CAS OpenIR
基于木聚糖资源的乙偶姻生物合成研究
贾晓静
Subtype博士
Thesis Advisor韩业君
2017-01
Degree Grantor中国科学院研究生院
Degree Name博士
Degree Discipline生物化工
Keyword木聚糖酶,乙偶姻,高温同步糖化发酵,高温酶,无细胞酶促合成
Abstract

木聚糖是大量存在于植物细胞壁中的半纤维素成分,也是自然界中五碳糖的主要来源,在生物炼制转化能源和化学品方面具有巨大的开发价值。乙偶姻是一种重要的平台化合物和多功能材料,被广泛应用于食品、化工、医药、烟草等行业。本文首先对能够直接利用天然生物质的极端嗜热厌氧细菌Caldicellulosiruptor lactoaceticus 6A(C. lac)降解木聚糖的酶解模式进行了解析,构建了体外天然木聚糖高温酶解体系;同时选育了能够同时利用五(六)碳糖合成乙偶姻的耐高温枯草芽孢杆菌,实现了从天然生物质到乙偶姻的高温同步糖化发酵转化;此外,以五(六)碳糖代谢过程和重组酶为基础,体外构建了从五(六)碳糖到乙偶姻的非细胞酶促合成体系。研究成果包括以下3个部分:1. 解析了极端嗜热菌C. lac对天然木聚糖的高温酶解模式:(1)胞内高温协同酶解体系的表征和构建:从C. lac基因组中分别克隆、异源表达和表征了胞内木聚糖降解酶系(GH10家族β-1,4-内切木聚糖酶Xyn10A、GH51家族α-L-阿拉伯呋喃糖苷酶Abf51A和GH67家族α-葡萄糖醛酸苷酶Agu67A),该酶系在75-80°C、pH 5.5-6.5条件下能够协同酶解天然木聚糖:Xyn10A能降解木聚糖主链,Abf51A和Agu67A能分别移除木聚糖侧链上的阿拉伯糖基和4-O-甲基-葡萄糖醛酸基团。(2)胞外多结构域β-1,4-内切木聚糖酶分子机理解析:C. lac胞外GH10家族β-1,4-内切木聚糖酶Xyn10B具有特异的CBM22/GH10/CBM9/SLH多结构域结构,其装配有不同结构域模块的重组酶在热稳定性、底物特异性和水解效率等方面有很大的差异。CBM22特别是CBM22c能够增强重组酶的热稳定性和催化活性,但这种正效应随着CBM9的存在而减弱。2. 选育了可同时利用五碳糖和六碳糖的耐高温乙偶姻高产枯草芽孢杆菌,实现了从生物质到乙偶姻高温同步糖化发酵转化:(1)菌种选育和发酵过程优化:利用菌种筛选和复合诱变选育获得耐高温的乙偶姻高产突变菌株Bacillus subtilis IPE5-4-UD-4;通过培养基组分和发酵条件的单因素及正交优化,以葡萄糖为底物,其37°C下摇瓶发酵乙偶姻最高产量达到26.09 ± 1.01 g/L;补料分批发酵时,以8 g/h的流加速度补料400 g葡萄糖,最终发酵60 h时乙偶姻浓度最高产量达到48.09 ± 0.63 g/L。(2)复合诱变菌株高温发酵单糖的研究:利用其静息细胞于50°C下催化10 mM D-葡萄糖反应24 h,最高可产生2.70 ± 0.02 mM乙偶姻。以葡萄糖和木糖的混合糖为底物,于50°C下摇瓶发酵72 h后乙偶姻最高产量达到17.78 ± 0.19 g/L,产率为0.38 g/g混合糖,生产速率为0.25 g/L/h。以葡萄糖为底物,于50°C下发酵罐分批发酵48 h后,乙偶姻最高产量达到28.83 ± 0.65 g/L,产率为0.34 g/g葡萄糖,生产速率为0.60 g/L/h。(3)高温同步糖化发酵碱处理玉米芯生产乙偶姻工艺的建立:以碱处理玉米芯为底物,利用商业纤维素酶-木聚糖高温酶系和耐高温菌株B. subtilis IPE5-4-UD-4,构建乙偶姻高温同步糖化发酵过程。于最优温度50°C下摇瓶同步糖化发酵72 h后,乙偶姻最高产量达到12.55 ± 0.28 g/L,产率为0.25 g/g底物,生产速率为0.17 g/L/h。同时原料中纤维素和半纤维素转化率的最高值分别达到96.34%和93.29%。进一步于50°C下发酵罐分批同步糖化发酵60 h后,乙偶姻最高产量达到22.76 ± 1.16 g/L,产率为0.46 g/g底物,生产速率为0.38 g/L/h,这是迄今为止以同步糖化发酵方式转化木质纤维素生物质获得的最高乙偶姻生产强度。3. 以微生物D-木糖和D-葡萄糖代谢为基础,从高温合成和常温合成的角度,解析了乙偶姻及其手性异构体(R)-乙偶姻的酶促合成过程,构建了人工体外无细胞酶促合成途径。(1)由丙酮酸高温无细胞酶促合成乙偶姻:基于丙酮酸代谢途径,以丙酮酸为底物,构建了一条包括两步连续反应的乙偶姻高温体外酶促合成途径。该途径包括2个高温重组催化酶,反应不需要ATP和辅酶I。从高温菌Caldicellulosiruptor owensensis OL和B. subtilis IPE5-4基因组中分别成功克隆、异源表达和表征了高温催化酶。在65°C、pH 6.5的最佳反应条件下,用1 U混合酶(coAHASL1/bsALDC 4:1,U/U)催化10 mM丙酮酸体外反应24 h可生成3.36 ± 0.26 mM乙偶姻,其生产速率达到0.14 mM/h,产率为33.92%,为理论产率的67.80%。(2)由D-木糖高选择性无细胞酶促合成(R)-乙偶姻:基于D-木糖代谢的Dahms途径,以D-木糖为底物,构建了一条高选择性无细胞酶促合成(R)-乙偶姻的途径。该途径包括7个重组催化酶,反应不需要ATP,且可以实现辅酶NAD+再生。从Escherichia coli W3110、B. subtilis shaijiu32和Caulobacter crescentus CB 2基因组中成功克隆、异源表达和纯化了所有重组催化酶。每步酶添加1 U,在30°C、pH 7.5的最佳反应条件下,催化10 mM D-木糖反应24 h可获得3.17 ± 0.06 mM (R)-乙偶姻,其对映体过量值达到99.07%,生产速率为0.13 mM/h,产率为18.75%,达到理论产率的63.89%。(3)由D-葡萄糖高温无细胞酶促合成乙偶姻探索:基于D-葡萄糖代谢的糖酵解途径,以D-葡萄糖为底物,构建了多酶高温催化合成乙偶姻途径。该途径共需要13个催化酶,能实现辅酶NAD+偶联再生。从高温菌C. owensensis OL、Thermobifida fusca DSM 43792和B. subtilis IPE5-4基因组中成功克隆、表达和纯化了相关高温催化酶。以D-葡萄糖或果糖-1,6-二磷酸为底物,利用由重组酶组成的无细胞酶促体系于50°C下催化可检测到乙偶姻生成。利用诱变菌IPE5-4-UD-4静息细胞的粗酶液以及粗酶上清催化液,于50°C下可转化葡萄糖生成乙偶姻,以粗酶液催化10 mM D-葡萄糖反应24 h最高可产生2.65 ± 0.59 mM乙偶姻。;Xylan, the most abundant hemicellulosic constituent of plant cell wall, has been considered as an abundant source of fermentable pentose (mainly xylose and arabinose) for biofuel and bio-chemical production by biorefinery. Acetoin (3-hydroxy-2-butanone) is a promising bio-based platform chemical with wide applications in food, chemical synthesis and multifunctional material. In present study, the enzymatic hydrolysis model of the natural biomass utilizing extremely thermophilic anaerobic bacterium Caldicellulosiruptor lactoaceticus 6A (C. lac) for natural xylan degradation was resolved, and the in vitro thermophilic enzymatic system for natural xylan degradation was constructed thereafter. Thermophilic Bacillus subtilis which can convert pentose and hexose simultaneously to acetoin was selected, and production of acetoin from natural biomass was realized through thermophilic simultaneous saccharification and fermentation (SSF). In addition, the cell-free enzyme catalytic biosystems for acetoin synthesis were constructed in vitro based on the metabolic pathway and recombinant enzymes. The results were summarized in the following three parts:1. The thermophilic enzymatic hydrolysis model of extremely thermophilic bacterium C. lac on natural xylan degradation was resolved:(1) Characterization of the synergy of intracellular xylanolytic glycoside hydrolases (GHs) and reconstruction of enzyme hydrolytic system for natural degradation in vitro: The intracellular xylanolytic GHs, GH10 β-1,4-endo-xylanase (Xyn10A), GH51 α-L-arabinofuranosidase (Abf51A) and GH67 α-glucuronidase (Agu67A), were cloned, heterologously expressed, and characterized. Natural xylan was degraded by the synergistic enzyme system under the optimal activity condition at 75-80°C and pH 5.5-6.5. Xyn10A acted on the backbone of xylan polymer, while Abf51A and Agu67A removed the L-arabinofuranosyl and 4-O-methyl-glucuronic acid side-chains of xylan, respectively. Therefore, synergistic application of these three enzymes significantly improved the amounts of xylose, xylobiose, small molecular oligosaccharides and arabinose in the end products of xylan.(2) Characterization of the molecular mechanism of extracellular multidomain β-1,4-endo-xylanase: The extracellular GH10 β-1,4-endo-xylanase (Xyn10B) contained CBM22/GH10/CBM9/SLH architecture. The truncated derivatives of Xyn10B with different module assembly modes displayed notable variation in thermostability, substrate specificity, and hydrolytic activity. The results indicated that CBM22s especially CBM22c promoted both thermostability and catalytic efficiency, while these positive effects were weakened with the presence of CBM9.2. Selection of thermotolerant acetoin producer, and bioconverting biomass to acetoin through thermophilic SSF process:(1) Selection of thermotolerant acetoin producer and optimization of fermentation process: A thermotolerant (up to 52°C) acetoin producer B. subtilis IPE5-4-UD-4 was isolated by compound mutagenesis. By single factor and orthogonal optimization of the medium components and fermentation conditions, the mutant produced 26.09 ± 1.01 g/L acetoin at 37°C with glucose as feedstock in shake flask fermentation. When fermenting at 37°C in a 5-L bioreactor, the acetoin concentration reached the maximum of 48.09 ± 0.63 g/L with feeding 400 g glucose at a flow rate of 8 g/h.(2) Study of the fermentation capacity of strain IPE5-4-UD-4 at high temperature: Using the resting cells, a concentration of 2.70 ± 0.02 mM acetoin was generated from 10 mM glucose at 50°C for 24 h. Besides, the acetoin concentration of 17.78 ± 0.19 g/L was achieved by IPE5-4-UD-4 in shake flask at 50°C with the mixture of glucose and xylose as substrate, and the acetoin yield and productivity came to 0.38 g/g mixed sugar and 0.25 g/L/h, respectively. When fermented at 50°C in a 5-L bioreactor with glucose as feedstock by IPE5-4-UD-4, the acetoin concentration reached 28.83 ± 0.65g/L with the acetoin yield and productivity of 0.34 g/g glucose and 0.60 g/L/h, respectively.(3) Constructing the thermophilic SSF process for acetoin production from alkali pretreated corncob (APC): Thermophilic SSF process was optimized and constructed by using commercial cellulase and thermophilic xylan enzymatic system and strain IPE5-4-UD-4 with APC as feedstock at 50°C. An acetoin concentration of 12.55 ± 0.28 g/L was achieved in shake flask SSF , and the acetoin yield and productivity came to 0.25 g/g APC and 0.17 g/L/h, respectively. Meanwhile, the utilization of cellulose and hemicellulose in the SSF approach reached the maximum of 96.34% and 93.29%, respectively. When further fermented at 50°C in a 5-L bioreactor for 60 h, 22.76 ± 1.16 g/L acetoin was obtained, the acetoin yield and productivity reached 0.46 g/g APC and 0.38 g/L/h, respectively. This was by far the highest acetoin yield from lignocellulosic biomass in SSF.3. Analysis of enzyme catalytic process of acetoin and (R)-acetoin at room or high temperature based on the microbial D-xylose and D-glucose metabolism, and construction of cell-free biosystems for acetoin and (R)-acetoin synthesis in vitro:(1) Thermophilic cell-free enzymatic synthesis of acetoin from pyruvate: Based on the metabolic pathway starting from pyruvate, a thermophilic cascade cell-free enzymatic reaction for acetoin synthesis was reconstructed in vitro. Two thermophilic catalytic elements were involved, and neither ATP nor coenzyme I was required. Recombinant thermophilic enzymes were cloned, heterologously expressed, and characterized from Caldicellulosiruptor owensensis OL and B. subtilis IPE5-4, respectively. Under optimal condition at 65°C and pH 6.5, a maximum concentration of 3.36 ± 0.26 mM acetoin corresponding to 67.80% of theoretical yield was obtained from 10 mM pyruvate within 24 h cell-free synthesis, and the acetoin productivity and yield reached 0.14 mM/h and 33.92%, respectively.(2) Cell-free enzymatic synthesis of (R)-acetoin from D-xylose: Based on the Dahms metabolic pathway starting from D-xylose, a NAD+-balanced synthetic pathway involving seven-step continuous reaction was constructed to produce (R)-acetoin from D-xylose in vitro. Recombinant enzymes were cloned, heterologously expressed, and characterized from Escherichia coli W3110, B. subtilis shaijiu32 and Caulobacter crescentus CB 2, respectively. Under optimal condition at 30°C and pH 7.5, a maximum concentration of 3.17 ± 0.06 mM (R)-acetoin with enantiomeric excess of 99.07% was obtained from 10 mM D-xylose within 24 h cell-free synthesis, and the (R)-acetoin productivity and yield reached 0.13 mM/h and 18.75%, respectively.(3) Thermophilic cell-free enzymatic synthesis of acetoin from D-glucose: Based on the Embdem-Meyerhof-Parnas pathway starting from D-glucose, A NAD+-balanced synthetic pathway involving thirteen-step continuous reaction was also constructed to synthetize acetoin from D-glucose in vitro at high temperature. Recombinant enzymes were cloned, heterologously expressed, and purified from C. owensensis OL, Thermobifida fusca DSM 43792 and B. subtilis IPE5-4, respectively. Using this cell-free enzyme catalytic biosystem, trace amount of acetoin was detected using D-glucose or D-fructose 1,6-bisphosphate as the substrate. Besides, the crude enzyme catalyst of B. subtilis IPE5-4-UD-4 resting cells and the supernatant of crude enzyme catalysts converted D-glucose to acetoin at 50°C in vitro, and a maximum concentration of 2.65 ± 0.59 mM acetoin was produced from 10 mM D-glucose by the crude enzyme catalyst of resting cells at 50°C for 24 h.

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/26830
Collection中国科学院过程工程研究所
研究所(批量导入)
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贾晓静. 基于木聚糖资源的乙偶姻生物合成研究[D]. 中国科学院研究生院,2017.
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