Engineering Rubisco for Food Security

Global demand for high quality digestible protein is expected to substantially outpace population growth over the coming decades, necessitating gains in feed-to-food conversion efficiency for sustainable food security. Toward these ends, identifying and producing new high quality protein feedstocks that supplement existing agriculture will be of major importance and have significant impacts that intersect with the energy and water infrastructure. RuBisCo, the enzyme complex that initiates carbon fixation, is often cited as the most abundant soluble protein on earth, typically representing 40 - 60% of the total protein content of alga and green leafy biomass. In addition to its abundance, RuBisCo is easy to extract and highly digestible, as well as having other favorable chemical and mechanical properties for food and feed applications, including foam formation and gelation. Although RuBisCo has a very high native amino acid score (PDCAAS ~ 98/100), we have recently demonstrated significant improvements in the amino acid value by increasing expression of two key amino acids, methionine (Met) and lysine (Lys), which are typically limiting and costly for feed applications, in the large subunit of the RuBisCo complex, RbcL, of Synechocystis sp. PCC6803. Enrichment targets for Met and Lys in RbcL were identified using Grantham's distance matrix, with avoidance of previously reported lethal mutations. Installation of the selected codon swaps was achieved using Circular Polymerase Extension Cloning (CPEC), yielding between 60-100% higher Met and Lys abundance, respectively. Expression of Met- and Lys- enriched RbcL in Synechocystis sp. PCC6803 showed no detriment to specific growth rate or total quantity of RbcL, indicating technological feasibility for a sustainable solution to global protein deficiency. Similar engineering strategies should be amenable for terrestrial food crops and green leafy biomass.