CAS OpenIR
大肠杆菌利用废弃生物质高效生产丁二酸的代谢工程研究
AYOBAMI MATTHEW OLAJUYIN
Thesis Advisor邢建民
2017-12
Degree Grantor中国科学院大学
Degree Name博士
Degree Discipline生物化工
Keyword大肠杆菌, 丁二酸,生物炼制,Strophanthus Preussii,可椰子水
Abstract

利用木质纤维素类生产生物化学品及生物燃料是生物炼制的核心内容。丁二酸是一种平台化合物,广泛应用于医药、食品、餐饮等行业,同时,丁二酸还可用于生产一些重要的聚酯、烷烃等。目前,利用木质纤维素生产丁二酸有很多挑战,比如木质纤维素预处理成本控制、基因工程菌的构建、五碳糖和六碳糖的利用等等。在本论文中,我们利用Palmaria palmata、Strophanthus preussii、Cocos nucifera water(可可椰子水)以及 elephant grass stalk(大象草茎)四种生物质为原料,开展了生物合成丁二酸的研究。首先,我们利用红巨藻P. palmata进行了微生物合成丁二酸的研究。在工程菌E. coli KLPPP中分别敲除了乳酸脱氢酶A(ldhA)、丙酮酸甲酸裂解酶(pflB)、磷酸转乙酰激酶A(pta-ackA)、丙酮酸氧化酶B(poxB)等基因,并且过表达了磷酸烯醇式丙酮酸羧化酶。P. palmata 水解产物含有葡萄糖(12.57 ± 0.17 g/L)和半乳糖(18.03 ± 0.10 g/L)。经过72小时得双阶段发酵,重组菌利用半乳糖(1.20 ± 0.02 mol/mol)作为底物比利用葡萄糖(0.48 ± 0.03 mol/mol)得到的丁二酸摩尔产率更高。丁二酸的浓度及摩尔产率分别达到22.40 ± 0.12 g/L和1.13 ± 0.02 mol/mol(总糖)。这一结果表明,P. palmata生物质是一种新型的、具有替代性的生物基化学品的发酵原料。第二,对E. coli工程菌利用S. preussii发酵生产丁二酸的生物炼制过程进行了研究。我们对S. preussii的甲醇浸提预处理工艺进行了优化。S. preussii甲醇浸提物主要含有葡萄糖(9.00 ± 0.02 g/L)、半乳糖(4.00 ± 0.02 g/L)、木糖(6.00 ± 0.02 g/L)、阿拉伯糖(0.50 ± 0.02 g/L)。工程菌E. coli K3OS中过表达了吡啶核苷酸转氢酶基因(sthA),同时敲除ldhA, pflB, pta-ackA, poxB。该菌有效利用了S. preussii的甲醇浸提产物。在M9培养基中,经过72小时的双阶段发酵,丁二酸浓度达到14.39 ± 0.02 g/L,产率为1.10 ± 0.01 mol/mol(总糖)。这一水平达到利用S. preussii甲醇浸提物发酵产生丁二酸最大理论值的64%。第三,我们构建了工程菌E. coli M6PM,该菌过表达了Bacillus subtilis的丙酮酸羧化酶基因(pyc)基因,并对ldhA、pflB、pta-ackA、poxB、pgi、murein cluster C (mreC)进行了突变,下调了磷酸烯醇式丙酮酸羧化酶(ppc)基因的活性。我们利用该菌进行了可可椰子水混合糖发酵生产丁二酸的研究。C. nucifera water主要含有葡萄糖(5.00 ± 0.02 g/L)、果糖(6.10 ± 0.01 g/L)、蔗糖(6.70 ± 0.02 g/L)。经过72小时得双阶段发酵,丁二酸最终浓度达到11.78 ± 0.02 g/L,产率为1.23 ± 0.01 mol/mol(总糖)。这一水平达到利用C. nucifera water发酵产生丁二酸最大理论值的72%。这一研究也显示了可可椰子水作为底物生产生物化学品的重要性。第四,利用大香草茎水解物进行了丁二酸发酵。大香草茎水解物主要含有葡萄糖(11.60 ± 0.04 g/L)、木糖(27.22± 0.04 g/L)、阿拉伯糖(0.65± 0.04 g/L)。工程菌E. coli M6PM可有效利用大香草茎水解物发酵生产丁二酸。该工程菌对ldhA、pflB、pta-ackA、poxB、pgi、mreC等多基因进行了突变、下调了ppc基因、过表达了B. subtilis的pyc。经过72小时双阶段发酵,产生的丁二酸最终浓度达到30.03 ± 0.02 g/L,产率为1.09 mol/mol。这一研究说明了工程菌利用可再生的生物质作为原料发酵生产丁二酸得可能性。最后,这些研究结果揭示了生物质可以作为一种可替代原料来生产生物化学品。在生物炼制中木质纤维素生物质的利用将有望提高生物质在生产生物化学品的使用,从而改善综合生物炼制中丁二酸的生产。 ;AbstractProduction of biochemicals and biofuels from lignocellulosic biomass has the potential to enhance the overall economic view of the lignocellulosic biorefinery. Succinic acid is a platform chemical for the production of some important polyester, alkyl resins, and it is also widely used in the pharmaceutical, medicine, food and beverage industry. Production of succinic acid from lignocellulosic biomass is laced with challenges including, the cost of biomass pretreatment, genetic engineering and utilization of cost-effective medium. Here, we investigated the biosynthesis of succinic acid from Palmaria palmata, Strophanthus preussii, Cocos nucifera water and elephant grass stalk.Firstly, microbial production of succinic acid from Palmaria palmata was investigated for the first time. In engineered Escherichia coli KLPPP, lactate dehydrogenase A (ldhA), phosphotransacetylase acetate kinase A (pta-ackA), pyruvate formate lyase B (pflB) and pyruvate oxidase B (poxB) genes were deleted while phosphoenolpyruvate carboxykinase was overexpressed. The recombinant exhibited higher molar yield of succinic acid on galactose (1.20 ± 0.02 mol/mol) than glucose (0.48 ± 0.03 mol/mol). The concentration and molar yield of succinic acid were 22.40 ± 0.12 g/L and 1.13 ± 0.02 mol/mol total sugars respectively, after 72 h dual-phase fermentation from P. palmata hydrolysate, which composed of glucose (12.57 ± 0.17 g/L) and galactose (18.03 ± 0.10 g/L). The results demonstrate that P. palmata red macroalgae biomass represents a novel and alternative feedstock for biochemical production.Secondly, a biorefinery process for high yield production of succinic acid from biomass sugars was investigated using recombinant E. coli. In this study, a novel biomass methanol extracts of Strophanthus preussii was used for the synthesis of succinic acid and optimization of the process parameters was carried out for pretreatment of biomass. Glucose (9.00 ± 0.02 g/L), galactose (4.00 ± 0.02 g/L), xylose (6.00 ± 0.02 g/L) and the arabinose (0.50 ± 0.02 g/L) were the major sugars present in methanol extracts of S. preussii. E. coli K3OS used methanol extracts of S. preussii effectively. E. coli K3OS with overexpression of soluble nucleotide pyridine transhydrogenase (sthA) and mutation of ldhA, pta-ackA, pflB and poxB, produced a final succinic acid concentration of 14.39 ± 0.02 g/L and yield of 1.10 ± 0.01 mol/mol total sugars after 72 h dual-phase fermentation in M9 medium. This value was 64 % of the maximum theoretical yield using methanol extracts of S. preussii.Thirdly, the genetically engineered strain with heterologous overexpression of pyc from Bacillus subtilis and ldhA, pflB, pta-ackA, poxB, pgi, murein cluster C (mreC) mutations and downregulation of phosphoenolpyruvate carboxylase (ppc) in E. coli were constructed and the mixed sugar fermentations was carried out with the best stains. C. nucifera water containing 5.00 ± 0.02 g/L glucose, 6.10 ± 0.01 g/L fructose and 6.70 ± 0.02 g/L sucrose was utilized for the production of succinic acid. Fermentation of C. nucifera water with E. coli M6PM produced a final succinic acid concentration of 11.78 ± 0.02 g/L and yield of 1.23 ± 0.01 mol/mol total sugars after 72 h dual-phase fermentation in M9 medium while modeled C. nucifera water was 0.38 ± 0.02 mol/mol total sugars. The novel substrate of C. nucifera water resulted in 72 % maximum theoretical yield of succinic acid. These investigations show the importance of C. nucifera water as a substrate for the production of biochemicals.Fourthly, efficient production of succinic acid from elephant grass stalk hydrolysate comprising of 11.60 ± 0.04 g/L glucose, 27.22 ± 0.04 g/L xylose and 0.65 ± 0.04 g/L arabinose was investigated. Fermentation of elephant grass stalk hydrolysate by engineered E. coli M6PM, involve the use of lactate dehydrogenase A, pyruvate formate lyase B, phosphotransacetylase-acetate kinase A, pyruvate oxidase B, phospho glucose isomerase, murein cluster genes mutations, down regulation of phosphoenolpyruvate carboxylase and overexpression of B. subtilis pyruvate carboxylase produced a final succinate concentration of 30.03 ± 0.02 g/L and a yield of 1.09 mol/mol during 72 h dual-phase fermentation. The high succinate yield from elephant grass stalk demonstrated possible application of renewable biomass as feedstock for the synthesis of succinic acid using recombinant E. coli.Conclusively, these results revealed that biomass represents an alternative feedstock for the production of biochemicals. The implementation of lignocellulosic biomass in biorefinery will enhance utilization of biomass for the production of biochemical and consequently improving succinic acid production in an integrated biorefinery. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/26935
Collection中国科学院过程工程研究所
Recommended Citation
GB/T 7714
AYOBAMI MATTHEW OLAJUYIN. 大肠杆菌利用废弃生物质高效生产丁二酸的代谢工程研究[D]. 中国科学院大学,2017.
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