基于代谢工程改造的链霉菌透明质酸合成体系构建

Metabolic engineering of Streptomyces for production of hyaluronic acid

摘要:

透明质酸是一种具有重要生物活性的糖胺聚糖，在化妆品和医药领域具有广泛应用。开发安全高效的底盘细胞以提升透明质酸的合成效率，已成为透明质酸生产的关键。本研究旨在构建以链霉菌为底盘细胞的高性能透明质酸合成体系，为透明质酸的微生物高效合成提供技术支撑。本研究主要聚焦链霉菌的代谢工程改造，通过强化透明质酸合成通路，结合培养条件的优化，最终实现透明质酸的高效合成。首先，在天蓝色链霉菌和小白链霉菌这2种宿主中，分别测试了4种不同来源的透明质酸合成酶的合成能力。结果表明：在这2种链霉菌中，源自化脓性链球菌的透明质酸合成酶的合成能力最强，且在小白链霉菌J1074中产量更高。随后，以小白链霉菌J1074为出发菌株，通过敲除透明质酸合成竞争代谢途径的关键基因，同时下调旁路途径相关基因的表达水平，构建了适用于透明质酸合成的底盘细胞。此外，对透明质酸前体合成途径中的关键基因进行了不同组合，并利用组成型强启动子强化其表达水平，获得的重组菌株的透明质酸产量最高达到2.62 g/L。最后，通过优化培养条件进一步释放工程菌株的合成能力，最终透明质酸产量提升至4.63 g/L。本研究构建的重组菌株，不仅为工程链霉菌的开发奠定了基础，也为透明质酸及类似生物产品的发酵制备提供了优异的底盘菌株。

Abstract:

Hyaluronic acid (HA) is a glycosaminoglycan with significant biological activities, which render it widely applicable in the cosmetics and pharmaceutical industries. The development of safe and efficient chassis cells to enhance HA synthesis efficiency has thus emerged as a key factor in HA production. Our study aims to construct a high-performance HA biosynthesis system using Streptomyces as the chassis cell, thereby providing technical support for the efficient microbial production of HA. Thus, our study focused on the metabolic engineering of Streptomyces for strengthening the HA synthesis pathway and then optimized the culture conditions for efficient HA synthesis. First, the HA-synthesizing capabilities of four hyaluronate synthases from different sources were evaluated in two host strains: Streptomyces coelicolor M1146 and Streptomyces albus J1074. The results indicated that the hyaluronate synthases derived from Streptococcus pyogenes exhibited the strongest HA synthesis capability. Notably, the HA yield in S. albus J1074 was higher. Building on this finding, S. albus J1074 was selected as the starting strain to construct a chassis strain tailored for HA synthesis: key genes in the competitive metabolic pathway of HA synthesis were knocked out, while the expression levels of genes associated with the bypass pathway were down-regulated. Furthermore, different combinations of key genes involved in the HA precursor synthesis pathway were designed, and their expression levels were enhanced via a constitutive strong promoter. The recombinant strain obtained therefrom achieved a maximum HA yield of 2.62 g/L. Finally, the synthetic capacity of this high-yield engineered strain was further unleased through the optimization of culture conditions, leading to a final HA yield of 4.63 g/L. The recombinant strain constructed in this study not only lays a foundation for the development of engineered Streptomyces but also provides an excellent chassis strain for the microbial production of HA and other related bioproducts.