Progress in synthetic biology research of Clostridium thermocellum for biomass energy applications

doi:10.12211/2096-8280.2023-046

Synthetic Biology Journal ›› 2023, Vol. 4 ›› Issue (6): 1055-1081.DOI: 10.12211/2096-8280.2023-046

• Invited Review • Previous Articles Next Articles

Progress in synthetic biology research of Clostridium thermocellum for biomass energy applications

Yan XIAO¹^,²^,³^,⁴, Yajun LIU¹^,²^,³^,⁴, Yin′gang FENG¹^,²^,³^,⁴, Qiu CUI¹^,²^,³^,⁴

^1.Shandong Provincial Key Laboratory of Synthetic Biology，CAS 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
^4.University of Chinese Academy of Sciences，Beijing 100049，China

Received:2023-07-02 Revised:2023-09-22 Online:2024-01-19 Published:2023-12-31
Contact: Yin′gang FENG, Qiu CUI

热纤梭菌在生物质能源开发中的合成生物学研究进展

肖艳¹^,²^,³^,⁴, 刘亚君¹^,²^,³^,⁴, 冯银刚¹^,²^,³^,⁴, 崔球¹^,²^,³^,⁴

^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，山东省合成生物学重点实验室，山东青岛 266101
^2.山东省能源研究院，山东青岛 266101
^3.青岛新能源山东省实验室，山东青岛 266101
^4.中国科学院大学，北京 100049

通讯作者: 冯银刚,崔球
作者简介:肖艳（1982—），女，博士，副研究员，硕士生导师。研究方向为能源微生物代谢机理与改造。E-mail：xiaoyan@qibebt.ac.cn
冯银刚（1977—），男，博士，研究员，博士生导师。研究方向为能源微生物的分子生理机制与合成生物学应用、工业酶催化机制与酶工程等。E-mail：fengyg@qibebt.ac.cn
崔球（1975—），男，博士，研究员，博士生导师。研究方向为基于代谢物组/代谢流组学的计算分析辅助能源微生物代谢工程设计、蛋白质结构功能研究。E-mail：cuiqiu@qibebt.ac.cn
基金资助:
国家自然科学基金(32070125);山东能源研究院(SEI I202106);山东省自然科学基金(ZR2022MC128)

Abstract

Abstract:

Biomass, including agricultural and forestry waste, energy plants, and microalgae, possesses both "energy" and "substance" properties, making it a promising renewable resource that can potentially replace fossil fuels. The efficient lignocellulose bioconversion relies on the development of effective biocatalysts. Clostridium thermocellum (also known as Ruminiclostridium thermocellum, Hungateiclostridium thermocellum, and Acetivibrio thermocellus) is a thermophilic anaerobic bacterium that can efficiently degrade lignocellulosic biomass. Over the past two decades, extensive research and development have led to the potential of using C. thermocellum as a cell factory to produce various energy and chemicals from lignocellulose. C. thermocellum has been used to produce ethanol, butanol, isobutanol, hydrogen, lactic acid, medium/short-chain fatty acid esters, and fermentable sugars from lignocellulosic biomass. The degradation and utilization process of lignocellulosic biomass by C. thermocellum mainly involves substrate recognition and hydrolysis through the cellulosome, hydrolysate uptake through ABC transporters, and intracellular metabolism via atypical glycolytic pathways. C. thermocellum possesses dynamic regulation of cellulosome production adapting extracellular substrates, which enables thehigh capability of degrading various lignocellulosic substrates. The cellulosome consists of non-catalytic scaffoldins and multiple enzymatic subunits with distinct catalytic activities and has broad applications in synthetic biology as well as lignocellulose degradation. In addition to lignocellulose refinery, the thermophilic C. thermocellum also has great potential in synthetic biology research under high-temperature conditions. Several genetic manipulation tools have been developed for C. thermocellum, although greater challenges have been encountered compared to model organisms such as Escherichia coli. The genetic tools include homologous recombination technology, Thermotargetron technology, and CRISPR/Cas systems, which enable gene knockout, insertion, replacement, mutation, and expression regulation of target genes in the strain. C. thermocellum has been used as the whole-cell biocatalyst for lignocellulose bioconversion through consolidated bioprocessing (CBP) and consolidated bio-saccharification (CBS). CBS follows the concept of sugar platform construction and shows great potential in real-world applications. The synthetic biology research targeting the CBS strategy still requires future development. For example, we need to explore new genetic tools and thermophilic functional elements for C. thermocellum and improve the efficiency of gene editing. We need to strengthen research on the genetic, physiological, and metabolic aspects of C. thermocellum, and the molecular mechanisms underlying lignocellulose degradation. It is noteworthy that, as a strict anaerobe, C. thermocellum cannot be used as the chassis for catalyzing oxygen-involved reactions. Selecting suitable metabolic pathways and target products will be the focus in future developments of synthetic biology based on C. thermocellum. Therefore, we need to investigate additional target pathways and products for synthetic biology development. In recent years, automation methods and artificial intelligence (AI) technologies are being developed rapidly and have been applied in various synthetic biology research fields. Such technologies may also be employed to promote research on thermophilic and anaerobic microorganisms.

Key words: bioenergy, Clostridium thermocellum, cellulose, biofuels, cellulosome

摘要：

农林废弃物、能源植物、微藻等生物质是唯一同时具备“能源”和“物质”双重属性的可再生资源，在替代不可再生的化石能源方面具有巨大的潜力。木质纤维素生物转化的核心之一在于高效生物催化剂的构建。热纤梭菌是高效降解木质纤维素的嗜热厌氧菌，是多种木质纤维素生物转化策略的理想底盘菌株，在生物质能源开发中具有重要价值。经过近二十年的研究和开发，针对热纤梭菌已经建立了多种遗传改造技术，并构建了可以生产多种能源分子及化学品的热纤梭菌细胞工厂。本文首先介绍了热纤梭菌及其纤维素降解与利用特性，简述了热纤梭菌的系统生物学研究和遗传改造工具开发的现状，随后重点回顾和总结了热纤梭菌在生产乙醇、丁醇、异丁醇、氢气、乳酸、中/短链脂肪酸酯和可发酵糖等生物能源开发中的合成生物学研究进展。最后对热纤梭菌的合成生物学发展方向进行了展望，并强调了合成生物学技术在未来生物质能源开发中的重要作用。

关键词: 生物能源, 热纤梭菌, 纤维素, 生物燃料, 纤维小体

CLC Number:

Q812

Yan XIAO, Yajun LIU, Yin′gang FENG, Qiu CUI. Progress in synthetic biology research of Clostridium thermocellum for biomass energy applications[J]. Synthetic Biology Journal, 2023, 4(6): 1055-1081.

肖艳, 刘亚君, 冯银刚, 崔球. 热纤梭菌在生物质能源开发中的合成生物学研究进展[J]. 合成生物学, 2023, 4(6): 1055-1081.

Figures/Tables 5

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热纤梭菌菌株	主要方法	研究内容、结果或结论	年份	文献
F7 (VKMB 2203)	蛋白质组	在热纤梭菌基因组草图中发现了超过71个编码纤维小体蛋白的基因；蛋白质组鉴定了纤维小体中含量较高的13个组分	2005	[37]
ATCC 27405	转录组	建立了有效的微阵列方法用于热纤梭菌的转录组研究	2007	[60]
ATCC 27405	蛋白质组	多通过蛋白质组分析了热纤梭菌纤维小体的组分以及在纤维素和二糖上生长时的变化	2007	[61]
ATCC 27405	构建代谢模型	构建了基因组尺度代谢模型iSR432，包含了577个反应	2010	[62]
DSM 1313	基因组	热纤梭菌DSM1313基因组测序结果	2011	[30]
ATCC 27405	转录组	对比纤维素和纤维二糖稳态培养的基因表达对比分析。3189个基因中检测到2846个，分析了底物依赖的基因表达变化	2011	[63]
ATCC 27405	转录组	在稀酸预处理的杨树和柳枝稷上生长的不同时间（12 h和37 h）的热纤梭菌的基因转录	2013	[64]
DSM 1313	代谢组	高纤维素载量（50～100 g/L）下的热纤梭菌胞外代谢产物分析	2014	[65]
ATCC 27405	通过机器学习预测转录单元	根据转录组数据，使用机器学习方法预测了2590个转录单元，44%有多基因	2015	[66]
DSM 1313突变株	转录组，蛋白质组	在热纤梭菌中敲除不同脚架蛋白后，检测热纤梭菌的各种基因表达变化，揭示各种脚架蛋白的重要性以及与其他基因之间的耦联关系	2016	[67]
ATCC 27405	转录组，代谢组，蛋白质组	稀酸预处理的柳枝稷上生长的不同时间下三种组学的情况，分析在降解过程中抑制物造成的代谢变化	2017	[68]
DSM 1313	构建代谢模型	建立基因组尺度代谢模型iCth446，并在此基础上构建了核心代谢动力学模型k-ctherm118	2017	[69]
ATCC 27405	转录组，蛋白质组	分析了纤维素附着细胞和游动细胞的基因表达和蛋白差异	2017	[70]
DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack	转录组，代谢组	分析了当时已知的产乙醇最高的菌株在不同pH稳态培养下的代谢物和基因表达变化	2018	[71]
KJ335(整合了木糖利用基因的工程菌株，来自DSM 1313)	转录组	分析了木糖利用工程菌株的在木糖和纤维二糖上的转录组差异，揭示了木糖的转运与代谢相关基因以及热纤梭菌趋化与运动相关基因的表达变化	2020	[72]
DSM 1313	代谢流分析、构建动力学模型	基于代谢流分析建立核心代谢动力学模型k-ctherm138，鉴定了限制乙醇生产的67种底物水平抑制机制	2022	[73]
DSM 1313	构建代谢模型	构建基因组尺度代谢模型iCTH669，包含了913个代谢反应，837种代谢物，669个基因，模型的可靠性得到巨大的提升	2023	[74]

热纤梭菌菌株	主要方法	研究内容、结果或结论	年份	文献
F7 (VKMB 2203)	蛋白质组	在热纤梭菌基因组草图中发现了超过71个编码纤维小体蛋白的基因；蛋白质组鉴定了纤维小体中含量较高的13个组分	2005	[37]
ATCC 27405	转录组	建立了有效的微阵列方法用于热纤梭菌的转录组研究	2007	[60]
ATCC 27405	蛋白质组	多通过蛋白质组分析了热纤梭菌纤维小体的组分以及在纤维素和二糖上生长时的变化	2007	[61]
ATCC 27405	构建代谢模型	构建了基因组尺度代谢模型iSR432，包含了577个反应	2010	[62]
DSM 1313	基因组	热纤梭菌DSM1313基因组测序结果	2011	[30]
ATCC 27405	转录组	对比纤维素和纤维二糖稳态培养的基因表达对比分析。3189个基因中检测到2846个，分析了底物依赖的基因表达变化	2011	[63]
ATCC 27405	转录组	在稀酸预处理的杨树和柳枝稷上生长的不同时间（12 h和37 h）的热纤梭菌的基因转录	2013	[64]
DSM 1313	代谢组	高纤维素载量（50～100 g/L）下的热纤梭菌胞外代谢产物分析	2014	[65]
ATCC 27405	通过机器学习预测转录单元	根据转录组数据，使用机器学习方法预测了2590个转录单元，44%有多基因	2015	[66]
DSM 1313突变株	转录组，蛋白质组	在热纤梭菌中敲除不同脚架蛋白后，检测热纤梭菌的各种基因表达变化，揭示各种脚架蛋白的重要性以及与其他基因之间的耦联关系	2016	[67]
ATCC 27405	转录组，代谢组，蛋白质组	稀酸预处理的柳枝稷上生长的不同时间下三种组学的情况，分析在降解过程中抑制物造成的代谢变化	2017	[68]
DSM 1313	构建代谢模型	建立基因组尺度代谢模型iCth446，并在此基础上构建了核心代谢动力学模型k-ctherm118	2017	[69]
ATCC 27405	转录组，蛋白质组	分析了纤维素附着细胞和游动细胞的基因表达和蛋白差异	2017	[70]
DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack	转录组，代谢组	分析了当时已知的产乙醇最高的菌株在不同pH稳态培养下的代谢物和基因表达变化	2018	[71]
KJ335(整合了木糖利用基因的工程菌株，来自DSM 1313)	转录组	分析了木糖利用工程菌株的在木糖和纤维二糖上的转录组差异，揭示了木糖的转运与代谢相关基因以及热纤梭菌趋化与运动相关基因的表达变化	2020	[72]
DSM 1313	代谢流分析、构建动力学模型	基于代谢流分析建立核心代谢动力学模型k-ctherm138，鉴定了限制乙醇生产的67种底物水平抑制机制	2022	[73]
DSM 1313	构建代谢模型	构建基因组尺度代谢模型iCTH669，包含了913个代谢反应，837种代谢物，669个基因，模型的可靠性得到巨大的提升	2023	[74]

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Progress in synthetic biology research of Clostridium thermocellum for biomass energy applications

热纤梭菌在生物质能源开发中的合成生物学研究进展

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