Economical Production of Phenazine-1-carboxylic Acid from Glycerol by Pseudomonas chlororaphis Using Cost-Effective Minimal Medium

Abstract

Phenazine compounds are widely used in agricultural control and the medicine industry due to their high inhibitory activity against pathogens and antitumor activity. The green and sustainable method of synthesizing phenazine compounds through microbial fermentation often requires a complex culture medium containing tryptone and yeast extract, and its cost is relatively high, which greatly limits the large-scale industrial production of phenazine compounds by fermentation. The aim of this study was to develop a cost-effective minimal medium for the efficient synthesis of phenazine compounds by Pseudomonas chlororaphis. Through testing the minimum medium commonly used by Pseudomonas, an ME medium for P. chlororaphis with a high production of phenazine compounds was obtained. Then, the components of the ME medium and the other medium were compared and replaced to verify the beneficial promoting effect of Fe²⁺ and NH₄⁺ on phenazine compounds. A cost-effective general defined medium (GDM) using glycerol as the sole carbon source was obtained by optimizing the composition of the ME medium. Using the GDM, the production of phenazine compounds by P. chlororaphis reached 1073.5 mg/L, which was 1.3 times that achieved using a complex medium, while the cost of the GDM was only 10% that of a complex medium (e.g., the KB medium). Finally, by engineering the glycerol metabolic pathway, the titer of phenazine-1-carboxylic acid reached the highest level achieved using a minimum medium so far. This work demonstrates how we systematically analyzed and optimized the composition of the medium and integrated a metabolic engineering method to obtain the most cost-effective fermentation strategy.

Keywords: Pseudomonas chlororaphis; fermentation optimization; genetic engineering; glycerol utilization; minimal medium; phenazine-1-carboxylic acid.

Figure 1

Comparison of fermentation performance of P. chlororaphis GP72-ANO (purple), P. chlororaphis LX24 (red), and P. chlororaphis HT66-H7 (green) in different minimal medium. (a) Phenazine compound titers of three strains in four minimal medium. (b) Cell growth (optical density at 600 nm, OD₆₀₀) of three strains in four minimal medium. The data represent the means ± SD for three independent cultures.

Figure 2

Effect of trace elements in ME medium on PCA titer. (a) Differences in trace elements of four minimal medium (ME, AB, MM, and M9). The purple area represents the components common to the trace elements of the ME, MM, and M9 medium. The areas with other colors represent components unique to the trace elements of each medium. (b) The PCA titer and cell growth (optical density at 600 nm, OD₆₀₀) of strain P. chlororaphis GP72-ANO in the AB; AB-ME (i.e., AB medium with the trace elements of the ME medium instead of the trace elements of the AB medium); MM; MM-ME M9; and M9-ME medium. (c) The PCA titer and cell growth (OD₆₀₀) of strain P. chlororaphis GP72-ANO in the ME, ME-MM, ME-M9, and ME-AB medium. ME-MM/M9/AB medium: ME medium with the trace elements of the MM/M9/AB medium instead of the trace elements of the ME medium. (d) The PCA titer and cell growth (OD₆₀₀) of strain P. chlororaphis GP72-ANO in the ME; ME(N) (i.e., ME medium without trace elements); ME(N)+CaCl₂ (i.e., ME(N) medium with the same CaCl₂ concentration as the ME medium); and ME(N)+FeSO₄ (i.e., ME(N) medium with the same FeSO₄ concentration as the ME medium) medium. The data represent the means ± SD for three independent cultures.

Figure 3

Effects of NH₄⁺ in ME medium. (a) Differences in NH₄⁺ concentration of four minimal medium (ME, AB, MM, and M9). The purple area represents the components common to the major elements of the ME, MM, and M9 medium. The areas with other colors represent components unique to the major elements of each medium. (b) The PCA titer and cell growth (optical density at 600 nm, OD₆₀₀) of P. chlororaphis GP72-ANO under different NH₄⁺ concentrations in the ME(N) + FeSO₄ (10 μM) medium. The data represent the means ± SD for three independent cultures.

Figure 4

Effects of the addition of different concentrations of Fe²⁺ (a) and Fe³⁺ (b) on PCA production and cell growth (optical density at 600 nm, OD₆₀₀) of P. chlororaphis GP72-ANO cultured in ME(N) medium. The data represent the means ± SD for three independent cultures.

Figure 5

(a) Functions of glycerol metabolism genes involved in modifications. (b) The PCA titer and cell growth (optical density at 600 nm, OD₆₀₀) in pure glycerol of engineered strains ANO, ANOE (strain ANO with empty plasmid), ANOF (strain ANO with glpF gene overexpression plasmid), ANOK (strain ANO with glpK gene overexpression plasmid), ANOFK (strain ANO with glpF and glpK gene co-overexpression plasmid), and ANOFK1 (strain ANOΔmgsA with glpF and glpK gene co-overexpression plasmid). (c) The differences in the PCA titer and cell growth (optical density at 600 nm, OD₆₀₀) in pure glycerol and crude glycerol of engineered strains ANO, ANOE, and ANOFK1. Abbreviations are as follows: DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; E4P, erythrose-4-phosphat. The data represent the means ± SD for three independent cultures. Note: ** p < 0.01, *** p < 0.001; N/S, not significant.