caa a22 4500 606212612 CHVBK 20210128101043.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s10295-015-1668-8 doi (NATIONALLICENCE)springer-10.1007/s10295-015-1668-8 Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis [Elektronische Daten] [Di Huang, Yongpeng Yao, Hang Zhang, Zhu Mei, Ru Wang, Lu Feng, Bin Liu] Terpenoids, a class of isoprenoids usually isolated from plants, are always used as commercial flavor and anticancer drugs. As a key precursor for triterpenes and sterols, biosynthesis of squalene (SQ) can be catalyzed by squalene synthase (SQS) from two farnesyl diphosphate molecules. In this work, the key SQS gene involved in sterols synthesis by Mortierella alpine, an industrial strain often used to produce unsaturated fatty acid such as γ-linolenic acid and arachidonic acid, was identified and characterized. Bioinformatic analysis indicated that MaSQS contained 416 amino acid residues involved in four highly conserved regions. Phylogenetic analysis revealed the closest relationship of MaSQS with Ganoderma lucidum and Aspergillus, which also belonged to the member of the fungus. Subsequently, the recombinant protein was expressed in Escherichia coli BL21(DE3) and detected by SDS-PAGE. To improve the expression and solubility of protein, 17 or 27 amino acids in the C-terminal were deleted. In vitro activity investigation based on gas chromatography-mass spectrometry revealed that both the truncated enzymes could functionally catalyze the reaction from FPP to SQ and the enzymatic activity was optimal at 37°C, pH 7.2. Moreover, based on the site-directed mutagenesis, the mutant enzyme mMaSQSΔC17 (E186K) displayed a 3.4-fold improvement in catalytic efficiency (k cat/K m) compared to the control. It was the first report of characterization and modification of SQS from M. alpine, which facilitated the investigation of isoprenoid biosynthesis in the fungus. The engineered mMaSQSΔC17 (E186K) can be a potential candidate of the terpenes and steroids synthesis employed for synthetic biology. Society for Industrial Microbiology and Biotechnology, 2015 Squalene synthase nationallicence Mortierella alpine nationallicence Truncated enzyme nationallicence GC-MS analysis nationallicence Site-directed mutagenesis nationallicence Huang Di TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut Yao Yongpeng TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut Zhang Hang TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut Mei Zhu TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut Wang Ru TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut Feng Lu TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut Liu Bin TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut Journal of Industrial Microbiology & Biotechnology Springer Berlin Heidelberg 42/10(2015-10-01), 1341-1352 1367-5435 42:10<1341 2015 42 10295 https://doi.org/10.1007/s10295-015-1668-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s10295-015-1668-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Huang Di TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut NATIONALLICENCE 700 1- Yao Yongpeng TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut NATIONALLICENCE 700 1- Zhang Hang TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut NATIONALLICENCE 700 1- Mei Zhu TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut NATIONALLICENCE 700 1- Wang Ru TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut NATIONALLICENCE 700 1- Feng Lu TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut NATIONALLICENCE 700 1- Liu Bin TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, People's Republic of China aut NATIONALLICENCE 773 0- Journal of Industrial Microbiology & Biotechnology Springer Berlin Heidelberg 42/10(2015-10-01), 1341-1352 1367-5435 42:10<1341 2015 42 10295