【作者】 刘慧敏；

【导师】 陈冠军； 祁庆生；

【作者基本信息】 山东大学 ， 微生物学， 2009， 博士

【摘要】 随着石油资源的不断枯竭,人们逐渐认识到作为众多化学中间体和终产物的生物基平台化合物的重要性。其中,羟基丙酸就是其中最重要的生物基平台化合物。羟基丙酸包括3-羟基丙酸和2-羟基丙酸(乳酸),二者均具有羟基和羧基两种官能团,是重要的化学中间体。3-羟基丙酸(3-HP)在工业上可以脱水生成丙烯酸,氧化生成丙二酸,与醇酯化作用生成酯,还可通过还原作用生成1,3-丙二醇等,是最具潜力的生物基平台化合物之一。目前,3-HP的生产方法主要是化学合成法,但其难度较大,且产品分离纯化复杂,生产成本相应较高,只有少量合成供实验室使用。而利用基因工程菌株生产3-羟基丙酸的方法主要包括两方面：Ⅰ、构建基因工程菌株,以葡萄糖为底物生产3-HP；Ⅱ、构建基因工程菌株,以甘油为底物生产3-HP。但上述两种方法的应用分别由于3-HP合成路径的构建存在较高难度和产量低、无法达到大规模生产的要求而受到限制。另一种羟基丙酸—乳酸是一种应用广泛的有机酸,可用于食品、医药、纺织业和化工等行业,自然界中产乳酸的微生物种类很多,与其它微生物相比,用大肠杆菌生产乳酸有培养周期短,易于控制乳酸的光学纯度,营养要求粗放等优势。随着基因工程的发展,通过改变菌株的代谢途径构建高产乳酸的重组大肠杆菌具有更加方便、实用的优点。而以乳酸为单体的聚合物—聚乳酸具有优良的生物可降解性和良好的生物相容性,被认为是最广泛和最有前景的高分子材料之一。目前,聚乳酸主要由乳酸经开环聚合法合成,但此类化学合成法通常需要添加有毒的金属催化剂,给人类和环境带来了危害。如何利用微生物进行聚乳酸的生物合成受到人们的广泛关注基于上述几个方面,本文对大肠杆菌中3-HP合成途径的构建,高产乳酸重组菌株的构建和发酵条件对乳酸产量的影响及PLA全生物合成的实现几方面进行了研究,主要的工作内容及结果如下：1.以葡萄糖为底物生产3-HP的重组大肠杆菌的构建及初步发酵研究橙色绿曲挠菌Chloroflexus aurantiacus中3-HP循环的第一步反应是由acetyl-CoA carboxylase催化acetyl-CoA同CO2羧化成malonyl-CoA,之后malonyl-CoA在malonyl-CoA reductase催化下被还原成3-HP。在E. coli中,同样的羧化反应是脂肪酸合成的第一步,由多组分的acetyl-CoA carboxylase (ACCase)催化。C. aurantiacus中的malonyl-CoA reductase是一个双功能酶,由mcr基因编码,含有N-端醇脱氢酶活性和C-端醛脱氢酶活性,催化malonyl-CoA经两步反应还原成3-HP。本论文从C. aurantiacus strain OK-70-fl (DSM636)基因组中经PCR扩增获得mcr基因,将其克隆到表达载体pET-28a上构建了重组质粒pET-28a-mcr,转化E. coli BL21(DE3)获得重组大肠杆菌DE3/pET-28a-mcr,并借助其自身脂肪酸合成的第一步反应,构建了一条发酵葡萄糖合成3-HP的代谢途径。重组大肠杆菌DE3/pET-28a-mcr及对照菌株BL21 (DE3)/pET-28a分别接种于50 ml LB(2%葡萄糖)液体培养基中好氧发酵60 h,用GC检测3-HP的生成。3-HP标准样品的保留时间是3.2 min,而重组菌DE3/pET-28a-mcr的发酵产物在相同位置也有一个产物峰,经计算其产量约为0.15g/L。上述代谢途径的构建在国内尚属首次,构建过程易于实现,并可进一步通过代谢工程技术来提高3-HP的产量,为实现高产3-HP奠定了坚实的理论基础。2.高产乳酸重组大肠杆菌的构建及其发酵研究厌氧条件下,大肠杆菌发酵葡萄糖产生乳酸、琥珀酸、乙酸、甲酸等一系列有机酸和乙醇。大肠杆菌代谢产物的分配主要由ldhA基因编码的D-乳酸脱氢酶基因,pfl基因编码的丙酮酸甲酸裂解酶,ppc基因编码的磷酸烯醇式丙酮酸羧化酶决定。为了维持氧化还原平衡,大肠杆菌中的乙酰辅酶A在乙酸激酶／磷酸转乙酰酶和乙醇脱氢酶(adhE)的催化下产生等量的乙酸和乙醇。本论文利用代谢工程技术将E. coli W3110葡萄糖发酵代谢途径中的pflB、adhE基因进行了敲除,获得了重组菌株SD2和SD4。两者在厌氧发酵中的乳酸产量分别达到了174.8 mM和178.3 mM/100 mM葡萄糖,接近2 mol乳酸/mol所消耗葡萄糖的最大理论产量。同时,对大肠杆菌中负责葡萄糖运输的磷酸转移酶系统(PTS)中的ptsG基因的进行改造,得到重组菌株SD6、SD8。这两株菌对葡萄糖的摄取速率变慢,生物量大大提高。琥珀酸的产量相比SD2、SD4提高了5.4倍以上,乳酸产量下降。培养基的不同对SD6和SD8发酵产物的组成也产生了较大的影响。SD6、SD8在LB培养基中发酵60 h时乳酸的产量低于16 mM,而在M9培养基中乳酸的产量可以达到100 mM以上。琥珀酸的产量则从LB培养基中的45.5 mM和42.5 mM分别下降到了M9培养基中的34.5 mM和31.4mM。推测M9培养基中K+的存在是促进乳酸产量提高的原因。用Na+取代M9培养基中的K+对SD4和SD8进行了培养,两者的乳酸产量均有所降低,琥珀酸产量相应升高。相比于LB培养基,M9培养基更有利于乳酸的产生。用甘油和山梨醇替代葡萄糖作为碳源、向M9培养基中添加还原剂L-Cysteine HCl和Na2S及对重组菌株SD4和SD8进行严格厌氧发酵均提高了两株重组菌株中乳酸的产量,并相应扩大了乳酸和琥珀酸之间的摩尔比。其中厌氧程度的控制起到了关键性的决定作用。本论文成功构建了两株高产乳酸菌株SD4、SD8,并从不同的方面对影响乳酸生产的因素进行了详细的研究,为更好地实现乳酸的可控性生产提供了重要的技术支撑。3.重组P (LA-co-HB)生产菌株的构建及初步发酵研究PHA合酶是PHAs(聚羟基脂肪酸酯)合成过程中的关键酶,具有广泛的底物特异性,其底物--羟基酸具有同乳酸类似的化学结构。以3HB为单体的PHB是最为常见的一种PHA。基于此,我们构建了一株重组大肠杆菌DH5a/pBBR1pctEC+pBHR69来合成PLA和PHB的共聚物P(LA-co-3-HB)。在重组菌株中,P(LA-co-3-HB)由三步反应催化获得：(i)乳酸在辅酶A转移酶PCT (Clostridium propionicum)的催化下被活化成lactyl-CoA, (ⅱ) acetyl-CoA经来源于Ralstonia eutropha的PhaA (β-ketothiolase)和PhaB (NADPH-dependent acetoacetyl-CoA reductase)的催化生成3-hydroxybutyryl-CoA (3HB-CoA), (ⅲ)来源于Allochromatium vinosum的PHA合酶PhaEC催化lactyl-CoA和3-hydroxybutyryl-Co A之间的聚合。重组大肠杆菌DH5α/pBBR1pctEC+pBHR69在添加1%乳酸的好氧发酵条件下能够合成P(LA-co-3-HB),其中的乳酸含量为0.22 mol%,3HB含量为99.78 mol%。厌氧发酵条件下合成的P(LA-co-3-HB)中,乳酸含量提高到了1.49 mol%。此代谢途径的构建在国内尚属首次,弥补了国内技术上的空白,在国际上亦属先进水平,突破了PLA一步法生物合成的难关,对于实现PLA的全生物合成具有重要的理论意义和应用价值。更多还原

【Abstract】 Considering the limited deposits of fossil fuels, bio-based platform chemicals, building blocks for numerous chemical intermediates and end products, are recognized as a burning issue in the last decade. Hydroxypropionic acid, consisting of 3-hydroxypropionic acid (3-HP) and 2-hydroxypropionic acid (lactate), was identified as one of the most important platform chemicals. The presence of two functional groups with different properties makes both of them suitable precursor for the synthesis of many optically active substances.3-Hydroxypropionic acid is a chemical reagent well known for its simple structure and its high reactivity.3-HP has utility for specialty synthesis and can be converted to commercially important intermediates by known art in the chemical industry, e.g., acrylic acid by dehydration, malonic acid by oxidation, esters by esterification reactions with alcohols, and reduction to 1,3-propanediol.3-HP for commercial use is now commonly produced by chemical syntheses, its use has remained on a laboratory scale due to its insufficient production, complex separation/purification and higher production costs. At present, the production of 3-HP by genetic engineering and microbial fermentation consists mainly of two parts:I, The genetic engineering progress of producing 3-HP from glucose;Ⅱ, The genetic engineering progress of producing 3-HP from glycerol. However, it’s certainly difficult to construct the certain former pathway mentioned above. And the production of 3-HP from glycerol was only 0.17 g/L and unable to meet the requirement for large-scale production.Lactate (2-hydroxypropionic acid) is the most widely occurring hydroxycarboxylic acid, having versatile applications in food, pharmaceutical, textile, and chemical industries. Although several lactic acid bacteria, such as Lactobacillus species, were able to produce lactic acid in a large quantity by fermentation of glucose and other renewable resources, Escherichia coli has many advantages as a host for production of lactic acid, including rapid growth under both aerobic and anaerobic conditions, the ability to produce optically pure lactate, and its simple nutritional requirements. Moreover, the ease of genetic manipulation of E. coli makes possible metabolic engineering strategies for improving lactate accumulation in E. coli. Polylactate (PLA), which is chemically synthesized by ring-opening polymerization of a cyclic diester (lactide) of lactate, has attracted considerable interest as a natural, biodegradable, and biocompatible plastic. However, as the chemo-process of PLA can be carried out via harmful metal catalysts, it often leaves chemical residues that are subject to health and safety concerns. The paradigm shift from the chemo-process to the bio-process for PLA production is thus preferable to overcome this problem.In this article, we constructed a genetic pathway for 3-HP production from glucose in E. coli. A series of recombinants defecting in competitive pathways aiming to produce lactate effectively were obtained. We also established a recombinant E. coli that allows the synthesis of LA-based polyester. The major results of the article are as follows:1. Construction of recombinant E. coli to accumulate 3-HP from glucoseThe initial step of 3-HP cycle in Chloroflexus aurantiacus is the acetyl-CoA carboxylation to malonyl-CoA catalyzed by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-HP. In E. coli, the formation of malonyl-CoA from acetyl-CoA plus CO2 occurs as the first committed step of the fatty acid synthetic pathway catalyzed by the multi-component acetyl-CoA carboxylase (ACCase). The biofunctional malonyl-CoA reductase from C. aurantiacus, encoded by mcr gene, consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain and catalyzes two-step reduction.The mcr gene was PCR amplified from C. aurantiacus strain OK-70-fl (DSM636) and inserted into pET-28a to give plasmid pET-28a-mcr. Recombinant DE3/pET-28a-mcr for production of 3-HP was constructed by transforming E. coli BL21 (DE3) with plasmid pET-28a-mcr. Recombinant DE3/pET-28a-mcr, together with BL21 (DE3)/pET-28a, were inoculated into 50 ml LB medium with 2% glucose and incubated for 60 h. Samples were removed for GC analysis of 3-HP. The retention time of 3-HP by GC is 3.2 min. There is a detectable peak at the same location for the fermentation supernatant of DE3/pET-28a-mcr, corresponding approximately to 0.15 g/L 3-HP.This novel biosynthetic pathways allowed us to achieve the biosynthesis of 3-HP at both the domestic and international level. With the help of metabolism engineering technology, we envision that it will be the solid theoretical basis to make the high yield of 3-HP.2. Construction of a series of recombinants defecting in competitive pathways to produce lactate effectivelyFermentation of sugars through native pathways in E. coli under anaerobic conditions produces a mixture of products consisting primarily of lactate, formate, acetate and ethanol, with smaller amounts of succinate. The relative proportions of these products varied with the relative in vivo enzyme activities such as lactate dehydrogenase (ldhA gene), pyruvate formate lyase (pfl gene) and phosphoenolpyruvate carboxylase (ppc gene). Meanwhile, this product ratio also changed with the growth conditions in order to balance the number of reducing equivalents generated during glycolytic breakdown of the substrate. Acetate and ethanol are typically produced from acetyl-CoA in approximately equimolar amounts, catalyzed by acetate kinase (ackA)/phosphostransacetylase (pta) and alcohol/aldehyde dehydrogenase (adhE) respectively, to provide redox balance.In this study, we constructed recombinant E. coli SD2 and SD4, defecting in pflB and adhE respectively, to improve the production of lactate. Anaerobic fermentation was performed in LB medium supplemented with 100 mM glucose. Deletion of pflB in SD2 obviously increased the lactate production to 174.8 mM, while inactivation of adhE led it to 178.3 mM, approaching the theoretical maximum of 2 mol of lactate per mol of glucose utilized. The mutation of ptsG in SD6 and SD8, derivatives of SD2 and SD4 respectively, altered the fermentative metabolism of E. coli and caused over five fold increase in the formation of succinate at the expense of lactate. Meanwhile, ptsG mutation led to reduced glucose uptake rate but improved biomass during the fermentation.The fermentation products of SD6 and SD8 varied with respect to the different composition of medium. Compared to no more than 16 mM in LB medium after 60 h fermentation, the formation of lactate in SD6 and SD8 with M9 medium largely improved to over 100 mM. Correspondingly, succiante produced by SD6 and SD8 dropped from 45.5 mM,42.5 mM with LB medium to 34.5 mM,31.4 mM with M9 medium, respectively. The existence of potassium ion in M9 was speculated to accounting for the increased lactate conversion. Replacement of potassium with sodium in M9 medium slightly reduced the accumulation of lactate in SD4 and SD8, accompanied by increased succinate production. It’s suggested that M9 medium is more conducive to lactate formation than LB.Influences of carbon sources with high degree of reduction, reducing agents, and oxygen availability on the contribution of products were tested. All three approaches expanded the production ratio of lactate to succinate and the dissolved oxygen tension was a key constraint.Through genetic manipulation, high-yield accumulation of lactate was achieved in engineered E. coli SD4 and SD8. Influences of carbon flow and the availability of reducing equivalents on lactate production provided an important technical support for controllable lactate production.3. Construction of a bioprocess for the production of LA-based polyester P(LA-co-3HB)Based on the substrate specificity of PHA synthase, a key enzyme for polymerization of various monomers to polyhydroxyalkanoate (PHAs), which has monomeric constituents share the common chemical structure, hydroxy acid, with 2-hydroxypropionate (the same as LA), we succeeded in creating a microbial biosynthetic system for LA-based polyesters, P(LA-co-3-HB), copolymerized with 3-hydroxybutyrate (3HB), which is a typical constituent of polyhydroxyalkanoates (PHAs). P(LA-co-3HB) is intracellularly synthesized by successive enzymatic reaction steps, as follows:(i) generation of lactyl-coenzyme A (LA-CoA) by propionyl-CoA transferase (PCT) from Clostridium propionicum, (ii) supply of 3-hydroxybutyryl-CoA (3HB-CoA) via the dimerization pathway catalyzed by PhaA (β-ketothiolase) and PhaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, and (iii) copolymerization of the CoA esters by PhaEC, the PHA synthase from Allochromatium vinosum. In our work, a copolymer consisting of 0.22 mol% of LA and 99.78 mol% 3HB was produced in recombinant Escherichia coli DH5a/pBBRl1pcrEC+pBHR69 under aerobic conditions supplied with 1% lactate. Furthermore, LA fraction in the copolymer was increased up to 1.49 mol% by conducting anaerobic culture preferable for LA production.Construction of this engineered system is the first of its kind in this country to make up the domestic technology gap in PLA production. It plays an important role in helping to understand the bio-process synthesis of PLA, thus has important theoretical significance and application value.更多还原