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

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.

Enhanced trans‐2,3‐dihydro‐3‐hydroxyanthranilic acid production by pH control and glycerol feeding strategies in engineered Pseudomonas chlororaphis GP72

“…In comparison with chemical‐based methods, microbial phenazine biosynthesis through biotechnological approaches has the economic advantages of using low‐cost substrate as carbon source. Besides ensuring a composition capable of satisfying microbial growth requirements, the substrate should be suitably conditioned in terms of pH and temperature for optimal efficiency . Several reports have explored the utilization of glucose as a carbon source to produce phenazine by different Pseudomonas strains.…”

“…Therefore, it gives the possibility to achieve high cell biomass together with elevated phenazine titer in a simple fermentation bioprocess. Importantly, it can considerably trim‐down the cost of DHHA production . Previously, Palmeri et al .…”

“…A non‐scar deletion method as reported earlier was followed to inactivate the target genes in strain GP72 . First, the upstream and downstream homologous arms ranging from 400–800 bp were amplified from the parent strain genomic DNA using two pairs of primers, rsmE ‐F1 ( Xba I)‐ rsmE ‐R1 and rsmE ‐F2‐ rsmE ‐R2 ( Eco RI).…”

“…Pseudomonas chlororaphis GP72 (hereafter, GP72) is a biocontrol strain which produces phenazine‐1‐carboxylic acid (PCA) and 2‐hydroxyphenazine (2‐OH‐PHZ) in high yields . Since GP72 ensures adequate metabolite flux in the phenazine biosynthesis pathway, higher production of DHHA may be achieved by this strain.…”

A decade of understanding secondary metabolism in Pseudomonas spp. for sustainable agriculture and pharmaceutical applications

“…P. putida X3 was engineered to bioremediate soil microcosms contaminated with methyl parathion and cadmium . Similarly, genetic engineering of P. chlororaphis GP72 enhanced the production of 2-hydroxyphenazine from 4.5 to 450.4 mg/L (Liu et al 2016). The same strain of P. chlororaphis was genetically manipulated for a reduced genome to observe an overall increase in the production of bioactive metabolites (Shen et al 2017).…”