6-磷酸果糖激酶基因pfkA在施氏假单胞菌中的异源表达及其表型分析

Heterologous Expression of 6-phosphofructo Kinase Gene pfk A from Escherichia coli in Pseudomonas stutzeri and Phenotype Analysis of Recombinant Strain

糖代谢在生命活动中具有重要意义,它保证了生物体生命活动所需的物质和能量的供应。葡萄糖作为自然界中普遍存在的最简单的单糖,是生物体的理想碳源。施氏假单胞菌(Pseudomonas stutzeri)A1501具有广泛的、典型的微生物代谢途径,但是该菌利用葡萄糖的效率很低。基因组分析表明A1501菌缺少糖酵解(EMP)途径中编码6-磷酸果糖激酶的基因pfk A,初步认为这可能是A1501菌不能高效利用葡萄糖的原因。利用穿梭质粒p LAFR3将大肠杆菌的pfk A基因导入到施氏假单胞菌中,通过pfk A基因的异源表达重建A1501菌的EMP途径。测定了重组菌株的糖类代谢能力,结果表明pfk A基因的导入并没有赋予重组施氏假单胞菌更强的葡萄糖及其他糖类的利用能力,说明A1501菌葡萄糖利用的低效率除了缺乏pfk A外,还有其他的因素。研究也发现,导入pfk A的重组施氏假单胞菌对H2O2高度敏感,推测pfk A导入重建后的EMP途径可能影响了该菌的还原力合成,导致菌体对氧化胁迫的耐受能力降低。

： Sugar metabolism is the process operated by nearly all organisms for the supplement of nutrition and energy. As one of the most widely used monosaccharides in nature, glucose is an ideal carbon source for microorganisms. Pseudomonas stutzeri A1501 has the canonica microbial metabolic pathways. However, it has a very low rate for glucose utilization. Genome analysis of P. stutzeri A1501 suggested that A1501 could not use glucose by the Embden-Meyerhof-Parnas （EMP） pathway, because it lacked the key glycolytic enzyme 6-phosphofructokinase encoded by pff, A. An Escherichia coli pfl^A gene was cloned and transferred to P. stutzeri A1501 to accomplish the reconstruction in vivo of the complete set of enzymes for enabling a functional EMP pathway in A1501. The expression of pfkA in P. stutzeri A1501 failed to glucose utilization by the EMP pathway, suggesting that there were other systems that might affect the glucose utilization except for the primA gene. It was also observed that the recombinant A1501- pfkA strain became more sensitive to hydrogen peroxide, compared to the wild type. We further hypothesized that the major reason for pfkA confers decreased oxidative tolerance to P. stutzeri A1501 was due to the influence of its NADPH formation.