Sugar Uptake Mechanism Of Biomass Degrading Bacterium Clostridium Thermocellum

Clostridium thermocellum is a kind of thermophilic anaerobic bacteria which can degrade lignocellulosic biomass efficiently. It has application value in the conversion and utilization of agricultural and forestry waste biomass. Recently, Cui Qiu, a researcher in the metabonomics research group of Qingdao bioenergy and Process Research Institute, Chinese Academy of Sciences, combined with in vivo and in vitro experiments, clarified the transporters responsible for the uptake of fibrooligosaccharides and glucose in Clostridium thermocellum and their structural and molecular mechanisms.

The cellulolytic celluloses are the main energy sources of cellulosic celluloses.

In 2009, Israeli researchers found five transporter gene clusters in the genome of Clostridium thermophilum, suggesting that they may be responsible for the transport of cellooligosaccharides, glucose and quinbuccal disaccharide, but there is no clear functional verification experiment. Due to the difficulty in genetic manipulation of Clostridium thermocellum, the mechanism of carbohydrate uptake by Clostridium thermocellum has not been clearly elucidated in the following decade except for some omic analysis involving these transporters.

The metabonomics research group of Qingdao Institute of energy has been carrying out research on Clostridium thermophilum for a long time, breaking through the bottleneck of genetic operation of Clostridium thermophilum, and developing a variety of genetic operation tools for Clostridium thermophilum, including rapid gene targeting system and accurate genome scarless editing system, providing effective means for in-depth study on the molecular mechanism of Clostridium thermocellum physiology and metabolism. On this basis, the researchers further studied the carbohydrate uptake mechanism of Clostridium thermocellum.

In this study, five knockout and complementing mutants of oligosaccharide transporter gene cluster were constructed. The growth phenotype test showed that transporter B was the only cellooligosaccharide transporter, while transporter A was the only glucose transporter.

The interaction between substrate binding subunits of transporters and various sugars were determined by isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) titration and X-ray crystallography. The mechanism of transporters A and B specifically binding to glucose and fibrooligosaccharides was elucidated. In addition, an isolated gene 2554 was identified as an ATPase subunit gene missing from the transporter B gene cluster, which complemented all components of the unique fibrooligosaccharide transporter.

Previous studies have shown that the fibrosome expression of Clostridium thermocellum is mainly regulated by a special sigma / anti sigma factor. It was found that the knockout of transporter B could significantly reduce the protrusion of fibrosomes on the cell surface, and the induction of fibrosome expression by fibrooligosaccharides disappeared, which indicated that there was a coupling relationship between transporter B and the regulation of fibrosome expression, which provided a new idea for the study of regulation mechanism of fibrosome expression.

Carbohydrate transporters are important targets for industrial microorganisms to carry out metabolic engineering transformation and synthetic biological development. It is of great potential application value to reveal the mechanism of carbohydrate uptake by industrial microorganisms. Clostridium thermocellum has potential application value in bioconversion of lignocellulose due to its unique cellulosome system. As a special thermophilic substrate cell, Clostridium thermophilum also has application potential in the development of synthetic biology. This study elucidated the oligosaccharide and glucose uptake mechanism of Clostridium thermocellum, and found the coupling relationship between oligosaccharide uptake and fibrosome expression regulation, which deepened the understanding of physiological and metabolic mechanism of Clostridium thermocellum

The results were published in the microbiology Journal mbio. The research was supported by Israel weizman Institute of science, Technion Technology Institute and Tianjin Industrial Biotechnology Institute of Chinese Academy of Sciences, as well as National Natural Science Foundation of China, Shandong Energy Research Institute, Chinese Academy of Sciences and Qingdao city.

Mechanism of carbohydrate uptake by Clostridium thermocellum revealed by Qingdao Energy Research Institute