Hao Jingjing, Liao Haoqian, Meng Shuhong, Guo Yan, Zhu Li, Wang Hengliang, Lyu Yufei
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Beijing 100071, China.
Life (Basel). 2025 May 26;15(6):858. doi: 10.3390/life15060858.
and are major contributors to the global challenge of antimicrobial resistance, posing serious threats to public health. Among current preventive strategies, conjugate vaccines that utilize bacterial surface polysaccharides have emerged as a promising and effective approach to counter multidrug-resistant strains. In this study, both the Wzy/Wzx-dependent and ABC transporter-dependent biosynthetic pathways for antigenic polysaccharides were introduced into W3110 cells. This dual-pathway engineering enabled the simultaneous biosynthesis of two structurally distinct polysaccharides within a single host, offering a streamlined and potentially scalable strategy for vaccine development. Experimental findings confirmed that both polysaccharide types were successfully produced in the engineered strains, although co-expression levels were moderately reduced. A weak competitive interaction was noted during the initial phase of induction, which may be attributed to competition for membrane space or the shared use of activated monosaccharide precursors. Interestingly, despite a reduction in plasmid copy number and transcriptional activity of the biosynthetic gene clusters over time, the overall polysaccharide yield remained stable with prolonged induction. This suggests that extended induction does not adversely affect final product output. Additionally, two glycoproteins were efficiently generated through in vivo bioconjugation of the synthesized polysaccharides with carrier proteins, all within the same cellular environment. This one-cell production system simplifies the workflow and enhances the feasibility of generating complex glycoprotein vaccines. Whole-cell proteomic profiling followed by MFUZZ clustering and Gene Ontology analysis revealed that core biosynthetic genes were grouped into two functional clusters. These genes were predominantly localized to the cytoplasm and were enriched in pathways related to translation and protein binding. Such insights not only validate the engineered biosynthetic routes but also provide a molecular basis for optimizing future constructs. Collectively, this study presents a robust synthetic biology platform for the co-expression of multiple polysaccharides in a single bacterial host. The approach holds significant promise for the rational design and production of multivalent conjugate vaccines targeting drug-resistant pathogens.
[细菌名称1]和[细菌名称2]是全球抗菌药物耐药性挑战的主要促成因素，对公众健康构成严重威胁。在当前的预防策略中，利用细菌表面多糖的结合疫苗已成为应对多重耐药菌株的一种有前景且有效的方法。在本研究中，抗原性多糖的Wzy/Wzx依赖性和ABC转运蛋白依赖性生物合成途径均被引入W3110细胞。这种双途径工程使得在单个宿主内能够同时生物合成两种结构不同的多糖，为疫苗开发提供了一种简化且可能可扩展的策略。实验结果证实，尽管共表达水平有所适度降低，但两种多糖类型均在工程菌株中成功产生。在诱导初期观察到微弱的竞争相互作用，这可能归因于对膜空间的竞争或活化单糖前体的共享使用。有趣的是，尽管随着时间推移生物合成基因簇的质粒拷贝数和转录活性降低，但延长诱导后多糖的总产量仍保持稳定。这表明延长诱导不会对最终产物产量产生不利影响。此外，通过在同一细胞环境中体内合成多糖与载体蛋白的生物缀合，高效产生了两种糖蛋白。这种单细胞生产系统简化了工作流程，提高了生产复杂糖蛋白疫苗的可行性。通过全细胞蛋白质组分析，随后进行MFUZZ聚类和基因本体分析，发现核心生物合成基因被分为两个功能簇。这些基因主要定位于细胞质，并且在与翻译和蛋白质结合相关的途径中富集。这些见解不仅验证了工程化的生物合成途径，还为优化未来构建体提供了分子基础。总体而言，本研究提出了一个强大的合成生物学平台，用于在单个细菌宿主中共表达多种多糖。该方法对于合理设计和生产针对耐药病原体的多价结合疫苗具有重大前景。
