生物
同工酶
生物化学
酵母
基因
异源表达
突变体
氨基酸
大肠杆菌
酿酒酵母
酶
分子生物学
重组DNA
作者
Yuxuan Liu,Meiru Zhang,Tianshi Wang,Xunxun Shi,Jie Li,Lu Jia,Hui Tang,Liping Zhang
标识
DOI:10.1007/s10529-015-2006-y
摘要
Two genes encoding two acetyl-CoA synthetase (ACS) isoenzymes have been identified in the marine yeast Rhodosporidium diobovatum MCCC 2A00023.ACS1 encoded a polypeptide with a sequence of 578 amino acid residues, a predicted molecular weight of 63.73 kDa, and pI of 8.14, while the ACS2 encoded a polypeptide containing 676 amino acid residues with a deduced molecular mass of 75.61 kDa and a pI of 5.95. Biological activity of Acs1p and Acs2p was confirmed by heterologous expression in Escherichia coli. A 1.5-kb DNA fragment of the ACS1 gene and a 2.7-kb DNA fragment of the ACS2 gene were deleted using the RNA guide CRISPR-Cas9 system. The strain lacking ACS1 was unable to grow on acetate and ethanol media, while the ACS2 deletant was unable to grow on glucose medium. ACS1-ACS2 double mutants of R. diobovatum were non-viable.ACS isoenzymes are essential to the yeast metabolism, and other sources of ACSs cannot compensate for the lack of ACSs encoded by the two genes.
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