电能细胞的合成生物学设计构建

doi:10.12211/2096-8280.2022-014

合成生物学 ›› 2022, Vol. 3 ›› Issue (5): 1031-1059.DOI: 10.12211/2096-8280.2022-014

• 特约评述 • 上一篇

电能细胞的合成生物学设计构建

由紫暄¹^,²^,³, 李锋¹^,²^,³, 宋浩¹^,²^,³

^1.天津大学化工学院，天津 300072
^2.天津大学合成生物学前沿科学中心和系统生物工程教育部重点实验室，天津 300072
^3.天津大学化工协同创新中心合成生物学研究平台，天津 300072

收稿日期:2022-03-01 修回日期:2022-04-19 出版日期:2022-10-31 发布日期:2022-11-16
通讯作者: 李锋,宋浩
作者简介:由紫暄（1997—），女，硕士研究生，研究方向为电能细胞的合成生物学研究。
由紫暄（1997—），女，硕士研究生，研究方向为电能细胞的合成生物学研究。E-mail：youzixuan_yzx@tju.edu.cn
李锋（1988—），男，助理教授，硕士生导师。研究方向为光电遗传学在物质、能量代谢中的应用。E-mail：messilifeng@163.com
宋浩（1973—），男，教授、博士生导师。研究方向为能量代谢与光-电遗传学，生物制药。E-mail：hsong@tju.edu.cn
基金资助:
国家重点研发计划(2018YFA0901300);国家自然科学基金(32071411);天津市科技计划(20JCQNJC00830);天津市教育部研究生科研创新项目(2020YJSB045)

Design and construction of electroactive cells by synthetic biology strategies

Zixuan YOU¹^,²^,³, Feng LI¹^,²^,³, Hao SONG¹^,²^,³

^1.School of Chemical Engineering and Technology，Tianjin University，Tianjin 300072，China
^2.Frontiers Science Center for Synthetic Biology （Ministry of Education），Tianjin University，Tianjin 300072，China
^3.Collaborative Innovation Center of Chemical Science and Engineering （Tianjin），School of Chemical Engineering and Technology，Tianjin University，Tianjin 300072，China

Received:2022-03-01 Revised:2022-04-19 Online:2022-10-31 Published:2022-11-16
Contact: Feng LI, Hao SONG

摘要/Abstract

摘要：

电能细胞具有与外界环境进行双向电子交换的能力，包括向外界环境释放电子的产电细胞，以及从外界环境获取电子的噬电细胞，在微生物电化学系统中发挥微生物电催化剂的核心作用。以电能细胞为核心的微生物电化学系统在生态环境治理、绿色能源开发、化学品高效合成等方面有着广泛应用。但是野生电能细胞因其摄取底物能力弱、胞内电子通量小、双向跨膜电子传递效率低、生物膜形成能力差等原因，化学能到电能的双向转化效率受到极大限制，是实现微生物电化学系统大规模产业化应用的核心瓶颈。本综述聚焦近5年电能细胞合成生物学改造的最新研究进展，通过分析双向电子传递的分子机制，分类汇总产电细胞和噬电细胞的合成生物学改造策略：①工程产电细胞（强化产电细胞的胞内电子生成、胞外传递效率，具体为拓宽底物谱、增强还原力转化，提高胞外传递能力、促进电极生物膜形成）；②工程噬电细胞（强化噬电细胞胞外电子摄取、还原力转化、产物合成效率，包括提高噬电细胞电子摄取和还原力转化，调控细胞代谢路径电合成化学品和生物燃料）。最后，展望了未来高效电能细胞和微生物电化学系统的设计与构建。

关键词: 电能细胞, 合成生物学, 生物电催化, 双向电子传递, 电活性生物膜

Abstract:

Bioelectrocatalytic systems (BESs) employ redox reactions of electroactive cells on electrodes to integrate biocatalysis and electrocatalysis, providing a green, economical and sustainable approach for energy and chemical production. Electroactive cells, including exoelectrogens that release electrons to the environment and electrotrophs that obtain electrons from the environment, have the ability to exchange electrons in inward and outward directions with the external environment and play a central role as microbial electrocatalysts in BESs. In the last decade, with in-depth researches on the electron transfer mechanisms including direct and indirect electron transfer pathways of electroactive cells, BESs with electroactive cells as the core component have been widely used in ecological environment management, green energy development, high-value chemical synthesis, and so on. However, the inefficient energy conversion rate of wild-type electroactive cells remains a major bottleneck for large-scale applications of BESs. This bottleneck is mainly caused by narrow available substrate spectrum, slow intracellular electron generation rate, weak bidirectional electron transfer capability, unstable biofilm structure, and low microbial electrosynthesis efficiency. Thus, this review focuses on the latest research progresses in the synthetic biology engineering of electroactive cells in the past five years. By disassembling and analyzing the bidirectional electron transfer mechanisms, the synthetic biology strategies are classified and summarized for electrogenic cells and electrophic cells, respectively. Electrogenic cells can be engineered via strengthening the intracellular electron production and extracellular transfer efficiency, with a special focus on broadening the substrate spectrum, improving intracellular electron release, accelerating extracellular electron transfer and strengthening electroactive biofilms formation. In terms of electrophic cells, they can be engineered via enhancing electrophic electron uptake and conversion as well as product synthesis efficiency, specifically promoting extracellular electron uptake and conversion and regulating cellular metabolic pathways to electrosynthesize chemicals and biofuels. Finally, perspectives on further engineering of electroactive cells and potential applications of BESs are proposed.

Key words: electroactive cells, synthetic biology, bioelectrocatalysis, bidirection electron transfer, electroactive biofilm

中图分类号:

Q819

由紫暄, 李锋, 宋浩. 电能细胞的合成生物学设计构建[J]. 合成生物学, 2022, 3(5): 1031-1059.

Zixuan YOU, Feng LI, Hao SONG. Design and construction of electroactive cells by synthetic biology strategies[J]. Synthetic Biology Journal, 2022, 3(5): 1031-1059.

图/表 11

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电能细胞	合成生物学策略	工程结果
底物代谢效率的合成生物学改造
S.oneidensis	强化乳酸利用关键因子CRP表达，增强菌株对乳酸的利用能力^[76]	最大电流密度约40 mA/m²
	异源表达视紫红质基因，通过光驱动改变膜电位，增强乳酸的摄取^[77]	平均电流密度达0.39 A/m²
	异源表达葡萄糖转运基因和葡萄糖激酶基因，增强菌株葡萄糖代谢能力^[78]	最大电流输出0.085 mA
	异源表达木糖转运蛋白、异构酶等构建木糖代谢途径^[79]	最大功率密度2.1 mW/m²
	异源表达乙酸辅酶A基因（ato1，ato2），强化柠檬酸合酶基因gtlA，构建乙酸代谢路径^[80]	最大功率密度8.3 mW/m²
胞内可释放电子池的合成生物学改造
P.aeruginosa	过表达NAD⁺合成酶基因nadE，扩充胞内NAD(H/⁺)总量^[81]	最大功率密度4.0 mW/m²
S.oneidensis	过表达ycel、pncB、nadM、nadD、nadE基因，增强NAD⁺的生物合成^[82]	最大功率密度162.8 mW/m²
	过表达gapA2、pflB、fdh、mdh*基因，强化NADH的合成^[83]	最大功率密度105.8 mW/m²
E.coli	破坏乳酸脱氢酶基因ldhA，释放储存在胞内中间代谢物的电子^[84]	最大功率密度3.0 mW/m²
	过表达非质子泵NADH脱氢酶NDH-2，使更多电子通过氧化呼吸链流入EET途径^[85]	输出电流4.7 μA
	构建C3N代谢通路，获得高效的NAD⁺代谢途径^[86]	NAD(H)浓度约6 mmol/L
胞外电子传递的合成生物学改造
E.coli	通过引入S.oneidensis的MtrCAB色素系统使得E.coli具备胞外电子传递能力^[87]	Fe(Ⅲ)还原速率达83 μmol/(L·d)
	构建S.oneidensis和E.coli细胞色素融合表达系统^[88]	电流密度达到25.32 mA/m²
	异源表达PCA合成路径phzA1B1C1D1E1F1G1基因簇，以提高间接胞外电子传递速率^[89]	最大功率密度为181.1 mW/m²
S.oneidensis	过表达内膜细胞色素CymA，加速电子传递至末端周质还原酶^[90]	功率达到0.13 mW
	敲除周质色素蛋白napB、fccA、tsdB并过表达细胞色素cctA，降低周质细胞色素复杂性^[91]	最高功率密度达到436.5 mW/m²
	异源表达来自B.subtilis的核黄素合成基因簇ribADEHC，以提高菌株的胞外电子传递速率^[74]	最大功率输出为233.0 mW/m²
	强化核黄素表达，结合孔蛋白增强分泌，添加材料rGO，构建三维杂合生物膜系统^[92]	最大功率输出达2630 mW/m²
	异源构建PCA合成路径和ombB-omaB-omcB-omcS直接电子传递链^[93]	最大电流密度达310.2 mA/m²
P.aeruginosa	通过截断PaPilA C端并增加N端 α-螺旋区的芳香族氨基酸含量，提高菌毛导电率^[94]	最大电流达130 μA
	过表达甲基转移酶基因phzM，增强菌株胞外电子传递载体-绿脓菌素的合成^[95]	最大功率密度达16.67 mW/m²
G.sulfurreducens	异源表达来自G.metallireducens的pilA基因，增强菌毛导电率^[96]	pili导电率为277 S/cm
电活性生物膜的合成生物学改造
S.oneidensis	过表达ydeH基因，增强菌株生物膜形成能力^[97]	最大功率密度达167.6 mW/m²
	过表达cyaC基因增加菌株胞内cAMP浓度，提高菌株电流输出密度^[98]	最大电流密度达356 mA/m²
G.sulfurreducens	过表达GSU1501基因促进胞外多糖分泌，促进生物膜形成，提高c型细胞色素丰度^[99]	Fe(Ⅲ)还原速率达1.75 mmol/(L·d)
P.aeruginosa	敲除RpoS基因促进吩嗪类物质合成，提高胞外电子通量^[100]	最大电流密度达4.5 μA/cm²
	过表达irrE基因，强化参与胞内代谢和EET过程^[101]	最大功率密度达56.0 mW/m²
	构建irrE突变基因文库，增强细胞环境适应性和产电性能^[102]	最大功率密度达149.2 mW/m²

电能细胞	合成生物学策略	工程结果
底物代谢效率的合成生物学改造
S.oneidensis	强化乳酸利用关键因子CRP表达，增强菌株对乳酸的利用能力^[76]	最大电流密度约40 mA/m²
	异源表达视紫红质基因，通过光驱动改变膜电位，增强乳酸的摄取^[77]	平均电流密度达0.39 A/m²
	异源表达葡萄糖转运基因和葡萄糖激酶基因，增强菌株葡萄糖代谢能力^[78]	最大电流输出0.085 mA
	异源表达木糖转运蛋白、异构酶等构建木糖代谢途径^[79]	最大功率密度2.1 mW/m²
	异源表达乙酸辅酶A基因（ato1，ato2），强化柠檬酸合酶基因gtlA，构建乙酸代谢路径^[80]	最大功率密度8.3 mW/m²
胞内可释放电子池的合成生物学改造
P.aeruginosa	过表达NAD⁺合成酶基因nadE，扩充胞内NAD(H/⁺)总量^[81]	最大功率密度4.0 mW/m²
S.oneidensis	过表达ycel、pncB、nadM、nadD、nadE基因，增强NAD⁺的生物合成^[82]	最大功率密度162.8 mW/m²
	过表达gapA2、pflB、fdh、mdh*基因，强化NADH的合成^[83]	最大功率密度105.8 mW/m²
E.coli	破坏乳酸脱氢酶基因ldhA，释放储存在胞内中间代谢物的电子^[84]	最大功率密度3.0 mW/m²
	过表达非质子泵NADH脱氢酶NDH-2，使更多电子通过氧化呼吸链流入EET途径^[85]	输出电流4.7 μA
	构建C3N代谢通路，获得高效的NAD⁺代谢途径^[86]	NAD(H)浓度约6 mmol/L
胞外电子传递的合成生物学改造
E.coli	通过引入S.oneidensis的MtrCAB色素系统使得E.coli具备胞外电子传递能力^[87]	Fe(Ⅲ)还原速率达83 μmol/(L·d)
	构建S.oneidensis和E.coli细胞色素融合表达系统^[88]	电流密度达到25.32 mA/m²
	异源表达PCA合成路径phzA1B1C1D1E1F1G1基因簇，以提高间接胞外电子传递速率^[89]	最大功率密度为181.1 mW/m²
S.oneidensis	过表达内膜细胞色素CymA，加速电子传递至末端周质还原酶^[90]	功率达到0.13 mW
	敲除周质色素蛋白napB、fccA、tsdB并过表达细胞色素cctA，降低周质细胞色素复杂性^[91]	最高功率密度达到436.5 mW/m²
	异源表达来自B.subtilis的核黄素合成基因簇ribADEHC，以提高菌株的胞外电子传递速率^[74]	最大功率输出为233.0 mW/m²
	强化核黄素表达，结合孔蛋白增强分泌，添加材料rGO，构建三维杂合生物膜系统^[92]	最大功率输出达2630 mW/m²
	异源构建PCA合成路径和ombB-omaB-omcB-omcS直接电子传递链^[93]	最大电流密度达310.2 mA/m²
P.aeruginosa	通过截断PaPilA C端并增加N端 α-螺旋区的芳香族氨基酸含量，提高菌毛导电率^[94]	最大电流达130 μA
	过表达甲基转移酶基因phzM，增强菌株胞外电子传递载体-绿脓菌素的合成^[95]	最大功率密度达16.67 mW/m²
G.sulfurreducens	异源表达来自G.metallireducens的pilA基因，增强菌毛导电率^[96]	pili导电率为277 S/cm
电活性生物膜的合成生物学改造
S.oneidensis	过表达ydeH基因，增强菌株生物膜形成能力^[97]	最大功率密度达167.6 mW/m²
	过表达cyaC基因增加菌株胞内cAMP浓度，提高菌株电流输出密度^[98]	最大电流密度达356 mA/m²
G.sulfurreducens	过表达GSU1501基因促进胞外多糖分泌，促进生物膜形成，提高c型细胞色素丰度^[99]	Fe(Ⅲ)还原速率达1.75 mmol/(L·d)
P.aeruginosa	敲除RpoS基因促进吩嗪类物质合成，提高胞外电子通量^[100]	最大电流密度达4.5 μA/cm²
	过表达irrE基因，强化参与胞内代谢和EET过程^[101]	最大功率密度达56.0 mW/m²
	构建irrE突变基因文库，增强细胞环境适应性和产电性能^[102]	最大功率密度达149.2 mW/m²

电能细胞	合成生物学策略	工程结果
工程强化噬电细胞电子摄取和转化
S.oneidensis	表达光驱动质子泵，强化丁二醇脱氢酶表达并敲除氢化酶基因hyaB、hydA，驱动丁二醇合成^[148]	2,3-丁二醇合成量10.06 mmol/L
E.coli	异源表达细胞色素蛋白MtrCAB、FccA、CymA碳酸氢盐转运蛋白和碳酸酐酶基因，构建产琥珀酸细胞工厂^[149]	琥珀酸产电达30.56 mmol/L
S.elongatus PCC 7942	异源表达色素蛋白OmcS和nif基因，促进N₂固定^[150]	NH₃ 20 h产率达295.7 μmol/L
工程噬电细胞代谢路径电合成化学品和生物燃料
R.eutropha	敲除聚羟基丁酸合成基因簇，异源表达异丁醇合成路径关键基因alsS、ilvC、ilvD、kivd和yqhD，使代谢流向异丁醇和3-甲基-1-丁醇合成^[73]	异丁醇、3-甲基-1-丁醇产量达140 mg/L
	破坏聚羟基丁酸酯合成，并强化异丙醇合成路径，使代谢流向异丙醇合成^[151]	异丙醇产量达216 mg/L
	异源表达核酮糖-1,5-二磷酸羧化酶，强化PHB合成^[152]	PHB产量达485 mg/L
	异源过表达的甲羟戊酸途径酶法尼基焦磷酸合酶ERG20、IPP异构酶fni和α-葎草烯合酶（ZSSI），促进α-葎草烯合成^[153]	α-葎草烯产量达10.8 mg/L
	表达番茄红素途径基因CrtEBI2，促进番茄红素合成^[154]	番茄红素产量达1.73 mg/L
S.cerevisiae	异源表达氧甾醇7α-羟化酶，促进7α-OH-DHEA合成^[155]	7α-OH-DHEA产量达288.6 mg/L
R.palustris	异源引入丁醇生物合成途径phaABJ、ter、adhE2基因，强化丁醇合成^[156]	丁醇产量达0.91 mg/L± 0.07 mg/L

电能细胞	合成生物学策略	工程结果
工程强化噬电细胞电子摄取和转化
S.oneidensis	表达光驱动质子泵，强化丁二醇脱氢酶表达并敲除氢化酶基因hyaB、hydA，驱动丁二醇合成^[148]	2,3-丁二醇合成量10.06 mmol/L
E.coli	异源表达细胞色素蛋白MtrCAB、FccA、CymA碳酸氢盐转运蛋白和碳酸酐酶基因，构建产琥珀酸细胞工厂^[149]	琥珀酸产电达30.56 mmol/L
S.elongatus PCC 7942	异源表达色素蛋白OmcS和nif基因，促进N₂固定^[150]	NH₃ 20 h产率达295.7 μmol/L
工程噬电细胞代谢路径电合成化学品和生物燃料
R.eutropha	敲除聚羟基丁酸合成基因簇，异源表达异丁醇合成路径关键基因alsS、ilvC、ilvD、kivd和yqhD，使代谢流向异丁醇和3-甲基-1-丁醇合成^[73]	异丁醇、3-甲基-1-丁醇产量达140 mg/L
	破坏聚羟基丁酸酯合成，并强化异丙醇合成路径，使代谢流向异丙醇合成^[151]	异丙醇产量达216 mg/L
	异源表达核酮糖-1,5-二磷酸羧化酶，强化PHB合成^[152]	PHB产量达485 mg/L
	异源过表达的甲羟戊酸途径酶法尼基焦磷酸合酶ERG20、IPP异构酶fni和α-葎草烯合酶（ZSSI），促进α-葎草烯合成^[153]	α-葎草烯产量达10.8 mg/L
	表达番茄红素途径基因CrtEBI2，促进番茄红素合成^[154]	番茄红素产量达1.73 mg/L
S.cerevisiae	异源表达氧甾醇7α-羟化酶，促进7α-OH-DHEA合成^[155]	7α-OH-DHEA产量达288.6 mg/L
R.palustris	异源引入丁醇生物合成途径phaABJ、ter、adhE2基因，强化丁醇合成^[156]	丁醇产量达0.91 mg/L± 0.07 mg/L

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电能细胞的合成生物学设计构建

Design and construction of electroactive cells by synthetic biology strategies

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