Ji Mengqi, Yao Buhan, Zhou Jingyu, Wang Yongzhong, Ding Qiang
School of Life Sciences, Anhui University, Hefei 230601, China.
Key Laboratory of Human Microenvironment and Precision Medicine of Anhui Higher Education Institutes, Anhui University, Hefei 230601, Anhui, China.
ACS Synth Biol. 2024 Dec 20;13(12):4180-4190. doi: 10.1021/acssynbio.4c00629. Epub 2024 Dec 4.
Microbial cell factories provide a nontoxic, economical way for the synthesis of various chemicals and drugs, garnering significant attention from researchers. However, excessive dispersion of enzymes and accumulation of intermediate metabolites in the production process will weaken the reaction efficiency of the pathway enzyme. In this study, a cellular compartment was constructed to isolate the enzyme reaction space and optimize the modular metabolic synthesis. First, a special spider silk protein was designed and constructed to form protein condensates in microbial cells, and its synthetic microcompartment effects were investigated. Second, the interaction of short peptide pairs or direct fusion based on the silk protein was used to recruit a variety of enzymes to improve the efficiency of enzyme catalysis. Third, the 2'-fucosyllactose (2'-FL) de novo synthesis pathway and its modular optimization were carried out to verify the mode. Finally, a synthetic compartment was introduced into the pathway to directly aggregate the 2'-FL synthesis pathway, thus obtaining synthetic-compartment-mediated multienzyme aggregates. The experimental results showed that the titer of 2'-FL was significantly improved compared with those of wild-type and modular-optimized free enzymes. The utilization of this cell microcompartment offers a novel avenue for the aggregation of diverse enzymes, thereby offering an innovative approach for enhancing the efficiency of the microbial modular metabolic pathway.
微生物细胞工厂为各种化学品和药物的合成提供了一种无毒、经济的方式，受到了研究人员的广泛关注。然而，生产过程中酶的过度分散和中间代谢物的积累会削弱途径酶的反应效率。在本研究中，构建了一个细胞区室来隔离酶反应空间并优化模块化代谢合成。首先，设计并构建了一种特殊的蜘蛛丝蛋白，使其在微生物细胞中形成蛋白质凝聚物，并研究其合成微区室效应。其次，基于丝蛋白利用短肽对的相互作用或直接融合来招募多种酶，以提高酶催化效率。第三，进行了2'-岩藻糖基乳糖（2'-FL）的从头合成途径及其模块化优化以验证该模式。最后，将一个合成区室引入该途径以直接聚集2'-FL合成途径，从而获得合成区室介导的多酶聚集体。实验结果表明，与野生型和模块化优化的游离酶相比，2'-FL的产量显著提高。这种细胞微区室的利用为多种酶的聚集提供了一条新途径，从而为提高微生物模块化代谢途径的效率提供了一种创新方法。
