Engineering RuBisCO to enrich essential amino acids in sustainable food applications

Plant-based proteins are more sustainable than animal-based proteins but are poorer in essential amino acids (EAA). This study reports Grantham’s distance matrix-guided targeted amino acid replacement to improve the EAA content of RuBisCO in Synechocystis sp. PCC 6803. Replaceable residues were identified from RuBisCO large subunit (RbcL), generating four in silico RbcL variants: RbcL1 and RbcL2 (methionine-enriched), RbcL3 and RbcL4 (lysine-enriched). Protein structures and folding were validated using SWISS-MODEL, ERRAT quality scores, and Ramachandran plots. PCC 6803 was engineered to individually express RuBisCO variants, with negligible differences in growth rate. Following RuBisCO activity results, only RbcL1, RbcL3, and RbcL4 were overexpressed in Escherichia coli BL21-DE3 for EAA quantification using GC–MS. RbcL1 exhibited ~2-fold (~100%) higher methionine, whereas RbcL3 and RbcL4 showed ~1.7-fold (~70%) higher lysine, relative to WT-RbcL. These EAA-fractions exceeded those from soy- and algae-derived proteins, demonstrating successful EAA-enrichment in a protein that could be extended to other photoautotrophs.