WO2018045591A1 - 一种能降解利用半纤维素的基因重组产朊假丝酵母及其应用 - Google Patents
一种能降解利用半纤维素的基因重组产朊假丝酵母及其应用 Download PDFInfo
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Definitions
- the invention relates to the field of genetic engineering and fermentation engineering, in particular to a genetically recombined Candida utilis which can degrade and utilize hemicellulose, and the application thereof is specifically for establishing a co-production by genetically recombinant Candida utilis A holistic bioprocess of xylooligosaccharides, arabinose, and xylitol.
- Xylooligosaccharides, xylitol and arabinose are prebiotics and have important physiological functions. Studies have shown that xylooligosaccharides, xylitol and arabinose have important physiological effects, including: (1) as a prebiotic, can not be digested and absorbed in the human body, can effectively promote probiotics such as bifidobacteria Proliferate, thereby inhibiting the growth of harmful bacteria, thereby improving the intestinal environment; (2) improving and inhibiting diarrhea and relieving constipation; (3) inhibiting the absorption of sucrose to inhibit the rise of blood sugar and obesity caused by ingestion of sucrose Such symptoms are conducive to the treatment of diabetes and weight control; (4) they supply little energy, can be used as a substitute for diabetes, for diabetic, hypoglycemic and obese patients; (5) will not cause dental caries; (6) oligomerization Xylose has an immunostimulating effect and can enhance the body's immunity.
- Xylo-oligosaccharides, xylitol and arabinose can be produced by hydrolyzing hemicellulose.
- Semi-fiber is one of the main components of lignocellulosic fiber. It is the second abundant renewable natural resource in addition to cellulose in nature. It is a complex of various components such as arabinose and glucose. Polysaccharide long chain mechanism.
- Methods for producing xylooligosaccharide, xylitol, arabinose, etc. by hydrolysis from hemicellulose include chemical extraction, acid-base hydrolysis, bio-enzymatic conversion, and the like. The chemical extraction or acid-base hydrolysis method is cumbersome to operate, has high energy consumption, and is easy to cause environmental pollution.
- the enzymatic hydrolysis method is relatively simple, and because it is not used in a large amount of chemicals, it is easy to purify, and it is advantageous to protect the environment, and the obtained product has high purity.
- enzymatic hydrolysis of hemicellulose requires the synergy of various hemicellulase such as xylanase.
- the prices of commercially available cellulase and hemicellulase are relatively expensive, which greatly increases the preparation cost and limits the industrial application of the enzymatic hydrolysis method.
- CBP consolidated bioprocessing
- CBP consolidated bioprocessing
- Hemicellulose is degraded by hemicellulase such as xylanase to produce a series of products such as xylooligosaccharide, xylose, and arabinose.
- hemicellulase such as xylanase
- xylooligosaccharide xylose
- arabinose a series of products
- xylooligosaccharide xylose
- Arabinose which produces relatively pure xylose and arabinose products
- the separation of xylose from hemicellulose hydrolysate has a very low input-to-output ratio. Therefore, it is crucial to find a simple, feasible and low-cost method for the separation, purification and preparation of xylooligosaccharides, xylose and arabinose.
- Candida utilis can convert xylose into xylitol, and has the characteristics of fast growth and high density culture. Therefore, the present invention selects Candida utilis (ATCC22023) as a host, and expresses a xylanase gene and an arabinosidase gene in the same. Can be used to assimilate the properties of xylose, and the xylose produced by hydrolysis of hemicellulose is converted into xylitol with higher economic value, which reduces the difficulty of separating xylose and xylose. And the cost also solves the problem that the arabinose and xylose are difficult to separate, and also can obtain xylitol and arabinose which have important physiological functions. It provides a new idea for the preparation and separation of xylooligosaccharides.
- the present invention provides a genetically recombined Candida utilis which can decompose hemicellulose.
- Another object of the present invention is to provide an integrated biological process for degrading hemicellulose to produce xylooligosaccharides, arabinose, xylitol.
- a genetically engineered Candida utilis that can decompose hemicellulose is constructed by cloning a xylanase gene and an arabinosidase gene into a multi-gene expression vector pScIKPr, and then transforming the recombinant expression vector into a production vector.
- Candida utilis can be recombinantly produced by Candida utilis; the multi-gene expression vector pScIKPr is obtained by removing the rDNA fragment of the multi-gene expression vector pScIKP.
- Yeast multi-gene co-expression vector pScIKP see patent number: ZL200810029630.6, a wine yeast Maternal multi-gene expression vector and its construction method and application.
- the expression vector pScIKPr was constructed by the following method:
- Saccharomyces cerevisiae multi-gene expression vector pScIKP was double-digested with Bsp119I and SacI;
- the double gene recombinant expression vector is constructed by the following method:
- the mature peptide fragment of the xylanase gene is linked to the GAP gene promoter of Candida utilis, the ⁇ -factor signal peptide fragment of Saccharomyces cerevisiae, and the CYC gene terminator of Saccharomyces cerevisiae to form a xylanase gene.
- arabinosidase gene and the xylanase gene expression vector are separately digested and ligated together to form a xylanase and arabinosidase double gene recombinant expression vector.
- the xylanase gene is derived from Trichoderma reesei, and after removing the signal peptide, mutating the 40th and 97th bases, and merging the Saccharomyces cerevisiae ⁇ signal peptide sequence, the nucleic acid sequence thereof is as shown in SEQ ID NO. Show.
- the arabinofuranosidase gene is derived from Trichoderma reesei and is mutated to the 131st base thereof, and the nucleic acid sequence thereof is shown in SEQ ID NO.
- the genetic recombination of Candida as described above is used in the degradation of hemicellulose to produce xylooligosaccharides, arabinose, xylitol.
- a process for degrading hemicellulose to produce xylooligosaccharide, arabinose, xylitol comprising the following steps:
- the genetically modified Candida glycerol strain (or the slant strain, the plate monoclonal strain) of claim 1 is inoculated with 1% yeast extract, 2% peptone, and 2 Activated culture in YPD medium containing % glucose;
- the growth of the cells reached a logarithmic growth phase, and the cells were inoculated to a YPD medium containing 2% hemicellulose at a seeding rate of 10%; and cultured at 30 ° C, 200 rpm.
- the present invention has the following beneficial effects:
- the invention constructs a genetically engineered strain of Candida utilis which secretes xylanase and arabinofuranosidase, and uses the strain as a fermentation bacterium to establish a hierarchical hemicellulose co-production of xylooligosaccharides, arabinose and xylose Integrated biological process for alcohols.
- This process is a new type of co-production process with high added value, rich product variety and easy realization.
- the semi-fiber degrading enzyme required for the process of the present invention is a fermenting strain, that is, Candida utilis genetically engineered bacteria Secretory expression production, no need to add additional commercial xylanase, which greatly saves production costs;
- chemical reagents such as acid and alkali are not used, the problem of chemical environmental pollution is avoided, and the follow-up is reduced.
- the step of chemical reagent recovery treatment not only saves the cost of chemical reagents and subsequent processing steps, but also simplifies the operation steps.
- the difficulty in the construction of recombinant Candida utilis in the present invention is how to successfully transfer the xylanase and arabinofuranosidase genes together into Candida utilis, and at the same time achieve secretion expression.
- the present invention clones the xylanase gene and the arabinofuranosidase gene together into the inventor's own proprietary Saccharomyces cerevisiae multi-gene expression vector, and converts the double gene recombinant expression vector into Candida utilis.
- a recombinant Candida utilis which simultaneously expresses xylanase and arabinofuranosidase is obtained.
- Figure 1 shows the construction of a Candida utilis expression vector for recombinant T. reesei xylanase gene and arabinofuranosidase gene.
- Figure 2 shows the results of recombinant Candida utilis degradation of bagasse hemicellulose; a: xylose change; b: xylitol change; c: xydisaccharide change; d: xylotriose change; : changes in arabinose; f: changes in glucose.
- Figure 3 is a result of thin layer chromatography of hemicellulose hydrolysate, wherein M: mixed standard, X1-X4 is xylose, xylobiose, xylotriose, and xylotetraose; 1-3: XA2 0h, 48h , 156h hydrolysate; 4-6: XYN4 0h, 48h, 156h hydrolysis product.
- pScIKP was digested with Bsp119I and SacI, and the conditions were as follows:
- the enzyme digestion reaction was carried out at 37 ° C for 2 hours, and about 5.8 kb fragment was recovered by agarose gel electrophoresis.
- reaction was carried out at 23 ° C for 10 minutes, and the reaction was terminated by adding 10 mM EDTA; and then purified by using a PCR product purification kit (Tiangen Biochemical Technology Co., Ltd.);
- step S3 The fragment obtained in the above step S2 was subjected to self-cyclization with T4 DNA ligase to obtain a modified vector pScIKPr.
- Primer for the xylanase mature peptide coding region according to the sequence of the xylanase gene xyn2 provided by Trichoderma reesei provided by NCBI, the oligo6 biological software was used to design primers for specific amplification of xyn2 and The primers for the cleavage site are ligated, and the corresponding cleavage site sequences are as follows:
- the above are the amplification primers of xyn2, and the 5' ends of the two primers are the restriction sites of XhoI and XbaI, respectively; the upstream primers simultaneously mutate the XhoI restriction site of the xyn2 coding region (bottom point).
- S12 Amplification primer for Candida utilis GAP promoter (CuGAP): According to the NCBI database, the Candida utilis GAP promoter sequence is referred to Kunigo et al. (2013). (Kunigo, M., et al. (2013). "Heterologous protein secretion by Candida utilis.” Applied Microbiology and Biotechnology 97(16): 7357-7368.), designed CuGAP amplification primers, and added corresponding restriction sites, the sequence is as follows:
- S13 Amplification primer for Saccharomyces cerevisiae ⁇ -factor secretion signal peptide: According to the Saccharomyces cerevisiae ⁇ -factor gene sequence obtained from the NCBI database, an amplification primer for the ⁇ -factor secretion signal peptide is designed, and the corresponding restriction sites are added, and the sequence is as follows:
- S14 Amplification primer for S. cerevisiae CYC gene terminator: According to the CYC gene terminator sequence of Saccharomyces cerevisiae obtained from NCBI database, the amplification primer of CYC gene terminator is designed, and the corresponding restriction sites are added, and the sequence is as follows:
- the xyn2 coding region fragment was amplified by a two-step PCR method. First, using the cDNA of T. reesei as template, the first part of xyn2 was amplified with primers Mxyn2-F and Mxyn2-SacImut-R, respectively. The second part of xyn2 was amplified with Mxyn2-SacImut-F and Mxyn2-R as primers. The two partial fragments are then ligated by overlapping PCR to obtain a complete fragment of the xyn2 coding region.
- the specific method is as follows:
- the first step of the PCR reaction system is as follows:
- PCR products were purified by PCR product purification kit (Tiangen Biochemical Technology Co., Ltd.); then the overlapping PCR reactions were carried out according to the following reaction system and reaction conditions, and the two fragments were overlapped and joined into one complete fragment:
- the second step of the PCR reaction system is as follows:
- the reaction was carried out for 5 cycles according to the reaction conditions of the first step PCR; then the upstream and downstream primers were added, and the reaction was continued for 30 cycles.
- the PCR product was identified by electrophoresis, purified and placed at -20 ° C until use.
- the genomic DNA of Candida utilis and the genomic DNA of Saccharomyces cerevisiae were used as templates to amplify the DNA fragments of the CuGAP promoter, the ⁇ -factor secretion signal peptide and the CYC terminator.
- the reaction system is as follows:
- PCR products were purified by PCR product purification kit (Tiangen Biochemical Technology Co., Ltd.) and placed at -20 ° C until use.
- the S32.xyn2 gene was cloned into an expression vector: the positive clone obtained by S31 was digested with restriction endonucleases (the restriction enzymes used in brackets): pMD19-T-XYN2 (XhoI and XbaI), pMD19 -T-CuGAP (SalI and Bsp119I), pMD19-T-aF (Bsp119I and XhoI) and pMD19-T-CYCTT (XbaI and NotI).
- the yeast multi-gene co-expression was digested with pScIKPr by SalI and NotI.
- Each of the target fragments xyn2, CuGAP, ⁇ -factor secretion signal peptide, CYC terminator and vector backbone fragment were separately recovered. Then, the recovered gene fragment and the pScIKPr backbone were ligated with T4 DNA ligase, and the ligated product was transformed into Escherichia coli, and the positive clone was identified by colony PCR method to obtain a recombinant xylanase Candida utilis expression vector (pCuGAPG ⁇ A). -XYN2).
- the above are amplification primers for abf1, and the 5' ends of the two primers are the cleavage sites of BamHI and SpeI, respectively.
- the abf1 coding region fragment was amplified by a two-step PCR method. Firstly, using the cDNA of Trichoderma reesei as template, the first part of abf1 was amplified by primers TrABF1-F and Abf1-SacImut-R, respectively. The second part of xyn2 was amplified by using Abf1-SacImut-F and TrABF1-R as primers. . The two partial fragments are then ligated by overlapping PCR to obtain a complete abf1 coding region fragment.
- the specific method is as follows:
- the first step of the PCR reaction system is as follows:
- PCR products were purified by PCR product purification kit (Tiangen Biochemical Technology Co., Ltd.); then the overlapping PCR reactions were carried out according to the following reaction system and reaction conditions, and the two fragments were overlapped and joined into one complete fragment:
- the second step of the PCR reaction system is as follows:
- the reaction was carried out for 5 cycles according to the reaction conditions of the first step PCR; then the upstream and downstream primers were added, and the reaction was continued for 30 cycles.
- the PCR product was identified by electrophoresis, purified and placed at -20 ° C until use.
- S2.abf1 gene was cloned into expression vector: pMD19-T-ABF1 and recombinant xylanase
- Candida utilis expression vector pCuGAPG ⁇ A-XYN2
- pCuGAPG ⁇ A-XYN2 were digested with restriction endonucleases BamHI and SpeI, respectively. Abf1 and vector backbone fragments.
- the recovered abf1 gene fragment and the pCuGAPG ⁇ A-XYN2 backbone were ligated with T4 DNA ligase, and the ligation product was transformed into Escherichia coli, and positive clones were identified by colony PCR to obtain recombinant xylanase and arabinosidase expression.
- Candida utilis expression vector pCuGAPG ⁇ A-XYN2-ABF1.
- Candida utilis electrotransformation The expression vectors pCuGAPG ⁇ A-XYN2-ABF1 obtained in Examples 1 and 2 were linearized with restriction endonuclease SacI; and then transformed into Candida utilis by electroporation. The transformed product was coated on YPD plate medium containing G418 antibiotic at the same time, and cultured at 30 ° C for 3 days. After the colony grew, colony PCR was performed to identify positive clones.
- Example 5 Process for degrading hemicellulose to produce xylooligosaccharide, arabinose, xylitol
- the cells When the cells were grown to the log phase, they were inoculated to a YPD medium containing 2% (dry weight) of hemicellulose at a 10% inoculation amount.
- the culture was carried out at 30 ° C, 200 rpm (two parallels per bacteria). 1.5 ml were sampled at three time points of 48h, 72h, and 156h. The sample was centrifuged at 12,000 rpm for 10 min, and the supernatant was taken out, filtered through a 0.45 ⁇ m filter, and analyzed for components by HPLC. Among them, the detection of arabinose and xylitol column BioRad HPX-87H, detection of xylooligosaccharide column BioRad HPX-42A.
- TLC Thin layer chromatography
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Abstract
一种能分解半纤维素的基因重组产朊假丝酵母及其应用。所述基因重组产朊假丝酵母能稳定表达来自里氏木霉的木聚糖酶和阿拉伯呋喃糖苷酶。利用该基因重组酵母,建立了一个能降解半纤维素联产低聚木糖、阿拉伯糖和木糖醇的统合生物工艺。运用该工艺,以20g/l的蔗渣半纤维素为底物,经培养156h后,中间过程所产生的木糖被酵母本身耗尽,低聚木糖得率为20%,此时木糖醇和阿拉伯糖分别达到最高得率为3.5%和2.7%,半纤维素水解率达到40.4%。
Description
本发明涉及基因工程及发酵工程领域,具体涉及一种能降解利用半纤维素的基因重组产朊假丝酵母及其应用,所述应用具体为以基因重组产朊假丝酵母建立一种联产低聚木糖、阿拉伯糖、木糖醇的统合生物工艺。
低聚木糖、木糖醇及阿拉伯糖均属于益生元,具有重要的生理功能。已有研究表明,低聚木糖、木糖醇及阿拉伯糖具有重要的生理作用,主要包括:(1)作为益生元,在人体内不能被消化吸收,可有效促进双歧杆菌等益生菌的增殖,从而抑制有害菌群的生长,进而改善肠道环境;(2)改善和抑制腹泻,缓解便秘;(3)可以抑制蔗糖的吸收,以抑制因摄入蔗糖而引起的血糖升高及肥胖等症状,有利于治疗糖尿病及控制体重;(4)它们供应的能量很少,可作为代糖,可供糖尿病,低血糖及肥胖病人食用;(5)不会引起龋齿;(6)低聚木糖具有免疫刺激作用,能够增强机体免疫力。
低聚木糖、木糖醇及阿拉伯糖可以通过水解半纤维生产。半纤维是木质纤维的主要成分之一,是自然界中除了纤维素之外的第二丰富的可再生自然资源;是以木聚糖为骨架,兼有阿拉伯糖、葡萄糖等多种成分构成的复杂多糖长链机构。从半纤维素中水解生产低聚木糖、木糖醇、阿拉伯糖等的方法包括化学提取、酸碱水解、生物酶解转化等方法。化学提取或酸碱水解方法操作繁琐,能耗高,且易造成环境污染。利用酶水解法操作相对简单,且因为没有大量化学物质的使用,易于纯化,而且有利于保护环境,得到的产物纯度较高。但是酶解半纤维素需要木聚糖酶(xylanase)等多种半纤维素酶(hemicellulase)的协同作用来完成。而目前商品化的纤维素酶、半纤维素酶的价格还比较昂贵,使得制备成本大幅上升,限制了酶水解法的工业应用。为解决以上这些困境,随着基因工程和代谢工程技术的进展,近几年来,统合生物工艺(consolidated bioprocessing,CBP)的概念获得了国内外生物质转化领域科学家的广泛关注。
Lynd等人(Kim S,Dale B E.Global potential bioethanol production from
wasted crops and crop residues[J].Biomass and bioenergy,2004,26(4):361-375.)提出了统合生物工艺方法(consolidated bioprocessing,CBP)的概念,即在不额外添加商业酶的情况下,由发酵微生物自身分泌表达的水解酶在同一个反应器中同时完成糖化和发酵的工艺过程。这种微生物通常是通过基因工程手段获得的重组菌。CBP工艺不需加入商业化酶,降低了生产成本,简化了操作步骤,有利于工业化生产。CBP工艺是实现半纤维素高效、绿色、环保利用的重要手段。
半纤维素经木聚糖酶等半纤维素酶降解后,可生成低聚木糖、木糖、阿拉伯糖等一系列产物,而目前还没有较为经济简便的方法能够很好地分离木糖和阿拉伯糖,得到较纯的木糖和阿拉伯糖产品;而且,由于木糖的经济价值较低,从半纤维素水解液中分离木糖,其投入产出比非常低。因此找到一个简便可行、低成本的低聚木糖、木糖和阿拉伯糖的分离纯化和制备方法在此类的研究中至关重要。
产朊假丝酵母能够将木糖转化为木糖醇,而且具有生长速度快,可以实现高密度培养等特点。因此,本发明选用产朊假丝酵母(ATCC22023)为宿主,在其体内表达木聚糖酶基因及阿拉伯糖苷酶基因。通过产朊假丝酵母能够同化木糖的性质,将半纤维素水解后产生的木糖利用,并转化为经济价值较高的木糖醇,既降低了低聚木糖和木糖分离的难度和成本,也解决了阿拉伯糖与木糖难分离的问题,同时也可以得到具有重要生理作用的木糖醇及阿拉伯糖。为低聚木糖的制备和分离提供了一个新的思路。
发明内容
本发明为了克服现有技术的上述不足,提供一种能分解利用半纤维素的基因重组产朊假丝酵母。
本发明的另一个目的是提供一种降解半纤维素联产低聚木糖、阿拉伯糖、木糖醇的统合生物工艺。
为了实现上述目的,本发明是通过以下方案予以实现的:
一种能分解利用半纤维素的基因重组产朊假丝酵母,通过以下方法构建得到:将木聚糖酶基因和阿拉伯糖苷酶基因克隆到多基因表达载体pScIKPr上,然后将重组表达载体转化产朊假丝酵母即得重组产朊假丝酵母;所述多基因表达载体pScIKPr是通过将多基因表达载体pScIKP的rDNA片段去除而得到。
酵母多基因共表达载体pScIKP见专利号:ZL200810029630.6,一种酿酒酵
母多基因表达载体及其构建方法与应用。
所述表达载体pScIKPr通过以下方法构建所得:
(1)将酿酒酵母多基因表达载体pScIKP用Bsp119I和SacI进行双酶切;
(2)将上述(1)双酶切所得片段用S1核酸酶进行双链DNA末端平滑处理;
(3)将上述(2)所得片段经T4连接酶自连接得到pScIKPr。
所述双基因重组表达载体通过以下方法构建所得:
(1)将木聚糖酶基因的成熟肽片段与产朊假丝酵母的GAP基因启动子、酿酒酵母的α因子信号肽片段以及酿酒酵母的CYC基因终止子连接在一起构成木聚糖酶基因表达盒;
(2)将上述木聚糖酶基因表达盒以及pScIKP载体分别双酶切后连接到一起,构成木聚糖酶基因表达载体;
(3)将阿拉伯糖苷酶基因以及上述木聚糖酶基因表达载体分别双酶切后连接到一起,构成木聚糖酶和阿拉伯糖苷酶双基因重组表达载体。
所述木聚糖酶基因来源于里氏木霉,经过去除其信号肽,突变第40位和97位碱基,并融合酿酒酵母α信号肽序列构成,其核酸序列如SEQ ID NO.1所示。
所述阿拉伯呋喃糖苷酶基因来源于里氏木霉,并突变其第131位碱基而成,其核酸序列如SEQ ID NO.2所示。
如上所述的基因重组产朊假丝酵母在降解半纤维素联产低聚木糖、阿拉伯糖、木糖醇中的应用。
一种降解半纤维素联产低聚木糖、阿拉伯糖、木糖醇的工艺,包括如下步骤:
(1)菌种活化:将权利要求1所述的基因重组产朊假丝酵母甘油菌种(或斜面菌种、平板单克隆菌种)接种于含1%酵母提取物、2%蛋白胨及2%葡萄糖的YPD培养基中进行活化培养;
(2)将经过活化的菌种以1%接种量接种至新鲜YPD培养基中进行扩大培养,作为种子培养液;
(3)待上步所得种子培养液中,菌体生长达到对数生长期,以10%的接种量接种至含有2%半纤维素的YPD培养基中;于30℃、200rpm培养。
(4)按时间间隔取样,检测木糖醇、阿拉伯糖及低聚木糖的生成情况。
与现有技术相比,本发明具有如下有益效果:
本发明构建能分泌表达木聚糖酶和阿拉伯呋喃糖苷酶的产朊假丝酵母基因工程菌株,并以该菌株为发酵菌,建立分级半纤维素联产低聚木糖、阿拉伯糖和木糖醇的统合生物工艺。此工艺为一种新型的联产工艺,具有产品附加值高、产品种类丰富、容易实现等特点。与现有低聚木糖、阿拉伯糖和木糖醇的生产工艺相比,具有以下特点:首先,本发明的工艺所需的半纤维降解酶由发酵菌株,即产朊假丝酵母基因工程菌分泌表达生产,不需要额外添加商品化的木聚糖酶,大大节约了生产成本;其次,与化学降解法相比,由于没有使用酸碱等化学试剂,避免了化学环境污染的问题,而且减少后续化学试剂回收处理的步骤,既节约了化学试剂及后续处理工序的成本,又简化了操作步骤。
运用所构建的工艺,以20g/l的蔗渣半纤维素为底物,经培养156h后,中间过程所产生的木糖被酵母本身耗尽,低聚木糖得率为20%,此时木糖醇和阿拉伯糖分别达到最高得率为3.5%和2.7%。应用本发明所述的基因重组产朊假丝酵母菌株,蔗渣半纤维素水解率达到40.4%。此基因工程重组菌株的构建以及本工艺的设计不但解决了木糖和阿拉伯糖难以分离的问题,而且同时得到了具有生理功能的活性物质低聚木糖、阿拉伯糖和木糖醇。
本发明在构建重组产朊假丝酵母时需要克服的难点在于如何将木聚糖酶和阿拉伯呋喃糖苷酶基因一起成功转入产朊假丝酵母中,并同时实现分泌表达。为了克服这一困难,本发明将木聚糖酶基因和阿拉伯呋喃糖苷酶基因一起克隆到发明人自有专利酿酒酵母多基因表达载体中,将双基因重组表达载体转化产朊假丝酵母就可以得到同时表达木聚糖酶和阿拉伯呋喃糖苷酶的重组产朊假丝酵母。
图1为重组里氏木霉木聚糖酶基因和阿拉伯呋喃糖苷酶基因的产朊假丝酵母表达载体的构建。
图2为重组产朊假丝酵母降解蔗渣半纤维素的结果;a:木糖变化情况;b:木糖醇变化情况;c:木二糖变化情况含量;d:木三糖变化情况;e:阿拉伯糖变化情况;f:葡萄糖变化情况。
图3为半纤维素水解产物薄层层析结果,其中,M:混合标准品,X1-X4依次为木糖,木二糖,木三糖,木四糖;1-3:XA2 0h,48h,156h的水解产物;4-6:
XYN4 0h,48h,156h的水解产物。
下面将结合说明书附图和具体实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤、条件所作的修改或替换,均属于本发明的范围。若无特别说明,实施例中所用的实验方法均为本领域技术人员所熟知的常规方法和技术,所使用的试剂配料或材料均为通过商业途径得到。
实施例1pScIKPr载体的构建
S1.将pScIKP用Bsp119I和SacI进行双酶切,酶切条件如下:
将上述各成分混匀后,于37℃进行酶切反应2小时,用琼脂糖凝胶电泳回收约5.8kb片段。
S2.将上述5.8kb片段用S1核酸酶进行末端平滑处理,条件如下:
将上述个反应组分混匀后,于23℃反应10分钟,加入10mM EDTA终止反应;然后用PCR产物纯化试剂盒(天根生化科技公司)纯化备用;
S3.将上述步骤S2所得片段用T4 DNA连接酶进行自环化,获得改造后的载体pScIKPr。
实施例2木聚糖酶基因(xyn2)表达盒的构建
S1.目的基因引物的设计:
S11.木聚糖酶成熟肽编码区的引物:根据NCBI中提供的里氏木霉的木聚糖酶基因xyn2的序列,运用oligo6生物软件设计用于特异性扩增xyn2的引物及
突变酶切位点的引物,并加上相应的酶切位点序列如下:
xyn2引物及突变XhoI和SacI位点的引物:
以上为xyn2的扩增引物,两引物的5’端分别为XhoI和XbaI的酶切位点;上游引物同时突变了xyn2编码区的XhoI酶切位点(下加点)。
以上为突变xyn2编码区SacI位点的引物(下加点)。
S12:产朊假丝酵母GAP启动子(CuGAP)的扩增引物:根据NCBI数据库所得产朊假丝酵母GAP启动子序列,参考Kunigo等的文献(Kunigo,M.,et al.(2013)."Heterologous protein secretion by Candida utilis."Applied Microbiology and Biotechnology 97(16):7357-7368.),设计CuGAP扩增引物,并添加相应的酶切位点,序列如下:
S13:酿酒酵母α因子分泌信号肽的扩增引物:根据NCBI数据库所得酿酒酵母α因子基因序列,设计α因子分泌信号肽的扩增引物,并添加相应的酶切位点,序列如下:
S14:酿酒酵母CYC基因终止子的扩增引物:根据NCBI数据库所得酿酒酵母的CYC基因终止子序列,设计CYC基因终止子的扩增引物,并添加相应的酶切位点,序列如下:
S2:目的基因片段的扩增:
S21.xyn2编码区片段的PCR扩增:以两步PCR的方法扩增xyn2编码区片段。首先以里氏木霉的cDNA为模板,分别用引物Mxyn2-F和Mxyn2-SacImut-R为引物扩增xyn2第一部分;以Mxyn2-SacImut-F和Mxyn2-R为引物扩增xyn2第二部分。然后采用重叠PCR的方法将前述两部分片段连接起来,获得完整的xyn2编码区片段。具体方法如下:
第一步PCR反应体系如下:
反应条件:
反应结束后,PCR产物分别用PCR产物纯化试剂盒(天根生化科技公司)纯化;然后按照以下反应体系和反应条件进行重叠PCR反应,将两片段重叠连接成为一个完整片段:
第二步PCR反应体系如下:
首先将除上下游引物之外的所有组分混匀,按照第一步PCR的反应条件反应5个循环;然后再加入上下游引物,继续反应30个循环。PCR产物经电泳鉴定,纯化后置于-20℃备用。
S22.CuGAP启动子、α因子分泌信号肽和CYC终止子的扩增:
分别以产朊假丝酵母的基因组DNA和酿酒酵母的基因组DNA为模板,扩增CuGAP启动子、α因子分泌信号肽和CYC终止子的DNA片段。反应体系如下:
反应条件:
反应结束后,PCR产物分别用PCR产物纯化试剂盒(天根生化科技公司)纯化后置于-20℃备用。
S3.xyn2基因表达盒克隆进表达载体
S31.基因片段的T-A克隆及测序:将上述S2所纯化得到的xyn2成熟肽、CuGAP启动子、α因子分泌信号肽和CYC终止子的DNA片段克隆进pMD19-T
载体,并转化进大肠杆菌DH5α。运用菌落PCR方法鉴定阳性克隆,并挑选阳性克隆进行测序分析。获得测序正确的阳性克隆,分别命名为:pMD19-T-XYN2、pMD19-T-CuGAP、pMD19-T-aF和pMD19-T-CYCTT。
S32.xyn2基因克隆进表达载体:分别用限制性内切酶对S31所得阳性克隆进行双酶切(括号中为所用的限制性内切酶):pMD19-T-XYN2(XhoI和XbaI)、pMD19-T-CuGAP(SalI和Bsp119I)、pMD19-T-aF(Bsp119I和XhoI)和pMD19-T-CYCTT(XbaI和NotI)。同时,用SalI和NotI对酵母多基因共表达载pScIKPr进行双酶切。分别回收各目的片段xyn2、CuGAP、α因子分泌信号肽、CYC终止子和载体骨架片段。然后用T4 DNA连接酶将回收的到的基因片段和pScIKPr骨架进行连接,并将连接产物转化大肠杆菌,利用菌落PCR方法鉴定阳性克隆,获得重组木聚糖酶产朊假丝酵母表达载体(pCuGAPGαA-XYN2)。
实施例3阿拉伯呋喃糖苷酶基因(abf1)表达载体的构建
S1.目的基因引物的设计及扩增:根据NCBI中提供的里氏木霉的阿拉伯呋喃糖苷酶基因abf1的序列,运用oligo6生物软件设计用于特异性扩增abf1的引物及突变酶切位点的引物,并加上相应的酶切位点序列如下:
abf1引物及abf1突变SacI位点的引物:
以上为abf1的扩增引物,两引物的5’端分别为BamHI和SpeI的酶切位点。
以上为突变xyn2编码区SacI位点的引物(下加点)。
S2.abf1编码区片段的PCR扩增:以两步PCR的方法扩增abf1编码区片段。首先以里氏木霉的cDNA为模板,分别用引物TrABF1-F和Abf1-SacImut-R为引物扩增abf1的第一部分;以Abf1-SacImut-F和TrABF1-R为引物扩增xyn2第二部分。然后采用重叠PCR的方法将前述两部分片段连接起来,获得完整的abf1编码区片段。具体方法如下:
第一步PCR反应体系如下:
反应条件:
反应结束后,PCR产物分别用PCR产物纯化试剂盒(天根生化科技公司)纯化;然后按照以下反应体系和反应条件进行重叠PCR反应,将两片段重叠连接成为一个完整片段:
第二步PCR反应体系如下:
首先将除上下游引物之外的所有组分混匀,按照第一步PCR的反应条件反应5个循环;然后再加入上下游引物,继续反应30个循环。PCR产物经电泳鉴定,纯化后置于-20℃备用。
S3.abf1基因克隆进表达载体
S31.abf1基因的T-A克隆及测序:将上述S2所纯化得到的abf1片段克隆进pMD19-T载体,并转化进大肠杆菌DH5α。运用菌落PCR方法鉴定阳性克隆,并挑选阳性克隆进行测序分析。获得测序正确的阳性克隆(pMD19-T-ABF1)。
S2.abf1基因克隆进表达载体:用限制性内切酶BamHI和SpeI分别对pMD19-T-ABF1和重组木聚糖酶产朊假丝酵母表达载体(pCuGAPGαA-XYN2)进行双酶切;分别回收abf1和载体骨架片段。然后用T4 DNA连接酶将回收的到的abf1基因片段和pCuGAPGαA-XYN2骨架进行连接,并将连接产物转化大肠杆菌,利用菌落PCR方法鉴定阳性克隆,获得重组木聚糖酶和阿拉伯糖苷酶双表达产朊假丝酵母表达载体(pCuGAPGαA-XYN2-ABF1)。
实施例4产朊假丝酵母电转化及重组基因工程菌株的筛选
S1.产朊假丝酵母电转化:用限制性内切酶SacI将实施例1和2所得的表达载体pCuGAPGαA-XYN2-ABF1进行线性化;然后电击转化产朊假丝酵母。转化产物涂布在同时含有G418抗生素的YPD平板培养基,30℃培养3天,待菌落长出后,进行菌落PCR鉴定,挑选阳性克隆。
S2.重组基因工程菌株的筛选:挑选6株菌落PCR鉴定为阳性的重组子,用YPD培养基活化后,以1%的接种量,接种至新的含有10ml YPD培养基的试管中,30℃,200rpm培养;在24h,48h,72h,各取一次样,每次取1.5ml菌液,离心收集上清,分别测木聚糖酶和阿拉伯呋喃糖苷酶酶活。挑选出酶活最高的重组子(命名为XA2),进行后续工艺构建。
实施例5一种降解半纤维素联产低聚木糖、阿拉伯糖、木糖醇的工艺
S1.甘蔗渣中半纤维素的提取及其含量的测定:根据N Jayapal等人(Jayapal N,Samanta A K,Kolte A P,et al.Value addition to sugarcane bagasse:Xylan extraction and its process optimization for xylooligosaccharides production[J].Industrial Crops and Products,2013,42:14-24.)提出的碱提取法提取蔗渣中的半纤维素,并参考Xiadi Gao等人(Gao X D,Kumar R,Charles E.Wyman.Fast hemicellulose quantification via a simple one-step acid hydrolysis[J].Biotechnology and Bioengineering,2014,9999:9.)的一步酸水解法测定所提取的半纤维素沉淀中干物质成分。
S2.低聚木糖、木糖醇及阿拉伯糖联产工艺的构建:取产朊假丝酵母野生菌,
及本实验保藏的表达里氏木霉木聚糖酶单基因的重组产朊假丝酵母菌株XYN4及上述实施例3中得到的同时表达木聚糖酶和阿拉伯呋喃糖苷酶的重组产朊假丝酵母菌株XA2进行活化。将活化过夜的菌种,以1%的接种量分别接种至5ml YPD培养基中,30℃,200rpm条件下培养。当菌体生长至对数期时,以10%的接种量接种至含有2%(干重)半纤维素的YPD培养基中。在30℃,200rpm条件下进行培养(每个菌做两个平行)。在48h,72h,156h三个时间点分别取样1.5ml。将所取样品12000rpm,离心10min,取上清,用0.45μm的过滤器过滤后,用HPLC检测分析其成分。其中,检测阿拉伯糖和木糖醇用色谱柱BioRad HPX-87H,检测低聚木糖用色谱柱BioRad HPX-42A。结果如附图3所示。从图中可以看出,产朊假丝酵母野生菌株,没有降解半纤维素的能力,整个培养过程中,没有低聚木糖,阿拉伯糖和木糖醇产生。而本发明所构建的重组菌XA2以及实验室之前构建的重组菌XYN4均能降解半纤维素产生低聚木糖和木糖醇。从图3-e可以看出,在整个培养过程中,重组菌XYN4没有产生阿拉伯糖,而重组菌XA2能降解半纤维素产生阿拉伯糖,这一结果证明了,阿拉伯糖苷酶能够降解木聚糖主链上的阿拉伯糖侧链,从而促进木聚糖酶对木聚糖的水解。
根据HPLC定量的结果显示,以20g/l的蔗渣半纤维素为底物,经培养156h后,中间过程所产生的木糖被酵母本身耗尽,低聚木糖得率为20%,此时木糖醇和阿拉伯糖分别达到最高得率为3.5%和2.7%。应用本发明所述的基因工程重组产朊假丝酵母菌株XA2,蔗渣半纤维素水解率达到40.4%。
S3.产物薄层层析(TLC)分析:为进一步分析上清液中低聚木糖的各种组分情况,参考Jingbo li等的方法,对产物进行薄层层析分析,将硅胶板在105℃活化30min后,取5μl样品上清液,点在事先在硅胶板上划好的点样点上,边点样,边用吹风机吹干,以避免样品扩散;在层析缸中加入适量展开剂,将硅胶板放入层析缸中,展开3h后,取出硅胶板,在通风橱内通风晾干,然后在硅胶板上均匀喷洒显色剂,通风晾干,然后于85℃,烘10min显色,观察条带并拍照。结果如附图4所示。
上述两个实验结果证明,本发明构建的低聚木糖,阿拉伯糖,木糖醇联产的统合生物工艺是有效的,能够同时产生上述三种产物,并消耗掉木糖,从而降低了木糖与阿拉伯糖分离的难度,同时也降低了生产成本。
Claims (9)
- 一种能分解利用半纤维素的基因重组产朊假丝酵母,其特征在于,将木聚糖酶基因和阿拉伯糖苷酶基因克隆到多基因表达载体pScIKPr上,然后将重组表达载体转化产朊假丝酵母即得重组产朊假丝酵母;所述多基因表达载体pScIKPr是通过将多基因表达载体pScIKP的rDNA片段去除而得到。
- 根据权利要求1所述的重组产朊假丝酵母,其特征在于,所述多基因表达载体pScIKPr构建方法如下:(1)将酿酒酵母多基因表达载体pScIKP用Bsp119I和SacI进行双酶切;(2)将上述(1)双酶切所得片段用S1核酸酶进行双链DNA末端平滑处理;(3)将上述(2)所得片段经T4连接酶自连接得到pScIKPr。
- 根据权利要求1所述的基因重组产朊假丝酵母,其特征在于,所述重组表达载体是通过以下方法构建得到的:(1)将木聚糖酶基因的成熟肽片段与产朊假丝酵母的GAP基因启动子、酿酒酵母的α因子信号肽片段以及酿酒酵母的CYC基因终止子连接在一起构成木聚糖酶基因表达盒;(2)将上述(1)木聚糖酶基因表达盒以及pScIKPr载体分别双酶切后连接到一起,构成木聚糖酶基因表达载体;(3)将阿拉伯糖苷酶基因以及上述(2)木聚糖酶基因表达载体分别双酶切后连接到一起,构成木聚糖酶和阿拉伯糖苷酶双基因重组表达载体。
- 根据权利要求2所述的重组表达载体,用于木聚糖酶基因表达盒以及pScIKP载体双酶切的限制性内切酶为SalI和NotI。
- 根据权利要求2所述的重组表达载体,用于阿拉伯糖苷酶基因以及木聚糖酶基因表达载体双酶切的限制性内切酶为BamHI和SpeI。
- 根据权利要求1所述的基因重组产朊假丝酵母,其特征在于,所述木聚糖酶基因来源于里氏木霉,经过去除其信号肽,突变第40位和97位碱基,并融合酿酒酵母α因子信号肽序列构成,其核酸序列如SEQ ID NO.1所示。
- 根据权利要求1所述的基因重组产朊假丝酵母,其特征在于,所述阿拉伯呋喃糖苷酶基因来源于里氏木霉,并突变其第131位碱基而成,其核酸序列如 SEQ ID NO.2所示。
- 权利要求1所述的基因重组产朊假丝酵母在降解半纤维素联产低聚木糖、阿拉伯糖、木糖醇中的应用。
- 一种降解半纤维素联产低聚木糖、阿拉伯糖、木糖醇的工艺,其特征在于,包括如下步骤:(1)菌种活化:将权利要求1所述的基因重组产朊假丝酵母甘油菌种(或斜面菌种、平板单克隆菌种)接种于含1%酵母提取物、2%蛋白胨及2%葡萄糖的YPD培养基中进行活化培养;(2)将经过活化的菌种以1%接种量接种至新鲜YPD培养基中进行扩大培养,作为种子培养液;(3)待上步所得种子培养液中,菌体生长达到对数生长期,以10%的接种量接种至含有2%半纤维素的YPD培养基中;于30℃、200rpm培养。(4)按时间间隔取样,检测木糖醇、阿拉伯糖及低聚木糖的生成情况。
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Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002262899A (ja) * | 2001-03-08 | 2002-09-17 | Godo Shusei Co Ltd | L−アラビノースの分離方法 |
CN101319225A (zh) * | 2008-07-22 | 2008-12-10 | 暨南大学 | 一种酿酒酵母多基因表达载体及其构建方法与应用 |
CN101952437A (zh) * | 2007-10-03 | 2011-01-19 | 维莱尼姆公司 | 木聚糖酶、编码它们的核酸以及其制备和应用方法 |
WO2012019175A2 (en) * | 2010-08-06 | 2012-02-09 | Mascoma Corporation | Production of malonyl-coa drived products via anaerobic pathways |
CN103725624A (zh) * | 2013-12-30 | 2014-04-16 | 广东启智生物科技有限公司 | 一种能降解利用餐厨废弃物的基因重组酿酒酵母 |
CN104039806A (zh) * | 2011-11-10 | 2014-09-10 | 马斯科马公司 | 被加工为发酵木糖和阿拉伯糖的啤酒酵母的遗传修饰菌株 |
CN105283546A (zh) * | 2013-05-10 | 2016-01-27 | 诺维信公司 | 具有木聚糖酶活性的多肽以及对其进行编码的多核苷酸 |
CN106520586A (zh) * | 2016-09-12 | 2017-03-22 | 广东启智生物科技有限公司 | 一种能降解利用半纤维素的基因重组产朊假丝酵母及其应用 |
-
2016
- 2016-09-12 WO PCT/CN2016/098748 patent/WO2018045591A1/zh active Application Filing
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002262899A (ja) * | 2001-03-08 | 2002-09-17 | Godo Shusei Co Ltd | L−アラビノースの分離方法 |
CN101952437A (zh) * | 2007-10-03 | 2011-01-19 | 维莱尼姆公司 | 木聚糖酶、编码它们的核酸以及其制备和应用方法 |
CN101319225A (zh) * | 2008-07-22 | 2008-12-10 | 暨南大学 | 一种酿酒酵母多基因表达载体及其构建方法与应用 |
WO2012019175A2 (en) * | 2010-08-06 | 2012-02-09 | Mascoma Corporation | Production of malonyl-coa drived products via anaerobic pathways |
CN104039806A (zh) * | 2011-11-10 | 2014-09-10 | 马斯科马公司 | 被加工为发酵木糖和阿拉伯糖的啤酒酵母的遗传修饰菌株 |
CN105283546A (zh) * | 2013-05-10 | 2016-01-27 | 诺维信公司 | 具有木聚糖酶活性的多肽以及对其进行编码的多核苷酸 |
CN103725624A (zh) * | 2013-12-30 | 2014-04-16 | 广东启智生物科技有限公司 | 一种能降解利用餐厨废弃物的基因重组酿酒酵母 |
CN106520586A (zh) * | 2016-09-12 | 2017-03-22 | 广东启智生物科技有限公司 | 一种能降解利用半纤维素的基因重组产朊假丝酵母及其应用 |
Non-Patent Citations (4)
Title |
---|
GONG, YINGXUE ET AL.: "Construction of Saccharomyces Cerevisiae integrated expression vector and its application in cellulose bioconvesion", JOURNAL OF SHENZHEN UNIVERSITY SCIENCE AND ENGINEERING, vol. 27, no. 1, 31 January 2010 (2010-01-31), pages 82 - 86 * |
SHAO, WEILAN ET AL.: "Molecular Biotechnology in exploiting the resource of hemicellulose", JOURNAL OF WUXI UNIVERSITY OF LIGHT INDUSTRY, vol. 21, no. 01, 31 January 2002 (2002-01-31), pages 88 - 92 * |
YANG, HONGLAN ET AL.: "Construction of integrative vector of candida utilis", ACTA MICROBIOLOGICA SINICA, vol. 49, no. 3, 4 March 2009 (2009-03-04), pages 316 - 323 * |
YIMING, LV. ET AL.: "The construction, expression, characterization of xyn2 and afb1 gene of trichoderma reesei in picha pastoris (Science paper online)", 22 April 2014 (2014-04-22) * |
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