(113e) Cyanobacterial Engineering and RNA-Sensor Based Rubisco Assay for Sustainable Production of Essential Amino Acids

The demand for protein-based foods is continuously increasing along with the overgrowing population. As a consequence, the next decade will witness a significant strain on natural resources if more sustainable and cheaper alternative protein resources aren’t developed. On the other hand, there is also an urgent need to produce nutritive and easily affordable protein food to overcome the global prevalence of undernourishment. Plant proteins are more sustainable and cheaper than animal-based proteins. However, plant proteins are low on certain essential amino acids. We propose that by engineering plant-based proteins to accumulate essential amino acids, we can overcome this limitation by retaining sustainability and cost-effectiveness. More specifically, to address this alarming issue we engineered RuBisCO, a key abundant soluble protein found in all photoautotrophs to accumulate methionine and lysine.

In this study the large sub-unit of RuBisCO in the model cyanobacterium Synechocystis sp. PCC 6803 was engineered to increase the content of two essential amino acids, lysine, and methionine by two-fold. Grantham’s distance was utilized to replace leucine and isoleucine with Methionine while Arginine and histidine were replaced with lysine. The protein structure of the RuBisCO variants were analyzed in silico to ensure proper folding. The engineered RuBisCO (encoded by rbcL) were integrated into the rbcL locus of Synechocystis 6803 genome through homologous recombination. Comparative growth studies showed that there was no significant difference in the growth rate of engineered strains compared to the wild-type (WT). Chlorophyll a and carotenoid pigments concentration were also estimated for the engineered and WT strains; results confirmed that there was no extra cellular stress or burden on the strains engineered with the RuBisCO variants.

Simultaneously, biochemical quantitative RuBisCO assay and novel RNA-sensor based fluorescence assay were developed to estimate the kinetic properties of the engineered protein variants. Both the assays confirmed that the function and properties of the RuBisCO variants were unaffected, indicating a successful protein engineering. In parallel, the engineered synthetic rbcL genes were expressed in E. coli BL21(DE3) cells under the control of P_T7 for overexpression and amino acids analysis of the protein variants. GC MS analysis of partly purified insoluble RuBisCO protein from E. coli cell lysate, showed ~75% increase in Methionine and ~65% increase in Lysine content as compared to that of WT RuBisCO.

In conclusion, we have successfully engineered and demonstrated the increased accumulation of essential amino acids in cyanobacteria by utilizing proteins as an amino acid sink. Cyanobacteria are edible and hence can themselves serve as nutritious protein food. Concurrently, the strategy can be translated to the plants, edible vegetables, crops, legumes to obtain the essential amino acid enriched proteins for humans and other animals.