BackgroundMussel foot proteins (Mfps) are considered as remarkable materials due to their extraordinary adhesive capability. Recombinant expression is an ideal way to synthesis these proteins at large scale. However, secretory expression of Mfps into culture medium has not been achieved in a heterologous host.Methods and ResultsHere, to realize the secretion of Mfp3 and Mfp5 in Bacillus subtilis, signal peptide screening was first performed. Minimal Mfp3‐6×His was targeted into the growth medium with AmyE signal peptide. We found that a small chaperone protein Spy was secreted efficiently in B. subtilis, and the fusion proteins Spy‐Mfp3‐6×His and Spy‐Mfp5‐6×His could also be delivered into growth medium well. The yield of Spy‐Mfp3‐6×His and Spy‐Mfp5‐6×His reached 255 and 119 mg L−1 at shake flask conditions, respectively. Mfp3‐6×His and Mfp5‐6×His were finally purified via TEV protease cleavage and NTA affinity chromatography.ConclusionMfp3‐6×His and Mfp5‐6×His could be efficiently secreted using a chaperone protein Spy as fusion tag in B. subtilis.

2,3,5-Trimethylhydroquinone (2,3,5-TMHQ) is the key precursor in the synthesis of vitamin E. It is still a major challenge to produce 2,3,5-TMHQ under mild reaction conditions by chemical methods. The monooxygenase system MpdAB can specifically catalyze the conversion of 2,3,6-trimethylphenol (2,3,6-TMP) to 2,3,5-TMHQ. However, the weak catalytic capacity of wild-type MpdA and the cytotoxicity of the substrate limited the production efficiency of 2,3,5-TMHQ. Here, homologous modeling and saturation mutation were performed to increase the catalytic activity of MpdA. Two variants, L128A and L128K, with higher activity toward 2,3,6-TMP (1.86–1.87-fold) were obtained. On the other hand, an evolved strain B5-4M-evolved with enhanced resistance to 2,3,6-TMP (8.15-fold higher for 1000 μM 2,3,6-TMP) was obtained through adaptive laboratory evolution. Subsequently, a 5.29-fold (or 4.87-fold) improvement in 2,3,5-TMHQ production was achieved by a strain B5-4M-evolved harboring L128K (or L128A) and MpdB, in comparison with that of the wild type (strain B5-4M expressing MpdAB). This study provides better genetic resources for producing 2,3,5-TMHQ and proves that the synthesis efficiency of 2,3,5-TMHQ can be improved through enzyme modification and adaptive laboratory evolution.