Key Laboratory of Microbial Metabolism, Ministry of Education, School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People's Republic of China.
Appl Microbiol Biotechnol. 2010 May;86(6):1761-73. doi: 10.1007/s00253-010-2464-z. Epub 2010 Feb 13.
Phenazine-1-carboxylic acid (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-beta-D-1-thiogalactopyranoside (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 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 based on the center composite design. The predicted PCA production was 6,335.2 mg/l after 60 h 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 1 g MgSO(4) per liter in the flask fermentations, with induction of 1.0 mmol/l IPTG 24 h after inoculation. In an experimental validation under these conditions, the maximum PCA production was 6,365.0 mg/l. This represents a approximately 60% increase over production by strain M18GQ in optimal conditions. The negative effect of plasmid pME6032 on the expression of chromosomally located phz gene cluster was found in Pseudomonas sp. M18GQ, and the possible reason was discussed in the text.
吩嗪-1-羧酸（PCA）的生产在野生型假单胞菌 M18 及其携带过表达 phz 基因簇的重组 pME6032Phz 的突变体中得到增强，其中携带 pME6032Phz 的 gacA 和 qscR 双基因染色体灭活突变体假单胞菌 M18GQ/pME6032Phz 表现出最高的 PCA 产量。在摇瓶实验中优化了 M18GQ/pME6032Phz 菌株的发酵和异丙基-β-D-1-硫代半乳糖吡喃糖苷（IPTG）诱导条件。通过单因素实验和部分因子设计确定了豆粕、玉米浆和乙醇是统计学上显著的因素。然后通过最陡上升法和基于中心复合设计的响应面法确定了这些因素的最佳浓度和相互作用。在最佳培养基中发酵 60 h 后，预测 PCA 的产量为 6335.2 mg/l，该培养基中含有 65.02 g 豆粕、15.36 g 玉米浆、12 g 葡萄糖、21.70 ml 乙醇和 1 g/L MgSO4，在接种后 24 h 用 1.0 mmol/L IPTG 诱导。在这些条件下进行实验验证时，PCA 的最大产量为 6365.0 mg/l。与 M18GQ 在最佳条件下的产量相比，这代表了大约 60%的增长。在假单胞菌 M18GQ 中发现质粒 pME6032 对染色体定位的 phz 基因簇表达有负面影响，文中讨论了可能的原因。
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