Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length

doi:10.1186/s13068-016-0596-9

QIBEBT-IR > 微生物代谢工程研究组

	Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length
	Bao,Luyao 1,3; Li,Jian-Jun 1,5; Jia,Chenjun 3,4; Li,Mei 4; Lu,Xuefeng 2
	2016-08-31
发表期刊	Biotechnology for Biofuels
ISSN	1754-6834
卷号	9 期号:1
摘要	AbstractBackgroundAldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C4 to C18 aldehydes, which is not suitable for producing fuels with different properties or different chain lengths.ResultsBased on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C14,16,18 aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C4,6,7 aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C6–10 aldehydes and L198F for C7–10 aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane.ConclusionsSome amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties.
关键词	Aldehyde-deformylating oxygenase Site-directed mutagenesis Structure-guided protein engineering Chain-length selectivity Synechococcus elongatus PCC7942
DOI	10.1186/s13068-016-0596-9
语种	英语
WOS记录号	BMC:10.1186/s13068-016-0596-9
出版者	BioMed Central
引用统计
文献类型	期刊论文
条目标识符	http://ir.qibebt.ac.cn/handle/337004/8419
专题	微生物代谢工程研究组
通讯作者	Li,Jian-Jun; Lu,Xuefeng
作者单位	1.Chinese Academy of Sciences; Key Laboratory of Biofuels 2.Chinese Academy of Sciences; Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology 3.University of Chinese Academy of Sciences 4.Chinese Academy of Sciences; National Laboratory of Biomacromolecules, Institute of Biophysics 5.Chinese Academy of Sciences; National Key Laboratory of Biochemical Engineering, Institute of Process Engineering
推荐引用方式 GB/T 7714	Bao,Luyao,Li,Jian-Jun,Jia,Chenjun,et al. Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length[J]. Biotechnology for Biofuels,2016,9(1).
APA	Bao,Luyao,Li,Jian-Jun,Jia,Chenjun,Li,Mei,&Lu,Xuefeng.(2016).Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length.Biotechnology for Biofuels,9(1).
MLA	Bao,Luyao,et al."Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length".Biotechnology for Biofuels 9.1(2016).