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 Km value 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.
p-Coumaric acid Error-prone PCR Escherichia coliFlavonoids Phenylpropanoids Random mutation
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This work was supported by the National High Technology Research and Development Program of China (863 Program, 2012AA022103), the National Natural Science Foundation of China (31370130), the Natural Science Foundation of Jiangsu Province (BK2011004), the Fundamental Research Funds for the Central Universities (JUSRP51307A), the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (FANEDD, 201256), the Program for New Century Excellent Talents in University (NCET-12-0876), and the 111 Project (111-2-06).
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no competing interests.
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