Recently, the team of Ma Qinyuan and Gao Xiuzhen from the College, based on the Synthetic Biology and Engineering Biotechnology Institute jointly established by Shandong University of Technology and Jin Cheng Pharmaceutical, has made another significant breakthrough in the field of the biological synthesis of the neuroprotective agent Citicoline (CDP-choline).
The related research results were published in the international top journal Chemical Engineering Journal (Impact Factor 13.2) under the title "AI-guided evolution of membrane-associated cytidylyltransferase and systems metabolic engineering enable high-titer Citicoline biosynthesis".
Citicoline has clinical application value in maintaining the integrity of cell membranes and in nerve repair, among other areas.
Synthesizing Citicoline using microbial cell factories such as Bacillus subtilis represents a promising green manufacturing approach.
However, the core rate-limiting enzyme in this synthetic pathway - phosphocholine cytidine transferase (CCT) - has the problem of slow kinetics, and due to its nature as a complex membrane-bound protein and the lack of high-quality crystal structures, traditional enzyme engineering modifications based on structure pose challenges.
To address this issue, the research team developed a comprehensive strategy that combines physical information assistance and evolutionary guidance.
The team first used multi-dimensional screening and deep learning tools to identify candidate enzymes MsCCT with good chassis compatibility.
Subsequently, a multi-view integrated protein language model (PLM) was introduced, which overcame the limitation of lacking structural templates and evolved a mutant MsCCTM3 with an 2.4-fold increase in activity.
After introducing this enzyme into the previously optimized Bacillus subtilis strain, multi-omics analysis revealed that the metabolic bottleneck had shifted to the upstream precursor supply.
Ultimately, the production of this engineered strain reached 5.65 g/L in a 5 L fermentation tank, with a 34.7% increase in space-time yield.
This work not only provides an efficient strain for the industrialization of Citicoline, but also offers a data-driven new approach for the modification of complex membrane proteins.
This research was supported by the Major Innovation and Technology Development Project of Shandong Province (2025CXGC010608).
Zhang Xinran, a 2023 master's degree student, and Yang Shaomei, a young teacher from the School of Life and Medicine of Shandong University of Technology, are the co-first authors of this paper.
Professor Ma Qinyuan and Professor Gao Xiuzhen from the School of Life and Medicine of Shandong University of Technology are the co-corresponding authors of this paper.
Shandong University of Technology is the sole corresponding unit.
Article DOI: https://doi.org/10.1016/j.cej.2026.178324