID NO.4中任一氨基酸序列，四种木糖异构酶分别来自于Acetanaerobacterium elongatum、Bacterium J10、Hallella seregens、Streptobacillus canis菌株，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。

Description

在酵母细胞中高活性表达的木糖异构酶及应用
技术领域
本发明属于生物技术领域，尤其涉及一种在酵母细胞中高活性表达的木糖异构酶，以及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
化石资源的过度开采和使用不仅使得这种不可持续资源存量不断减少，而且化石资源的过度使用还带来二氧化碳大量排放及其引起的全球变暖、极端气候多发等问题。木质纤维素类生物质是地球上最丰富的可再生有机资源，全球每年光合作用生产约1500～2000亿吨生物质，其中80％以上为木质纤维素类生物质，包括常见的农业秸秆、林业废弃物等。利用木质纤维素生产乙醇等燃料或化学品，对于缓解能源危机、减轻环境污染和温室效应具有重要作用(Vu et al.,Science of The Total Environment,2020,743:140630)。
根据组成的结构单元不同，木质纤维素主要分为三种主要成分：纤维素、半纤维素和木质素。根据植物物种的不同，纤维素、半纤维素和木质素约占木质纤维素总质量的30-50％、 15-35％和10-30％(Ragauskas et al.,Science,2014,344:6185)。木质纤维素生物质经过预处理、酶水解后，其纤维素和半纤维素组分分别被水解成葡萄糖和木糖两种主要单糖。许多微生物，例如酿酒酵母，可以利用木质纤维素水解液中的葡萄糖发酵生产包括乙醇在内的多种化学品，但野生的酿酒酵母缺乏对木糖的转化能力，导致木质纤维素中的木糖不能被有效转化为目标化学品。基于半纤维素组分在木质纤维素生物质中占有的高比例，假如半纤维素水解出的木糖同样能够被微生物转化为相关产品，可以使得木质纤维素为原料生产乙醇等化学品的转化率得到大幅度提高(Lee et al.,Current Opinion inBiotechnology,2021,67:15-25)。
许多微生物，例如酿酒酵母，具有完整的木酮糖代谢系统，木酮糖在木酮糖激酶的作用下生成5-磷酸木酮糖，进入非氧化磷酸戊糖途径，进而可以继续转化为多种化学品。因此，如何将木糖转化为木酮糖则成为了木糖利用的关键。目前微生物中发现的木糖转为木酮糖的途径主要有两种，第一种是存在于毕赤酵母等真菌的木糖还原酶-木糖醇脱氢酶途径，其中木糖还原酶首先将木糖转化成木糖醇，继而木糖醇被木糖醇脱氢酶转化为木酮糖。但是这个过程中木糖还原酶和木糖醇脱氢酶的偏好性并不一致：木糖还原酶更偏好NADPH作为辅酶，木糖醇脱氢酶则偏好NAD+作为辅酶。因为木糖还原酶和木糖醇脱氢酶辅因子偏好性的不同，导致木糖在利用这个途径转化的过程中有大量副产物木糖醇的积累，降低了目标产物的产率 (Cunha et al.,Biotechnology for Biofuels,2019,12(1):1-14)。第二种微生物木糖利用途径是大部分存在于细菌的木糖异构酶途径。在该途径中只涉及到木糖异构酶一个关键酶，它可以直接将木糖异构生成木酮糖，期间不依赖于辅因子(Hou etal.,Journal of Bioscience and Bioengineering,2016,121(2):160-165；Brat et al.,Applied and Environmental Microbiology,2009, 75(8):2304-2311)。但是目前只有极少数木糖异构酶能够在酿酒酵母中表现出活性，限制了基于木糖异构酶的木糖代谢途径的应用。
一些研究尝试将木糖异构酶基因在酿酒酵母等酵母细胞中进行表达，但是大多数表达的木糖异构酶都没有活性，推测原因可能是蛋白的错误折叠、翻译后修饰、二硫键形成。对在酿酒酵母中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，但也不是在酿酒酵母中活性表达的充分条件。目前公布的在酿酒酵母中有活性的木糖异构酶包括来自真菌的Piromyces sp.E2、Orpinomyces sp.ukk1、Termite gut (unspecified)，细菌的Thermus thermophilus、Clostridiumphytofermentans、Soil—xym1 (unspecified)、Soil—xym2(unspecified)、Bacteroidesstercoris、Ruminococcus flavefaciens、 Prevotella ruminicola、Burkholderiacenocepacia、Bacteroides vulgatus、Bovine rumen(unspecified)、Sorangiumcellulosum、Uncultured Lachnospira sp.clone XI58444和Passalid beetle gut—8054_2 (unspecified)。但是仅有Piromyces sp.E2、Clostridium phytofermentans和Bovine rumen (unspecified)在酿酒酵母等酵母细胞中表现出较高的活力，发掘更多的在酿酒酵母等酵母细胞中有活力的木糖异构酶对于木糖转化，尤其是木质纤维素资源中木糖的转化具有重要意义。
发明内容
针对现有技术的不足，本发明提供了一种在酵母细胞中高活性表达的木糖异构酶及应用。
本发明的目的是通过以下技术方案实现的：一种在酵母细胞中高活性表达的木糖异构酶，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.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8所示的核苷酸序列；
(2)SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8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.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8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.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8所示核苷酸序列的核苷酸序列。
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)、克鲁维酵母(Kluyveromyces)。
进一步地，所述宿主细胞优选为酿酒酵母细胞。
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，其中共同表达的形式包括SEQ ID NO.5+SEQ ID NO.6(组合1)、SEQ ID NO.5+SEQID NO.7 (组合2)、SEQ ID NO.5+SEQ ID NO.8(组合3)、SEQ ID NO.6+SEQ ID NO.7(组合4)、 SEQ ID NO.6+SEQ ID NO.8(组合5)、SEQ ID NO.7+SEQ ID NO.8(组合6)、SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.7(组合7)、SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.8(组合 8)、SEQ ID NO.5+SEQ ID NO.7+SEQ ID NO.8(组合9)、SEQ ID NO.6+SEQ ID NO.7+SEQ IDNO.8(组合10)、SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.7+SEQ ID NO.8(组合11)。
进一步地，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。
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。这四种木糖异构酶分别来自于Acetanaerobacterium elongatum、 Bacterium J10、Hallella seregens、Streptobacillus canis菌株，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是四种木糖异构酶在酿酒酵母中游离表达时，重组酿酒酵母CRD3AE、CRD3BJ、CRD3HS、CRD3SC以初始40g/L木糖为碳源发酵192小时后的发酵液成分柱状图；
图2是四种木糖异构酶整合至酿酒酵母染色体时重组酿酒酵母CRD5AE、CRD5BJ、CRD5HS、CRD5SC以初始40g/L木糖进行发酵的发酵曲线图，其中，(a)是CRD4AE的发酵曲线图、(b)是CRD4BJ的发酵曲线图、(c)是CRD4HS的发酵曲线图、(d)是CRD4SC 的发酵曲线图；
图3是四种木糖异构酶整合至酿酒酵母染色体时重组酿酒酵母CRD5AE、CRD5BJ、CRD5HS、CRD5SC以初始80g/L葡萄糖、40g/L木糖的混糖培养基进行发酵的发酵曲线图，其中，(a)是CRD4AE的发酵曲线图、(b)是CRD4BJ的发酵曲线图、(c)是CRD4HS的发酵曲线图、(d)是CRD4SC的发酵曲线图；
图4是四种木糖异构酶整合至酿酒酵母染色体并经过驯化后重组酿酒酵母CRD5AE、 CRD5BJ、CRD5HS、CRD5SC以初始40g/L木糖的培养基进行发酵的发酵曲线图，其中，(a) 是CRD4AE的发酵曲线图、(b)是CRD4BJ的发酵曲线图、(c)是CRD4HS的发酵曲线图、 (d)是CRD4SC的发酵曲线图；
图5是四种木糖异构酶整合至酿酒酵母染色体并经过驯化后重组酿酒酵母CRD5AE、 CRD5BJ、CRD5HS、CRD5SC以初始80g/L葡萄糖、40g/L木糖的培养基进行发酵曲线图，其中，(a)是CRD4AE的发酵曲线图、(b)是CRD4BJ的发酵曲线图、(c)是CRD4HS的发酵曲线图、(d)是CRD4SC的发酵曲线图；
图6是重组酿酒酵母CRD5AE、CRD5BJ、CRD5HS、CRD5SC细胞破碎后，获得的粗酶液的木糖异构酶活力柱状图；
图7是重组酿酒酵母CRD5AE、CRD5BJ、CRD5HS、CRD5SC以30％(w/w)底物浓度DLCA(ch)预处理的玉米秸秆水解液为底物进行发酵实验曲线图，其中，(a)是CRD4AE 的发酵实验曲线图、(b)是CRD4BJ的发酵曲线图、(c)是CRD4HS的发酵实验曲线图、(d) 是CRD4SC的发酵实验曲线图；
图8是重组酿酒酵母CRD5AE、CRD5BJ、CRD5HS、CRD5SC以30％(w/w)底物浓度 DLCA(sa)预处理的玉米芯为底物进行发酵实验曲线图，其中，(a)是CRD4AE的发酵实验曲线图、(b)是CRD4BJ的发酵曲线图、(c)是CRD4HS的发酵实验曲线图、(d)是CRD4SC 的发酵实验曲线图。
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。
实施例1：四种木糖异构酶在酿酒酵母中的游离表达
1.1、游离表达载体的构建
委托金斯瑞生物科技股份有限公司对SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8的木糖异构酶核苷酸序列分别进行合成。之后将合成的四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 ID NO.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-AE、 pESC-BJ、pESC-HS、pESC-SS。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、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-AE、pESC-BJ、pESC-HS、pESC-SS转化至双倍体酿酒酵母CRD3(ATCC 26603,MATa/α,△Gre3， 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CRD3AE、 CRD3BJ、CRD3HS、CRD3SC。
1.3、重组菌株利用木糖能力测定
将酵母CRD3AE、CRD3BJ、CRD3HS、CRD3SC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1：
如图1所示，YPX培养基初始木糖浓度为40g/L，当酿酒酵母CRD3AE、CRD3BJ、CRD3HS、 CRD3SC在其中培养192h后，培养基中剩余的木糖含量分别为24.02、8.84、8.09、9.67g/L 木糖，并且伴随着菌体的生长和乙醇的生成。该结果表明，本实验室中涉及的四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。
实施例2：四种木糖异构酶基因在酿酒酵母染色体整合表达
2.1、基于Crispr-Cas9系统的染色体整合系统构建
将G418抗性基因克隆至pML104载体的HindIII-EcoRI位点，获得质粒pML-G418。通过http://crispr.dbcls.jp/查询酿酒酵母delta序列的20bp靶点序列，构建至pML-G418质粒 sgRNA表达框的5’端，获得pML-delta质粒。以酿酒酵母基因组为模板，PCR扩增得到delta 序列的上下游片段、TDH3启动子、CYC1终止子，重叠PCR得到含有delta序列上游片段、 TDH3启动子、木糖异构酶基因、CYC1终止子、delta序列下游片段的基因片段，将其和质粒pML-delta转化至酿酒酵母CRD3，转接至YPX液体培养基(400μg/mL G418)，30℃、150 rpm培养至培养液稍显浑浊。
2.2、筛选染色体整合木糖异构酶基因的酿酒酵母
取YPX液体培养基中生长的酵母涂布于YPX平板培养，30℃静置培养至有单菌落出现。分别挑取YPX平板上的单菌落至酵母裂解缓冲液，85℃处理30min，然后以其为模板，PCR扩增相应的木糖异构酶基因，获得相应大小PCR产物，测序确定木糖异构酶基因整合至酵母染色体。将含有SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8对应的木糖异构酶核苷酸片段的酵母命名为CRD4AE、CRD4BJ、CRD4HS、CRD4SC。
2.3、CRD4AE、CRD4BJ、CRD4HS、CRD4SC酵母木糖利用的测定
将酵母CRD4AE、CRD4BJ、CRD4HS、CRD4SC在YPD液体培养基(2％蛋白胨、1％酵母提取物、2％葡萄糖)中30℃、150rpm过夜培养作为种子液，之后以初始OD600为1.0 接入YPDX(2％蛋白胨、1％酵母提取物、8％平葡萄糖、4％木糖)、YPX(2％蛋白胨、1％酵母提取物、4％木糖)培养基，30℃、150rpm进行厌氧发酵实验。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：
以YPX(2％蛋白胨、1％酵母提取物、4％木糖)培养基进行发酵时，CRD4HS、CRD4BJ、CRD4AE、CRD4SC分别在72h、84h、108h、108h消耗完40g/L木糖，其乙醇得率分别为0.38、0.38、0.40和0.39g乙醇/g木糖(图2)。以YPDX(2％蛋白胨、1％酵母提取物、8％平葡萄糖、4％木糖)培养基进行发酵时，CRD4HS、CRD4BJ、CRD4AE、CRD4SC 4种重组酿酒酵母均在12h内将葡萄糖消耗完全，120h利用10g/L左右的木糖(图3)。
实施例3：通过菌株驯化提高CRD5HS、CRD5BJ、CRD5AE、CRD5SS利用木糖的能力
将酵母CRD4HS、CRD4BJ、CRD4AE、CRD4SC在YPX(2％蛋白胨、1％酵母提取物、 4％木糖)培养基中连续传代培养，其木糖利用速率和生长速率随着传代不断提升，最终获得稳定的驯化酵母，命名为CRD5HS、CRD5BJ、CRD5AE、CRD5SC。
分别将CRD5HS、CRD5BJ、CRD5AE、CRD5SC酵母在YPX(2％蛋白胨、1％酵母提取物、4％木糖)培养基中过夜培养，以初始OD600为1.0接入YPDX(2％蛋白胨、1％酵母提取物、8％葡萄糖、4％木糖)、YPX(2％蛋白胨、1％酵母提取物、4％木糖)培养基，30℃、 150rpm进行厌氧发酵实验。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3：
YPX(2％蛋白胨、1％酵母提取物、4％木糖)培养基进行发酵时，驯化后的菌株CRD5HS、 CRD5BJ在14h剩余7.54、5.09g/L木糖，16h基本利用完40g/L木糖。CRD5AE、CRD5SC在14h剩余11.87、10.76g/L木糖，18h基本利用完40g/L木糖(图4)。以YPDX(2％蛋白胨、1％酵母提取物、8％葡萄糖、4％木糖)培养基进行发酵时，CRD5HS、CRD5BJ、CRD5AE、 CRD5SC在14h消耗完80g/L葡萄糖。10h时，木糖开始利用，18h分别剩余4.45、4.02、 25.21、20.40g/L木糖(图5)。上述结果表明导入四种木糖异构酶的重组酿酒酵母菌株经过菌株驯化后利用木糖的速度明显加快。
实施例4：木糖异构酶酶活测量
4.1、重组酿酒酵母细胞破碎
将重组酿酒酵母细胞接种于YPD(2％蛋白胨、1％酵母提取物、2％葡萄糖)液体培养基， 150rpm、30℃培养至培养液OD600达到3.0时，将菌液低温离心收集菌体至离心管。向离心管中加入含有蛋白酶抑制剂PMSF和NaF的100mM Tris-Hcl(pH7.5)缓冲液和一定量的酸洗玻璃珠(2g/g细胞湿重)，使用旋涡震荡仪混匀并破碎细胞。4℃、12000rpm离心10min，收集上清液。使用考马斯亮蓝法测量上清液在595nm处的吸收值来确定蛋白浓度。
4.2、木糖异构酶酶活测量
酶活测量体系为：66mM木糖，10mM MgCl2，1/3体积的粗酶液，100mM Tris-HCl缓冲液(pH7.5)。30℃反应30min，沸水浴10min终止反应。使用高效液相色谱(HPLC)测量木糖的消耗和木酮糖的生成。每分钟催化产生1μM木酮糖的酶量为1个酶活力单位。
结果4：
酿酒酵母CRD5AE、CRD5BJ、CRD5HS和CRD5SC均表现出高的木糖异构酶活性，其活性分别为0.76、3.30、1.05和2.02U/mg蛋白质。
实施例5：CRD5HS、CRD5BJ、CRD5AE、CRD5SC酵母以DLCA(ch)玉米秸秆水解液为底物进行发酵
5.1、DLC(ch)预处理玉米秸秆：
DLC(ch)预处理按照文献(Chen et al.,Green Chemistry,2021,23:4828-4839)中描述进行，具体地，首先将玉米秸秆水洗至洗涤水颜色接近无色，将水洗后的秸秆放至60℃烘箱烘干至水分为10％-20％。对烘干后的玉米秸秆进行DLC(ch)预处理(densifyinglignocellulosic biomass with calcium hydroxide,氢氧化钙辅助密化预处理)，即先将氢氧化钙溶液均匀的喷洒至玉米秸秆上，其中氢氧化钙和水的加量分别为0.15、0.5g/g玉米秸秆，然后使用造粒机将秸秆制粒。将制备成颗粒状的玉米秸秆晾干后于室温储存待用。
5.2、DLCA(ch)玉米秸秆水解：
在酶水解之前，首先使用高温灭菌锅对DLC(ch)玉米秸秆进行进一步处理，条件为：秸秆底物浓度为25％(w/w)、121℃反应60min。待处理后的DLC(ch)玉米秸秆温度降至室温后，使用硫酸调节pH为中性，于通风橱晾干，直至水分为10％左右。使用30％(w/w)底物浓度的DLCA(ch)玉米秸秆进行水解，纤维素酶为
Figure BDA0003627644390000081
CTec2(87mg蛋白/mL)，酶加量为 20mg蛋白/g葡聚糖。秸秆和纤维素酶分两批加入，即初始加入50％质量的秸秆和纤维素酶， 4h后加入剩余的秸秆和纤维素酶。水解条件为pH 4.8，50℃、250rpm水解72h。
5.3、DLCA(ch)玉米秸秆酶水解液发酵：
CRD5HS、CRD5BJ、CRD5AE、CRD5SC酵母在YPX(2％蛋白胨、1％酵母提取物、4％木糖)培养基中以30℃、150rpm进行种子培养。培养好的种子液以初始OD600为2.0接入 30％(w/w)底物浓度DLCA(ch)玉米秸秆水解液，并添加5g/L酵母粉和10g/L蛋白胨，调节pH为5.5，30℃、150rpm进行厌氧发酵实验。
结果5：
图7显示了含有木糖异构酶的重组菌株CRD5HS、CRD5BJ、CRD5AE、CRD5SC利用 DLCA(ch)玉米秸秆水解液进行发酵时的葡萄糖、木糖和乙醇浓度变化。DLCA(ch)玉米秸秆水解液中初始葡萄糖和木糖浓度分别为116.31、42.90g/L。四株重组酵母均在24h消耗完所有葡萄糖。120h时，CRD5HS、CRD5BJ、CRD5AE、CRD5SC菌株分别消耗39.86、34.46、 31.40、20.86g/L木糖，并伴随着73.72、71.00、67.68、65.85g/L乙醇生成。以上结果说明含有木糖异构酶的酿酒酵母菌可以利用玉米秸秆水解液中的葡萄糖和木糖两种糖进行发酵，并生成乙醇。
实施例6：CRD5HS、CRD5BJ、CRD5AE、CRD5SC酵母以DLCA(sa)玉米芯水解液为底物进行发酵
6.1、DLC(sa)预处理：
DLC(sa)预处理按照文献(Yuan et al.,Renewable Energy,2022,182:377-389)中描述进行，具体地，首先将玉米芯水洗至洗涤水颜色接近无色，将水洗后的玉米芯放至60℃烘箱烘干至水分为10％-20％。对烘干后的玉米芯进行DLC(sa)预处理(densifyinglignocellulosic biomass with sulfuric acid,硫酸辅助密化预处理)，即首先将硫酸溶液均匀的喷洒在秸秆上面，硫酸和水的加量分别为0.075、0.5g/g玉米芯，然后使用造粒机将玉米芯制粒。将制备成颗粒状的玉米芯晾干后于室温储存待用。
6.2、DLCA(sa)玉米芯水解：
在酶水解之前，首先使用高温灭菌锅对DLC(sa)玉米芯进行进一步处理，条件为：秸秆底物浓度为30％(w/w)、121℃反应20min。待处理后的DLC(ch)玉米芯温度降至室温后，使用氢氧化钙调节pH为中性，于通风橱晾干，直至水分为10％左右。使用30％(w/w)底物浓度的DLCA(sa)玉米芯进行水解，其中使用的纤维素酶为
Figure BDA0003627644390000091
CTec2(87mg蛋白/mL)，酶加量为20mg蛋白/g葡聚糖。秸秆和纤维素酶分两批加入，即初始加入50％质量的秸秆和纤维素酶，4h后加入剩余的秸秆和纤维素酶。水解条件为pH 4.8，50℃、250rpm水解72h。
6.3、DLCA(sa)玉米芯酶水解液发酵：
CRD5HS、CRD5BJ、CRD5AE、CRD5SC酵母在YPX(2％蛋白胨、1％酵母提取物、4％木糖)培养基中以30℃、150rpm进行种子培养。将培养好的种子液以初始OD600为2.0接入 30％(w/w)底物浓度DLCA(sa)玉米芯酶水解液中，并添加5g/L酵母粉和10g/L蛋白中。调节pH为5.5，30℃、150rpm进行厌氧发酵实验。
结果6：
图8显示了含有木糖异构酶的重组菌株CRD5HS、CRD5BJ、CRD5AE、CRD5SC利用 DLCA(sa)玉米芯酶水解液发酵时水解液中葡萄糖、木糖和乙醇浓度变化。DLCA(sa)玉米芯水解液中初始葡萄糖和木糖浓度分别为96.27g/L和94.09g/L木糖。四株重组酵母均在24h消耗完所有葡萄糖。120h时，CRD5HS、CRD5BJ、CRD5AE、CRD5SC菌株分别消耗69.04、 57.97、45.04、25.04g/L木糖，伴随着76.78、69.38、64.09、55.21g/L乙醇的生成。以上结果说明含有木糖异构酶的酿酒酵母菌可以利用玉米芯水解液中的葡萄糖和木糖两种糖进行发酵，并生成乙醇。
序列表
<110> 南京理工大学
<120> 在酵母细胞中高活性表达的木糖异构酶及应用
<141> 2022-05-05
<160> 8
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gcctttgctc tcggcctgat taaggctgct gcagctatcg aagacggcag actggataag 1140
tttgttgaaa accgttatgc ctcctacaaa accggcatcg gtaaagacat tgttgacggt 1200
aaggttaccc tcgagcagct cgaagcctac accctcaaga acggcgagcc caagatggaa 1260
agcggcaagc aggaatacct tgagagcgta ctcaacgaga ttatctttgg ttaa 1314
<210> 6
<211> 1317
<212> DNA
<213> Bacterium J10（2018）
<400> 6
atggcaacaa aagagtattt tcccgggata ggtaaaatcc agtttgaagg caaagacagc 60
aagaatccgc tggcttaccg ctactatgac gccgaaaagg taatcctcgg caaaaaaatg 120
aaagattggc tgaagtttgc catggcatgg tggcacactc tttgcgccga gggcggcgac 180
cagttcggcg gcggcacaaa gaaatttcct tggaacgagg gcaacgatgc cgtagaaatc 240
gcaaaacaaa aagtcgatgc aggcttcgaa ttcatgcaga agatgggcat cgaatacttc 300
tgcttccatg atgtcgacct cgtaagcgaa ggcaattctg tagaagaata tgaaagcaac 360
ctcaaggcta ccgttgctta tctcaaggaa aagatggccg agaccggcat caaaaacctc 420
tggggtacag ccaatgtgtt cggcaacggc cgttacatga acggagcagc caccaaccct 480
gactttgacg tagtagcacg cgctgccgtt cagatcaaga acgccattga cgccactatc 540
gagctcggcg gtaccaacta cgtattctgg ggtggtcgcg aaggctacat gtcactgctc 600
aataccgacc agaagcgtga gaaagagcac ttggccatga tgcttaccat agcacgcgac 660
tacgcacgcg ccaaaggctt cacaggcaca ttcctcatcg agcccaagcc catggaaccc 720
tccaagcacc agtatgatgt tgacagcgaa accgtaatag gcttcctcaa ggctcacggt 780
cttgacaagg acttcaagct caacatcgag gtaaaccacg ctactctcgc cggccacaca 840
ttcgagcacg aactcgcagt agctgtcgac aacaacatgc tcggctctat cgacgccaac 900
cgcggtgact accagaacgg atgggatacc gaccagttcc ccatcgacaa cttcgagctc 960
attcaggcca tgatgcagat catacgcaac ggcggcctcg gcaacggcgg taccaatttc 1020
gacgcaaaaa cccgtcgcaa ctctaccgat ctcgaagaca tattcatcgc ccatatcgcc 1080
ggcatggacg caatggcacg cgcactcgag agcgcagccg ctctgctcga agagtcgccc 1140
tacaagaaga tgcttgccga ccgctacgca tcgtttgaca gcggcaaggg caaggaattc 1200
gaggaaggca agctcacact cgaggaagta gccgcctacg gcaaagaggt caacgaaccc 1260
aagcagacca gcggcaagca ggaactttat gaggctatcg tagccatgta ctgctaa 1317
<210> 7
<211> 1320
<212> DNA
<213> Hallella seregens
<400> 7
atgactaaag aatacttccc agaaatcggc aagattccat tcgaaggtac cgaatccaag 60
accccaatgg ctttccacta ctacgaacca gaaagagttg ttaagggtaa gaagatgaag 120
gactggttga agttcgctat ggcttggtgg cacaccttag gtcaagccag tggtgaccaa 180
ttcggtggtc aaaccagaaa ctatgcttgg gacgctgacg ccaacccagt tcaaagagct 240
aaagacaagg tcgacgctgg tttcgaaatt atgaccaaac ttggtatcga atacttctgt 300
ttccatgatg tcgatttggt tgacccagac gatgacattg acagatacga agctaacatg 360
gctgccgtta ctgactactt gaaggaaaag atggccgccg acccaaccaa gaaattgttg 420
tggggtactg ctaacgtttt ctctgacaag agatacatga acggtgctgc taccaaccca 480
aactttgatg ttgtcgctcg tgctgctgtc caaatcaaga acgccattga cgcgaccatc 540
aagctgggtg gtcaaaacta cgtcttctgg ggtggaagag aaggttacat gtctttgtta 600
aacactcaaa tgcaaagaga aaagaaccat ttggctagaa tgttgactgc cgctagggac 660
tacgctagag ctcaaggttt caagggcact tttttgattg aaccaaagcc atgtgaacca 720
actaagcacc aatacgacgt tgacactgaa accgttattg gtttcttgag agctaatggt 780
ttagataagg atttcaaggt taacattgaa gtcaaccacg ctactttggc cggtcacact 840
ttcgaacacg aattagctgt tgctgtcgat aacggtatgt tgggttctat cgacgctaac 900
agaggtgatg ctcaaaacgg ttgggatacc gatcaattcc caattgacaa ctttgagcta 960
acccaagcta tgatgcaaat catcagaaat ggtggtttgg gtaacggtgg ttccaacttc 1020
gatgcaaaga tcagaagaaa ctccacagat cctgaagata ttttcttggc tcacatctcc 1080
ggtatggacg ccatggctcg tgctttattg aacgctgccg ctatcttgga cgaatctgaa 1140
ttgccacaaa tgctaagaga tcgttacgct tctttcgatg aaggtcaagg taaggctttt 1200
gaagaaggta gattgtcttt ggaagacttg gtggattacg ccaagcaaaa tggtgaacct 1260
agacagatct ctggtaagca agaattgtat gaaactattg tcgctttgta ctccaaatga 1320
<210> 8
<211> 1314
<212> DNA
<213> Streptobacillus canis
<400> 8
atgaaggaat atttcgtcaa catcccaaag attaagttcg aaggtccaga ctctaagaat 60
ccattgtctt tcaagtacta cgacgctgaa agagttatca acggtaagaa gatgaaggac 120
cacttgaaat tcgctatgtc ttggtggcac accatcgttg ctgaaggtgt tgacccattc 180
ggtagaggta ccatcgacag aaagtacggt gaaattgatg aaatcgccag atccaaagcc 240
aaggttgacg ctggctttga attgatggaa aaattaggta tcgaatactt ctgtttccat 300
gatgtcgata ttgctgtcga aggtaacaac tttaaggaat acagaaagaa cttgaaggaa 360
atcgtccaat acatcaaggg taagatggaa aacactaaca tcaagttgtt gtggggtact 420
gccaattgtt tctctaaccc aatttacatg cacggtgctg ctacctcttg taacgttgat 480
gcctttgctc acgctgcttc tcaaatcaaa aattccattg atgcaactat tgaattgaac 540
ggttcaggat acgttttctg gggtgggaga gaaggttatg aaactttatt aaacaccgat 600
atgggtttcg aattagacaa cttagctaga ttgatgaaga tggctgttaa gtacgctaga 660
gacaagggtt tcaagggtga tttctacatc gaacctaagc caaaagaacc aactaagcac 720
caatatgact tcgatgttgc taccaccttg gctttcttga gaaagtacgg tttggaaaac 780
gattttaaga tgaacattga agctaaccat gctactttgt ccggtcacac tttccaacac 840
gaattgaacg ttgcccgtgt taacaacgtc ttcggttcca ttgatgctaa ccaaggtgac 900
atgttgttgg gttgggacac tgaccaattc ccatccaaca tttacgatgc cacactagcc 960
atgtacgaag tcattaaggc tggtggtttc accaacggtg gtttgaactt cgacgctaag 1020
gtgagaagag gttccttcac tttcgaagac atcgtcttgg cttacatctt gggtatggac 1080
acctttgcca agggtctgat taaggctttc gagattatag aagatggtag aatcgaagag 1140
aacatcaaga acagatactc ttcttacaac tctgaaattg gtaagaaaat cttggacgaa 1200
aataccaact tggaagaatt ggaaaactac attgaaaaca aggaaaagat aactatggaa 1260
agtggtcgtc aagaatacct agaatctatc ttgaaccaaa tcattttgcg ttga 1314

Claims (8)

1.一种在酵母细胞中高活性表达的木糖异构酶，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所述在酵母细胞中高活性表达的木糖异构酶，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.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8所示的核苷酸序列；
(2)SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8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.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8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.5、SEQ ID NO.6、SEQ ID NO.7、SEQ IDNO.8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)。
4.根据权利要求3所述的木糖异构酶，其特征在于，所述宿主细胞优选为酿酒酵母细胞。
5.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。
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，其中共同表达的形式包括SEQ ID NO.5+SEQ IDNO.6(组合1)、SEQ ID NO.5+SEQ ID NO.7(组合2)、SEQ ID NO.5+SEQ ID NO.8(组合3)、SEQID NO.6+SEQ ID NO.7(组合4)、SEQ ID NO.6+SEQ ID NO.8(组合5)、SEQ ID NO.7+SEQ IDNO.8(组合6)、SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.7(组合7)、SEQ ID NO.5+SEQ IDNO.6+SEQ ID NO.8(组合8)、SEQ ID NO.5+SEQ ID NO.7+SEQ ID NO.8(组合9)、SEQ IDNO.6+SEQ ID NO.7+SEQ ID NO.8(组合10)、SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.7+SEQID NO.8(组合11)。
7.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。
8.一种权利要求1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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