Robert C. Byrd Biotechnology Science Center, Progenesis Technologies, LLC, One John Marshall Drive, Suite 314, Huntington, WV, 25755, USA.
Los Alamos National Laboratory, Biosecurity and Public Health, PO Box 1663 M888, Los Alamos, NM 87545, NM, USA.
Microb Biotechnol. 2020 Jan;13(1):162-175. doi: 10.1111/1751-7915.13411. Epub 2019 Apr 21.
Alginate is an important polysaccharide that is commonly used as a gelling agent in foods, cosmetics and healthcare products. Currently, all alginate used commercially is extracted from brown seaweed. However, with environmental changes such as increasing ocean temperature and the increasing number of biotechnological uses of alginates with specific properties, there is an emerging need for more reliable and customizable sources of alginate. An alternative to seaweed for alginate production is Pseudomonas aeruginosa, a common Gram-negative bacterium that can form alginate-containing biofilms. However, P. aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. Therefore, we sought to engineer a non-pathogenic P. aeruginosa strain that is safe for commercial production of alginate. Using a homologous recombination strategy, we sequentially deleted five key pathogenicity genes from the P. aeruginosa chromosome, resulting in the marker-free strain PGN5. Intraperitoneal injection of mice with PGN5 resulted in 0% mortality, while injection with wild-type P. aeruginosa resulted in 95% mortality, providing evidence that the systemic virulence of PGN5 is highly attenuated. Importantly, PGN5 produces large amounts of alginate in response to overexpression of MucE, an activator of alginate biosynthesis. The alginate produced by PGN5 is structurally identical to alginate produced by wild-type P. aeruginosa, indicating that the alginate biosynthetic pathway remains functional in this modified strain. The genetic versatility of P. aeruginosa will allow us to further engineer PGN5 to produce alginates with specific chemical compositions and physical properties to meet different industrial and biomedical needs.
藻酸盐是一种重要的多糖，通常用作食品、化妆品和保健产品中的胶凝剂。目前，商业上使用的所有藻酸盐都是从褐藻中提取的。然而，随着环境的变化，如海洋温度升高以及对具有特定性质的藻酸盐的生物技术应用的增加，对更可靠和可定制的藻酸盐来源的需求日益增加。藻酸盐生产的海藻替代品是铜绿假单胞菌，一种常见的革兰氏阴性菌，它可以形成含有藻酸盐的生物膜。然而，铜绿假单胞菌是一种机会性病原体，会导致免疫功能低下的患者发生危及生命的感染。因此，我们试图构建一种非致病性的铜绿假单胞菌菌株，用于商业生产藻酸盐。我们使用同源重组策略，从铜绿假单胞菌染色体上依次缺失了五个关键的致病性基因，得到了无标记的 PGN5 菌株。将 PGN5 菌株腹腔注射到小鼠体内，小鼠的死亡率为 0%，而野生型铜绿假单胞菌的死亡率为 95%，这证明了 PGN5 的全身毒力高度减弱。重要的是，PGN5 在过量表达 MucE 时会大量产生藻酸盐，MucE 是藻酸盐生物合成的激活剂。PGN5 产生的藻酸盐与野生型铜绿假单胞菌产生的藻酸盐在结构上完全相同，表明该修饰菌株的藻酸盐生物合成途径仍然具有功能。铜绿假单胞菌的遗传多样性将使我们能够进一步工程化 PGN5 菌株，以生产具有特定化学组成和物理性质的藻酸盐，以满足不同的工业和生物医学需求。
