产油核桃内生细菌乙酰辅酶A羧化酶acb基因 克隆及优化表达Cloning and expression optimization of acetyl-CoA carboxylase acb gene from oil-producing walnut endophyte

Abstract

为了探究细菌异质性乙酰辅酶A羧化酶β亚基生物活性及其对乙酰辅酶A羧化酶酶活影响，以高产油核桃内生细菌B. subtilis HB1310基因组DNA为模板，利用PCR技术扩增其乙酰辅酶A羧化酶羧基转移酶β亚基（β-CT）基因（acb基因），构建pET-28a-acb载体并在E. coli BL21中表达，进一步对乙酰辅酶A羧化酶acb基因的诱导表达条件进行了优化。结果表明：乙酰辅酶A羧化酶acb基因在E. coli BL21中实现了表达，分子质量在25~35 kDa之间；重组蛋白最佳诱导表达条件为诱导剂异丙基-β-D-硫代半乳糖苷（IPTG）浓度1.0 mmol/L、诱导时间6 h、诱导初始pH 8.0，在此条件下工程菌的乙酰辅酶A羧化酶活性可达（4.11±0.03） U/mL，较野生菌提高39.7%。综上，乙酰辅酶A羧化酶β-CT为具有较好生物活性的乙酰辅酶A羧化酶亚基。 To explore the biological activity of the bacterial heterogeneous acetyl-CoA carboxylase β subunit and its influence on the acetyl-CoA carboxylase activity, the acetyl-CoA carboxylase carboxyltransferase subunit beta(β-CT) gene(acb gene) was amplified by PCR technology using the genomic DNA of a high oil-producing walnut endophyte B. subtilis HB1310 as template, the expression vector pET-28a-acb was constructed and expressed in E. coli BL21, and the induced expression conditions of this acb gene were further optimized. The results showed that the acetyl-CoA carboxylase acb gene was expressed in E. coli BL21 with a molecular weight of 25-35 kDa. Moreover, the optimal induced expression conditions were determined as follows: isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration 1.0 mmol/L, induction time 6 h, and induction initial pH 8.0. Under the optimal conditions, the acetyl-CoA carboxylase activity of the engineering bacteria reached (4.11±0.03) U/mL, which was 39.7% higher than that of the wild bacteria. In conclusion, acetyl-CoA carboxylase β-CT is an acetyl-CoA carboxylase subunit with good biological activity.

Keywords

• 核桃内生细菌；产油；乙酰辅酶a羧化酶；克隆；优化表达
• walnut endophyte; oil-producing; acetyl-coa carboxylase; cloning; expression optimization