Production of glutamate and stereospecific flavors, (S)-linalool and (+)-valencene, by Synechocystis sp. PCC6803

Abstract

Cyanobacteria can grow photoautotrophically, producing a range of substances by absorbing sunlight and utilizing carbon dioxide, and can potentially be used as industrial microbes that have minimal sugar requirements. To evaluate this potential, we explored the possibility of l-glutamate production using the Synechocystis sp. PCC6803. The ybjL gene encoding the putative l-glutamate exporter from Escherichia coli was introduced, and l-glutamate production reached 2.3 g/L in 143 h (34°C, 100 μmol m^-2 s^-1). Then, we attempted to produce two flavor substances, (S)-linalool, a monoterpene alcohol, and the sesquiterpene (+)-valencene. The Synechocystis sp. PCC6803 strain in which the linalool synthase gene (LINS) from Actinidia arguta (AaLINS) was expressed under control of the tac promoter (GT0846K-P_tac-AaLINS) produced 11.4 mg/L (S)-linalool in 160 h (30°C, 50 μmol m^-2 s^-1). The strain in which AaLINS2 and the mutated farnesyl diphosphate synthase gene ispA∗ (S80F) from E. coli (GT0846K-P_psbA2-AaLINS-ispA∗) were expressed from the P_psbA2 promoter accumulated 11.6 mg/L (S)-linalool in 160 h. Genome analysis revealed that both strains had mutations in slr1270, suggesting that loss of Slr1270 function was necessary for high linalool accumulation. For sesquiterpene production, the valencene synthase gene from Callitropsis nootkatensis and the fernesyl diphosphate synthase (ispA) gene from E. coli were introduced, and the resultant strain produced 9.6 mg/L of (+)-valencene in 166 h (30°C, 50 μmol m^-2 s^-1). This study highlights the production efficiency of engineered cyanobacteria, providing insight into potential industrial applications.

Keywords: (+)-Valencene; (S)-Linalool; Glutamate; Synechocystis sp.; TolC; Two-phase fermentation.