Rational design of signal peptides for improved MtC1LPMO production in Bacillus amyloliquefaciens.

Affiliation

Guo X(1), Chai C(1), An Y(1), Peng C(1), Shi N(1), Wang W(1), Lu F(1), Dai Y(2), Liu F(3).
Author information:
(1)Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin 300457, PR China; Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, PR China.
(2)Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin 300457, PR China; Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, PR China. Electronic address: [Email]
(3)Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin 300457, PR China; Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, PR China. Electronic address: [Email]

Abstract

A high-throughput screening system was established by employing enhanced green fluorescent protein as a screenable fusion tag to evaluate the expression and secretion of a lytic polysaccharide monooxygenase (MtC1LPMO) using 20 Sec-type signal peptides (SPs) from Bacillus amyloliquefaciens 111018. Among these, 10 SPs were found to be better than the native SP of MtC1LPMO. The protein expression and secretion levels using SP12 (MNITNWAAILQLQSMALQSISNTGTASS) were the highest among all SPs, with 4.1- and 2.1-fold increases over the native SP, respectively. Then, the amino acids of the 10 best SPs were analyzed, and the results indicated that the most abundant amino acid of the N-region was K, those of the H-region were L, F, A and V, and the C-region contained an AXA motif. Additionally, we found that the protein expression level gradually improved along with the increasing folding free energies of the SP-encoding part of the mRNA. Finally, the SPs were rationally designed to improve the expression and secretion level of MtC1LPMO. An increased positive charge of the SP N-region was found to enhance the protein expression and secretion level, as long as the folding free energy of the mRNA did not change significantly.