State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian, 223003, China.
World J Microbiol Biotechnol. 2018 Aug 9;34(9):129. doi: 10.1007/s11274-018-2501-0.
Phenazine-1-carboxamide (PCN) is one of the major biocontrol agents produced by plant growth-promoting rhizosphere (PGPR) pseudomonads including Pseudomonas chlororaphis. In this study, a combined strategy of genetic modification and statistical experimental designs was applied to obtain mutants of P. chlororaphis strains with high-yield PCN production. To achieve this, the lon gene was knocked out in wild-type P. chlororaphis HT66 and the breeding mutant P3 strain with a non-scar deletion strategy. The resulting HT66Δlon and P3Δlon mutants produced a significantly higher PCN production in shake-flask cultures which was 5- and 9-folds greater than their native counterparts. The potential ability of strain P3Δlon for PCN production was further optimized by statistical designs. A two-level Plackett-Burman (PB) experimental design with six variables was employed to scrutinize medium components that significantly influence PCN production. Notably, glycerol, tryptone, and soy peptone were identified to be the most significant factors (p < 0.05). Response surface methodology (RSM) based on the central composite design (CCD) was adopted to determine these factors optimal levels and their interactive effects between culture components for PCN production. The predicted maximum PCN production was 9002 mg/L, whereas an actual PCN production of 9174 mg/L was recorded in the validation experiments using the optimal medium containing glycerol 37.08 mL/L, tryptone 20.00 g/L, and soy peptone 25.03 g/L, which was nearly threefolds higher than without optimization and 20-folds higher than the wild-type strain. In conclusion, the results revealed that P. chlororaphis display a high potential for industrial-scale production for phenazine biopesticides.
苯并吡嗪-1-甲酰胺（PCN）是植物促生根际（PGPR）假单胞菌产生的主要生物防治剂之一，包括绿脓假单胞菌。在这项研究中，采用遗传修饰和统计实验设计相结合的策略，获得了高产 PCN 的绿脓假单胞菌菌株突变体。为此，采用非粘性缺失策略敲除野生型绿脓假单胞菌 HT66 中的 lon 基因，并培育出突变株 P3。摇瓶培养中，HT66Δlon 和 P3Δlon 突变株的 PCN 产量明显提高，分别比其天然菌株高出 5 倍和 9 倍。进一步采用统计设计优化菌株 P3Δlon 的 PCN 生产能力。采用六变量两级 Plackett-Burman（PB）实验设计来仔细研究对 PCN 生产有显著影响的培养基成分。值得注意的是，甘油、胰蛋白胨和大豆蛋白胨被鉴定为最重要的因素（p<0.05）。采用基于中心复合设计（CCD）的响应面法（RSM）来确定这些因素的最佳水平及其对培养物成分之间的相互作用，以提高 PCN 的产量。预测的最大 PCN 产量为 9002mg/L，而在验证实验中，使用含有 37.08mL/L 甘油、20.00g/L 胰蛋白胨和 25.03g/L 大豆蛋白胨的最佳培养基，实际 PCN 产量为 9174mg/L，这比未优化时提高了近 3 倍，比野生型菌株提高了 20 倍。总之，结果表明，绿脓假单胞菌具有很高的工业生产潜力，可以生产苯并吡嗪生物农药。
