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

Abstract

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.

Figure 1

Confirmation of deletion of five gene sequences in P. aeruginosa strain PGN5. A–E. Sanger sequencing results of each gene deletion in the PGN5 strain. Panels A, B and E show plus strand sequence obtained. Panels C and D show minus strand sequence obtained. Underlined sequence is upstream of the start codon of each gene. Bold sequence is downstream of the stop codon of each gene. Location of deleted sequence is shown by red bracket. F. Image of agarose gel electrophoresis from PCR amplification of deleted gene regions. Odd lanes are PCR products from P. aeruginosa strain PAO1, and even lanes are PCR products from strain PGN5.

Figure 2

Confirmation of loss of deleted gene products in P. aeruginosa strain PGN5. A. Western blot analysis of Exotoxin A from the concentrated extracellular fraction of Exotoxin A‐positive P. aeruginosa PA103, wild‐type P. aeruginosa PAO1 and P. aeruginosa PGN5. PIB was used as a negative control. B. Haemolysis on blood agar observed with P. aeruginosa strains PAO1 and VE2, but not PGN5 or E. coli strain BL21. Areas of β‐haemolysis shown with white arrows. C. Pyocyanin concentrations detected in PAO1 and PGN5 media. D. Silver‐stained SDS‐PAGE (left) and Western blot analysis (right) against O‐antigen on cell lysates of P. aeruginosa strains PAO1_wbpL, PAO1, VE2 and PGN4. O‐antigen regions indicated by red brackets. Antibodies used are indicated. E. ELISA of cell lysates of PAO1 and PGN5 for 3‐phosphoshikimate 1‐carboxyvinyltransferase (AroA). ***P < 0.001, determined by two‐tailed Student's t‐test.

Figure 3

Phenotype and alginate characterization of P. aeruginosa strain PGN5. A. Non‐mucoid phenotype of P. aeruginosa strains PAO1 and PGN5 on PIA. B. Mucoid phenotype of PGN5+mucE and VE2 on PIAGm300. C. Uronic acid (carbazole) assay was used to measure alginate production on VE2 and PGN5+mucE over 72 h. Mean ± SD shown. D. Overlay of HPLC chromatograms of alginate prepared from a seaweed alginate control (red), and P. aeruginosa strains VE2 (blue) and>PGN5+mucE (green). Only area of interest is shown. Determined M:G ratios shown in table inlay. X‐axis = retention time; Y‐axis = absorbance units. E. Measured viscosity of 2% sodium alginate gels prepared from seaweed alginate samples Sigma 2158 and Sigma 180947, and alginate produced by VE2 and PGN5+mucE. Mean ± SD shown. F. Measured shear stress/shear rate measured from 2% sodium alginate gels prepared from seaweed alginate samples Sigma 2158 and Sigma 180947, and alginate produced by VE2 and PGN5+mucE. Mean ± SD shown.

Figure 4

Per cent survival of male and female C57BL/6 mice after injections with P. aeruginosa strains VE2, PGN5+mucE or E. coli BL21. A. Per cent survival of male mice n = 10. B. Per cent survival of female mice, n = 10. C. Per cent survival of combined survival of both male and female, n = 20.

Figure 5

Images of mice injected with bioluminescent‐labelled P. aeruginosa strains. A,B. Mice injected with bioluminescent PAO1+mucE at A. 0 h post‐injection and B. 9 h post‐injection (n = 5). C,D. Mice injected with bioluminescent PGN5+mucE at C. 0 h post‐injection and D. 9 h post‐injection (n = 5). Note: different mice were imaged at each time point to avoid overdose of anaesthetic.