Efficient whole-cell-catalyzing cellulose saccharification using engineered Clostridium thermocellum

Cost-efficient saccharification is one of the main bottlenecks for industrial lignocellulose conversion. Clostridium thermocellum naturally degrades lignocellulose efficiently using the cellulosome, a multiprotein supermolecular complex, and thus can be potentially used as a low-cost catalyst for lignocellulose saccharification. The industrial use of C. thermocellum is restrained due largely to the inhibition of the hydrolysate cellobiose to its cellulosome. Although the supplementation of beta-glucosidase may solve the problem, the production of the enzymes greatly complicates the process and may also increase the cost of saccharification. To conquer the feedback inhibition and establish an efficient whole-cell catalyst for highly efficient cellulose saccharification, we constructed a recombinant strain of C. thermocellum ∆pyrF::CaBglA which produced a secretory exoglucanase CelS-bearing heterologous BGL using a newly developed seamless genome editing system. Without the extra addition of enzymes, the relative saccharification level of ∆pyrF::CaBglA was stimulated by over twofolds compared to its parent strain ∆pyrF through a two-stage saccharification process with 100 g/L Avicel as the carbon source. The production of reducing sugars and the relative saccharification level were further enhanced to 490 mM and 79.4%, respectively, with increased cell density. The high cellulose-degrading ability and sugar productivity suggested that the whole-cell-catalysis strategy for cellulose saccharification is promising, and the C. thermocellum strain ∆pyrF::CaBglA could be potentially used as an efficient whole-cell catalyst for industrial cellulose saccharification.