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. Finally, the SPs were rationally designed to improve the expression and secretion level of MtC1LPMO. 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. A and V, and the C-region contained an AXA motif. 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. with 4.1- and 2.1-fold increases over the native SP, respectively. The protein expression and secretion levels using SP12 (MNITNIINAAILQLQSMALQSISNTGTASS) were the highest among all SPs. Among these, 10 SPs were found to be better than the native SP of MtC1LPMO. 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.

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