Modularized reconstruction of pyrimidine pathway in Escherichia coli for enhanced pyrimidine nucleosides biosynthesis

Pyrimidine is a fundamental component of genetic material and a precursor for synthesis of various antiviral and antitumor drugs. In this study, we constructed a plasmid-free Escherichia coli strain HD11 as a platform for cytidine production. Compared to strain CR23-p21-p31, a higher titer of cytidine was achieved by overexpressing uridylate kinase from Sulfurovum lithotrophicum and the newly discovered cytidine triphosphate synthetase of Aquifex aeolicus in HD11. To enable efficient cytosine biosynthesis, a modularized metabolic pathway was integrated into HD11. Two different pathways based on the type of precursor were compared, and ribosome binding sites were optimized to enhance cytosine production. For pyrimidine ribonucleotide de novo biosynthesis pathway, an enzyme constrained metabolic model was utilized to guide media optimization. Finally, HD11pDA20 produced 3.6 g/L cytosine and HD11 produced 8.1 g/L cytidine in fed-batch fermentation. The application of an enzyme-constrained metabolic model in cytidine/cytosine producing strain represents a novel approach to modular engineering.