Metabolic Engineering of Clostridium sp. to Increase Hydrogen Production Using the CRISPR/Cas System

Biofuels are eco-friendly since they do not contaminate the environment nor contribute to global warming, but present the disadvantage that their production costs are higher than those for fossil fuels and sometimes compete for food resources.

To reduce fuel-associated production costs, organic wastes can be used to produce biogas by microbial fermentation. A promising biogas is hydrogen since it is the fuel with the highest energetic capacity known and its combustion product is just water. Anaerobic Clostridia are potential producers of this fuel, and biotechnological techniques can be applied to improve fuel production as is the case of metabolic engineering. Previously, Gonzalez-Garcia et al. (2017) proposed a metabolic model of a mixed culture, containing Clostridium sp., which suggests that a theoretical yield as high as 10 mol H₂/mol of substrate can be achieved if H₂ pressure is sufficiently low. The same study suggests that H₂ yields can be improved by redirecting the metabolic flux towards the pentose phosphate pathway instead of glycolysis, thus increasing the reduction power necessary for a higher hydrogen production. Knocking out competing reactions for byproduct production are also suggested.

CRISPR/Cas system is a novel genome editing approach that has been widely used in several organisms and research areas. It offers various advantages over traditional editing methods as can be a high genome editing efficiency, a simple and easy way for its implementation and maybe the most important, the simultaneous modification at different loci of the same organism. With those advantages, the numerous modifications required for metabolic engineering approaches can be easily performed.

In the present study, a core metabolic flux model of Clostridium pasteurianum ATCC 6013 aimed to increase hydrogen production is proposed. Knockouts at the phosphoglucose isomerase; ethanol, lactate and acetate dehydronenase genes are expected in the model as potential genetic modifications in Clostridium pasteurianum. Those modifications are feasible by using the Clostridium endogenous CRISPR/Cas system as proven by Pyne et. al (2016) who worked on the same microorganism to the one considered in this study.

Via metabolic engineering and CRISPR/CAs editing, we are developing an engineered strain of Clostridium pasteurianum ATCC 6013 with a higher yield of hydrogen. This development will improve the knowledge of hydrogen-producing organisms.