CN101633928B - Recombinant expression of aldehyde reductase and application thereof in bioconversion of glycerol into 1,3-propylene glycol - Google Patents

Recombinant expression of aldehyde reductase and application thereof in bioconversion of glycerol into 1,3-propylene glycol Download PDF

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CN101633928B
CN101633928B CN 200810228268 CN200810228268A CN101633928B CN 101633928 B CN101633928 B CN 101633928B CN 200810228268 CN200810228268 CN 200810228268 CN 200810228268 A CN200810228268 A CN 200810228268A CN 101633928 B CN101633928 B CN 101633928B
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glycol
propylene
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expression
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CN101633928A (en )
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修志龙
张乐
马成伟
戴建英
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大连理工大学
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P20/52Improvements relating to the production of products other than chlorine, adipic acid, caprolactam, or chlorodifluoromethane, e.g. bulk or fine chemicals or pharmaceuticals using catalysts, e.g. selective catalysts

Abstract

The invention discloses recombinant expression of aldehyde reductase and an application thereof in bioconversion of glycerol into 1, 3-propylene glycol, belonging to the technical field of biological engineering. The invention is characterized in that a recombinant expression vector containing aldehyde reductase gene sequence is used to express the enzyme to a host cell for preparing 1, 3-propylene glycol. The beneficial effects and benefits of the invention are that recombinant Klebsiella (Pdk-yqhD) takes glycol as substrate to ferment for 30h, and the concentration of 1, 3-propylene glycol in the fermentation liquor is improved by 17.1% in comparison with a comparison strain; compared with the original strain, by using the recombinant restrain in the invention for fermentation, the yield of 1, 3-propylene glycol is improved by 10-50%, which lays a foundation on constructing genetically engineered microorganism of the high yield of the 1, 3-propylene glycol taking glycol as substrate.

Description

一种醛还原酶的重组表达及其在甘油生物转化为1,3_丙二醇中的应用 Recombinant reductase and an aldehyde in the bioconversion of glycerol propylene glycol application 1,3_

技术领域 FIELD

[0001] 本发明属于生物工程技术领域,涉及到含有醛还原酶基因的重组表达载体,以及用其转化的宿主细胞发酵甘油高产1,3_丙二醇的应用。 [0001] Application of the present invention belongs to the technical field of biological engineering, related to a recombinant expression vector containing aldehyde reductase gene and used to transform a host cell fermentation yield 1,3_ glycerol propylene glycol.

背景技术 Background technique

[0002] 1,3_丙二醇(l,3-pr0panedi0l,简称1,3_PD)是无色、无味的粘稠液体,是一种重要的化工原料,可用作溶剂、抗冻剂或保护剂、精细化工原料以及新型聚酯——聚对苯二甲酸丙二醇酯(PTT)和聚氨酯的单体。 [0002] 1,3_-propanediol (l, 3-pr0panedi0l, referred 1,3_PD) is a colorless, odorless viscous liquid, is an important chemical raw material used as a solvent, antifreeze or protective agent, fine chemicals and new polyester - polyethylene terephthalate monomer (PTT), and polyurethane. PTT是一种新型聚酯材料,可用于地毯、工程塑料、膜及纺织业的服装材料等,而且PTT具有优异的回弹性、染色性、抗污性、较好的抗紫外变色性能以及不易起静电、吸水较少等特点,1998年被评为美国六大石化新产品之一。 PTT is a new polyester material, can be used in carpets, engineering plastics, textile garment material film and the like, and PTT having excellent resilience, dyeing, stain resistance, good color properties, and not easy to UV static, less absorbent, etc., in 1998 was named one of America's top six petrochemicals new products. 1,3_丙二醇还可用于生产热塑性聚氨酯和用作PVC的高分子型增塑剂。 1,3_ glycol also for the production of thermoplastic polyurethane and polymeric plasticizers for PVC. 作为二元醇,它还能代替1, 4-丁二醇和新戊二醇作为中间体。 As the diol, it can instead of 1, 4-butanediol and neopentyl glycol as an intermediate. 美国Chem Systems公司研究发现,1,3-丙二醇的二醇官能团,使其用于聚氨酯的生产具有很多潜力,如用于聚酯多元醇的生产和作为链增长剂。 American Chem Systems Research found that 1,3-propanediol diol functional groups, so as to produce polyurethanes having a lot of potential, as in the manufacture of polyester polyols and a chain extender. 在医药合成领域,1,3_丙二醇己得到应用,在该领域中一些新用途也正在开发。 In the field of pharmaceutical synthesis, 1,3_ glycol has been applied, some of the new uses in the art are also being developed. 近年,以1, 3-丙二醇作为有机合成原料己越来越受重视,如1,3-丙二醇经空气氧化可合成3-羟基丙酸和丙二酸,与尿素反应可合成环状碳酸酯。 In recent years, 1, 1,3-propanediol as a starting material for organic synthesis have more and more attention, such as 1,3-propanediol can be synthesized by air oxidation of 3-hydroxypropionic acid and malonic acid, with urea can be synthesized cyclic carbonate. 1,3-丙二醇被认为是本世纪最具有广阔应用前景的化工原料。 1,3-propanediol is considered chemical raw materials most of this century has broad application prospects.

[0003] 鉴于1,3_丙二醇的广泛市场前景,其生产方法多年来受到国内外大型企业和科研机构的广泛关注。 [0003] Given the broad market prospects 1,3_ propylene glycol, the production method over the years attracted widespread attention at home and abroad large enterprises and scientific research institutions. 几家世界著名的化学工业公司,例如荷兰Siel 1公司,德国Degussa公司相继投入规模化生产1,3-丙二醇并加紧了应用研究和开发。 Several world-renowned chemical industry companies, such as the Netherlands Siel 1, Germany's Degussa company have been put into large-scale production of 1,3-propanediol and stepped up research and application development. 其生产1,3-丙二醇的方法主要是化学合成法。 The method of production of 1,3-propanediol is mainly chemical synthesis. 用化学法合成1,3-丙二醇需要在高温、高压及使用贵重催化剂才能实现,成本较高,设备投资大,技术难度高,产品分离纯化困难,特别是催化剂的制备较难,且产生CO等污染环境的废气。 By chemical synthesis of 1,3-propanediol requires high temperature, high pressure and using a catalyst to achieve valuable, high cost, large investment in equipment, high technical difficulty, the product separation and purification difficult, especially in the difficult preparation of the catalyst, and the like to produce CO exhaust pollution of the environment. 随着现代生物技术的发展,人们开始尝试用微生物发酵法生产1,3_丙二醇的研究。 With the development of modern biotechnology, people began to try 1,3_ propanediol Production by microbial fermentation. 微生物发酵法具有条件温和、操作简单、副产物少、绿色环保等优点,这方面的研究成为当前国内外研究的热点。 Microbial fermentation method has mild condition, simple operation, less by-products, environmental protection, etc., research in this area has become a research focus at home and abroad.

[0004] 微生物法发酵甘油生产1,3-丙二醇的菌种包括克雷伯氏菌属(Klebsiella)、柠檬酸菌属(Citrobacter)、梭菌属(Clostridium)等。 [0004] The microbial method of glycerol fermentation production of 1,3-propanediol comprising species Klebsiella (Klebsiella), citric acid genus (Citrobacter), Clostridium (Clostridium) and the like. 克雷伯氏菌(Klebsiellapneumoniae) 具有较高的甘油耐受力,较高的转化率和1,3_丙二醇生产能力,因此受到更多的关注。 Klebsiella (Klebsiella pneumoniae) glycerol with higher tolerance, high conversion rate and production capacity 1,3_ propylene glycol, so more attention. 而且克雷伯氏菌属兼性菌,其生化特性与大肠杆菌(E. coli)非常相近,这就为菌种的基因改良和利用基因工程构建新的菌种提供了便利。 And Klebsiella facultative bacteria, biochemical properties of E. coli (E. coli) is very similar, which provides a convenient and build a new genetically modified strains of bacteria using genetic engineering. 克雷伯氏菌以甘油为底物厌氧发酵的代谢途径涉及氧化途径和还原途径两条平行的路线。 Klebsiella glycerol metabolic pathway substrates anaerobic fermentation pathway involving oxidative and reductive pathways two parallel routes. 通过氧化途径,甘油被与NAD+相连的甘油脱氢酶(GDH)催化脱氢生成二羟基丙酮(DHA),然后进一步代谢为丙酮酸,生成能量ATP和还原当量NAD+/NADH以及乙酸、乙醇等副产物,并伴随着微生物细胞的生长;而通过还原途径, 甘油则被与维生素B12相关联的甘油脱水酶(GDHt)催化脱水生成3-羟基丙醛(3-HPA),再进一步由与NADH相连的1,3_丙二醇氧化还原酶(PDOR)还原为产物1,3-丙二醇,同时消耗了氧化途径中生成的过量的还原型辅酶I (NADH)。 By oxidation pathway, glycerol is coupled to NAD + and glycerol dehydrogenase (GDH) catalytic dehydrogenation dihydroxyacetone (DHA), and then further metabolized to pyruvate, ATP and reducing energy generation sub equivalents NAD + / NADH and acetic acid, ethanol, etc. the product, along with the growth of the microbial cells; through reductive pathway, glycerol were associated with vitamin B12 glycerol dehydratase (GDHt) catalytic dehydration of 3-hydroxypropionaldehyde (3-HPA), and further connected by NADH 1,3_ the propanediol oxidoreductase (PDOR) reduction of the product 1,3-propanediol, while consuming excessive amounts of reduced coenzyme I (NADH) generated in the oxidation pathway. 氧化途径和还原途径通过还原当量NAD+/ NADH相连接,使甘油歧化与细胞生长相偶联。 Oxidation pathway by reduction and reductive pathways equivalents NAD + / NADH is connected to the Ganyou Qi of the cell growth phase coupling.

[0005] 近年来,已有许多公司和科研机构针对生物转化法生产1,3-丙二醇进行了研发。 [0005] In recent years, there have been many companies and research institutions production of 1,3-propanediol has been developed for the biotransformation. 生物转化法生产1,3-丙二醇的一种常用策略是利用原始菌株,通过优化发酵工艺条件来提高1,3-丙二醇的产量。 1,3-propanediol production Biotransformation One common strategy is to use the original strain, to improve the yield of 1,3-propanediol by optimizing fermentation conditions. 如大连理工大学采用微氧条件下发酵生产1,3-丙二醇并完成中试放大(中国专利ZL01117282.7);清华大学采用外源添加维生素C、维生素E或反丁烯二酸来促进1,3-丙二醇生产(中国专利ZL03121946. 2;中国专利ZL200510011917. 2);东南大学采用在发酵液中添加非离子表面活性剂的方法来改变细胞膜的通透性,从而提高1, 3-丙二醇的产量(中国专利CN200810020203. 1)。 Dalian University of Technology The use of 1,3-propanediol fermentation under microaerobic conditions and scale-up is completed (Chinese Patent No. ZL01117282.7); Tsinghua using exogenously added vitamin C, vitamin E or a fumaric acid to promote, production of 1,3-propanediol (China Patent ZL03121946 2;.. Chinese Patent ZL200510011917 2); Southeast University using a method of adding a non-ionic surfactant in the fermentation broth to change the permeability of cell membranes, thereby improving 1, 1,3-propanediol yield (Chinese patent CN200810020203. 1). 通过优化发酵工艺条件虽然可以在一定程度上提高1,3_丙二醇的产量,但由于受到原始菌株自身的生产能力等诸多因素限制,1, 3-丙二醇的发酵产量无法获得大幅度增加。 Although it is possible to increase production 1,3_ propylene glycol to a certain extent by optimizing the fermentation conditions, but due to the original strain their productive capacity, and many other factors limit, 1, 1,3-propanediol fermentation yield is unable to obtain a substantial increase. 为了打破原始菌株自身生产能力的限制,人们逐渐转向另一种策略,即采用重组菌株发酵生产1,3-丙二醇。 In order to break the original strain limit their production capacity, it gradually turned to another strategy, i.e. using recombinant fermentation production of 1,3-propanediol. 在采用重组菌株方面,美国Dupont公司与世界第二大工业酶生产商Genencor国际有限公司申请了以可发酵碳源为底物用基因工程菌直接生产1,3_丙二醇的专利(USPatent7067300 ;US Patent6514733),1, 3-丙二醇最高产量可达到135g/L。 In terms of using recombinant strains, American Dupont company and the world's second-largest producer of industrial enzymes Genencor International Limited applied for a fermentable carbon source to produce 1,3_ propylene glycol as the substrate directly with genetically engineered bacteria patent (USPatent7067300; US Patent6514733 ), 1, 1,3-propanediol highest yield can reach 135g / L. 国内各科研院所也积极开展重组菌株构建方面的研究, 如江南大学构建了一株重组大肠杆菌,该菌株在初始甘油浓度为50g/L,单批发酵成熟发酵液中1,3-丙二醇的最终浓度可达到35〜42g/L (中国专利ZL200610039670. X)。 Domestic research institutes are also active in the construction of recombinant strains of research, such as Jiangnan University constructed a recombinant E. coli, the strain in the initial glycerol concentration of 50g / L, mature single batch fermentation broth of 1,3-propanediol The final concentration of up to 35~42g / L (Chinese Patent ZL200610039670. X). 北京化工大学在克雷伯氏杆菌中同时高表达甘油脱水酶和来自大肠杆菌的醛还原酶,使1,3-丙二醇的产量得到提高(中国专利CN200710176065. 1)。 Beijing University of Chemical Technology in Klebsiella while high expression of glycerol dehydratase and aldehyde reductase from Escherichia coli, the yield of 1,3-propanediol improved (Chinese patent CN200710176065. 1). 在关键酶改造方面,上海理工大学采用易错PCR技术获得了1,3_丙二醇氧化还原酶同工酶变体酶(中国专利CN200710171758. 1 ; 中国专利CN200710171759. 6)。 The key enzyme in the transformation of Shanghai University of Technology using error-prone PCR technology to obtain 1,3_ propanediol oxidoreductase isoenzyme variant enzymes (Chinese patent CN200710171758 1;.. Chinese patent CN200710171759 6). 除Dupont公司采用重组大肠杆菌生产1,3_丙二醇产量得到很大提升外,目前采用重组菌株生产1,3_丙二醇仍存在产物浓度低、甘油转化率低、生产强度低等问题。 In addition to use in recombinant Escherichia Dupont Company 1,3_ greatly enhance the yield of propylene glycol, the production of recombinant strains currently used 1,3_ glycol product concentration remains low, low conversion rate of glycerol production strength is poor.

[0006] 研究发现在厌氧批式发酵中,当起始甘油浓度达到44. 2g/L时,3-羟基丙醛在细胞中积累,导致细胞停止生长,甘油消耗停滞(Barbirato,F. et al.,Appl. Environ. Microbiol. 1996,62(4) :1448-1451)。 [0006] found in anaerobic batch fermentation, when the starting glycerol concentration of 44. 2g / L, 3- hydroxypropionaldehyde accumulation in cells, leading to cell has stopped growing, glycerol stagnation consumption (Barbirato, F. Et .... al, Appl Environ Microbiol 1996,62 (4): 1448-1451). 同时也反映出此时菌体内1, 3-丙二醇氧化还原酶的相对缺乏。 In this case also reflects a 1,3-propanediol oxidoreductase relative lack of bacteria in vivo. 但即使在菌体内高表达1,3_丙二醇氧化还原酶仍无法得到预期的效果Cheng, P. etal. , Process Biochem. 2006,41(10) :2160-2169)。 However, the high expression of 1,3_ propanediol oxidoreductase still can not give the desired effect Cheng, P. etal even in bacterial cells, Process Biochem 2006,41 (10):.. 2160-2169). 1,3-丙二醇氧化还原酶是一个双向酶,既能催化3-羟基丙醛转化为1,3-丙二醇,又能将生成的1,3-丙二醇转化为3-羟基丙醛,这样对1,3_丙二醇的积累是不利的。 1,3-propanediol oxidoreductase enzyme is a two-way, both the catalytic conversion of 3-hydroxypropionaldehyde to 1,3-propanediol, 1,3-propanediol can be generated converted to 3-hydroxypropionaldehyde, so that the 1 , 3_ propylene glycol accumulation is unfavorable. 而克雷伯氏菌DSM2(^6(德国国家微生物菌种保藏中心,编号20¾)中的醛还原酶,具有逆反应为零的性质,能将3-羟基丙醛转化为1, 3-丙二醇,从而解除3-羟基丙醛的毒害作用,有利于1,3-丙二醇的合成。 And Klebsiella DSM2 (^ 6 (German National Collection of Microorganisms, the aldehyde reductase number 20¾), has a property of zero reverse reaction can be converted to 3-hydroxypropionaldehyde 1, 1,3-propanediol, thereby releasing the toxic effects of 3-hydroxypropionaldehyde, it is conducive to the synthesis of 1,3-propanediol.

[0007] 由生产1,3-丙二醇的微生物甘油代谢途径可见,菌体内NADH的量是限制1,3_丙二醇生成的一个重要因素。 [0007] apparent from microbial metabolic pathway glycerol 1,3-propanediol production, the amount of NADH bacterial cell is an important factor in generating 1,3_ glycol limitation. 考虑到菌体内存在大量的NADPH,而某些微生物,比如克雷伯氏菌属(Klebsiella)、柠檬酸菌属(Citrobacter)、梭菌属(Clostridium)等中的菌种,其中的1,3_丙二醇氧化还原酶不能有效利用NADPH合成1,3_丙二醇。 Considering the large number of memory cells of NADPH, and some microbes, such as Klebsiella (Klebsiella), citric acid genus (Citrobacter), Clostridium (Clostridium) bacteria and the like, wherein the 1,3 _ propanediol oxidoreductase can not effectively use NADPH synthesis 1,3_ glycol. 而醛还原酶是天然的可以利用NADPH作为辅酶的,同时我们克隆得到的醛还原酶既可以利用NADPH,又可以利用NADH,从而加强了醛还原酶的催化能力。 The aldehyde reductase is natural can utilize NADPH as a coenzyme, and we cloned aldehyde reductase can use both NADPH, and can take advantage of NADH, thereby strengthening the ability to catalyze aldehyde reductase. 如果使用醛还原酶在这些微生物体内实现高效表达,这样不仅能够维系氧化途径与还原途径的平衡,而且能够有效利用菌体内还原力,有利于1,3-丙二醇的浓度和生产强度的提高。 If efficient aldehyde reductase in vivo expression in these microorganisms, so that not only maintain the oxidation and reduction equilibrium pathway pathway, and can be effectively utilized in vivo bacteria reducing power, it is conducive to concentration and the strength to improve production of 1,3-propanediol.

发明内容 SUMMARY

[0008] 本发明要解决的技术问题是提供一种能够高产1,3-丙二醇的重组菌株,提高1, 3-丙二醇的产量。 [0008] The present invention is to solve the technical problem of providing a recombinant strain capable of high yield of 1,3-propanediol, increased by 1, 3-propanediol production.

[0009] 本发明的技术方案如下: [0009] aspect of the present invention is as follows:

[0010] 第一步:本发明所述醛还原酶的基因来自于克雷伯氏菌属(Klebsiella)、 大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、痢疾杆菌(Shigellaflexneri)或沙门氏菌属(Salmonella)。 [0010] First Step: The present invention is an aldehyde reductase gene from Klebsiella (Klebsiella), E. coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Shigella flexneri (Shigellaflexneri) or Salmonella genus (Salmonella). 所述醛还原酶具有如SEQ ID NO :1所示的氨基酸序列或者因取代、缺失、插入和/或添加一或几个氨基酸残基而与SEQ ID NO :1 的序列有所不同,但仍具有相同酶活性的酶的氨基酸序列。 The aldehyde reductase having the SEQ ID NO: 1 or the amino acid sequence shown by substitution, deletion, insertion and / or addition of one or several amino acid residues of SEQ ID NO: 1 sequence is different, but still It has the same enzymatic activity of the amino acid sequence. 所述醛还原酶基因具有如SEQ ID NO :2所示的核苷酸序列,其简并性序列、或者编码因取代、缺失、插入和/或添加一或几个氨基酸残基而与SEQ ID NO :2所编码序列有所不同,但仍具有相同酶活性的酶的核苷酸序列。 As the aldehyde reductase gene having SEQ ID NO: 2 nucleotide sequence thereof, a degenerated sequence, or the coding by substitution, deletion, insertion and / or addition of one or several amino acid residues in SEQ ID NO: 2 encoded by different sequences, but still having the same enzymatic activity of the nucleotide sequence.

[0011] 第二步:本发明提供了一种重组表达载体,其中包含一个或多个拷贝的在启动子控制下的醛还原酶基因。 [0011] Step Two: The present invention provides a recombinant expression vector, which contains one or more copies of aldehyde reductase gene under control of a promoter.

[0012] 所述启动子为本领域技术人员所熟知的能够应用于原核基因表达的合适启动子。 [0012] The promoters are known to those skilled in the art can be applied to gene expression in a suitable prokaryotic promoter. 所述启动子选自Pk(蛋白激酶)启动子、nif (固氮)启动子或dha(二羟丙酮)启动子之中的组成型启动子,或者是选自Iac(乳糖)启动子、T7(噬菌体)启动子、tac启动子(乳糖和色氨酸的杂合启动子)或trp (色氨酸)启动子之中的可诱导型启动子。 Pk of said promoter is selected from (protein kinase) promoter, among NIF (nitrogen fixation) promoter or DHA (dihydroxyacetone) promoter, a constitutive promoter, or is selected from the group of Iac (lactose) promoter, the T7 ( phage) promoter, tac promoter (a hybrid lactose and tryptophan promoter) or Trp (tryptophan) promoter being inducible promoter.

[0013] 所述重组载体具有任何合适的载体骨架,例如可选自pBR322、pUC系列、pET系列或pDK系列载体。 [0013] The recombinant vector having any suitable vector backbone, for example, from pBR322, pUC series, the pET series or series pDK carrier.

[0014] 第三步:本发明提供了一种含有前述重组表达载体的宿主细胞。 [0014] The third step: The present invention provides a host cell containing the recombinant expression vector.

[0015] 所述宿主细胞选自克雷伯氏菌属(Klebsiella)、柠檬酸菌属(Citrobacter)或梭菌属(Clostridium)等。 [0015] The host cell is selected from Klebsiella (Klebsiella), citric acid genus (Citrobacter) or Clostridium (Clostridium) and the like.

[0016] 第四步:本发明提供了上述重组菌株在制备1,3_丙二醇中的应用。 [0016] Step IV: The present invention provides the use of the above recombinant strain prepared 1,3_ propylene glycol.

[0017] 发酵方式采用批式发酵、批式流加发酵或连续发酵等。 [0017] Fermentation embodiment uses batch fermentation, fed batch fermentation or continuous fermentation.

[0018] 其中所述发酵是在微生物培养过程中通入空气进行的有氧发酵、微氧发酵或者在微生物培养过程中通入氮气进行的厌氧发酵。 [0018] wherein the fermentation process is fed microbial culture aerobic fermentation of air, or microaerobic fermentation of a microorganism culture into nitrogen during anaerobic fermentation.

[0019] 种子及发酵培养基中含有碳源、氮源、无机盐、维生素以及菌株生长所需的其它各种成分。 [0019] The seed and fermentation medium containing a carbon source, a nitrogen source, inorganic salts, vitamins and other ingredients required for the growth of the strain.

[0020] 发酵接种量1〜12%,培养温度20〜50°C,发酵时通气量为0. 1〜lvvm,调节pH 维持在5. 0〜9. 0,搅拌转速为80〜350rpm,培养时间10〜50h。 [0020] 1~12% inoculum fermentation, culture temperature 20~50 ° C, aeration rate of the fermentation 0. 1~lvvm, adjusting the pH was maintained at 5. 0 ~ 9. 0, stirring speed 80~350rpm, culture time 10~50h.

[0021] 本发明的效果和益处是:同原始菌株相比,利用本发明提供的重组菌株进行发酵, 1,3-丙二醇的产量提高10〜50 %。 [0021] The effects and benefits of the present invention are: compared with the original strain, the recombinant strains of the present invention provides the use of fermented, 1,3-propanediol production increased by 10~50%.

具体实施方式 detailed description

[0022] 以下结合技术方案详细叙述本发明的具体实施例。 [0022] The following detailed description of embodiments in conjunction with the specific aspect of the present invention.

[0023] 实施例: [0023] Example:

[0024] (1)醛还原酶基因yqhD的克隆: [0024] (1) an aldehyde reductase gene yqhD Cloning:

[0025] 根据GenBank中公开的克雷伯氏菌(Klebsiella pneumoniae) ASl. 1736的醛还原酶基因序列(yqhD, ID :EU012494)设计引物如下: [0025] The disclosed in GenBank Klebsiella (Klebsiella pneumoniae) ASl aldehyde reductase gene sequence 1736 (yqhD, ID: EU012494). Primers were designed as follows:

[0026] Pl : 5' -ATGAATAATTTCGACCTGCATACCC-3' (SEQ ID NO :3) [0026] Pl: 5 '-ATGAATAATTTCGACCTGCATACCC-3' (SEQ ID NO: 3)

[0027] P2 :5,-TTAGCGTGCAGCCTCGTAAATAC-3,(SEQ ID NO :4) [0027] P2: 5, -TTAGCGTGCAGCCTCGTAAATAC-3, (SEQ ID NO: 4)

[0028] 以克雷伯氏菌(Klebsiella pneumoniae)DSM2(^6基因组(制备方法见F.奥斯伯等著,《精编分子生物学实验指南》,科学出版社,1998)为模板完成PCR反应(参见Sambrook, J.,Russel, DW,Molecular Cloning :A Laboratory Maunal3rd ed.,Cold Spring Harbor, New York : Co Id Spring Harbor Laboratory Press, 2001)。在PCR 反应管中加入以下成分:基因组DNAl μ 1,dNTPs4l·! 1,引物Pl和P2 (由宝生物工程(大连)有限公司合成)各1 μ 1,5 μ IlOXEx Taq buffer, IU Ex Taq DNA聚合酶,补水至50 μ 1 ;反应条件:95°C变性60s,经95°C 30s,58 °C lmin、72°C anin30个循环。获得的PCR产物经电泳分析确认,经凝胶回收试剂盒纯化后,回收后的产物连入载体PMD18-T,获得重组质粒pMD18-T_yqhD。热击法转化Ε. coli DH5 α,在含有氨苄青霉素的抗性平板上对阳性重组体进行筛选,然后挑取单克隆,提取质粒,对双酶切鉴定正确的重组质粒,送 [0028] In Klebsiella (Klebsiella pneumoniae) DSM2 (^ 6 genome (preparation F. Aosi Bo waiting "for fine in Molecular Biology", Science Press, 1998) as a template PCR was performed reaction (see Sambrook, J., Russel, DW, Molecular Cloning: A Laboratory Maunal3rd ed, Cold Spring Harbor, New York:. Co Id Spring Harbor Laboratory Press, 2001) by adding the following ingredients in the PCR reaction tube: genomic DNAl μ ! 1, dNTPs4l · 1, the primers Pl and P2 (by Takara Biotechnology (Dalian) Co., Ltd. synthesis) each 1 μ 1,5 μ IlOXEx Taq buffer, IU Ex Taq DNA polymerase, water to 50 μ 1; reaction conditions: 95 ° C denaturation 60s, was 95 ° C 30s, 58 ° C lmin, 72 ° C anin30 cycles. PCR product obtained was confirmed by electrophoresis, the gel was recovered the reagent cartridge purification, the recovered product was ligated into vector PMD18 -T, recombinant plasmid pMD18-T_yqhD. Ε heat shock transformation method. coli DH5 α, of the positive recombinants were screened on plates containing ampicillin resistance, and then monoclonal picked, plasmid was extracted, double digestion of the recombinant plasmid, send 测序。 Sequencing.

[0029] (2)醛还原酶基因yqhD的亚克隆: [0029] (2) Subcloning of aldehyde reductase gene yqhD:

[0030] 利用测序正确的重组载体pMD18-T-yqhD为模板,通过PCR引入酶切位点,设计引物如下: [0030] The correct sequence using recombinant vector pMD18-T-yqhD as a template, introducing restriction sites by the PCR, the following primers were designed:

[0031] P3 : 5 ' -CCTGCAGGTCGACGCATATGAATAATT-3 ' (SEQ ID NO :5) [0031] P3: 5 '-CCTGCAGGTCGACGCATATGAATAATT-3' (SEQ ID NO: 5)

[0032] P4 : 5 ' -ACCCGGGGATCCTCTAGAGATTTTAGCGTG-3 ' (SEQ ID NO :6) [0032] P4: 5 '-ACCCGGGGATCCTCTAGAGATTTTAGCGTG-3' (SEQ ID NO: 6)

[0033] 引物两端分别引入Nde I和BamH I酶切位点。 Both ends of the [0033] primers introduced Nde I and BamH I restriction sites. 以重组质粒pMD18_T-yqhD为模板, 以P3和P4为引物,扩增得到带有酶切位点的yqhD基因。 PMD18_T-yqhD recombinant plasmid as template and primers P3 and P4, amplified yqhD gene with restriction sites. 获得的PCR产物经电泳分析确认, 经凝胶回收试剂盒纯化后双酶切,回收后的酶切产物连入经过同样酶切的载体pET23a(+), 获得重组质粒pET23a(+)-yqhD。 PCR products were analyzed by electrophoresis obtained confirmed by double digestion after purification Gel Extraction Kit, the digestion product was recovered and ligated into similarly digested pET23a vector after (+), recombinant plasmid pET23a (+) - yqhD. 热击法转化Ε. coli DH5 α,在含有氨苄青霉素的抗性平板上对阳性重组体进行筛选,然后挑取单克隆,提取质粒,对双酶切鉴定正确的重组质粒,送样测序。 Heat shock conversion method Ε. Coli DH5 α, of the positive recombinants were screened on plates containing ampicillin resistance, and then monoclonal picked, plasmids were extracted for double digestion of recombinant plasmids, sequencing sample delivery.

[0034] (3)醛还原酶在大肠杆菌中的表达: [0034] (3) an aldehyde reductase expression in E. coli:

[0035] 将测序正确的重组质粒pET23a(+)-yqhD热击转化E. coli BL21 (DE3),挑取单克隆接至LB培养基,37°C下170r/min培养12小时,转接培养至OD600为0. 6,加入IPTG至终浓度lmM,20°C下150r/min诱导12小时。 [0035] The sequenced recombinant plasmid pET23a (+) - yqhD heat shock transformed E. coli BL21 (DE3), connected to the monoclonal picked LB medium, 170R at 37 ° C / min cultured for 12 hours, transfer the culture to an OD600 of 0.6, IPTG was added to a final concentration of lmM, 150r at 20 ° C / min induced 12 hours. 取4ml菌液,4°C,12000r/min离心15min收集菌体,重悬,超声破碎,4°C,8000r/min离心20min,取上清进行酶活力测定和SDS-PAGE。 Take 4ml broth, 4 ° C, 12000r / min 15min cells were harvested by centrifugation, resuspended, sonicated, 4 ° C, 8000r / min centrifugal 20min, supernatant enzyme activity assay and SDS-PAGE. 浓缩胶浓度为4%,分离胶浓度为12%,电泳缓冲液采用Tris-甘氨酸系统。 Concentration of 4% stacking gel, 12% separation gel concentration, using electrophoresis Tris- Glycine buffer system.

[0036] (4)醛还原酶的纯化及酶活测定: [0036] (4) Purification and aldehyde reductase activity assay:

[0037] 醛还原酶的纯化首先采用离子交换层析Οί-S印harose FF)。 [0037] The first aldehyde reductase purified by ion exchange chromatography Οί-S plate harose FF). 样品为细胞破碎获取的细胞粗提液,上样前需用0. 45 μ m的微孔滤膜过滤处理。 Cell disruption sample was obtained the crude cell extract, filter membrane required processing before 0. 45 μ m on the sample. 用PH7. 4的50mM Tris-HCl 缓冲液(含0. ImM Mn2\2mM DTT)上样,流速lml/min,洗去未紧密结合的蛋白质,用含有IM KCl的上述Tris-HCl缓冲液进行线性梯度洗脱,流速为5ml/min。 With 50mM Tris-HCl buffer (0. ImM Mn2 \ 2mM DTT) PH7. 4 samples with a flow rate lml / min, wash away the non-bound proteins close, the above-described linear Tris-HCl buffer containing IM KCl in gradient elution, flow rate 5ml / min. 采用部分收集器连续收集洗脱液,每管收集:3ml。 Fraction collector using a continuous eluate collected per tube: 3ml. 层析过程中通过观察^Onm下吸光度来判断蛋白质洗脱情况,选择有吸收峰的部分进行酶活力测定。 During chromatography by observing the absorbance at ^ Onm eluted protein is determined, the selected portion of the absorption peak of the enzyme activity was measured. 蛋白质浓度需要采用考马斯亮蓝法定量测量。 The protein concentration using Coomassie need Quantitative measurements.

[0038] 测定离子交换层析所收集的洗脱液,取其酶活较为显著的部分合并起来,用凝胶过滤层析(kphacryl S-300)进一步分离纯化。 [0038] Determination of ion exchange chromatography eluate collected, whichever is the more significant part of the enzymatic activity combined, is further isolated and purified by gel filtration chromatography (kphacryl S-300). 用含有pH7. 4的50mMTris-HCl缓冲液(含0. ImM Mn2+、2mM DTT)进行洗脱,流速为0. 5ml/min,分步收集每管1ml。 Containing 50mMTris-HCl buffer (0. ImM Mn2 +, 2mM DTT) and eluted at a flow rate of 0. 5ml pH7. 4 min to, fractional collection / each tube 1ml.

[0039] 经过纯化后,得到电泳条带单一的纯酶。 [0039] After purification, give pure single electrophoretic bands enzyme. 以3-羟基丙醛为底物,测得的该酶的比活力为3.8U/mg;而以1,3_丙二醇为底物时,检测不到酶活力。 3-hydroxypropionaldehyde to as a substrate, the specific activity of the enzyme was measured 3.8U / mg; and when to 1,3_-propanediol as substrate, enzyme activity is not detected. 同时,发现该酶在辅酶的利用上具有灵活性,既能利用NADPH,又能利用NADH。 At the same time, we found that the enzyme has flexibility in the use of coenzyme, both the use of NADPH, but also the use of NADH.

[0040] 初速度法测定酶活力:光径0. 5cm的石英比色皿中,1. 5mL反应液(27mM3_羟基丙醛、0.37mM NADH 或NADPH、1 μ M ZnCl2UOOmM Tris-HCl buffer, ρΗ7·4),加入0. Iml 的酶液启动反应,立即计时测定反应液的吸光度变化值。 Enzyme activity assay [0040] The initial velocity method: the optical path of 0. 5cm quartz cuvette, 1 5mL reaction solution (27mM3_ hydroxypropionaldehyde, 0.37mM NADH or NADPH, 1 μ M ZnCl2UOOmM Tris-HCl buffer, ρΗ7 1.4), the enzyme solution was added 0. Iml start of the reaction, the absorbance was measured immediately change the timing of the reaction solution. 酶活定义为:37°C下,催化3-羟基丙醛转化为1,3-丙二醇时,340nm处每分钟NADH或NADPH消耗的微摩尔数。 Activity is defined as: the time at 37 ° C, catalytic conversion of 3-hydroxypropionaldehyde to 1,3-propanediol, at 340nm micromoles per minute NADH or NADPH consumed.

[0041] (5)重组克雷伯氏菌(Klebsiellapneumoniae)的构建: [0041] (5) a recombinant Klebsiella (Klebsiella pneumoniae) Construction:

[0042] 1)重组表达载体的构建: Construction [0042] 1) The recombinant expression vector:

[0043] 重组质粒pET23a(+)-yqhD经Xba I酶切后,克隆至经同样酶切后的表达载体pDK 中,热击法转化E. coli DH5a。 [0043] The recombinant plasmid pET23a (+) - yqhD after digestion by Xba I, cloned into the expression vector digested likewise by the pDK, the heat shock conversion method E. coli DH5a. 挑取单克隆,提取质粒pDK-yqhD,进行双酶切鉴定。 Pick a single colony, plasmid was extracted pDK-yqhD, for double digestion.

[0044] 2)电转化克雷伯氏菌: [0044] 2) electrically converted Klebsiella:

[0045] 将双酶切鉴定正确的重组质粒pDK-yqhD电击转化克雷伯氏菌(Klebsiellapneumoniae)DSM2026。 [0045] The double digestion of the recombinant plasmid electroporation pDK-yqhD Klebsiella (Klebsiellapneumoniae) DSM2026.

[0046] 3)重组克雷伯氏菌的表达: [0046] 3) The recombinant expression Klebsiella:

[0047] 挑取单克隆接至LB培养基,37°C下170r/min培养至12小时,转接一次培养至OD600为0. 6,加入IPTG至终浓度ImM, 370CT 120r/min诱导5小时。 [0047] Pick a single colony into LB medium connected, 170R at 37 ° C / min to 12 hr culture, primary transfer to an OD600 of 0.6 the culture was added IPTG to a final concentration of ImM, 370CT 120r / min 5 hours of induction . 分别对诱导后全细胞蛋白和破碎后的上清液进行SDS-PAGE。 Whole cell proteins were induced and the supernatants were crushed for SDS-PAGE. 电泳结束后,有42. 3kD蛋白亚基的条带出现,说明醛还原酶在克雷伯氏菌中实现了表达。 After the run, there is Article 42. 3kD protein subunit bands appear, indicating aldehyde reductase achieve expression in Klebsiella.

[0048] (6)重组菌发酵甘油生产1,3_丙二醇: [0048] (6) Production of recombinant fermentation of glycerol propylene glycol 1,3_:

[0049] 1)菌种:克雷伯氏菌(Klesiella pneumoniae)DSM2026,重组克雷伯氏菌(Klebsiella pneumoniae)(pDK-yqhD)。 [0049] 1) Strain: Klebsiella (Klesiella pneumoniae) DSM2026, recombinant Klebsiella (Klebsiella pneumoniae) (pDK-yqhD).

[0050] 2)培养基: [0050] 2) medium:

[0051]种子培养基(/1):甘油 20g、K2HPO4 · 3Η204· 454g, KH2PO4L 3g、(NH4) 2S042. 0g、 MgSO4 · 7H200. 2g、酵母粉1. 0g、CaC032g、Fe2+ 溶液1ml、Ca2+ 溶液1ml、微量元素A2ml。 [0051] The seed medium (/ 1): Glycerol 20g, K2HPO4 · 3Η204 · 454g, KH2PO4L 3g, (NH4) 2S042 0g, MgSO4 · 7H200 2g, yeast 1. 0g, CaC032g, Fe2 + solution 1ml, Ca2 + solution 1ml, trace elements A2ml.

[0052]发酵培养基(/1):甘油 50g、K2HPO4 · 3Η204· 454g, KH2PO4L 3g、(NH4) 2S042. 0g、 MgSO4 · 7H200. 2g、酵母粉1. 0g、CaC032g、Fe2+ 溶液1ml、Ca2+ 溶液1ml、微量元素A2ml。 [0052] Fermentation medium (/ 1): Glycerol 50g, K2HPO4 · 3Η204 · 454g, KH2PO4L 3g, (NH4) 2S042 0g, MgSO4 · 7H200 2g, yeast 1. 0g, CaC032g, Fe2 + solution 1ml, Ca2 + solution. 1ml, trace elements A2ml.

[0053] Fe2+ 溶液(/1) =FeSO4 · 7H205g、饱和盐酸4ml。 [0053] Fe2 + solution (/ 1) = FeSO4 · 7H205g, hydrochloric acid-saturated 4ml.

[0054] Ca2+溶液(/1) :CaCl220g。 [0054] Ca2 + solution (/ 1): CaCl220g.

[0055]微量元素 A 溶液(/1) =ZnCl2O. 07g、MnCl2 · 4Η200· lg、CoCl2 · 6Η200· 2g、 NiCl2 · 6Η200· 025g、CuCl2 · 2Η200· 02g、NaMoO4 · 2Η200· 035g、H3BO3O. 06g、饱和盐酸0· 9ml。 [0055] A trace element solution (/ 1) = ZnCl2O. 07g, MnCl2 · 4Η200 · lg, CoCl2 · 6Η200 · 2g, NiCl2 · 6Η200 · 025g, CuCl2 · 2Η200 · 02g, NaMoO4 · 2Η200 · 035g, H3BO3O. 06g, saturated hydrochloric acid 0 · 9ml.

[0056] 3)培养方式: [0056] 3) culture methods:

[0057] A.种子培养 [0057] A. Seed culture

[0058] 从固体培养基上挑取单克隆,37°C下使用种子培养基活化。 [0058] Pick a single colony from a solid medium, the use of activated seed medium at 37 ° C. 种子培养使用50ml三角瓶,装液量10ml。 Seed culture flasks using 50ml, liquid volume 10ml. 培养温度37°C,摇床转速170rpm,培养时间12小时。 Culture temperature 37 ° C, shaking speed of 170rpm, 12 hours culture.

[0059] B.发酵培养 [0059] B. Fermentation

[0060] 对照组使用克雷伯氏菌作为菌种,实验组使用重组克雷伯氏菌(pDK-yqhD)作为菌种。 [0060] The control group using recombinant Klebsiella Klebsiella (pDK-yqhD) as strains, experimental strains as a group. 发酵使用250ml三角瓶,装液量100ml,接种量2%,培养温度37°C,摇床转速120rpm。 250ml flask used fermentation, liquid volume 100ml, 2% inoculation, culture temperature 37 ° C, shaking speed of 120rpm. 发酵开始2小时后实验组加入IPTG诱导表达。 Beginning of the fermentation the experiment group was added after 2 hours and induced with IPTG.

[0061] 4)发酵结果: [0061] 4) Fermentation Results:

[0062] 发酵共进行30小时,发酵结束时对照组发酵液中1,3_丙二醇浓度为16. 89g/l,实验组发酵液中1,3_丙二醇浓度为19. 77g/l。 [0062] The total fermentation 30 hours, at the end of fermentation 1,3_ glycol group concentration of 16. 89g fermentation broth / l, the experimental group 1,3_ propanediol fermentation broth at a concentration of 19. 77g / l. 由此可见,使用重组克雷伯氏菌作为发酵菌种进行发酵,1,3-丙二醇浓度比对照提高17. 1 %。 Thus, using recombinant Klebsiella strains for fermentation as a fermentation, the concentration of 1,3-propanediol 17.1% increase compared with the control.

[0063] 序列表 [0063] SEQUENCE LISTING

[0064] SEQ ID NO :1 [0064] SEQ ID NO: 1

[0065] [0065]

MNNFDLHTPTRILFGKGAIEKLREQIPAEARVLITYGGGSVKKTGVLDQVLTA MNNFDLHTPTRILFGKGAIEKLREQIPAEARVLITYGGGSVKKTGVLDQVLTA

LNGLDVLEFGGIEPNPSYETLMNAVKLAREEKVTFLLAVGGGSVLDGTKFIA LNGLDVLEFGGIEPNPSYETLMNAVKLAREEKVTFLLAVGGGSVLDGTKFIA

AAAHYDADIDPWEILETYGSKIASAIPMGSVLTLPATGSESNKGAVISRKTTG AAAHYDADIDPWEILETYGSKIASAIPMGSVLTLPATGSESNKGAVISRKTTG

DKRAFMSSHVQPQFAILDPVYTYTLPPRQVANGVVDAFVHrVEQYVTYPVD DKRAFMSSHVQPQFAILDPVYTYTLPPRQVANGVVDAFVHrVEQYVTYPVD

GKIQDRFAEGILLTLIEDGPKALQEPENYNVRANIMWAATQALNGLIGAGVP GKIQDRFAEGILLTLIEDGPKALQEPENYNVRANIMWAATQALNGLIGAGVP

QDWATHMLGHELTAMHGLDHAQTLAIVLPALWNEKRDAKREKLLQYAER QDWATHMLGHELTAMHGLDHAQTLAIVLPALWNEKRDAKREKLLQYAER

VWNITEGSDDQRIDAAIAATRQFFEQMGVPTRLSDYGLDGSSIPALLAKLEEH VWNITEGSDDQRIDAAIAATRQFFEQMGVPTRLSDYGLDGSSIPALLAKLEEH

GMTKLGEHQDITLDVSRRIYEAAR GMTKLGEHQDITLDVSRRIYEAAR

[0066] SEQ ID NO :2 [0066] SEQ ID NO: 2

[0067] [0067]

5'-atgaataatttcgacctgcataccccaacccgcattctgtttggcaaaggcgcgattgaaaagctgcgtgaacagatcc 5'-atgaataatttcgacctgcataccccaacccgcattctgtttggcaaaggcgcgattgaaaagctgcgtgaacagatcc

cggcggaagcccgcgtactgatcacctacggcggcggcagcgtcaaaaaaacaggcgtcctggatcaggtcctcaccg cggcggaagcccgcgtactgatcacctacggcggcggcagcgtcaaaaaaacaggcgtcctggatcaggtcctcaccg

ctctgaatggcctggatgtccttgaatttggcggcatcgagccgaatccgtcttacgaaaccctgatgaatgcggtgaaact ctctgaatggcctggatgtccttgaatttggcggcatcgagccgaatccgtcttacgaaaccctgatgaatgcggtgaaact

cgcccgggaagagaaagtgaccttcctgctggcggtcggcggcggttcggtgctggatggcaccaagtttatcgctgca cgcccgggaagagaaagtgaccttcctgctggcggtcggcggcggttcggtgctggatggcaccaagtttatcgctgca

gcagcccactacgacgcggatatcgatccgtgggaaattctggaaacctatggcagcaaaattgccagcgccattccaat gcagcccactacgacgcggatatcgatccgtgggaaattctggaaacctatggcagcaaaattgccagcgccattccaat

gggctcggtactgactctgccggcgaccggttctgaatccaacaaaggcgcggtcatatcgcggaaaaccaccggcga gggctcggtactgactctgccggcgaccggttctgaatccaacaaaggcgcggtcatatcgcggaaaaccaccggcga

caaacgcgcgtttatgtcttcgcacgtccagccgcagttcgcgatcctcgatccggtttatacctataccctgccgccgcgc caaacgcgcgtttatgtcttcgcacgtccagccgcagttcgcgatcctcgatccggtttatacctataccctgccgccgcgc

caggtcgcgaacggcgtggttgacgccttcgtccataccgtcgagcagtacgtgacctacccggttgacggcaaaatcca caggtcgcgaacggcgtggttgacgccttcgtccataccgtcgagcagtacgtgacctacccggttgacggcaaaatcca

ggaccgcttcgccgaaggcattctcctgaccctgatcgaggatggcccgaaagccctgcaggaaccggagaactataac ggaccgcttcgccgaaggcattctcctgaccctgatcgaggatggcccgaaagccctgcaggaaccggagaactataac

gtgcgcgccaatattatgtgggcggcgacgcaggcgctgaacggcctgatcggcgcaggcgtgccacaggactgggc gtgcgcgccaatattatgtgggcggcgacgcaggcgctgaacggcctgatcggcgcaggcgtgccacaggactgggc

gacgcatatgctcggccacgagctgacggcgatgcacggcctggatcacgcccagacgctggctatcgtgctgccggc gacgcatatgctcggccacgagctgacggcgatgcacggcctggatcacgcccagacgctggctatcgtgctgccggc

gctgtggaatgagaaacgcgatgccaagcgcgagaagctgctgcagtatgccgagcgcgtgtggaatattaccgaagg gctgtggaatgagaaacgcgatgccaagcgcgagaagctgctgcagtatgccgagcgcgtgtggaatattaccgaagg

ctctgacgaccaacgtatcgatgccgccatcgccgcaacgcgtcagttcttcgagcagatgggcgtgccgacccgccttt ctctgacgaccaacgtatcgatgccgccatcgccgcaacgcgtcagttcttcgagcagatgggcgtgccgacccgccttt

ccgattacggtctcgacggtagctccatcccggcgctgctggcgaaactggaagagcacggcatgactaaacttggcga ccgattacggtctcgacggtagctccatcccggcgctgctggcgaaactggaagagcacggcatgactaaacttggcga

[0068] [0068]

Figure CN101633928BD00091

[0069] SEQ ID NO :3 [0069] SEQ ID NO: 3

[0070] [0070]

5,- ATGAATAATTTCGACCTGCATACCC -3, 5, - ATGAATAATTTCGACCTGCATACCC -3,

[0071] SEQ ID NO :4 [0071] SEQ ID NO: 4

[0072] [0072]

5'- TTAGCGTGCAGCCTCGTAAATAC -3' 5'- TTAGCGTGCAGCCTCGTAAATAC -3 '

[0073] SEQ ID NO :5 [0073] SEQ ID NO: 5

[0074] [0074]

5'- CCTGCAGGTCGACGCATATGAATAATT -3' 5'- CCTGCAGGTCGACGCATATGAATAATT -3 '

[0075] SEQ ID NO :6 [0075] SEQ ID NO: 6

[0076] [0076]

5'- ACCCGGGGATCCTCTAGAGATTTTAGCGTG -3' 5'- ACCCGGGGATCCTCTAGAGATTTTAGCGTG -3 '

Claims (1)

  1. 1. 一种醛还原酶的重组表达在甘油生物转化为1,3_丙二醇中的应用,其特征在于:(1)使用的醛还原酶基因的核苷酸序列如SEQ ID NO :2所示或者为SEQ ID NO :2的简并性序列;(2)使用的重组表达载体含有一个拷贝的在启动子控制下的上述醛还原酶基因;其中启动子选自组成型启动子或可诱导型启动子;其中组成型启动子选自蛋白激酶启动子、固氮启动子或二羟丙酮启动子;可诱导型启动子选自乳糖启动子、噬菌体启动子、乳糖和色氨酸的杂合启动子或色氨酸启动子;载体的骨架选自PBR322、pUC系列、pET系列或pDK系列载体;(3)使用的宿主细胞为克雷伯氏菌属;(4)发酵方式采用批式发酵、批式流加发酵或连续发酵;发酵是在微生物培养过程中通入空气进行的微氧发酵或者在微生物培养过程中通入氮气进行的厌氧发酵;种子及发酵培养基中含有碳源、氮源、无机盐 An aldehyde reductase expression in recombinant bioconversion of glycerol application 1,3_ propanediol, wherein: (1) a nucleotide sequence of using aldehyde reductase gene as SEQ ID NO: 2 shown in FIG. or of SEQ ID NO: 2, degenerate sequences; (2) a recombinant expression vector containing a copy of the aldehyde reductase gene under control of a promoter; wherein the promoter is selected from a constitutive promoter or an inducible promoter; wherein the constitutive promoter is selected from protein kinase promoter, nitrogenase promoter, or a promoter dihydroxyacetone; inducible promoter is selected from the lactose promoter, hybrid promoters phage promoter promoter, lactose and tryptophan or tryptophan promoter; backbone vector is selected from PBR322, pUC series, the pET vector series or series pDK; host cell (3) used is Klebsiella; (4) fermenting the batch fermentation way using batch formula fed or continuous fermentation; fermenting microorganism during the culture is passed through a micro air or oxygen in the fermentation microorganism culture into the process of anaerobic fermentation nitrogen; and seed fermentation medium containing a carbon source , inorganic salts 维生素;发酵接种量1〜12%,培养温度20〜50°C,发酵时通气量为0. 1〜1. Ovvm,调节pH维持在5. 0〜9. 0,搅拌转速为80〜350rpm,培养时间10〜50h。 Vitamins; 1~12% inoculum fermentation, the culture temperature is 20~50 ° C, aeration of the fermentation in an amount of 0. 1~1 Ovvm, adjusting the pH maintained at 0 5. 0 ~ 9, stirring speed 80~350rpm,.. incubation time 10~50h.
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