Abstract
BackgroundThe biocontrol strain Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere synthesizes three antifungal phenazine compounds, 2-Hydroxyphenazine (2-OH-PHZ), 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) and phenazine-1-carboxylic acid (PCA). PCA has been a commercialized antifungal pesticide registered as “Shenqinmycin” in China since 2011. It is found that 2-OH-PHZ shows stronger fungistatic and bacteriostatic activity to some pathogens than PCA. 2-OH-PHZ could be developed as a potential antifungal pesticide. But the yield of 2-OH-PHZ generally is quite low, such as P. chlororaphis GP72, the production of 2-OH-PHZ by the wide-type strain is only 4.5 mg/L, it is necessary to enhance the yield of 2-OH-PHZ for its application in agriculture.ResultsDifferent strategies were used to improve the yield of 2-OH-PHZ: knocking out the negative regulatory genes, enhancing the shikimate pathway, deleting the competing pathways of 2-OH-PHZ synthesis based on chorismate, and improving the activity of PhzO which catalyzes the conversion of PCA to 2-OH-PHZ, although the last two strategies did not give us satisfactory results. In this study, four negative regulatory genes (pykF, rpeA, rsmE and lon) were firstly knocked out of the strain GP72 genome stepwise. The yield of 2-OH-PHZ improved more than 60 folds and increased from 4.5 to about 300 mg/L. Then six key genes (ppsA, tktA, phzC, aroB, aroD and aroE) selected from the gluconeogenesis, pentose phosphate and shikimate pathways which used to enhance the shikimate pathway were overexpressed to improve the production of 2-OH-PHZ. At last a genetically engineered strain that increased the 2-OH-PHZ production by 99-fold to 450.4 mg/L was obtained.ConclusionsThe 2-OH-PHZ production of P. chlororaphis GP72 was greatly improved through disruption of four negative regulatory genes and overexpression of six key genes, and it is shown that P. chlororaphis GP72 could be modified as a potential cell factory to produce 2-OH-PHZ and other phenazine biopesticides by genetic and metabolic engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0529-0) contains supplementary material, which is available to authorized users.
Highlights
The biocontrol strain Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere synthesizes three antifungal phenazine compounds, 2-Hydroxyphenazine (2-OH-PHZ), 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) and phenazine-1-carboxylic acid (PCA)
Disruption of four negative regulatory genes to enhance 2‐OH‐PHZ production In order to obtain a high yield of 2-OH-PHZ, we firstly chose to inactivate the gene pykF of GP72, and obtained the mutant strain GP72Δpyk (Figs. 2, 3)
The strain GP72Δpyk was constructed with the non-scar deletion method (Additional file 1: Fig. S1)
Summary
The biocontrol strain Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere synthesizes three antifungal phenazine compounds, 2-Hydroxyphenazine (2-OH-PHZ), 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) and phenazine-1-carboxylic acid (PCA). The biocontrol strain Pseudomonas chlororaphis GP72, isolated from the green pepper rhizosphere, has broadspectrum antifungal activity against many agricultural phytopathogens [1, 2]. This capability depends primarily on the following three phenazine compounds: 2-Hydroxyphenazine (2-OH-PHZ), 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) and phenazine1-carboxylic acid (PCA) [1, 3]. PCA is an effective antifungal pesticide and was registered as “Shenqinmycin” in 2011 by the Ministry of Agriculture of China as a biologically synthesized fungicide, which. P. chlororaphis GP72 is a 2-OH-PHZ producing strain, the production by the wild type is only 4.5 mg/L [1], it is possible to improve the 2-OH-PHZ production largely for its industrial production by genetic engineering and regulation
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