From lignocellulose to whole-cell catalysts

How to achieve the high-value utilization of this low-value raw material, lignocellulosic biomass, has always been a research hotspot both at home and abroad. The Metabolomics Team of the Qingdao Institute of Bioenergy and Bioprocess Technology of the Chinese Academy of Sciences aimed to break the foreign technological monopoly and overcome the bottleneck of lignocellulose saccharification technology. They have been dedicated to the genetic modification and metabolic engineering research of cellulose-degrading bacteria such as Thermotoga, using a series of gene manipulation tools developed by the team in the past. Through the thermotoga and its cellulose-degrading enzyme system, - — The directional modification of fibril bodies has resulted in the construction of a new type of engineered strain, which can serve as a whole-cell catalyst to achieve efficient conversion of lignocellulosic substrates to fermentable sugars, significantly promoting the industrialization process of lignocellulosic bioconversion. Lignocellulosic biomass has attracted attention due to its reserves and renewability, but the unreasonable disposal of agricultural and forestry waste will greatly increase environmental pressure and cause serious environmental pollution problems such as water pollution and haze from incineration. Therefore, the efficient utilization of non-food lignocellulosic materials is an urgent global issue that has significant strategic importance for achieving sustainable economic development. However, the industrialization, large-scale application, and commercialization of lignocellulosic biomass have not truly been carried out, and the failure to break through the bottleneck step of efficient and low-cost conversion of lignocellulosic biomass to fermentable sugars is the main reason. Fibril bodies are currently one of the most efficient cellulose-degrading molecular machines known in nature. As a typical cellulose-producing fibril body strain, Thermotoga thermophila has the natural characteristic of highly efficient degradation of cellulose substrates, and is therefore considered to be the most promising strain that can achieve efficient biocatalytic conversion of lignocellulosic biomass through the strategy of integrating bioprocessing technology. However, the existing wild strains and their fibril bodies have shortcomings such as substrate hydrolysis activity being inhibited by the feedback of enzyme catalytic products, and cannot meet the requirements of industrialization. Based on this current research situation, the metabolomics team has carried out targeted directional modification of Thermotoga thermophila and its fibril bodies. By establishing a scar-free genome editing system, β-glucosidase CaBglA from an extreme thermophilic bacterium and the key fibril body enzyme Cel48S were fused and expressed and assembled onto the extracellular fibril body. Using this recombinant strain as a whole-cell catalyst for saccharification reactions, it was found that when using 100 g/L microcrystalline cellulose as the substrate, the reducing sugar yield reached 489 mM (converted to approximately 88 g/L based on the molecular weight of glucose). The ability of this bacterium to efficiently degrade cellulose and produce fermentable sugars has preliminarily proved the feasibility of the lignocellulosic whole-cell catalytic saccharification strategy in industrial applications. This research expands the new perspective of lignocellulosic saccharification and strongly promotes the use of cellulose sugar as a carbon source for starch sugar in the industrial fermentation field.