构建假单胞菌突变株m18gq携带重组质粒pme6032phz的工程菌株及响应面法优化吩嗪-1-羧酸发酵条件的研究-construction of pseudomonas mutant m18gq engineering strain carrying recombinant plasmid pme 6032 phz and optimization of phenazine - 1 - carboxylic acid fermentatio.docxVIP

第 第 PAGE IV 页 第 第 III 页 design, CCD）的响应面方法（response surface methodology, RSM），研究 了这些因子的相互作用关系，并确定了最优浓度。优化后的发酵培养基 配方为黄豆粉 65.02 g/l，玉米浆 15.36 g/l，葡萄糖 12 g/l，乙醇 21.70 ml/l， MgSO4 1 g/l。在此发酵培养基中，IPTG 在接种后 24 小时以终浓度 1.0 mmol/l 进行诱导，经 60 小时发酵，PCA 预测产量为 6335.2 mg/l。经实 验验证，PCA 产量达到 6365.0 mg/l，与预测结果吻合，比 M18GQ 菌株 在优化发酵培养基中的产量提高了约 60%，是 M18 野生型在基础培养基 中产量的 60 倍。 关键词：吩嗪-1-羧酸，过表达，假单胞菌株 M18GQ，响应面方法，优化 Construction of A genetically modified strain of Pseudomonas sp. M18GQ harboring pME6032Phz and optimization of phenazine-1- carboxylic acid production using response surface methodology ABSTRACT Pseudomonas sp. stain M18, a new kind of plant growth-promoting rhizobacteria isolated from the melon roots in the suburb of Shanghai, would be a promising strain as a powerful and effective bio-control agent due to its secondary metabolite phenazine-1-carboxylic acid (PCA), which has a broad-spectrum antifungal activity. PCA production was enhanced in Pseudomonas sp. M18 wild strain and its mutants carrying recombinant pME6032Phz for phz gene cluster overexpression, among which Pseudomonas sp. strain M18GQ/pME6032Phz, a gacA and qscR double gene chromosomally inactivated mutant harboring pME6032Phz, showed the highest PCA yield. The conditions for fermentation and isopropyl-β-D-1-thio- galactopyranoside (IPTG) induction were optimized for strain M18GQ/pME6032Phz in shake flask experiments. A one-factor-at-a-time approach, followed by a fractional factorial design (FFD) identified soybean meal, corn steep liquor, and ethanol as statistically significant factors. Optimal concentrations and mutual interactions of the factors were then determined by the method of steepest ascent and by response surface methodology (RSM) based on the center composite design (CCD). The predicted PCA production was 6335.2 mg/l after 60 hour fermentation in the optimal medium of 65.02 g soybean meal, 15.36 g corn steep liquor, 12 g glucose, 21.70 ml ethanol, and 1g MgSO4 per liter in the flask fermenta