Systems metabolic engineering of a plasmid-free Escherichia coli cell factory for high-level cytidine production.

Authors

Runze Zhao, Changgeng Li, Chaoye Xu, Zhichao Chen, Haibo Xiong, Zichen Yu, Tang'en Shi, Lanxiao Li, Meng Yuan, Ling Ma, Siyu Zhao, Hongyu Deng, Qingyang Xu

Published in

Synthetic and systems biotechnology. Volume 19. Pages 21-32. Epub Sep 22, 2026.

Abstract

Cytidine is an important precursor for nucleoside pharmaceuticals, but its fermentative production is constrained by competing degradation, poor selectivity of pyrimidine flux, insufficient precursor supply, and energy-redox imbalance. In this study, a plasmid-free E. coli cell factory was constructed through systems metabolic engineering. Sequential deletion of cytidine degradation, salvage, and precursor-consuming pathways enabled initial cytidine accumulation. The conversion from UMP to CMP was strengthened by introducing feedback-deregulated pyrH ^D93A and pyrG ^{D160E, E162A, E168K} _cgl mutants and nudG, while the heterologous expression of PHM8 _sce improved CMP-to-cytidine conversion selectivity. Scaffold-based assembly of PyrF, PyrH^D93A, NudG, and PHM8_sce using CipA/CipB further improved local pathway channeling and reduced uridine formation by 98.70%. To reinforce upstream flux, de novo UMP biosynthesis was reconstructed with the B. subtilis pyr operon, PRPP synthesis was enhanced, and nitrogen-donor and pyrimidine-ring precursor supply was improved through glnA, ppc, and aspC expression. Finally, an ATP-responsive ydaO motif regulatory module was used to dynamically control respiratory-chain and oxygen-utilization modules, coordinating ATP supply with NAD⁺ regeneration. The final strain cyt 23 produced 96.20 g/L cytidine in 50 h in a 5 L fed-batch bioreactor, with a glucose conversion yield of 0.38 g/g and productivity of 1.92 g/(L·h), representing the highest reported titer, yield and productivity for microbial cytidine production to date. This work provides an integrated strategy for high-level microbial cytidine production and nucleoside cell-factory development.

PMID:
42861367
Bibliographic data and abstract were imported from PubMed on 12 Oct 2026.

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