Genetic engineering of Pseudomonas chlororaphis GP72 for the enhanced production of 2-Hydroxyphenazine

Abstract

Background: 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). 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.

Results: Different 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.

Conclusions: The 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.

Keywords: 2-Hydroxyphenazine; Non-scar deletion; Overexpression; Phenazine-1-carboxylic acid; Pseudomonas chlororaphis GP72.

Fig. 1

Genes selected from the gluconeogenesis, pentose phosphate and shikimate pathway to increase the phenazine yield. DHAP, dihydroxyacetone phosphate; Gly3P, Glycerol 3-phosphate; G6P, glucose 6-phosphate; F16BP, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; F6P, fructose 6-phosphate; 6PGNL, 6-phosphogluconolactone; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xu5P, xylulose 5-phosphate; PEP, phosphoenolpyruvate; E4P, erythrose 4-phosphate; ACoA, acetyl-coenzyme A; PYR, pyruvate; OAA, oxaloacetate; CIT, citrate; DHQ, 3-dehydroquinic acid; DAHP, 3-deoxy-Darabinoheptulosonate7-phosphate; QA, quinic acid; DHS, 3-dehydroshikimic acid; SA, shikimic acid; GA, gallic acid; PCA, phenazine-1-carboxylic acid; CHO, chorismate; 2-OH-PCA 2-hydroxy-phenazine-1-carboxylic acid. Gene coding for enzymes not named in the figure: pgi phosphoglucose isomerase; glk glucokinase; eno enolase; gapA glyceraldehyde 3-phosphate dehydrogenase; glpK glycerol kinase; glpF glycerol facilitator; fda fructose-1,6-diphosphate aldolase; glpD glycerol-3-P dehydrogenase; fbp fructose 1,6-bisphosphatase; tpiA triosephosphate isomerase; talB transaldolase; zwf G6P dehydrogenase; pck PEP carboxykinase; ppc PEP carboxylase; pgm phosphoglyceromutase; pgk phosphoglycerate kinase

Fig. 2

Growth curves, 2-OH-PHZ and PHZ production of the GP72 mutant derivative strains. a 2-OH-PHZ production. b Phenazine production. c Growth curves. The error bars indicate standard deviations from triplicate experiments

Fig. 3

A summary of steps in the genetic and metabolic engineering of GP72 for 2-OH-PHZ production

Fig. 4

2-OH-PHZ and PHZ production in the mutimutant strain GP72-ND3 with different over-expressing plasmids. a 2-OH-PHZ production of different single genes overexpressing in GP72ND-3. b PHZ production of different single genes overexpressing in GP72ND-3. c 2-OH-PHZ production of different multiple genes overexpressing in GP72ND-3. d PHZ production of different multiple genes overexpressing in GP72ND-3. The error bars indicate standard deviations from triplicate experiments

Fig. 5

Proposed model for the regulation of phenazine biosynthesis by the TCST system in GP72. Solid straight arrows point to genes that are positively regulated. Blunt lines point to genes that are negatively affected. A dashed arrow indicates an unknown or as-yet uncharacterized regulatory pathway. In GP72, the sensors GacS and RpeB are activated by a putative environmental factor. Lon protease negatively affects GacA by controlling its protein stability. GacA positively controls the expression of rsmX, rsmY, and rsmZ, which in turn activates phenazine production by titrating the translation suppressor RsmE. In the absence of RpeA, RpeB is possibly over-phosphorylated by small phospho-donors (PD), resulting in the increased expression of the pip, phzR/phzI and the phenazine biosynthetic genes

Fig. 6

The BglBrick plasmid of pBbB5K-aroE-aroD-aroB-phzC-tktA-ppsA, which was overexpressed to enhance the production of 2-OH-PHZ. The gene order in the plasmid was aroE, aroD, aroB, phzC, tktA and ppsA, which is a reverse of the expression pathway