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生物法合成香叶醇及碳原子高效利用研究
Alternative Title
刘炜
Thesis Advisor咸漠
2016-05
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline化学工程
Keyword香叶醇 Mva途径 香叶醇乙酸酯 联产 厌氧发酵
Abstract香叶醇是一种非环单萜醇类化合物,作为玫瑰系香精的主剂,香叶醇广泛应用于日化、烟草和食品等领域。此外,还可用作天然低毒防虫剂及新型的化学防癌制剂。国内对香叶醇的年需求量超过1000吨,供不应求,缺口达上百吨。目前市场上香叶醇的主要来源是从天然植物中提取挥发油。但是由于植物中香叶醇含量很低,导致提取成本过高,且由植物挥发油中含有多种成分,提取出来后与其他挥发油成分较难分离。化学法也可合成出香叶醇,但化学合成法又存在石油基原料不可持续,反应过程环境污染严重,选择性差等瓶颈问题,且化学合成产物“非天然”,消费者的认可度低。 以葡萄糖等廉价的生物质为原料,利用基因工程菌合成香叶醇已成为未来发展趋势。本研究利用代谢工程手段,在大肠杆菌体内构建了香叶醇生物合成途径,并研究了磷酸酶和香叶醇合成酶这两种类型酶对催化香叶基焦磷酸GPP合成香叶醇的作用,最后利用发酵工程技术,实现了香叶醇的高效合成。取得主要进展如下: (1)探索了磷酸酶对香叶醇合成的作用。萜类化合物的生物合成一般需要特异性的萜类合成酶。已报道的香叶醇生物合成都是利用植物来源的香叶醇合酶催化底物GPP获得。然而,由于香叶醇的特殊结构,推测还可利用磷酸酶,通过水解去磷酸化直接形成香叶醇,但至今没有相关研究。本研究通过对来源于酿酒酵母的磷酸酶DPP1和LPP1,以及来源于大肠杆菌的ADP核糖焦磷酸水解酶 NudF 和碱性磷酸酶PhoA 进行体外酶反应研究,首次发现PhoA可在体外催化香叶基焦磷酸GPP合成香叶醇。随后,通过在大肠杆菌中同时过表达杂合的MVA途径、香叶基焦磷酸合成酶基因以及碱性磷酸酶基因phoA,建立了香叶醇生物合成途径,并在摇瓶水平合成出5.3 mg/l 香叶醇。相关成果为香叶醇及其他类似结构萜醇类化合物的生物合成提供了新的思路。 (2)研究了大肠杆菌对香叶醇和香叶醇乙酸酯的生物转化。本研究首次证明大肠杆菌可催化香叶醇和香叶醇乙酸酯相互转化。大肠杆菌E. coli BL21 (DE3) 可将 40% 以上外源香叶醇转化为香叶醇乙酸酯;同时,大肠杆菌中的乙酰酯酶AES 可水解香叶醇乙酸酯形成香叶醇,经过2h 的体外反应,75%的香叶醇乙酸酯转化为香叶醇。 (3)构建了高效的香叶醇合成菌并优化了香叶醇发酵体系。通过在大肠杆菌中共表达了杂合甲羟戊酸合成途径(MVA途径)、香叶基焦磷酸合成酶GPPS2和香叶醇合成酶GES,构建了高效工程大肠杆菌LWG6,在摇瓶水平可合成出68.6mg/L香叶醇。在5L发酵体系中,首先通过添加肉豆蔻异丙酯,形成双相发酵系统,有效解决了香叶醇在发酵罐中的挥发问题,其次,通过发酵调控实现了香叶醇乙酸酯转化为香叶醇,使香叶醇的终产量提高到2.0 g/L,该产量是已有微生物法合成香叶醇报道的11倍。 以葡萄糖为原料合成香叶醇等萜类化合物的过程中有二氧化碳的产生,不仅造成碳损失,降低了理论产率,同时,会增加二氧化碳排放量,导致温室效应的加剧。此外,已有异戊二烯、香叶醇等萜类化合物的微生物合成都是在有氧条件下进行。由于萜类化合物多数具有强挥发性,有氧发酵会加速产物的挥发,增加了产物收集的难度和成本。而厌氧发酵合成萜类化合物的报道很少,其主要原因是厌氧条件下缺少电子受体,过表达外源途径往往会造成细胞体内的氧化还原不平衡,从而导致目标产物产率较低。针对上述问题,本研究以MVA合成途径中的重要中间体甲羟戊酸 (MVA)生物合成为代表,在厌氧条件下开展了碳原子高效利用研究。主要进展如下: (1)利用NOG途径厌氧发酵合成MVA。 非氧化的糖酵解途径(NOG途径)是2013年新发现的一条途径,与传统糖酵解途径(EMP途径)相比,NOG可在厌氧条件下将1分子葡萄糖转化为3分子乙酰磷酸,转化过程中没有二氧化碳产生,可有效提高碳原子的利用。本研究在大肠杆菌BL21(DE3)中构建了利用NOG途径合成MVA的代谢途径,试图提高MVA的产率。然而,和预期结果相反,在大肠杆菌体内当引入NOG途径后,在厌氧发酵条件下,MVA的产率不仅没有提高反而下降到原有的1/3。过表达磷酸乙酰转移酶基因pta和去除P-loop NTPase部分的ptaF3后,还是不能提高MVA产率,反而使MVA产率下降到对照菌的0.053倍和0.09倍。 (2)通过联产琥珀酸和MVA提高原料碳的综合利用。通过过表达pyc基因和MVA上游途径,构建出了甲羟戊酸和琥珀酸的联产途径。在厌氧条件下,联产菌株LWPYC1合成出1.8g/L MVA和5.1g/L琥珀酸,两个产物的总碳摩尔产率提高到对照菌株的2倍。在此基础上,在厌氧发酵过程中,利用CO2代替为N2,利用K2CO3+KOH代替氨水,通过外源碳源的补加,使联产菌株LWPYC1合成MVA的产量提高到5.9 g/L, 琥珀酸产量为6g/L,两个产物的总碳摩尔产率提高到对照菌株的3倍。最后,进一步敲除乳酸脱氢酶基因ldhA,使MVA的产量提高到12.2 g/L, 琥珀酸的终浓度达7.8g/L,两个产物的总碳摩尔产率提高到对照菌株的9倍。
Other AbstractThe monoterpene geraniol, which is emitted from flowers, takes an important role in flavor and fragrance industries due to its pleasant rose-like odor. Geraniol also exhibits huge potential in pharmacy and agrochemistry. Growing world demand for aroma chemicals and fuels has led to an increased demand for geraniol. Fractional distillation of plant essential oils is the major method for geraniol manufacture, but high cost and other limitations, such as weather dependence and plant diseases, limited the supplies of geraniol. For the chemical-synthesis method, synthesis from petrochemicals is not sustainable and often lacks of substrate selectivity, which may cause the formation of undesirable racemic mixtures. The disadvantages of both methods and the rising interest in natural products have sparked people to seek sustainable technologies for geraniol production. Converting renewable resources into monoterpene products by engineered microorganisms was interesting technology and developed quickly, which have the advantages of fast growth, no need for land during their growth and sustainable development. In this study, we tried to assemble a new pathway for geraniol products in E. coli, and several strategies to increase the production efficiency and selectivity were tested. (1) Utilization of phosphatase in the bioproduction of geraniol. Geraniol is likely to be synthesized from geranyl diphosphate (GPP). It has been hypothesized that phosphatases can catalyze geranyl diphosphate (GPP) into geraniol. But, whether and which phosphatases can transform GPP to geraniol has remained unanswered up to now. In this paper,the catalysis ability of four different types of phosphatases were studied with GPP as substrate in vitro, and just alkaline phosphatase (PhoA) from Escherichia coli can catalyze GPP into geraniol. Moreover, in order to confirm the ability of PhoA in vivo, the heterologous mevalonate pathway and geranyl diphosphate synthase gene from Abies grandis were co-overexpressed in E. coli with PhoA gene and 5.3±0.2 mg/l geraniol was produced from glucose in flask-culture. (2) Biotransformation between geranyl acetate and geraniol by E.coli. For the first time, the biotransformation between geranyl acetate and geraniol by E.coli was proved. More than 40% of fed geraniol was converted into geranyl acetate by E. coli BL21 (DE3). Moreover, we revealed the role of acetylesterase (Aes, EC 3.1.1.6) from E. coli in hydrolyzing of geranyl acetate to geraniol and about 75% of geranyl acetate was converted into geraniol after 2 h of incubation in vitro. (3) Engineering E. coli for high-yield geraniol production. Recombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6±3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol production to 78.8 mg/L. To further improve the production yield, the fermentation conditions were optimized. Firstly, 81.4% of fed geraniol was lost during the first 5 h of fermentation in a solvent-free system. Hence, isopropyl myristate was added to the culture medium to form an aqueous-organic two-phase culture system, which effectively prevented volatilization of geraniol. Secondly, fermentation condition were optimized and geraniol production reached up to 2.0 g/L with biotransformation of 88.8% geranyl acetate to geraniol by our strategy. Mevalonate is an intermediate metabolites in MVA pathway, which is the precursor of geraniol, isoprenen, carotenoids, artemisinin, paclitaxel and other high value-added products. MVA can be biosynthesized from acetyl coenzyme A, which is produced from glucose through EMP pathway. However, there is carbon dioxide producted in this process and caused carbon loss. In addition, the pervious studies on terpenoid biosynthesis were most carried out under aerobic conditions, which will increase the difficulty and costs of terpenoid collection for its volatility. Overexpression of heterologous genes maybe lead to the unblance of cell under anaerobic conditions, which will result in the lower production and productivity. According to the above problems, this study explores the ways for carbon utilization under the anaerobic condition. The main results as follows: (1) Use NOG pathway for MVA biosynthesis under anaerobic fermentation. NOG pathway was discovered in 2013. Compared with the traditional EMP pathway, 1 mol glucose can be convert to 3 mol acetyl phosphate and without carbon dioxide produced under anaerobic conditions by NOG pathway. In this study, we tried to improve the yield of the MVA and MVA biosynthesis with NOG pathway was constructed in E. coli BL21 (DE3). However, when introducing NOG way in E. coli, the yield of the MVA decreased to 1/3 of control. Overexpression of acetyl transferase gene pta or ptaF3, which removal of P - loop NTPase part of pta, MVA yeild down to 0.053 times and 0.09 times of control. (2) Co-production of succinic acid and MVA to increase the comprehensive utilization of carbon. pyc gene and MVA upstream were co-expression in E.coli to co-production of succinic acid and MVA. Under anaerobic conditions, the co-production strains LWPYC1 synthetic 1.8 g/L MVA and 5.1 g/L succinic acid. The total production carbon conversion rate increase to 2 times of control. Moreover, adding exogenous carbon dioxide to fermentation process and MVA production increased to 5.9 g/L and succinic acid production reached 6 g/L while total carbon conversion rate increased to three times of control. Finally, lactic acid dehydrogenase gene ldhA was knockout, which lead to the total carbon conversion rate increase to 10 times of control while MVA prodction up to 12.2 g/L and succinic acid reach 7.8 g/L.
Department生物基材料所
Subject Area生物化工
Date Available2021-06-01
Subtype博士 ; 学位论文
Language中文
Document Type学位论文
Identifierhttp://ir.qibebt.ac.cn/handle/337004/9758
Collection生物基材料组群
Affiliation中国科学院青岛生物能源与过程研究所
Recommended Citation
GB/T 7714
刘炜. 生物法合成香叶醇及碳原子高效利用研究[D]. 北京. 中国科学院研究生院,2016.
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