3.1 Construction of the bicistronic T7 expression system
The artificial bicistronic structure in prokaryotes has been shown to enhance the production of recombinant proteins.[20] In this study, to further improve the expression performance of the T7 promoter in E. coli , we constructed the T7 in a bicistronic manner. We selected 8 well-performed fore-cistrons from the Pbtac system based on our previous study in C. glutamicum ,[19] and optimized their sequences according to the codon preference of E. coli , thus generating 8 additional sequences. In total, 16 fore-cistron sequences were used to construct bicistronic T7 systems, and the sequence information was presented in Table 1 . According to the characteristics of the BCD expression cassette, a conserved SD sequence (the second SD motif, SD2) and a translation coupling frame were inserted between the fore-cistron and the reporter gene EGFP (Fig. 1A and 1B ).[20] The LacI gene located upstream of the T7 promoter confers the inducible characteristic of the BCD system, so the expression of recombinant proteins in bicistronic T7 systems requires the addition of IPTG.