Genetic engineering of Pseudomonas chlororaphis GP72 for the enhanced production of 2-Hydroxyphenazine.
Genetic engineering of Pseudomonas chlororaphis GP72 for the enhanced production of 2-Hydroxyphenazine.
复制标题
用于提高 2-羟基吩嗪产量的绿针假单胞菌 GP72 的基因工程
DOI：
10.1186/s12934-016-0529-0
复制
发表时间：
2016-07-28
影响因子：
6.4
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Liu K;Hu H;Wang W;Zhang X
背景从绿色辣椒根际分离的生防菌绿针假单胞菌（Pseudomonashlororaphis）GP 72能合成3种抗真菌吩嗪类化合物：2-羟基吩嗪（2-OH-PHZ）、2-羟基吩嗪-1-羧酸（2-OH-PCA）和吩嗪-1-羧酸（PCA）。五氯苯甲醚是一种商业化的抗真菌农药，自2011年起在中国注册为“申秦霉素”。结果表明，2-OH-PHZ对某些病原菌的抑菌活性比PCA强。2-OH-PHZ可作为一种潜在的抗真菌农药开发。但2-OH-PHZ的产量普遍较低，如绿针假单胞菌GP 72，其野生型菌株2-OH-PHZ的产量仅为4.5mg/L，因此，提高2-OH-PHZ的产量是其在农业上应用的必要条件。敲除负调控基因，增强莽草酸途径，删除基于分支酸的2-OH-PHZ合成竞争途径，提高催化PCA转化为2-OH-PHZ的PhzO活性，虽然后两种策略并没有给我们带来令人满意的结果。本研究首次从菌株GP 72基因组中逐步敲除了4个负调控基因（pykF、rpeA、rsmE和lon）。2-OH-PHZ的产量提高了60倍以上，从4.5 mg/L提高到300 mg/L左右。在此基础上，选择了莽草酸合成途径、戊糖磷酸途径和莽草酸途径中的6个关键基因ppsA、tktA、phzC、aroB、aroD和aroE，通过过量表达来提高2-OH-PHZ的产量。结论通过对4个负调控基因的敲除和6个关键基因的过表达，使绿针假单胞菌GP 72的2-OH-PHZ产量提高了99倍，达到450.4mg/L。通过基因工程和代谢工程改造绿针假单胞菌GP 72，可作为生产2-OH-PHZ等吩嗪类生物农药的潜在细胞工厂。
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.