Genomic integration of sucrose catabolism into Escherichia coli for sustainable hydrogen production

Addressing society's energy needs to achieve low greenhouse gas emissions requires the production of renewable energy through innovative methods. Hydrogen gas is a potential fuel that, when produced renewably, could facilitate the energy sector to become carbon neutral. In our research group, Escherichia coli has been engineered to produce hydrogen gas at unprecedented rates. The current feedstock for this hydrogen production mechanism is glucose. To improve the economics and sustainability of this hydrogen production method, alternative feedstocks need to be sought such as sucrose as derived from sugarcane. However, the ability for E. coli to utilise sucrose as a carbon source is uncommon. Here, synthetic biology techniques were employed to integrate a synthetic sucrose utilising gene cluster (SSGC) into the genome of our hydrogen-producing K-12 derivative DH5α strain. Integration of large gene pathways into E. coli have not been studied extensively. Here, the optimization of genomic integration at multiple chromosomal loci using λ-Red and CRISPR/Cas9 guided recombineering approaches was examined. The results showed that incorporation of the SSGC conferred E. coli the ability to grow on sucrose as sole carbon source. This achievement now allows our E. coli strain to generate hydrogen from sucrose as an alternative feedstock to glucose.