(498b) De Novo Biosynthesis of the Non-Standard Amino Acid Para-Nitrophenylalanine
AIChE Annual Meeting
2022
2022 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Engineering bacteria for novel chemistry and interactions
Wednesday, November 16, 2022 - 12:48pm to 1:06pm
Initially, we constructed a de novo biosynthesis pathway for pN-Phe based upon a literature identified amine oxidizing diiron monooxygenase enzyme, achieving a modest 24h titer of ~150 µM in M9 minimal media. We then performed genome engineering to increase the flux to the pN-Phe pathway, leading to improved 24h titers of ~250 µM in M9 minimal media. However, because the metabolic intermediate pA-Phe was the dominant heterologous product, we further screened a range of putative natural diiron monooxygenases. By expressing one of these diiron monooxygenases within our pathway, we increased pN-Phe titers to 330 µM, which was a 2-fold improvement over our starting titers. By fermenting this strain using a MOPS EZ Rich glucose media, we were able to obtain even higher titers of 820 µM. Given these near millimolar levels of pNPhe obtained from this biosynthetic pathway, we looked to employ strategies to achieve paired biosynthesis and site-specific incorporation of pN-Phe within target proteins. Through combination of these technologies within a strain, it would enable autonomous utilization of pN-Phe within proteins and create an E. coli chassis with an expanded 21 amino acid genetic code.