摘要
作为新型Ⅲ型聚酮合酶,米曲霉来源的CsyB能够依次接受一个起始单元为短链脂肪酰辅酶A、一个延伸单元为丙二酰辅酶A和另一个延伸单元为乙酰乙酰辅酶A的3个底物形成短链的csypyrone B1-3。基于CsyB的晶体结构分析,显示它的活性中心存在一个长约16?的能够接受脂肪酰辅酶A结合通道,这个通道很可能能够接受多种底物。为了检测该酶的底物多样性,将CsyB基因导入到存在长链脂肪酰辅酶A前体的大肠杆菌中表达。高效液相结果显示,相比对照菌株,重组菌株产生了一系列长链的csypyrone衍生物。利用紫外可见光特征吸收值和高分辨液相色谱-质谱联用仪对这些新产物作了初步分析。对3个具有羟基的csypyrone产物的结构进行了核磁共振一维谱和二维谱的详细鉴定,确定了其羟基的位置。上述结果显示,CsyB具有广泛的底物特异性,不但可以接受多种长链饱和或不饱和脂肪酰辅酶A,还可以接受具有羟基修饰的长链脂肪酰辅酶A作为底物。
As a novel fungal type Ⅲ polyketide synthase, CsyB from Aspergillus oryzae can sequentially accept one molecular short chain fatty acyl CoA as start unit, one molecular malonyl-CoA and one molecular acetoacetyl-CoA as extend unit to produce the short chain csypyrone B1-3. On the basis of crystal structure of CsyB, a fatty acyl CoA binding tunnel of a length of about 16 A is located in its active center that is proposed to accept diversified start units. In order to examine the substrate diversity of CsyB, CsyB gene was introduced and expressed in Escherichia coli that contained a number of precursors of long chain fatty acyl CoA in vivo. The results of HPLC revealed that a series of long chain csypyrone derivatives were detected in the recombinant strain in comparison with the control strain. These new csypyrone compounds were preliminarily analyzed by UV-visible spectroscopy and LC-HRMS. Three hydroxylated csypyrones were intensively determined by 1D and 2D NMR experiments, especially the position of the hydroxyl group in these compounds. These results demonstrate that CsyB exhibits a broad substrate specificity, which not only can accept the long chain saturated or unsaturated fatty acyl CoA as substrate, but also accept hydroxylated long chain fatty acyl CoA.
作者
潘丽霞
朱婧
王青艳
申乃坤
李亿
杨登峰
Lixia Pan;Jing Zhu;Qingyan Wang;Naikun Shen;Yi Li;and Dengfeng Yang(Guangxi Key Laboratory ofBiorefinery, Guangxi Biomass Industrialization Engineering Institute, National Engineering Research Center of Non-food Biorefinery, State Key Laboratory of Non-Food Biomass and Enzyme Technology, Guangxi Academy of Sciences, Nanning 530007, Guangxi, China;Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning 530007, Guangxi, China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2018年第7期1137-1146,共10页
Chinese Journal of Biotechnology
基金
国家自然科学基金(No.31660251)
广西自然科学基金(No.2017GXNSFAA198010)资助~~