title: 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.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Kaiquan
authors.initials: K
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China. xuehzhang@sjtu.edu.cn.
authors.forename: Xuehong
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000935:Antifungal Agents / Q000378:metabolism* / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005818:Genetic Engineering; D010619:Phenazines / Q000378:metabolism / Q000494:pharmacology; D000070096:Pseudomonas chlororaphis / Q000235:genetics* / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D010619:Phenazines; C088013:2-hydroxyphenazine
keywords: 2-Hydroxyphenazine; Non-scar deletion; Overexpression; Phenazine-1-carboxylic acid; Pseudomonas chlororaphis GP72