(113d) Engineering of Fast Growing Cyanobacteria Synechococcus Elongatus sp. PCC 11801 for Phenylalanine Production

Cyanobacteria as microbial factories are being increasingly explored due to their ability to directly convert CO₂ to useful bio-chemicals and biofuels. However, initial engineering efforts have resulted in low productivity compared to heterotrophs primarily due to slow growth. Recently, a robust fast-growing strain resistant to abiotic stresses, Synechococcus elongatus sp. PCC 11801 (hereafter PCC 11801) was discovered with growth rates comparable to yeasts. We engineered PCC 11801 for production of the valuable amino acid phenylalanine (Phe). Due to the limited availability of synthetic biology tools, random mutagenesis coupled with amino acid analog selection is an attractive option for the development of Phe overproducers. In this work, we use ultraviolet (UV) irradiation or chemical as mutagens followed by selection on 3-(2-thienyl)-DL-alanine. The mutants resistant to the analog were characterized for Phe production to identify the best producer M14, which can accumulate 554 ± 34 mg/L in 3 days. Genes coding putative bottleneck enzymes in the shikimate pathway were sequenced to identify single nucleotide polymorphisms that could be responsible for the Phe overproducing phenotypes. A second round of UV mutagenesis and selection resulted in the isolation of M14.2, which can produce 1.2 ± 0.1 g/L of Phe in 3 days. Additionally, mutants show no reduction in growth but achieve Phe overproduction by fixing carbon at a higher rate. This work highlights the advantages of using fast growing cyanobacteria as systems for biochemical production and provides a starting point for the development of strains to produce Phe as a feed supplement.