Journal
METABOLIC ENGINEERING
Volume 40, Issue -, Pages 115-123Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2017.01.006
Keywords
Biosensor; p-Coumarate: coenzyme A ligase; High-throughput screening; Phenylpropanoids pathway; Protein engineering; Resveratrol
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Funding
- Ministry of Science and Technology of China [2013CB734003]
- National Natural Science Foundation of China [21472234, 31500054, 31160017]
- CAS/SAFEA International Partnership Program for Creative Research Teams
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Overexpressing key enzymes of biosynthetic pathways for overproduction of value-added products usually imposes metabolic burdens on cells, which can be circumvented by improving the key enzyme activities. p-Coumarate: CoA ligase (4CL) is a critical enzyme in the phenylpropanoid pathway that synthesizes various natural products. To screen for 4CL with improved activity, a biosensor of resveratrol whose biosynthetic pathway involves 4CL was designed by engineering the TtgR regulatory protein. The biosensor exhibited good specificity and robustness, allowing rapid and sensitive selection of resveratrol hyper-producers. A 4CL variant with improved activity was selected from a 4CL mutagenesis library constructed in the resveratrol biosynthetic pathway in Escherichia coli. This mutant led to increased production of not only resveratrol but also the flavonoid naringenin, when introduced in their corresponding biosynthetic pathways. These findings demonstrate the feasibility of improving key enzyme activities in important biosynthetic pathways with the aid of designed biosensors of pathway products.
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