Design and Assembly of a Biofactory for (2S)-Naringenin Production in Escherichia coli: Effects of Oxygen Transfer on Yield and Gene Expression

Abstract

The molecule (2S)-naringenin is a scaffold molecule with several nutraceutical properties. Currently, (2S)-naringenin is obtained through chemical synthesis and plant isolation. However, these methods have several drawbacks. Thus, heterologous biosynthesis has emerged as a viable alternative to its production. Recently, (2S)-naringenin production studies in Escherichia coli have used different tools to increase its yield up to 588 mg/L. In this study, we designed and assembled a bio-factory for (2S)-naringenin production. Firstly, we used several parametrized algorithms to identify the shortest pathway for producing (2S)-naringenin in E. coli, selecting the genes phenylalanine ammonia lipase (pal), 4-coumarate: CoA ligase (4cl), chalcone synthase (chs), and chalcone isomerase (chi) for the biosynthetic pathway. Then, we evaluated the effect of oxygen transfer on the production of (2S)-naringenin at flask (50 mL) and bench (4 L culture) scales. At the flask scale, the agitation rate varied between 50 rpm and 250 rpm. At the bench scale, the dissolved oxygen was kept constant at 5% DO (dissolved oxygen) and 40% DO, obtaining the highest (2S)-naringenin titer (3.11 ± 0.14 g/L). Using genome-scale modeling, gene expression analysis (RT-qPCR) of oxygen-sensitive genes was obtained.

Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution

“…Naringenin chalcone, synthesized by CHS, is unstable but can be converted efficiently in vivo to stable (2S)-naringenin by chalcone isomerase (CHI). 6,23 Therefore, overexpressing CHI may increase the (2S)-naringenin titer by immediately converting naringenin chalcone before it can degrade. The TtgR-based biosensor was used to screen CHS mutants for increased catalytic activity because of the high specificity, S/N ratio, and broad detection range of the biosensor for (2S)-naringenin.…”