Generation of a highly attenuated strain of Pseudomonas aeruginosa for commercial production of alginate

Abstract

SummaryAlginate 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.

Bacterial biopolymers: from pathogenesis to advanced materials

“…Also, it is clear that development of safe bacterial cell factories (for example, novel endotoxinfree and non pathogenic strains) through synthetic biology and bio engineering as well as efficient purification methods can lead to a plethora of new polymers and highvalue biomaterials. Successful examples include generation of endotoxinfree E. coli ClearColi 150 and the commercial use of highly attenuated P. aeruginosa PGN5 for produc tion of alginate 152 and nonpyogenic S. zooepidemicus for production of hyaluronate, which was purified through extensive filtration and diverse adsorbents to eliminate impurities 153 (TAblE 1).…”

Whole Genome Analysis of Environmental Pseudomonas mendocina Strains: Virulence Mechanisms and Phylogeny

“…However, the missing genes responsible for the core minor pili subunits for Type IV pili (T4P) could avoid the attachment of these strains to host epithelial cells [ 58 , 59 , 60 ]. It is the same for the missing mucE gene, an activator of alginate biosynthesis, which could explain, joined to other mechanisms, the non-mucoid phenotype observed in the P. mendocina strains [ 61 ]. Regarding the secretion pathway H1-T6SS, whose complete structure was only present in four genomes, including the Ps799, it can act as an antibacterial weapon, giving the bacteria the capacity to inject different effectors to compete with other bacterial cells for the same ecological niche, even the environmental strains [ 62 ].…”

Industrial biotechnology of Pseudomonas putida: advances and prospects

“…Alginate is a common additive to cosmetics and foods and has many medical applications. However, up to now, alginate production has been mainly studied in the pathogenic P. aeruginosa and there has been no commercial application associated to these efforts (Liu et al 2019 ; Valentine et al 2020 ).…”