pXMJ19

In terms of protein production, the host's internal environment affects the activity of expression elements, thereby affecting the expression level of the target protein. Native expression elements from a specific strain always work well in the original host. In this study, in order to improve the endoxylanase (XynA) production level of C. glutamicum CGMCC1.15647 and its natural expression element, methods to reduce host expression barriers and promote expression were evaluated. Researchers identified the CspB2 signal peptide in C. glutamicum CGMCC1.15647 by MALDI-TOF and applied it together with its promoter for the production of endoxylanase (XynA) in this strain. For XynA expression in C. glutamicum CGMCC1.15647, the native cspB2 promoter and cspB2 signal peptide are better than the commonly used cspB1 promoter and cspA signal peptide, and expression in this strain is superior to the expression in C. glutamicum ATCC13032. The highest levels of XynA secretion efficiency at the deep 24-well plate level were achieved by disruption of the cell wall protein CspB2 and the protease ClpS, chromosomal integration of xynA and coexisting plasmid expression, which increased expression 11.43- and 1.35-fold compared to that of chromosomal expression and pXMJ19-xynA-mediated expression in the original strain, respectively.

XynA plays a key role in the degradation of xylan, one of nature's most renewable biomass energy sources. In this study, researchers used the endogenous cspB2 signal peptide and its promoter to produce XynA. The wild-type C. glutamicum CGMCCl.15647 was transformed with the pXMJ19 and pXMJ19-xynA plasmid to obtain Cg47+P19-0 (C. glutamicum CGMCCl.15647 carrying pXMJ19) and Cg47+P19-X (C. glutamicum CGMCCl.15647 carrying pXMJ19-xynA), respectively. After 48 h of culture, the XynA activity in the culture supernatant of Cg47+P19-X reached 1849.03 U/mL, which was higher than the production (1504.27 U/mL) of Cg47+P19-cspB1X (C. glutamicum CGMCCl.15647 carrying pXMJ19-cspB1-cspA-xynA) (Figure 1). This result shows that the endogenous cspB2 promoter, 5'UTR and signal peptide of C. glutamicum CGMCCl.15647 can successfully express and secrete XynA, and the endogenous cspB2 promoter and cspB2 signal peptide is superior to the cspB1 promoter and cspA signal peptide for XynA production in C. glutamicum CGMCCl.15647 under the same culture conditions.

Figure 1. Comparison of the production of XynA using different promoters, signal peptides and hosts. (Zhang W, et al., 2019)