Cell-Free production of soybean leghemoglobins and non-symbiotic hemoglobin

ABSTRACT Hemoglobins are heme proteins and are present in some microorganisms, higher plants and mammals. In legume nodules there are two types: leghemoglobin (LegH) or symbiotic and non-symbiotic (nsHb). LegHs are present in high amounts at legumes roots and are responsible together with bacteroides for the nitrogen fixation process. Non-symbiotic hemoglobins Class 1 protein have very high affinity for O2 and are found in monocotyledons and legumes. LegH has aroused great interest in the vegetable meat industry due to its organoleptic and nutritional properties. Here, we demonstrated that soybean LegH A, C1, C2, C3 and nsHb are produced by E. coli-based cell-free protein synthesis (CFPS) and correctly synthesized in its amino acids sequence. In addition, it was also possible to reproduce some post-translational modifications confirmed by LC/MS analysis. All LegHs produced in this system showed peroxidase activity and heme binding correlated with its concentration in the assays. Furthermore, all proteins were readily digested by pepsin within 1 minute in analog digestion conditions. Therefore, LegHs and nsHb proteins were synthesized using cell-free systems (CFSs), maintaining their functionality and being digestible. These findings suggest that they could serve as viable alternative food additives for plant-based meat. Competing Interest Statement The authors have declared no competing interest.