Metabolic engineering of Escherichia coli to enhance hydrogen production from glycerol

Glycerol is an attractive carbon source for biofuel production since it is cheap and abundant due to the increasing demand for renewable and clean energy sources, which includes production of biodiesel. This research aims to enhance hydrogen production by Escherichia coli from glycerol by manipulating its metabolic pathways via targeted deletions. Since our past strain, which had been engineered for producing hydrogen from glucose, was not suitable for producing hydrogen from glycerol, we rescreened 14 genes related to hydrogen production and glycerol metabolism. We found that 10 single knockouts are beneficial for enhanced hydrogen production from glycerol, namely, frdC (encoding for furmarate reductase), ldhA (lactate dehydrogenase), fdnG (formate dehydrogenase), ppc (phosphoenolpyruvate carboxylase), narG (nitrate reductase), focA (formate transporter), hyaB (the large subunit of hydrogenase 1), aceE (pyruvate dehydrogenase), mgsA (methylglyoxal synthase), and hycA (a regulator of the transcriptional regulator FhlA). On that basis, we created multiple knockout strains via successive P1 transductions. Simultaneous knockouts of frdC, ldhA, fdnG, ppc, narG, mgsA, and hycA created the best strain that produced 5-fold higher hydrogen and had a 5-fold higher hydrogen yield than the parent strain. The engineered strain also reached the theoretical maximum yield of 1 mol H₂/mol glycerol after 48 h. Under low partial pressure fermentation, the strain grew over 2-fold faster, indicating faster utilization of glycerol and production of hydrogen. By combining metabolic engineering and low partial pressure fermentation, hydrogen production from glycerol was enhanced significantly.