Long Chuannan, Liu Mengmeng, Chen Xia, Wang Xiaofang, Ai Mingqiang, Cui Jingjing, Zeng Bin
1Jiangxi Key Laboratory of Bioprocess Engineering, Jiangxi Science and Technology Normal University, Nanchang, 330013 People's Republic of China.
2School of Life Science, Jiangxi Science and Technology Normal University, Nanchang, 330013 People's Republic of China.
3 Biotech. 2018 Feb;8(2):121. doi: 10.1007/s13205-018-1147-9. Epub 2018 Feb 9.
The present study verified whether acyl-coenzyme A (acyl-CoA)-binding protein (ACBP) affected the production of pigments (MPs) in CICC41233 (MrACBP). Phylogenetic analysis revealed that the cloned gene, which encoded the MrACBP protein, exhibited the closest match (99% confidence level) to the gene from . The MrACBP and maltose-binding protein (MBP) were simultaneously expressed in Rosetta DE3 in the form of a fusion protein. The microscale thermophoresis binding assay revealed that the purified MBP-MrACBP exhibited a higher affinity for myristoyl-CoA (Kd = 88.16 nM) than for palmitoyl-CoA (Kd = 136.07 nM) and octanoyl-CoA (Kd = 270.9 nM). Further, the gene was homologously overexpressed in CICC41233, and a positive transformant ACBP5 was isolated. The fatty acid myristic acid in ACBP5 was lower than that in the parent strain CICC41233. However, when compared with the parent strain, the production of total MPs, water-soluble pigment, and ethanol-soluble pigment in ACBP5 increased by 11.67, 9.80, and 12.70%, respectively, after 6 days. The relative gene expression level, as determined by a quantitative real-time polymerase chain reaction analysis, of the key genes , , , , and increased by 4.03-, 3.58-, 1.67-, 2.11-, and 2.62-fold after 6 days. These data demonstrate the binding preference of MrACBP for myristoyl-CoA, and its influence on MPs production.
本研究验证了酰基辅酶A（acyl-CoA）结合蛋白（ACBP）是否影响CICC41233（MrACBP）中色素（MPs）的产生。系统发育分析表明，克隆的编码MrACBP蛋白的基因与来自[具体来源未提及]的基因表现出最接近的匹配（置信水平99%）。MrACBP和麦芽糖结合蛋白（MBP）以融合蛋白的形式在Rosetta DE3中同时表达。微量热泳结合试验表明，纯化的MBP-MrACBP对肉豆蔻酰辅酶A（Kd = 88.16 nM）的亲和力高于棕榈酰辅酶A（Kd = 136.07 nM）和辛酰辅酶A（Kd = 270.9 nM）。此外，该基因在CICC41233中同源过表达，并分离出阳性转化体ACBP5。ACBP5中的脂肪酸肉豆蔻酸低于亲本菌株CICC41233。然而，与亲本菌株相比，ACBP5中总MPs、水溶性色素和乙醇溶性色素的产量在6天后分别增加了11.67%、9.80%和12.70%。通过定量实时聚合酶链反应分析确定，关键基因[具体基因未提及]、[具体基因未提及]、[具体基因未提及]、[具体基因未提及]和[具体基因未提及]的相对基因表达水平在6天后分别增加了4.03倍、3.58倍、1.67倍、2.11倍和2.62倍。这些数据证明了MrACBP对肉豆蔻酰辅酶A的结合偏好及其对MPs产生的影响。
