Abstract
Dark fermentation is considered as one of the most practical biological hydrogen production methods. However, current productivity and yield are still not economically viable for industrial applications. This biological process must be improved through multiple strategies, of which screening for more effective microbial strains is an important aspect. Here, based on the hydrogen production pathway of E. aerogenes, we describe three strategies to improve hydrogen production by effectively regulating the anaerobic metabolism of E. aerogenes through genetic modification. This protocol describes in detail how to obtain NADH dehydrogenase-damaged mutants and overexpress Nad synthase genes using the CRISPR-Cas9 gene editing system. In addition, the overexpression of small RNA RyhB was achieved and verified by Northern Blot. This protocol is of great significance for the study of genetic engineering operation in E. aerogenes and other bacteria, and also provides theoretical guidance and technical support for the study of E. aerogenes biological hydrogen production.