Extracellular Expression of Feruloyl Esterase and Xylanase in Escherichia coli for Ferulic Acid Production from Agricultural Residues.

There is still a large amount of ferulic acid (FA), an outstanding antioxidant, present in agricultural residues. Enzymatic hydrolysis has been regarded as the most effective way to release FA. This present study therefore selected feruloyl esterase (FAE) and xylanase (XYN) from the metagenomes of a cow rumen and a camel rumen, respectively, for their recombinant expression in Escherichia coli BL21 and further application in releasing FA. After screening the candidate signal peptides, the optimal one for each enzyme, which were selected as SP1 and SP4, respectively, was integrated into the vectors pET22b(+) and pETDuet-1. Among the generated E. coli strains SP1-F, SP4-X, and SP1-F-SP4-X that could express extracellular enzymes either separately or simultaneously, the latter one performed the best in relation to degrading the biomass and releasing FA. Under the optimized culture and induction conditions, the strain SP1-F-SP4-X released 90% of FA from 10% of de-starched wheat bran and produced 314.1 mg/L FA, which was deemed to be the highest obtained value to the best of our knowledge. This result could pave a way for the re-utilization of agricultural residues and enhancing their add-value.

Synthesis and in vitro antitumour activity of 4(R)-methyl-3-O-phosphonomethyl-α-l-threose nucleosides.

A series of novel α-l-threose nucleoside phosphonate analogs, 4(R)-methyl-3-O-phosphonomethyl-α-l-threose nucleosides, were synthesized in multistep sequences starting from d-xylose. The synthetic sequence consisted of the following key stages: (i) the multistep synthesis of 1,2-O-isopropylidenyl-4(R)-methyl-3-O-phosphonomethyl-l-threose, (ii) the transformation of 1,2-O-isopropylidenyl sugar into suitable 1,2-di-O-acyl l-threose precursor, and (iii) the construction of target α-l-threose nucleoside phosphonate analogs by Vorbrüggen glycosidation reaction, deprotection of acyl group, and hydrolysis of diethyl group on phosphonate. The target nucleoside phosphonates were evaluated for their antitumour activities in cell culture-based assays. Compound 8g, 2-fluroadenosine phosphonate, showed remarkable activity against human breast cancer cell lines (MCF-7 and MDA-MB-231) with IC50 values of 0.476 and 0.391 µM, corresponding to 41- and 47-fold higher potency than the reference compound 5-FU, respectively. Subsequent investigations found that the compound 8g can inhibit the proliferation of breast cancer cells and cell cloning. The mechanistic studies indicated that compound 8g could cause DNA damage to breast cancer cells through the ATM-Chk1/Chk2-cdc25c pathway, leading to blockage of the G2/M phase cycle of breast cancer cells, which ultimately led to apoptosis. Moreover, 8g could inhibit the PI3K/AKT signaling pathway and induce apoptosis. These results indicate that compound 8g holds promising potential as an antitumour agent.