Characterization, functional analysis and application of 4-Coumarate: CoA ligase genes from Populus trichocarpa.

• Populus trichocarpa genome sequence data from NCBI was analyzed and 17 putative 4-coumarate: CoA ligase (4CL) or 4CL-like proteins were obtained. • Ptr4CL4, Ptr4CL5 and Ptr4CL7 were selected for investigating their properties and functions. • A cinnamyl alcohol biosynthesis pathway was constructed in Escherichia coli with Ptr4CL5, PtrCCR2 and endogenous reductases. • 4.8 mM cinnamyl alcohol was obtained from trans -cinnamic acid and the titer is higher than other reports. 4-Coumarate: CoA ligase (4CL) is an important branch point directing metabolites to flavonoid or monolignol pathways in plants. It plays a vital role in the biosynthesis of plant nature products in microbes. Herein, Ptr4CL4, Ptr4CL5 and Ptr4CL7 from Populus trichocarpa were cloned and expressed in Escherichia coli. Two recombinant proteins Ptr4CL4 and Ptr4CL5 showed distinct activities for different substrates. The Ptr4CL4, not previously reported, showed the highest affinity and activity for p -coumaric acid, but a unique substrate self-inhibition was observed at high concentration of p -coumaric acid. Ptr4CL5 was suitable for pathway construction due to no self-substrate inhibition and high initial reaction rate. To explore the potential of Ptr4CL5 in biosynthesis of cinnamyl alcohol, a biosynthesis pathway established with Ptr4CL5, PtrCCR2, endogenous reductases was constructed in E. coli and the titer of cinnamyl alcohol reached 4.8 mM which is higher than other reports. The result indicates that the wood-derived Ptr4CL5 has signification potential in the biosynthesis of cinnamyl alcohol and other monolignol derivatives. [ABSTRACT FROM AUTHOR]