LAP activity and expression by full-length scmp promoter in PG medium
We have reported that SCMP, which is a metalloendoprotease from S. cinnamoneus TH-2 strain controlled under the scmp promoter, required 2% glucose and 0.8% phosphate to be secreted into the culture as an active form (Hatanaka et al. 2005). Based on our findings, we examined four media for secreted LAP expression using the transformant harboring the pTONA5a-lap. After 4 days of culture at 30°C, the LAP activity of the supernatant was analyzed, and protein expression was detected by SDS-PAGE. The supernatant of the culture in PG medium showed LAP activity and protein expression (Fig. 1). In contrast, low phosphate medium (0.1% K2HPO4 in PG), Pgly medium (2% glycerol instead of glucose in PG), and TSB showed lower LAP activity and expression than PG medium (Fig. 1).
LAP activity and expression by truncated scmp promoters
To identify the essential region of the scmp promoter, we constructed expression vectors combined with truncated promoters and the lap gene (Fig. 2). pTONA5a-lap, which included the full-length scmp promoter (FL), and expression vectors with four types of truncated scmp promoters (A, B, C, and D) were transformed into the S. lividans 1326 strain, and the obtained transformants were cultivated in PG medium. As shown in Fig. 3A, the A promoter, which lacks 148 bp of the 5′ region of the scmp promoter, showed three-fold higher LAP activity than FL. In addition, promoter C, which lacked 358 bp of the 5′ region of the scmp promoter, showed four-fold higher activity than that of FL. Promoter D, which harbors 46 bp of the 3′ region in the scmp promoter, also showed two-fold higher LAP activity than FL. Promoter B lacked 206 bp of the 3 region of the scmp promoter. The secreted LAP proteins were detected except for promoter B, and the content of LAP proteins increased in order from A and C to D compared with FL (Fig. 3B).
Truncated promoter C required no glucose and phosphate for lap expression
From the results of the truncated promoter in PG medium (Fig. 3), we expected that promoter C would require no glucose and phosphate and would be easy to use. Thus, we compared the secreted LAP activities using FL and truncated promoters A and C under the conditions of low phosphate concentration (0.1% phosphate), the exchanged carbon source of glucose with glycerol (Pgly), and TSB. The LAP activity using the FL promoter was the highest in the PG medium compared to the other three cultivation conditions (Fig. 4A). The LAP activity of promoter A was remarkably increased in the low-phosphate medium. This value was three times higher than that of FL. Furthermore, the LAP activity of promoter C was higher than that of FL and A under all conditions (Fig. 4A). Moreover, the protein expression levels under the four conditions by the C promoter were also increased compared with those under the FL promoter under PG (Fig. 4B). In particular, the LAP protein was strongly secreted by the combination of the C promoter and Pgly medium (Fig. 4B). In addition, the levels of other secreted proteins were reduced by using promoters C and TSB, although the content of secreted LAP protein was slightly lower than that in PG or Pgly (Fig. 4B).
Expression of other actinomyces proteins by promoter C
We attempted to produce other proteins with promoter C. FAE is an enzyme that releases ferulic acid from plant biomass (Uraji et al. 2013). When the Streptomyces FAE gene (Uraji et al. 2018) was cloned downstream of promoter C and expressed in PG and TSB media, enzyme production was confirmed in both (Fig. 5). To utilize the full-length scmp promoter, it was necessary to use PG medium; however, when cultured in PG medium, the expression of proteins other than the target protein was induced, and multiple proteins were often expressed simultaneously. It was necessary to purify the culture supernatant and extract only the target protein. In the case of FAE expressed by promoter C, the expression of contaminating proteins was suppressed in both PG and TSB medium, and the expression of other proteins was particularly reduced in TSB medium (Fig. 5). TG mainly connects proteins (glutamine side chain) to proteins (lysine side chains) by covalent bonds; by utilizing this property, it can be used in the processing of marine products, modification of meat, and processing of dairy products (Tokai et al. 2020). Similar to the FAE, TG was expressed by promoter C in PG or other media. The enzyme is released into the culture’s supernatant due to its secretory signal; there is a prosequence downstream of the secretory signal, which is cleaved by another enzyme (peptidase) secreted after TG. By removing the prosequence, the enzyme is converted into an active mature form of TG. When TG was expressed by promoter C in PG medium, a mature protein mixed with the prosequence form was expressed (Fig. 5). On the other hand, when TG was expressed in Pgly medium, conversion to the mature form of TG was completed. These results indicate that using promoter C facilitates the secretion of several types of proteins because the promoter does not restrict the carbon source.