(554b) Engineering of Cyanobacterial Rubisco for Sustainable Production of Essential Amino Acids

Global demand for high quality digestible protein is expected to significantly exceed population growth over the coming decades. To address this alarming issue we engineered RuBisCO, a key abundant soluble protein found in all photoautotrophs to act as a sink for the accumulation of two essential amino acids methionine and lysine. Specifically, the large sub-unit of RuBisCO in the model cyanobacterium Synechocystis sp. PCC 6803 was engineered for this purpose.

First, by utilizing Grantham’s distance matrix, leucine and isoleucine in the RuBisCO amino acids sequence were replaced with methionine, while arginine and histidine were replaced with lysine. Towards this, four RuBisCO variants, cbbL1, cbbL2, cbbL3, and cbbL4 were created in silico. Further, in silico studies revealed that more than 80 % sequence similarity and 0.98 structure similarity were maintained by all protein variants. The rbcL variants encoding the engineered RuBisCO were integrated into the rbcL locus of Synechocystis 6803. Comparative growth studies of the engineered strains showed that there was no significant difference in the growth rate compared to the wild type. Estimation of chlorophyll a and carotenoid pigments also confirmed that there was no extra cellular stress on the engineered strains.

Secondly, a quantitative absorbance-based assay and a novel RNA-sensor based fluorescence assay for RuBisCO were developed. Both the assays confirmed that the function and properties of the RuBisCO variants were unaffected, indicating successful protein engineering. In parallel, the engineered rbcL genes were expressed in Escherichia coli cells to conduct amino acids analysis of the protein variants. GC MS analysis of the partly purified insoluble RuBisCO protein from the E. coli cell lysate exhibited ~75% increase in methionine and ~65% increase in lysine content as compared to that of the wild type. In conclusion, we have successfully engineered and demonstrated the increased accumulation of essential amino acids in cyanobacteria by utilizing a protein as an amino acid sink.