苜蓿中华根瘤菌fabA和fabB基因功能的鉴定

在大肠杆菌(Escherichia coli)脂肪酸合成酶体系中，fabA基因编码有双功能的3-羟基脂酰ACP脱水异构酶，其异构产物能被fabB基因编码的3-酮基脂酰ACP合成酶Ⅰ延伸，合成不饱和脂肪酸，该FabA-FabB途径被认为是缺氧条件下不饱和脂肪酸合成的经典途径．生物信息学分析发现，苜蓿中华根瘤菌(Sinorhizobium meliloti)的SmFabA与EcFabA相似性达到60.6%，具有相同的保守活性位点和两个保守的α螺旋结构；SmFabB与EcFabB相似性达到61.1%，具有相同的Cys-His-His活性中心．用携带SmfabA和SmfabB的质粒载体遗传互补大肠杆菌温度敏感突变株CY57和CY242，在添加三氯森(TCL)抑制烯脂酰ACP还原酶活性的条件下，转化子能在42℃恢复生长，且放射性薄层层析能检测到转化子中不饱和脂肪酸棕榈油酸(Δ9C16:1)和十八碳烯酸(Δ11C18:1)的合成．体外重建脂肪酸合成反应表明，SmFabA能催化羟脂酰ACP的脱水反应且能够使反-2-癸烯酰ACP异构化，SmFabB能催化不同链长的脂酰ACP和丙二酸单酰ACP的聚合反应．另外，未得到SmFabA和SmFabB的突变株，表明SmFabA和SmFabB可能是苜蓿中华根瘤菌脂肪酸合成酶系中必不可少的关键蛋白．上述结果证实了苜蓿中华根瘤菌fabA和fabB两个基因在不饱和脂肪酸合成中的功能．

Identification and Function Reasearch of fabA and fabB of Sinorhizobium meliloti

In E. coli, FabA, a bifunctional enzyme, is the key enzyme of the classic anaerobic pathway of unsaturated fatty acid synthesis and introduces the cis double bond into a 10-carbon intermediate. This intermediate is then elongated by FabB (one of long chain 3-ketoacyl-ACP synthases) to form the unsaturated fatty acids found in the membrane phospholipids. Sequence alignments indicated that Sinorhizobium meliloti SmFabA and SmFabB are 60.6% and 61.1% identical to E. coli FabA and FabB, respectively. Further analysis showed that the conservative active-site histidine residue in EcFabA and the Cys-His-His catalytic triads in EcFabB, are also found in SmFabA and SmFabB. The genetic complementary revealed that SmfabA is able to restore the growth and the fatty acid synthesis of the E. coli temperature sensitive mutant CY57 at nonpermissive temperature under addition low concentration triclosan to inhibit enoyl-acyl carrier protein reductases. Moreover, SmfabB is able to complement temperature sensitive mutant CY242. In vitro assay identifies that SmfabA, like E. coli FabA, is able to introduce the cis double bond into a 10-carbon intermediate, and SmfabB, like FabB, is able to condense the acyl-ACPs with malonyl-ACP to long-chain acyl-ACPs. However, we also attempted to inactivate the fabA and fabB genes by allelic replacement but none of the fabA and fabB deletion mutant was obtained, and it seemed likely that fabA and fabB are essential genes in S. meliloti. These results demonstrated that SmFabA and SmFabB are key enzymes in unsaturated fatty acid synthesis in S. meliloti.