恶臭假单胞菌中3-酮脂酰ACP还原酶FabG5是脂肪酸合成关键酶

3-Ketoacyl ACP Reductase FabG5 is Essential for Fatty Acid Synthesis in Psedomonas putida

3-酮脂酰ACP还原酶(FabG)催化脂肪酸合成中的第一步还原反应,是细菌生长的关键酶之一.恶臭假单胞菌在环境污染治理和工业聚羟基脂肪酸(PHA)的生产中,都具有重要的应用价值.生物信息学分析显示,恶臭假单胞菌基因组编码6个FabG同源蛋白质,与大肠杆菌FabG相比较,PpFabG5序列相似性最高(76.5%),其他几个PpFabG也都具有较高的序列相似性(约50%).除PpFabG4之外,其他的同源蛋白质都具有催化活性位点和N端辅因子结合位点.为研究恶臭假单胞菌中这6个FabG同源蛋白质的生物学功能,本文进行了异体遗传互补、体外酶学活性分析、体内基因敲除与突变株性状分析等研究.结果显示,只有PpfabG1、PpfabG3、PpfabG5能恢复大肠杆菌fabG温度敏感突变株CL104在42℃时生长,其中PpfabG1互补株生长较弱.而在体外活性检测中,PpFabG1、PpFabG3和PpFabG5在脂肪酸合成起始反应和延伸反应中都具有催化活性,但PpFabG1活性较弱,PpFabG6仅在起始反应中具有催化活性. PpfabG5是恶臭假单胞菌生长的必需基因,不能被敲除,而其他几个PpfabG基因敲除后不影响菌体的生长,突变株的脂肪酸组成与野生菌也无差异.但PpfabG1、PpfabG2敲除后菌体的运动性下降,PpfabG3、PpfabG6突变影响了生物被膜的合成量,而PpfabG4、PpfabG6敲除突变株对H2O2的耐受性增强,表明这些基因具有不同的生理功能,可能在菌体的不同逆境中发挥作用.

3-Ketoacy ACP reducatase(FabG), the key enzyme for bacteria growth, catalyzes the first reduction step in fatty acid synthesis. Pseudomonas putida has important application values in environmental pollution bioremediation and industrial polyhydroxy fatty acid(PHA) production. Bioinformatics analysis showed Pseudomonas putida genome encodes six FabG homologues with E. coli FabG, of which PpFabG5 shows the highest sequence similarity(76.5%) and other five PpFabGs also have high similarity(about 50%). Except PpFabG4, the other homologs have the conserved catalytic activity sites and N-terminal cofactor binding sites. So the paper used different methods, including the genetic complementary, catalytic activity analysis in vitro, gene deletion in vivo, and mutant characteristic analysis, to study the biological functions of the six homologs. The results showed that only PpfabG1, PpfabG3 and PpfabG5 could restore the growth of E.coli fabG temperaturesensitive mutant CL104 at 42℃, and the complementary strain of PpfabG1 grew weakly. While in vitro analysis,PpFabG1, PpFabG3 and PpFabG5 also showed the catalytic activities in the initial and extension reactions of fatty acid synthesis, although the activity of PpFabG1 was really weak. PpFabG6 only had catalytic activity in the initial reaction. PpfabG5 is an essential gene for growth, which can’t be deleted in the genome. While the deletion of the other PpfabG individually does not affect bacteria growth or the compositions of fatty acids comparing with wild type strain. However, the motility of PpfabG1 or PpfabG3 deletion mutant decreased, PpfabG3 or PpfabG6 deletion affected the biofilm formation, and PpfabG3 or PpfabG6 deletion mutant showed higher tolerance to H2O2. These results proved different FabG homologs contained different biological functions,especially when countered with stressful conditions.