Progress of cyanobacterial synthetic biotechnology for efficient light-driven carbon fixation and ethanol production

doi:10.12211/2096-8280.2023-051

Synthetic Biology Journal ›› 2023, Vol. 4 ›› Issue (6): 1161-1177.DOI: 10.12211/2096-8280.2023-051

• Invited Review • Previous Articles Next Articles

Progress of cyanobacterial synthetic biotechnology for efficient light-driven carbon fixation and ethanol production

Huili SUN¹^,²^,³, Jinyu CUI¹^,²^,³, Guodong LUAN¹^,²^,³, Xuefeng LYU¹^,²^,³

^1.Key Laboratory of Biofuels，Qingdao Institute of Bioenergy and Bioprocess Technology，Chinese Academy of Sciences，Qingdao 266101，Shandong，China
^2.Shandong Energy Institute，Qingdao 266101，Shandong，China
^3.Qingdao New Energy Shandong Laboratory，Qingdao 266101，Shandong，China

Received:2023-07-17 Revised:2023-08-24 Online:2024-01-19 Published:2023-12-31
Contact: Xuefeng LYU

面向高效光驱固碳产醇的蓝细菌合成生物技术研究进展

孙绘梨¹^,²^,³, 崔金玉¹^,²^,³, 栾国栋¹^,²^,³, 吕雪峰¹^,²^,³

^1.中国科学院青岛生物能源与过程研究所，中国科学院生物燃料重点实验室，山东青岛 266101
^2.山东能源研究院，山东青岛 266101
^3.青岛新能源山东省实验室，山东青岛 266101

通讯作者: 吕雪峰
作者简介:孙绘梨（1995—），女，博士后。研究方向为蓝细菌合成生物技术研究，包括光驱固碳细胞工厂的构建和底盘细胞生理功能认识与改造。E-mail：sunhl@qibebt.ac.cn
吕雪峰（1974—），男，研究员，博士生导师，中国科学院青岛生物能源与过程研究所所长。研究方向为合成生物学与绿色生物制造，在光驱固碳产能蓝细菌的人工设计与构建及真菌天然产物药物等。E-mail：lvxf@qibebt.ac.cn
基金资助:
国家重点研发计划(2021YFA0909700);国家自然科学基金(32270103);山东省博士后创新项目(SDCX-ZG-202202036);中国博士后科学基金第70 批面上项目(2021M703320)

Abstract

Abstract:

The utilization of solar energy and carbon dioxide by cyanobacterial cell factories for photosynthetic ethanol production represents a promising and sustainable route towards green biofuels. Ethanol is one of the most representative products of the cyanobacterial photosynthetic biomanufacturing technology. Cyanobacterial ethanol production systems could serve as models for developing and optimizing advanced synthetic biology and metabolic engineering strategies. While most known cyanobacterial species lack the ability to synthesize and accumulate ethanol, the introduction and overexpression of heterologous pyruvate decarboxylase and alcohol dehydrogenase (heterologous or native) are required to enable ethanol synthesis in cyanobacteria. In the past decades, the performance of ethanol-producing cyanobacterial cell factories has been significantly improved through systematic optimization of proteins, pathways, chassis cells, and cultivation techniques. Cyanobacterial ethanol production technology has yielded the highest titer, productivity, and carbon partitioning ratio among all current cyanobacterial biomanufacturing systems. Recent advances have led to further improved efficiency of cyanobacterial ethanol photosynthetic production. Based on extensive systems biology data and rapidly developing computer modeling technologies, more accurate simulations of cyanobacterial physiological characteristics and metabolic networks have become possible. These simulations facilitate the identification of potential modification targets, thereby enhancing ethanol production capacity and guiding the design of next-generation alcohol-producing cell factories. With a more comprehensive understanding of cyanobacteria physiology and metabolism, systematic genome modifications and pathway optimizations have been performed, resulting in further improved ethanol productivity and final titers. Concurrently, efforts have been made to improve model strains and evaluate newly emerging non-conventional strains to establish more robust and efficient ethanol production processes. In conclusion, this review summarizes and compares three technological routes of light-driven carbon fixation and ethanol production in cyanobacteria, introduces the technological development trajectory and basic status of efficient light-driven carbon fixation for ethanol synthesis by cyanobacteria, provides valuable and up-to-date insights to facilitate the development of more promising cyanobacterial ethanol photosynthetic production technologies and explores future challenges and directions in this dynamic field.

Key words: cyanobacteria, photosynthetic biomanufacturing, synthetic biology, metabolic engineering, ethanol

摘要：

蓝细菌能够直接利用二氧化碳和太阳能通过光合作用生产乙醇，为提供绿色生物燃料提供了一条有前途的可持续路线。光驱固碳合成乙醇是最具代表性的蓝细菌光合生物制造技术。乙醇并不属于典型的蓝细菌天然代谢物，蓝细菌产醇细胞工厂的构建需要通过向基因组中导入异源的丙酮酸脱羧酶并结合异源/内源醇脱氢酶的过量表达来实现；在过去二十多年间，通过蛋白、途径、底盘、工艺层面的系统优化，蓝细菌产醇细胞工厂的效能得到有效提高，乙醇成为目前产量最高、产率最高、碳流分配率最高的蓝细菌代谢工程产物。本文总结并比较了“蓝细菌生物质炼制产醇”“蓝细菌固碳产糖-产醇”“蓝细菌固碳直接产醇”等三种光驱固碳产醇技术路线，并以构建光合细胞工厂驱动二氧化碳一站式转化为乙醇的技术路线为主，从乙醇合成途径优化与强化、蓝细菌光合碳代谢网络的调节与重塑、代谢网络模型与计算机辅助设计引导细胞工厂构建和优化三个方面对蓝细菌高效光驱固碳合成乙醇的技术发展历程和基本现状进行了介绍，特别是强调了计算生物学、系统代谢工程、生物材料嵌合等研究手段在本领域的应用进展；在此基础上，也从新型底盘开发、高通量筛选技术应用、细胞工厂的稳定性与鲁棒性优化等角度对蓝细菌固碳产醇的未来发展方向进行了展望。

关键词: 蓝细菌, 光合生物制造, 合成生物学, 代谢工程, 乙醇

CLC Number:

Q815

Huili SUN, Jinyu CUI, Guodong LUAN, Xuefeng LYU. Progress of cyanobacterial synthetic biotechnology for efficient light-driven carbon fixation and ethanol production[J]. Synthetic Biology Journal, 2023, 4(6): 1161-1177.

孙绘梨, 崔金玉, 栾国栋, 吕雪峰. 面向高效光驱固碳产醇的蓝细菌合成生物技术研究进展[J]. 合成生物学, 2023, 4(6): 1161-1177.

Figures/Tables 3

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策略	菌株	操作	效果	参考文献
优化Adh2的活性	PCC 6803	采用集胞藻PCC 6803来源的偏好使用NADPH为辅因子的slr1192基因替代运动发酵单胞菌adh2基因	增强了乙醛还原催化步骤与蓝细菌代谢背景的适配性，将乙醇产率提高了50%	[41]
优化Pdc与Adh2的实际丰度和配比	PCC 6803	使用5种强度不同的RBS分别控制pdc与adh2的表达，并进行组合，评价其乙醇产量及酶活	发现乙醇合成通量更强烈地决定于pdc而非adh2的表达强度和丰度，在5种RBS控制的不同pdc表达强度的突变体中，pdc表达强度最高的藻株乙醇合成能力最强，培养7 d时的乙醇含量约为1 g/L	[44]
强化卡尔文循环以提高蓝细菌固碳效率并加强乙醇合成	PCC 6803	分别将RuBisCo、SBPase、FBA或TK基因与Pdc-Adh2途径共表达	分别将乙醇产量提高了55%、67%、37%和69%，同时，总生物量也分别增加7.7%、15.1%、8.8%和10.1%	[45]
		在表达Pdc-Adh2途径的基础上，共表达FBA与TK基因	相比于单独表达FBA藻株的乙醇产量提高9倍以上	[46]
		在表达Pdc-Adh2途径的基础上，共表达SBPase与FBA基因	相比FBA单独表达藻株的乙醇产量提高2.5倍	[46]
加强蓝细菌底盘藻株对无机碳源的吸收	PCC 7942	在表达Pdc-Adh2途径的基础上，过量表达ictB、ecaA以及groESL	提高了在模拟烟道气条件下藻株的生物质含量（提高约4倍，0.9 g/L，72 h）和乙醇含量（提高约20倍，0.2 g/L，72 h）	[47]
敲除丙酮酸消耗途径，并将TCA循环中的碳流重新导回丙酮酸节点以增强乙醇合成前体供应	PCC 6803	在表达Pdc-Adh2途径的基础上，敲除了PEP合成酶（PpsA）基因，催化糖原合成的关键酶（GlgC）基因，并过量表达大肠杆菌来源的苹果酸酶（MaeB）基因	乙醇产量提升至1.09 g/L（7 d）	[48]
阻断糖原合成途径	PCC 7002	将两个乙醇合成途径（Pdc-Adh2）拷贝整合至基因组中两个糖原合成酶（GlgA）编码基因的位点，从而同时加强乙醇合成通量并阻断糖原合成途径竞争	在实验室环境柱式反应器中乙醇产量达到2.2 g/L（10 d），在户外挂袋式培养中乙醇产量达到0.8 g/L（7 d）	[49]
阻断糖原和PHB的合成途径	PCC 6803	敲除糖原合成关键基因glgC	乙醇产量从0.212 g/L（3 d）提高至0.297 g/L（3 d）	[50]
		在以上基础上进一步敲除phaCE基因以阻断PHB合成路径	乙醇产量提高至0.332 g/L（3 d）	[50]
		对以上藻株进行缺氮处理	乙醇产量达到0.6 g/L（3 d）	[50]
共培养“碳汇”工程策略	PCC 6803	将敲除glgC和phaA基因以阻断糖原和PHB合成途径的藻株和整合了Pdc-Adh2途径的工程藻株进行共培养	双菌体系中的乙醇产量达到4.6 g/L（25 d），而单平台藻株（基因组同时进行乙醇合成途径整合和糖原、PHB合成途径阻断）的产量4.1 g/L（25 d）	[51]
补充还原力供应	PCC 6803	过量表达内源G6PDH编码基因zwf，并导入Pdc-Adh2途径	乙醇产量从0.44 g/L增加到0.59 g/L（14 d），同时生物量积累增加了50%	[42]
向蓝细菌培养体系中添加金属氧化物以介导NADPH再生	PCC 6803	在培养体系中添加MgO或Fe₂O₃	乙醇产量达到5.1 g/L或4.851 g/L（25 d）	[52]
区室化合成乙醇并靶向性模拟缺氮环境	PCC 7120	在异形胞中使用特异性启动的hupS启动子控制Pdc-Adh2的表达	乙醇产量达到1.68 g/L（23 d）	[53]
区室化合成乙醇并靶向性模拟缺氮环境	PCC 7120	在以上基础上，使用特异性靶向异形胞的CRISRPi基因表达系统抑制glnA的表达	乙醇产量提高了27%	[54]
通过优化的基因组尺度代谢网络模型，预测乙醇/生物量耦合突变体	PCC 6803	预测最优突变体为通过13个基因的敲除来达到耦联乙醇合成和细胞生长的效果	预测乙醇产量为3.498 g/L（4 d）	[55]

策略	菌株	操作	效果	参考文献
优化Adh2的活性	PCC 6803	采用集胞藻PCC 6803来源的偏好使用NADPH为辅因子的slr1192基因替代运动发酵单胞菌adh2基因	增强了乙醛还原催化步骤与蓝细菌代谢背景的适配性，将乙醇产率提高了50%	[41]
优化Pdc与Adh2的实际丰度和配比	PCC 6803	使用5种强度不同的RBS分别控制pdc与adh2的表达，并进行组合，评价其乙醇产量及酶活	发现乙醇合成通量更强烈地决定于pdc而非adh2的表达强度和丰度，在5种RBS控制的不同pdc表达强度的突变体中，pdc表达强度最高的藻株乙醇合成能力最强，培养7 d时的乙醇含量约为1 g/L	[44]
强化卡尔文循环以提高蓝细菌固碳效率并加强乙醇合成	PCC 6803	分别将RuBisCo、SBPase、FBA或TK基因与Pdc-Adh2途径共表达	分别将乙醇产量提高了55%、67%、37%和69%，同时，总生物量也分别增加7.7%、15.1%、8.8%和10.1%	[45]
		在表达Pdc-Adh2途径的基础上，共表达FBA与TK基因	相比于单独表达FBA藻株的乙醇产量提高9倍以上	[46]
		在表达Pdc-Adh2途径的基础上，共表达SBPase与FBA基因	相比FBA单独表达藻株的乙醇产量提高2.5倍	[46]
加强蓝细菌底盘藻株对无机碳源的吸收	PCC 7942	在表达Pdc-Adh2途径的基础上，过量表达ictB、ecaA以及groESL	提高了在模拟烟道气条件下藻株的生物质含量（提高约4倍，0.9 g/L，72 h）和乙醇含量（提高约20倍，0.2 g/L，72 h）	[47]
敲除丙酮酸消耗途径，并将TCA循环中的碳流重新导回丙酮酸节点以增强乙醇合成前体供应	PCC 6803	在表达Pdc-Adh2途径的基础上，敲除了PEP合成酶（PpsA）基因，催化糖原合成的关键酶（GlgC）基因，并过量表达大肠杆菌来源的苹果酸酶（MaeB）基因	乙醇产量提升至1.09 g/L（7 d）	[48]
阻断糖原合成途径	PCC 7002	将两个乙醇合成途径（Pdc-Adh2）拷贝整合至基因组中两个糖原合成酶（GlgA）编码基因的位点，从而同时加强乙醇合成通量并阻断糖原合成途径竞争	在实验室环境柱式反应器中乙醇产量达到2.2 g/L（10 d），在户外挂袋式培养中乙醇产量达到0.8 g/L（7 d）	[49]
阻断糖原和PHB的合成途径	PCC 6803	敲除糖原合成关键基因glgC	乙醇产量从0.212 g/L（3 d）提高至0.297 g/L（3 d）	[50]
		在以上基础上进一步敲除phaCE基因以阻断PHB合成路径	乙醇产量提高至0.332 g/L（3 d）	[50]
		对以上藻株进行缺氮处理	乙醇产量达到0.6 g/L（3 d）	[50]
共培养“碳汇”工程策略	PCC 6803	将敲除glgC和phaA基因以阻断糖原和PHB合成途径的藻株和整合了Pdc-Adh2途径的工程藻株进行共培养	双菌体系中的乙醇产量达到4.6 g/L（25 d），而单平台藻株（基因组同时进行乙醇合成途径整合和糖原、PHB合成途径阻断）的产量4.1 g/L（25 d）	[51]
补充还原力供应	PCC 6803	过量表达内源G6PDH编码基因zwf，并导入Pdc-Adh2途径	乙醇产量从0.44 g/L增加到0.59 g/L（14 d），同时生物量积累增加了50%	[42]
向蓝细菌培养体系中添加金属氧化物以介导NADPH再生	PCC 6803	在培养体系中添加MgO或Fe₂O₃	乙醇产量达到5.1 g/L或4.851 g/L（25 d）	[52]
区室化合成乙醇并靶向性模拟缺氮环境	PCC 7120	在异形胞中使用特异性启动的hupS启动子控制Pdc-Adh2的表达	乙醇产量达到1.68 g/L（23 d）	[53]
区室化合成乙醇并靶向性模拟缺氮环境	PCC 7120	在以上基础上，使用特异性靶向异形胞的CRISRPi基因表达系统抑制glnA的表达	乙醇产量提高了27%	[54]
通过优化的基因组尺度代谢网络模型，预测乙醇/生物量耦合突变体	PCC 6803	预测最优突变体为通过13个基因的敲除来达到耦联乙醇合成和细胞生长的效果	预测乙醇产量为3.498 g/L（4 d）	[55]

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Progress of cyanobacterial synthetic biotechnology for efficient light-driven carbon fixation and ethanol production

面向高效光驱固碳产醇的蓝细菌合成生物技术研究进展

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