A codon-based live-cell biomonitoring system for assessing intracellular phenylalanine bioavailability in cyanobacteria

Phenylalanine, as an essential aromatic amino acid, is not only needed for protein and vital molecules such as neurotransmitter and hormone synthesis but also a substrate for the biosynthesis of phenylpropanoids and various bioactive compounds. The metabolism of phenylalanine is dynamic and transitory, which would otherwise inhibit cell growth. Therefore, it is challenging and imperative to monitor intracellular phenylalanine bioavailability in real time, which has great significance for evaluating the effectiveness of introducing pathway-specific genetic modifications to enhance phenylalanine generation. In this study, we proposed a live-cell bio-monitoring system to assess phenylalanine bioavailability in real time in cyanobacteria based on codon de-generacy and species-specific usage bias. The biomonitoring system was generated through genetic modification of phenylalanine codons in the chloramphenicol antibiotic resistance gene to wholly preferred and rare codons, in combination with an orthogonal constitutive promoter Trc to express these genes. Cyanobacterial cells equipped with a preferred codon-based gene showed a significant growth advantage over those with rare codons under antibiotic pressure, while the delayed growth caused by rare codon-based genes could be rescued by supplementing phenylalanine in the cultivation medium. Increasing intracellular phenylalanine bioavailability could promote rare codon-based gene containing cell growth to a similar level as wild-type strains harboring preferred codon-based gene, providing a live-cell visualized screening method to relatively define phenylalanine content from either random mutation libraries or pathway-specific engineering cyanobacterial chassis before conducting labor-intensive quantitative measurements.

Automated summary

In this study, a live-cell bio-monitoring system was proposed to assess phenylalanine bioavailability in real time in cyanobacteria based on codon de-generacy and species-specific usage bias. Increasing intracellular phenylalanine bioavailability could promote cell growth and provide a screening method for phenylalanine content.