WO2016015469A1 - 一种生产n-乙酰氨基葡萄糖的枯草芽孢杆菌及其构建方法和应用 - Google Patents

一种生产n-乙酰氨基葡萄糖的枯草芽孢杆菌及其构建方法和应用 Download PDF

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WO2016015469A1
WO2016015469A1 PCT/CN2015/073498 CN2015073498W WO2016015469A1 WO 2016015469 A1 WO2016015469 A1 WO 2016015469A1 CN 2015073498 W CN2015073498 W CN 2015073498W WO 2016015469 A1 WO2016015469 A1 WO 2016015469A1
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acetylglucosamine
bacillus subtilis
gene
producing
phosphate
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张帆
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张帆
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  • the invention belongs to the construction and application of genetic engineering bacteria, in particular to a Bacillus subtilis producing N-acetylglucosamine and a construction method and application thereof.
  • N-acetylglucosamine is a derivative of glucose which is usually polymerized into chitin by beta-1,4-glycosidic linkages.
  • Chitin is the second largest class of carbohydrates in the natural world, second only to cellulose. It is widely found in fungi, algae, shells of shrimps, crabs, insects, and cell walls of higher plants. Therefore, N-acetylglucosamine is The stock of nature is huge.
  • N-acetylglucosamine forms a repeating disaccharide unit with D-glucuronic acid to form hyaluronic acid.
  • Hyaluronic acid is a key substance for lubricating joints between joints and has an important protective effect on bone joints.
  • N-acetylglucosamine can be used as an effective drug for the treatment and prevention of joint diseases in the elderly, and experiments have shown that even large doses of N-acetylglucosamine have no side effects on the human body, and are a safe and reliable health care product.
  • drugs N-acetylglucosamine can also produce glucosamine by enzymatic hydrolysis or acid hydrolysis, which is also a widely used middle-aged and elderly joint health care product, and the market is huge.
  • N-acetylglucosamine is mainly obtained by chemical condensation reaction of glucosamine and acetic anhydride.
  • the raw material glucosamine is mainly obtained by acid hydrolysis of chitin. This process is accompanied by the discharge of a large amount of acid-containing wastewater, and The supply of raw materials for crab shells is limited by seasonal and production factors, resulting in excessive N-acetylglucosamine production costs. Therefore, the work of producing N-acetylglucosamine by microbial fermentation has been carried out at home and abroad.
  • Patent WO2004003175 discloses a series of metabolic engineering E. coli construction methods. After fermentation, the yield of N-acetylglucosamine reaches up to 110 g/L, and the patents CN102268399 and CN102286420 disclose two kinds of Escherichia coli engineering. The method of bacteria and the yield of 70-g/L by fermentation of N-acetylglucosamine.
  • the above patents are genetically engineered to produce high-yield N-acetylglucosamine genetically engineered bacteria, but E. coli produces endotoxin during fermentation, which makes the fermented products in health products and foods. The application is limited, so the production of glucosamine or N-acetylglucosamine by safe and non-toxic microorganisms has become a requirement for industrial production.
  • N-acetylglucosamine-producing Bacillus subtilis engineering bacteria construction methods are disclosed in the patents CN103045527A, CN102978149A and CN103060252A.
  • Several strains of N-acetylglucosamine-producing Bacillus subtilis are genetically engineered, and the yields after fermentation are respectively 115 mg/ L, 415mg / L and 1.23g / L, initially solved the problem of the safety of N-acetylglucosamine production by fermentation, but the above fermentation yield only reached 1.23g / L, the production is too low, resulting in increased production costs, which is not conducive to industrialization. Production applications.
  • One of the objects of the present invention is to provide a Bacillus subtilis producing N-acetylglucosamine and a construction method and application thereof, which utilize the constructed high-yield N-acetylglucosamine genetically engineered Bacillus subtilis to achieve through biological fermentation engineering.
  • the purpose of producing high-safety N-acetylglucosamine which can be applied in the field of health care products.
  • a Bacillus subtilis producing N-acetylglucosamine the preservation number is CCTCC M 2014342.
  • CCTCC The above-mentioned strain applicants have been deposited on July 16, 2014 at the China Center for Type Culture Collection in Wuhan University, Wuhan, Hubei province.
  • the depositary unit is referred to as CCTCC.
  • the method for constructing N-acetylglucosamine to produce Bacillus subtilis is to efficiently express the Bacillus subtilis host bacteria by encoding 6-phosphomeglucosidase and 6-phosphosamine glucoamylase, and knock out or inactivate the grass.
  • 6-phosphate glucosamine deaminase gene nagB and gamA 6-phosphate-N-acetylglucosamine deacetylase gene nagA
  • the acetylglucosamine transporter gene nagP was constructed.
  • the 6-phosphate glucosamine synthase gene and the 6-phosphate glucosamine acetylase gene are introduced into Bacillus subtilis for overexpression, and the two genes can be cloned into an expression vector, respectively, in a plasmid manner in Bacillus subtilis.
  • the expression vector of the present invention selects the pHT01 vector.
  • the nagB, nagA, nagP, gamA, and gamP genes are knocked out by homologous recombination Completed.
  • nagA and nagB are linked in a row on the Bacillus subtilis chromosome, which can be knocked out at one time.
  • the gamA and gamP are linked in a chain and can be knocked out at one time. Therefore, the knockout of the above five genes can be completed by three experimental operations or by five experiments.
  • the knockout of Bacillus subtilis can be accomplished by pMAD vectors and molecular biology experiments.
  • the construction method includes the following process steps:
  • the nagAB gene cluster, the gamAP gene cluster and the nagP gene in Bacillus subtilis are respectively knocked out to obtain a genetically engineered host;
  • the 6-phosphate glucosamine synthase gene is derived from Bacillus subtilis or other microorganisms having the same functional enzyme.
  • the 6-phosphate glucosamine synthase catalyzes the reaction of fructose 6-phosphate and glutamine to synthesize glucosamine 6-phosphate, which is feedback-suppressed by the reaction product 6-glucosamine, so the reaction is N-acetylglucosamine
  • the rate-limiting step in the synthetic pathway, high expression of glucosamine 6-phosphate synthase helps to increase the supply of the intermediate 6-glucosamine glucophosphate, thereby increasing the concentration of the final product N-acetylglucosamine.
  • the enzyme may be derived from Bacillus subtilis, and the gene may be obtained by whole gene synthesis according to the glmS gene sequence of the Bacillus subtilis 168 strain genome sequence GenBank No. NC_000964, or by PCR using the genomic DNA of Bacillus subtilis 168 strain as a template. obtain.
  • the 6-phosphoglucosamine acetylase gene is derived from Saccharomyces cerevisiae or other microorganisms having the same functional enzyme.
  • the 6-phosphoglucosylacetylase catalyzes the synthesis of 6-phosphate-N-acetylglucosamine by glucosamine 6-phosphate and acetyl CoA.
  • the enzyme is not present in Bacillus subtilis and is genetically engineered. Into the high expression, thereby achieving the purpose of synthesizing N-acetylglucosamine.
  • the enzyme is derived from Saccharomyces cerevisiae, and the gene can be obtained by whole-gene synthesis according to the GenBank No. NM_001179949 sequence, or by PCR amplification using the S. cerevisiae S288c strain genomic DNA as a template.
  • the knockout or inactivation of the 6-phosphate glucosamine deaminase gene nagB and gamA in the N-acetylglucosamine catabolic pathway in Bacillus subtilis is simultaneously performed.
  • the invention constructs a new metabolic pathway from glucose to N-acetylglucosamine by genetic engineering operation in Bacillus subtilis, enhances the expression of rate-limiting enzyme gene in the N-acetylglucosamine synthesis pathway, and simultaneously knocks
  • the engineered strain constructed by the present invention can accumulate a higher concentration of N-acetylglucosamine than the prior art (detected The concentration of N-acetylglucosamine in the final fermentation product can reach 38.3g/L, which has high industrial utilization value.
  • the microorganism used in the present invention has been deposited with the China Center for Type Culture Collection on July 16, 2014, and the depository unit is referred to as CCTCC for short.
  • Figure 1 is a related metabolic pathway for producing an engineered strain of N-acetylglucosamine in the present invention.
  • the plasmid extraction, the genomic extraction, the PCR reagent, and the like used in the present embodiment are commercial products, and the specific operation is carried out in accordance with the instructions. Other unspecified experimental procedures were carried out in accordance with the method of operation of the Molecular Cloning Experiment Guide, Third Edition (translated by J. Shambrook Huang Peitang).
  • Bacillus subtilis electrotransformation method reference Meddeb Mouelhi F et al, High Transformation efficiency of Bacillus subtilis with integrative DNA using glycine betaine as osmoprotectant, Anal Biochem. 2012 May 15;424(2):127-9.
  • the pMAD- ⁇ nagAB plasmid was transformed into Bacillus subtilis 168 strain, and coated with 50 ug/mL of X-gal (5-bromo-4-chloro-3-indol- ⁇ -D-galactoside) and red mold. Plain plate, cultured at 30 degrees overnight;
  • the pMAD- ⁇ gamAP plasmid was transformed into the Bacillus subtilis Bs168/ ⁇ nagAB strain of Example 1, and the positive colonies were screened by the same procedure as above, and the primer was selected to be F-gamAP-up-BamHI (SEQ ID NO. 5). And R-gamAP-down-NcoI (SEQ ID NO. 8), can amplify a 2 kb colony that is a positive colony knocking out the gamAP gene Bs168/ ⁇ nagAB / ⁇ gamAP.
  • the above fragment was purified and inserted into the vector pMAD by conventional molecular cloning techniques to obtain the knockout vector pMAD- ⁇ nagP;
  • the pMAD- ⁇ nagP plasmid was transformed into the Bacillus subtilis Bs168/ ⁇ nagAB/ ⁇ gamAP strain of Example 2, and positive colonies were screened by the same procedure as above, and the primer was selected for F-nagP-up-BamHI (SEQ ID NO. 9). Shown and R-nagP-down-NcoI (shown in SEQ ID NO. 12), a colony of 2 kb in size can be amplified as a positive colony Bs168/ ⁇ nagAB/ ⁇ gamAP/ ⁇ nagP that knocks out the nagP gene.
  • GNA1 gene was synthesized based on the GNA1 gene sequence of Saccharomyces cerevisiae GenBank No.NM_001179949, and BamHI and XbaI sites were added at both ends (shown below). The specific sequence is as follows (SEQ ID NO.13).
  • the GNA1 gene was ligated to the BamHI and XbaI sites of the pHT01 vector (MoBiTec, Germany) using conventional molecular biology techniques to obtain the vector pHT01-GNA1.
  • the double expression vector pHT01-GNA1-glmS constructed above was electrotransformed into the modified Bacillus subtilis Bs168/ ⁇ nagAB/ ⁇ gamAP/ ⁇ nagP, coated with chloramphenicol (10 mg/L) LB plate, and cultured overnight at 37° to obtain the gene.
  • the seed medium is LB medium (ingredients: peptone 10 g/L, yeast powder 5 g/L, sodium chloride 10 g/L); when cultured in solid or liquid, chloramphenicol is added to a final concentration of 10 mg/L.
  • the fermentation medium consists of the following components: peptone 1-10g/L, yeast powder 1-5g/L, dipotassium hydrogen phosphate 1-5g/L, potassium dihydrogen phosphate 1-8g/L, ammonium sulfate 1-5g/ L, glucose 5-20 g / L, the balance is water.
  • the feed medium is a 40% to 60% by mass glucose solution.
  • the seed culture medium was cultured overnight, and Bacillus subtilis CCTCC M2014342 was transferred to the fermentation medium at a dose of 2-5%, and cultured at 33-37 ° C.
  • - ⁇ -D-thiogalactopyranoside (IPTG) induces protein expression, continues to culture until the end of fermentation, and samples are detected by high performance liquid chromatography (HPLC).
  • the specific test conditions are:
  • Detection wavelength 210 nm.
  • the concentration of N-acetylglucosamine in the fermentation broth can reach above 38.3g/L.

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Abstract

本申请提供了一种生产N-乙酰氨基葡萄糖的枯草芽孢杆菌及其构建方法和应用。该基因工程菌通过在枯草芽孢杆菌中表达编码6-磷酸氨基葡萄糖合成酶基因、6-磷酸氨基葡萄糖乙酰化酶基因，同时敲除或失活枯草芽孢杆菌中N-乙酰氨基葡萄糖分解代谢途径中的6-磷酸氨基葡萄糖脱氨酶基因nagB和gamA、6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶基因nagA、氨基葡萄糖转运蛋白基因gamP和N-乙酰氨基葡萄糖转运蛋白基因nagP构建而成。

Description

一种生产N-乙酰氨基葡萄糖的枯草芽孢杆菌及其构建方法和应用 技术领域
本发明属于基因工程菌的构建及应用，特别是指一种生产N-乙酰氨基葡萄糖的枯草芽孢杆菌及其构建方法和应用。
背景技术
N-乙酰氨基葡萄糖是葡萄糖的衍生物，通常以beta-1,4-糖苷键聚合成几丁质。几丁质是自然界中数量仅次于纤维素的第二大类碳水化合物，广泛存在于菌类、藻类，虾、蟹、昆虫的外壳和高等植物的细胞壁等，因此，N-乙酰氨基葡萄糖在自然界的存量巨大。
N-乙酰氨基葡萄糖与D-葡萄糖醛酸形成重复的二糖单位而构成透明质酸，透明质酸是关节间起润滑作用的关键物质，对于骨关节具有重要的保护作用。实验已证明N-乙酰氨基葡萄糖可作为治疗和预防中老年人关节疾病的有效药物，并且实验证明即使大剂量的N-乙酰氨基葡萄糖对人体也无任何毒副作用，是一种安全可靠的保健品和药物。N-乙酰氨基葡萄糖也可以通过酶水解或酸水解产生氨基葡萄糖，后者也是一种已得到广泛应用的中老年人关节保健品，市场巨大。
N-乙酰氨基葡萄糖的生产目前主要是利用氨基葡萄糖和乙酸酐经化学缩合反应而成，原料氨基葡萄糖主要通过酸水解几丁质而获得，这个过程伴随着大量含酸废水的排放，而且由于虾蟹壳几丁质的原料供应受季节和产地因素的限制，导致最终N-乙酰氨基葡萄糖生产成本过高。因此，国内外开展了利用微生物发酵法生产N-乙酰氨基葡萄糖的工作。
专利WO2004003175(同族专利包括US7332304和CN101365785等)中公开了一系列代谢工程大肠杆菌的构建方法，发酵后N-乙酰氨基葡萄糖产量最高达到110g/L，专利CN102268399和CN102286420公开了两种构建大肠杆菌工程菌的方法，并通过发酵N-乙酰氨基葡萄糖产量达到70g/L。上述专利都是通过遗传改造大肠杆菌得到能高产N-乙酰氨基葡萄糖的基因工程菌，但是大肠杆菌在发酵过程中会产生内毒素，这使得其发酵产品在保健品和食品中 的应用受到限制，因此采用安全无毒的微生物生产氨基葡萄糖或N-乙酰氨基葡萄糖成为工业生产的需要。
专利CN103045527A，CN102978149A和CN103060252A中公开了一系列产N-乙酰氨基葡萄糖的枯草杆菌工程菌构建方法，通过遗传改造构建了几株产N-乙酰氨基葡萄糖的枯草芽孢杆菌，发酵后产量分别达到115mg/L，415mg/L和1.23g/L，初步解决了发酵法生产N-乙酰氨基葡萄糖安全性的问题，但以上发酵产量最高只达到1.23g/L，产量过低导致生产成本增高，不利于工业化生产的应用。
发明内容
本发明的目的之一在于提供一种生产N-乙酰氨基葡萄糖的枯草芽孢杆菌及其构建方法和应用，利用所构建的高产N-乙酰氨基葡萄糖的基因工程枯草芽孢杆菌，通过生物发酵工程，达到生产可以在保健品领域应用的高安全性N-乙酰氨基葡萄糖的目的。
本发明的整体技术构思是：
一种生产N-乙酰氨基葡萄糖的枯草芽孢杆菌Bacillus subtilis，其保藏编号为CCTCC M 2014342。
上述菌种申请人已于2014年7月16日提交位于湖北省武汉市武汉大学内的中国典型培养物保藏中心保藏，保藏单位简称为CCTCC。
生产N-乙酰氨基葡萄糖的枯草芽孢杆菌的构建方法，是通过将编码6-磷酸氨基葡萄糖合成酶、6-磷酸氨基葡萄糖乙酰化酶引入枯草芽孢杆菌宿主菌高效表达，并敲除或失活枯草芽孢杆菌中N-乙酰氨基葡萄糖分解代谢途径中的6-磷酸氨基葡萄糖脱氨酶基因nagB和gamA、6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶基因nagA、氨基葡萄糖转运蛋白基因gamP和N-乙酰氨基葡萄糖转运蛋白基因nagP构建而成。
所述6-磷酸氨基葡萄糖合成酶基因和6-磷酸氨基葡萄糖乙酰化酶基因引入枯草芽孢杆菌超表达，可将这两个基因分别克隆到表达载体上后，以质粒的方式在枯草芽孢杆菌中串联表达，本发明中表达载体选择pHT01载体。
所述nagB、nagA、nagP、gamA和gamP基因的敲除，是通过同源重组系 统完成。五个基因中nagA和nagB在枯草杆菌染色体上连锁排列，可一次性敲除，gamA和gamP连锁排列，可一次性敲除。因此，上述5个基因的敲除可以通过三次实验操作完成，也可以通过五次实验分别完成。枯草芽孢杆菌的基因敲除工作可通过pMAD载体和分子生物学实验完成。
生产N-乙酰氨基葡萄糖的枯草芽孢杆菌在N-乙酰氨基葡萄糖中的应用，将生产N-乙酰氨基葡萄糖的枯草芽孢杆菌CCTCC M 2014342接种至以葡萄糖为碳源的无菌培养基，在37℃条件下通气发酵70小时。
本发明的具体技术构思还有：
所述的构建方法包括如下工艺步骤：
A、分别敲除枯草芽孢杆菌中的nagAB基因簇、gamAP基因簇和nagP基因，获得基因工程菌宿主；
B、构建GNA1和glmS双表达载体；
C、将双表达载体转入1)所得宿主菌，得到最终生产N-乙酰氨基葡萄糖的枯草芽孢杆菌。
所述的6-磷酸氨基葡萄糖合成酶基因来源于枯草芽孢杆菌或其它具有相同功能酶的微生物。
所述6-磷酸氨基葡萄糖合成酶催化6-磷酸果糖和谷氨酰胺合成6-磷酸氨基葡萄糖的反应，该酶受反应产物6-磷酸氨基葡萄糖的反馈抑制，因此该反应为N-乙酰氨基葡萄糖合成途径中的限速步骤，高表达6-磷酸氨基葡萄糖合成酶有助于增加中间产物6-磷酸氨基葡萄糖的供应量，从而会增加终产物N-乙酰氨基葡萄糖的浓度。所述酶可来源于枯草芽孢杆菌，基因的获得可根据枯草芽孢杆菌168菌株基因组序列GenBank No.NC_000964中glmS基因序列全基因合成，或利用枯草芽孢杆菌168菌株的基因组DNA为模板通过PCR扩增获得。
所述的6-磷酸氨基葡萄糖乙酰化酶基因来源于酿酒酵母或其它具有相同功能酶的微生物。
所述6-磷酸氨基葡萄糖乙酰化酶催化6-磷酸氨基葡萄糖和乙酰CoA合成6-磷酸-N-乙酰氨基葡萄糖，该酶在枯草芽孢杆菌中不存在，通过基因工程引 入并高表达，从而实现合成N-乙酰氨基葡萄糖的目的。该酶来源于酿酒酵母，基因的获得可根据GenBank No.NM_001179949序列，经全基因合成得到，或利用酿酒酵母S288c菌株基因组DNA为模板通过PCR扩增获得。
所述的敲除或失活枯草芽孢杆菌中N-乙酰氨基葡萄糖分解代谢途径中的6-磷酸氨基葡萄糖脱氨酶基因nagB和gamA同时进行。
本发明所具备的实质性特点和取得的显著技术进步在于：
本发明通过在枯草芽孢杆菌中进行基因工程操作，构建了一条新的从葡萄糖到N-乙酰氨基葡萄糖的代谢通路，增强了N-乙酰氨基葡萄糖合成途径中的限速酶基因表达，同时，敲除导致N-乙酰氨基葡萄糖消耗和回流的基因，阻止N-乙酰氨基葡萄糖的回流和消耗，相比现有技术，本发明构建的工程菌株能积累更高浓度的N-乙酰氨基葡萄糖(经检测，发酵终产物中N-乙酰氨基葡萄糖浓度可达38.3g/L)，具有较高的工业化利用价值。
本发明中所采用的微生物已于2014年7月16日提交中国典型培养物保藏中心保藏，保藏单位简称为CCTCC。
附图说明
图1是本发明中生产N-乙酰氨基葡萄糖工程菌株的相关代谢途径。
具体实施方式
以下结合附图对本发明的实施例做进一步描述，但不作为对本发明的限定，本发明的保护范围以权利要求记载的内容为准，任何依据说明书做出的等效技术手段替换，均不脱离本发明的保护范围。
本实施例中使用的质粒抽提、基因组抽提、PCR试剂等采用商业产品，具体操作按照说明书进行。其他未注明的实验操作按照《分子克隆实验指南第三版》(〔美〕J.莎姆布鲁克黄培堂译)操作方法进行。
枯草芽孢杆菌实验操作及pMAD基因敲除原理和方法见文献(Maryvonne Arnaud et al，New vector for efficient allelic replacement in naturally nontransformable，low-GC-content，gram-positive bacteria，APPLIED AND ENVIRONMENTAL MICROBIOLOGY，Nov.2004，p.6887–6891)。
枯草芽孢杆菌电转化方法参照文献Meddeb Mouelhi F et al，High  transformation efficiency of Bacillus subtilis with integrative DNA using glycine betaine as osmoprotectant,Anal Biochem.2012May 15；424(2):127-9.
实施例1
一、敲除枯草芽孢杆菌168菌株中nagAB基因簇
1、根据枯草芽孢杆菌168菌株基因组(Genbank No.NC_000964)序列，设计引物：上游引物F-nagAB-up-BamHI:CTGGATCCGACTGCAAGATTTCGGCCTGGG(SEQ ID NO.1所示)和下游引物R-nagAB:CATAAGTCAGCATGTTCCTTTCACATAGATGATCCGCCTTTCTGG(SEQ ID NO.2所示)。以枯草芽孢杆菌168菌株基因组DNA为模板，经PCR扩增得到nagAB基因簇上游1000bp片段。
2、根据枯草芽孢杆菌168菌株基因组(Genbank No.NC_000964)序列，设计引物：上游引物F-nagAB:CCAGAAAGGCGGATCATCTATGTGAAAGGAACATGCTGACTTATG(SEQ ID NO.3所示)和下游引物R-nagAB-down-NcoI:GCTCCATGGTAACGTATATACCAATGAAGAG(SEQ ID NO.4所示)。以枯草芽孢杆菌168菌株基因组DNA为模板，经PCR扩增得到nagAB基因簇下游1000bp片段。
3、将上述两种PCR产物纯化后各取5ul作为模板，以引物F-nagAB-up-BamHI(SEQ ID NO.1所示)和R-nagAB-down-NcoI(SEQ ID NO.4所示)做overlap PCR扩增，获得nagAB基因簇中上下游各1000bp，且中间缺失了nagAB编码区的DNA片段。
4、纯化上述片段，利用常规分子克隆技术将其插入载体pMAD，得到敲除载体pMAD-ΔnagAB；
5、将pMAD-ΔnagAB质粒转化进枯草芽孢杆菌168菌株，涂布含有50ug/mL的X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)和红霉素平板，30度过夜培养；
6、挑取显蓝色转化子，30度过夜培养，按5％接种量转接至5mL红霉素抗性的LB培养基，30度孵育2小时，升温至42度继续孵育6小时，稀释(10-2-10-5)涂布含有X-gal和红霉素的平板，42度过夜培养；
7、挑取蓝色单菌落于30度和无抗性LB培养基中孵育6小时，升温至42 度继续孵育3小时，稀释(10-2-10-5)涂布含有X-gal的无抗性LB平板，42度过夜培养；
8、挑取显白斑的单菌落分别在红霉素和无抗LB培养基过夜培养，选择无红霉素抗性的单菌落做菌落PCR验证，引物选择F-nagAB-up-BamHI(SEQ ID NO.1所示)和R-nagAB-down-NcoI(SEQ ID NO.4所示)，能扩增出大小为2kb的菌落即为敲除了nagAB基因簇的阳性菌落Bs168/ΔnagAB。
实施例2
二、继续敲除枯草芽孢杆菌中的gamAP基因簇
1、根据枯草芽孢杆菌168菌株基因组(Genbank No.NC_000964)序列，设计引物：上游引物F-gamAP-up-BamHI:CTGGATCCACTGCTCCCCACAGCACTTTTCC(SEQ ID NO.5所示)和下游引物R-gamAP:CGCAGCAGGGGGGACTTTTTTACATGTGACACCCCCTCAAAGAG(SEQ ID NO.6所示)。以枯草芽孢杆菌168菌株基因组DNA为模板，经PCR扩增得到gamAP基因簇上游1000bp片段。
2、根据枯草芽孢杆菌168菌株基因组(Genbank No.NC_000964)序列，设计引物：上游引物F-gamAP:CTCTTTGAGGGGGTGTCACATGTAAAAAAGTCCCCCCTGCTGCG(SEQ ID NO.7所示)和下游引物R-gamAP-down-NcoI:GCTCCATGGATACCACTCGTTTGGGACAGCC(SEQ ID NO.8所示)。以枯草芽孢杆菌168菌株基因组DNA为模板，经PCR扩增得到gamAP基因簇下游1000bp片段。
3、将上述两种PCR产物纯化后各取5ul作为模板，以引物F-gamAP-up-BamHI(SEQ ID NO.5所示)和R-gamAP-down-NcoI(SEQ ID NO.8所示)扩增，获得gamAP基因簇中上下游各1000bp，且中间缺失了gamAP编码区的DNA片段。
4、纯化上述片段，利用常规分子克隆技术将其插入载体pMAD，得到敲除载体pMAD-ΔgamAP；
5、将pMAD-ΔgamAP质粒转化进实施例1中的枯草芽孢杆菌Bs168/ΔnagAB菌株，采用与上述同样的步骤筛选阳性菌落，引物选择F-gamAP-up-BamHI(SEQ ID NO.5所示)和R-gamAP-down-NcoI(SEQ ID NO.8所示)，能扩增出大小为2kb的菌落即为敲除了gamAP基因的阳性菌落Bs168/ΔnagAB /ΔgamAP。
实施例3
三、继续敲除枯草芽孢杆菌中的nagP基因
1、根据枯草芽孢杆菌168菌株基因组(Genbank No.NC_000964)序列，设计引物：上游引物F-nagP-up-BamHI:CTGGATCCCAAGACCTCCTCGTACAGAATAATG(SEQ ID NO.9所示)和下游引物R-nagP:GGTTGCCCTCTCCGCTTTTTTACATACCCATCCCCCTCATACCC(SEQ ID NO.10所示)。以枯草芽孢杆菌168菌株基因组DNA为模板，经PCR扩增得到nagP基因上游1000bp片段。
2、根据枯草芽孢杆菌168菌株基因组(Genbank No.NC_000964)序列，设计引物：上游引物F-nagP:GGGTATGAGGGGGATGGGTATGTAAAAAAGCGGAGAGGGCAACC(SEQ ID NO.11所示)和下游引物R-nagP-down-NcoI:GCTCCATGGTTCCGGCGATTCTGAAGTCTAAG(SEQ ID NO.12所示)。以枯草芽孢杆菌168菌株基因组DNA为模板，经PCR扩增得到nagP基因下游1000bp片段。
3、将上述两种PCR产物纯化后各取5ul作为模板，以引物F-nagP-up-BamHI(SEQ ID NO.9所示)和R-nagP-down-NcoI(SEQ ID NO.12所示)扩增，获得nagP基因中上下游各1000bp，且中间缺失了nagP编码区的DNA片段。
4、纯化上述片段，利用常规分子克隆技术将其插入载体pMAD，得到敲除载体pMAD-ΔnagP；
5、将pMAD-ΔnagP质粒转化进实施例2中的枯草芽孢杆菌Bs168/ΔnagAB/ΔgamAP菌株，采用与上述同样的步骤筛选阳性菌落，引物选择F-nagP-up-BamHI(SEQ ID NO.9所示)和R-nagP-down-NcoI(SEQ ID NO.12所示)，能扩增出大小为2kb的菌落即为敲除了nagP基因的阳性菌落Bs168/ΔnagAB/ΔgamAP/ΔnagP。
实施例4
四、GNA1和glmS基因双表达载体的构建
1、根据酿酒酵母GNA1基因序列GenBank No.NM_001179949全合成GNA1基因，两端加BamHI和XbaI位点(如下划线所示)，具体序列如下所示(SEQ  ID NO.13)。
ggatccatgagcttacccgatggattttatataaggcgaatggaagagggggatttggaacaggtcactgagacgctaaaggttttgaccaccgtgggcactattacccccgaatccttcagcaaactcataaaatactggaatgaagccacagtatggaatgataacgaagataaaaaaataatgcaatataaccccatggtgattgtggacaagcgcaccgagacggttgccgctacggggaatatcatcatcgaaagaaagatcattcatgaactggggctatgtggccacatcgaggacattgcagtaaactccaagtatcagggccaaggtttgggcaagctcttgattgatcaattggtaactatcggctttgactacggttgttataagattattttagattgcgatgagaaaaatgtcaaattctatgaaaaatgtgggtttagcaacgcaggcgtggaaatgcaaattagaaaatagtctaga
2、利用常规分子生物学技术将GNA1基因连接至pHT01载体(MoBiTec公司，德国)的BamHI和XbaI位点上，得到载体pHT01-GNA1。
3、根据枯草芽孢杆菌168菌株基因组(Genbank No.NC_000964)序列中glmS序列设计引物，正向引物F-glmS-XbaI：CGCTCTAGAGGAGGAAGAAAAATATGTGTG(SEQ ID NO.14所示)和反向引物R-glmS-AatII：GTAGACGTCTTACTCCACAGTAACACTCTTCGC(SEQ ID NO.15所示)，该引物中的两端增加了XbaI和AatII位点；
4、以枯草芽孢杆菌168菌株的总DNA为模板，以上述引物(SEQ ID NO.14-15所示)PCR扩增得到glmS基因及其上游核糖体结合位点的DNA片段，通过常规克隆技术将片段插入上述构建好的载体pHT01-GNA1的XbaI和AatII位点之间，得到双表达载体pHT01-GNA1-glmS。
实施例5
五、最终工程菌的构建
将上述构建的双表达载体pHT01-GNA1-glmS电转化改造过的枯草芽孢杆菌宿主菌Bs168/ΔnagAB/ΔgamAP/ΔnagP，涂布氯霉素(10mg/L)LB平板，37度培养过夜，获得基因工程菌株Bs168/ΔnagAB/ΔgamAP/ΔnagP/pHT01-GNA1-glmS，即为能合成N-乙酰氨基葡萄糖的枯草芽孢杆菌，命名为BsNAG01。
实施例6
六、工程菌株发酵
1、种子和发酵培养基：
种子培养基为LB培养基(成分为蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L)；固体或液体培养时，补加氯霉素至终浓度10mg/L。
发酵培养基由如下组份组成：蛋白胨1-10g/L，酵母粉1-5g/L，磷酸氢二钾1-5g/L，磷酸二氢钾1-8g/L，硫酸铵1-5g/L，葡萄糖5-20g/L，余量为水。补料培养基为质量百分比为40-60％的葡萄糖溶液。
2、发酵过程：
先过夜培养种子培养基，按2-5％的接种量将枯草芽孢杆菌CCTCC M2014342转接至发酵培养基，33-37℃好气培养，通过通气量、罐压和搅拌保持溶氧维持在20％以上，用氨水控制pH＝6.8-7.0并补充氮源，初始葡萄糖耗完后开始按照3-6g/L·h速率补加碳源，发酵液OD600达到15后添加0.01-0.05mM的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导蛋白表达，继续培养至发酵结束，取样利用高效液相色谱(HPLC)检测。
具体检测条件为：
检测柱：Bio-Rad AMINEX HPX 87H Organic Analysis Column(300×7.8mm)；
柱温：60℃；
流动相：6mM硫酸，流速：0.6ml/min；
检测波长：210nm。
经检测，72-84小时发酵后，发酵液中N-乙酰氨基葡萄糖浓度可达38.3g/L以上。

Claims (7)

  1. 一种生产N-乙酰氨基葡萄糖的枯草芽孢杆菌(Bacillus subtilis)，其特征在于其保藏编号为CCTCC M 2014342。
  2. 生产N-乙酰氨基葡萄糖的枯草芽孢杆菌的构建方法，其特征在于是通过将编码6-磷酸氨基葡萄糖合成酶、6-磷酸氨基葡萄糖乙酰化酶引入枯草芽孢杆菌宿主菌高效表达，并敲除或失活枯草芽孢杆菌中N-乙酰氨基葡萄糖分解代谢途径中的6-磷酸氨基葡萄糖脱氨酶基因nagB和gamA、6-磷酸-N-乙酰氨基葡萄糖脱乙酰酶基因nagA、氨基葡萄糖转运蛋白基因gamP和N-乙酰氨基葡萄糖转运蛋白基因nagP构建而成。
  3. 根据权利要求2所述的生产N-乙酰氨基葡萄糖的枯草芽孢杆菌的构建方法，其特征在于所述的构建方法包括如下工艺步骤：
    A、分别敲除枯草芽孢杆菌中的nagAB基因簇、gamAP基因簇和nagP基因，获得基因工程菌宿主；
    B、构建GNA1和glmS双表达载体；
    C、将双表达载体转入A所得宿主菌，得到最终生产N-乙酰氨基葡萄糖的枯草芽孢杆菌。
  4. 根据权利要求2所述的生产N-乙酰氨基葡萄糖的枯草芽孢杆菌的构建方法，其特征在于所述的6-磷酸氨基葡萄糖合成酶基因来源于枯草芽孢杆菌或其它具有相同功能酶的微生物。
  5. 根据权利要求2所述的生产N-乙酰氨基葡萄糖的枯草芽孢杆菌的构建方法，其特征在于所述的6-磷酸氨基葡萄糖乙酰化酶基因来源于酿酒酵母或其它具有相同功能酶的微生物。
  6. 根据权利要求2所述的生产N-乙酰氨基葡萄糖的枯草芽孢杆菌的构建方法，其特征在于所述的敲除或失活枯草芽孢杆菌中N-乙酰氨基葡萄糖分解代谢途径中的6-磷酸氨基葡萄糖脱氨酶基因nagB和gamA同时进行。
  7. 生产N-乙酰氨基葡萄糖的枯草芽孢杆菌在N-乙酰氨基葡萄糖中的应用，其特征在于将生产N-乙酰氨基葡萄糖的枯草芽孢杆菌CCTCC M 2014342接种至以葡萄糖为碳源的无菌培养基，在37℃条件下通气发酵70小时。
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