title: Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis.
abstract: In the phenylpropanoid production process, p-coumaric acid is the most important intermediate metabolite. It is generally accepted that the activity of tyrosine ammonia-lyase (TAL), which converts L-tyrosine to p-coumaric acid, represents the rate-limiting step. Therefore, an error-prone PCR-based random mutagenesis strategy was utilized for screening variants with higher catalytic activity. After rounds of screening, three variant enzymes were obtained, exhibiting improved production rates of 41.2, 37.1, and 38.0 %, respectively. Variants associated with increased expression level (S9N), improved catalytic efficiency (A11T), and enhanced affinity between TAL and L-tyrosine (E518V) were identified as beneficial amino acid substitutions by site-directed mutagenesis. Combining all of the beneficial amino acid substitutions, a variant, MT-S9N/-A11T/-E518V, exhibiting the highest catalytic activity was obtained. The Kmvalue of MT-S9N/-A11T/-E518V decreased by 25.4 % compare to that of wild-type, while the activity, kcat/Km, and p-coumaric-acid yield were improved by 36.5, 31.2, and 65.9 %, respectively. Furthermore, the secondary structure of the 5'-end of MT-S9N mRNA and the three-dimensional protein structure of MT-E518V were modeled. Therefore, two potential mechanisms were speculated: (1) a simplified mRNA 5'-end secondary structure promotes TAL expression and (2) anchoring the flexible loop region (Glu325-Arg336) to maintain the active-site pocket opening ensures easy access by the L-tyrosine to the active site and thus improves p-coumaric acid yields.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.
authors.forename: Shenghu
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.
authors.forename: Peiran
authors.initials: P
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China. hzhli@jiangnan.edu.cn.
authors.forename: Huazhong
authors.initials: H
authors.lastname: Li
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China. zhoujw1982@jiangnan.edu.cn.
authors.forename: Jingwen
authors.initials: J
authors.lastname: Zhou
mesh_terms: D019943:Amino Acid Substitution; D000642:Ammonia-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001711:Biotransformation; D003373:Coumaric Acids / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011422:Propionates; D011487:Protein Conformation; D012248:Rhodotorula / Q000201:enzymology*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D003373:Coumaric Acids; D050505:Mutant Proteins; D011422:Propionates; D014443:Tyrosine; D000642:Ammonia-Lyases; C023208:L-tyrosine ammonia-lyase; C495469:p-coumaric acid
keywords: Error-prone PCR; Escherichia coli; Flavonoids; Phenylpropanoids; Random mutation; p-Coumaric acid