谷氨酸棒杆菌GPAT功能基因鉴定与分析

Identification and Analysis of GPAT Functional Genes of Corynebacterium glutamicum

甘油-3-磷酸酰基转移酶(GPAT)是三酰基甘油(TAG)合成过程中的关键酶。以谷氨酸棒杆菌(Corynebacterium glutamicum)CG14为研究对象,利用生物信息学预测了Corynebacterium glutamicum ATCC13032编码GPAT的多个候选功能基因,并选择其中之一cg2777基因进行后续验证,该基因具有典型的GPAT的基因结构域,包含4个跨膜结构域,其编码的氨基酸序列与多种产油细菌来源的GPAT的基因序列具有很高的相似度;通过构建基因cg2777敲除菌株和过表达菌株,结合发酵验证分析,推测基因cg2777行使GPAT功能,但是油脂合成是一个多酶作用的复杂生物催化过程,单一过表达基因cg2777并不能显著提高胞内油脂含量。

Glycerol-3-phosphate acyltransferase(GPAT)is a key enzyme in the synthesis of triacylglycerols(TAGs).GPAT catalyzes the formation of lysophosphatidic acid(LPA)from glycerol-3-phosphate and fatty acyl-CoA,which is the initial reaction of the synthesis of TAG and glycerol-phospholipids.However,previous research showed that there was no definite gene ontology(GO)annotations of the GPAT gene in the complete genome sequence of Corynebacterium glutamicum ATCC13032.With the aid of bioinformatics and literature mining,several candidate genes encoding GPAT in Corynebacterium glutamicum ATCC13032 have been predicted.cg2777,a putative membrane protein gene,has been selected for the subsequent analysis and verification.The cg2777 gene has typical GPAT functional domain and four typical transmembrane helix regions.Compared with the GPAT gene sequences derived from a variety of oil-producing bacteria,there are several deletion and insertion regions in the amino acid sequence.By constructing the knockout and overexpression strain of the gene cg2777,fermentation verification analysis implies that cg2777 performs partial GPAT function with low activity.As lipid synthesis is a complex biocatalytic process involving multiple enzymes,the single overexpression of cg2777 gene cannot significantly increase the intracellular lipid content.