热纤梭菌在生物质能源开发中的合成生物学研究进展

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

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。

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 the high 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.