【作者】 刘丹；

【导师】 曹英秀； 肖敏；

【作者基本信息】 天津大学 ， 工程硕士（专业学位）， 2021， 硕士

【摘要】 CO₂的捕获与转化在将CO₂转化为化学原料或生物燃料的同时,有利于实现人工碳循环闭环。其中微生物的CO₂固定因其代谢途径灵活多样、还原产物附加值高的特点正成为一种优选策略。然而受限于固碳途径限速步骤的核心酶,微生物的固碳效率尚不能满足大规模应用。如何提高CO₂固定的效率是微生物固碳技术需要解决的关键问题之一。本研究选用化能自养微生物罗尔斯通氏菌为底盘细胞,构建了一个强化CO₂转运和固定能力的微生物电合成系统,通过固碳强化与还原力供给并行,实现从无机碳到多碳化合物的生物转化。本研究,首先从碳酸氢根转运、碳酸酐酶、前体物供应三大功能模块不断优化罗尔斯通氏菌内的卡尔文循环,并采用模块间组合优化策略强化微生物的固碳能力,筛选出的异源表达碳酸酐酶与CO₂固定限速酶1,5-二磷酸核酮糖羧化酶/加氧酶的工程菌株,借助微生物电合成的丰富还原力实现胞内聚羟基丁酸酯积累量达190.45±12.36 mg/g DCW,是对照菌（空表达载体）的1.41倍（135.54±13.84 mg/g DCW）。其次通过对微生物电合成体系的不断优化,确定了本系统的最优电合成条件:阴极电势为-0.8 V（vs.Ag/AgCl）,电子载体中性红浓度为0.1 mM,磷酸盐浓度为50 mM,（NH₄）₂SO₄浓度为0.2g/L,工作电极为经单壁碳纳米管（SWCNTs）修饰的碳布电极,反应96 h后,工程菌株胞内的聚羟基丁酸酯积累量达到本研究最高产量:211.54±7.18 mg/g DCW,是对照菌（空表达载体）的1.56倍。最后对工程菌株的细胞活性和胞内活性氧进行了探索,证明了工程菌株能够在仅供给电能和CO₂下进行正常的生长和代谢。这项研究阐明了一种微生物电合成体系下强化CO₂转运与固定的策略,将CO₂高效转化为聚羟基丁酸酯,为增强罗尔斯通氏菌的固碳能力及目标产物合成开拓了新思路。更多还原

【Abstract】 The capture and conversion of CO₂ not only converts CO₂ into chemical raw materials or biofuels but also helps realize the closed loop of the artificial carbon cycle.Microbial CO₂ fixation is becoming a preferred strategy due to its flexible and diverse metabolic pathways and the ability to produce high value-added products.However,due to the core enzymes of the rate-limiting step of the CO₂fixation pathway,the carbon fixation efficiency of microorganisms is still not enough for large-scale applications.Therefore,a key issue is how to enhance biological CO₂-fixing efficiency for value added chemical production.In this study,the chemoautotrophic microorganism Cupriavidus necator H16 was used as the chassis cells to construct a microbial electrosynthesis system with enhancing CO₂transport and fixation,the conversion of CO₂ into polymer was realized through the strengthening of carbon fixation and the supply of reducing power.The research firstly continuously optimized the Calvin-Benson-Bassham cycle from the bicarbonate transport module,carbonic anhydrase module and precursor supply module.Furthermore,the combination optimization strategy between modules was used to enhance the carbon fixation ability of C.necator.The heterologous expression of carbonic anhydrase and CO₂ fixed rate-limiting enzyme 1,5-bisphosphate ribulose carboxylase/oxygenase were selected.With the abundant reducing power of microbial electrosynthesis,the intracellular poly（3-hydroxybutyrate）（PHB）accumulation of the engineered strain reached 190.45±12.36 mg/g DCW,which was 1.41 times higher than of the control strain（135.54±13.84 mg/g DCW）.Then through the optimization of microbial electrosynthesis operation parameters,the optimal electrosynthesis conditions of the system were finally determined.The intracellular PHB accumulation of the engineered strain reached the highest yield in this study with the cathode potential poised at-0.8 V（vs.Ag/AgCl）,the electron shuttle of 0.1 mM neutral red,the phosphate concentration of 50 mM,nitrogen content of 0.2 g/L,and single-walled carbon nanotubes（SWCNTs）modified carbon cloth electrode.The highest PHB yield was211.54±7.18 mg/g DCW,which was 1.56 times higher than of the control strain.Finally,the cell activity and intracellular reactive oxygen species of the engineered strain were characterized,which proved that the engineered strain could grow and metabolize normally under the condition of only electricity and CO₂.This study clarifies a strategy for enhancing CO₂ transport and fixation under a microbial electrosynthesis system,which converts CO₂ into PHB efficiently,and opens up new ideas for enhancing the ability of carbon fixation and synthesis of desired products.更多还原