Effect of Glucose or Glycerol as the Sole Carbon Source on Gene Expression from the Salmonella prpBCDE Promoter in Escherichia coli Sung Kuk Lee ² and Jay D. Keasling* ,²,‡,§ Departments of Chemical Engineering and Bioengineering, University of California, and Synthetic Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 We have developed an expression system (Salmonella-based pPro system) containing the Salmonella enterica prpBCDE promoter (P prpB ) and prpR encoding the positive transcriptional regulator of this promoter. In this study, the transcriptional efficiency of the pPro expression system was measured by placing the gene encoding the green fluorescent protein (gfp) under the control of P prpB and growing cells containing this construct in minimal medium supplemented with glucose or glycerol as a sole carbon source. In wild-type Escherichia coli (E. coli) BL21, the system exhibited high induced expression as well as high background expression; however, in E. coli JSB, a sbm-ygfDGHI deletion mutant of E. coli BL21(DE3), the system showed low background expression and high induced expression. The system exhibited homogeneous expression at the single-cell level, highly regulatable expression over a wide range of propionate concentrations, and fully induced expression at a low propionate concentration relative to that needed to induce the system in rich, undefined medium. The expression system is comparable to the widely used T7 promoter-driven expression systems in glucose or glycerol minimal medium. Introduction Escherichia coli (E. coli) has been used for the overproduction of recombinant proteins, because of its ability to grow rapidly, its well-characterized genetics, and the availability of an increasingly large number of expression vectors and mutant host cells (1, 9, 25). To date, a number of strong promoters are available for high-level expression in E. coli (1, 6, 11, 17, 26). Promoters typically used for high-level protein production have high maximal expression, low un-induced expression, consistent expression across all cells in a culture, and use of an inexpensive inducer. The T7 expression systems have been widely used for the overexpression of recombinant proteins in bacterial cells (21, 23). In the pET system, target genes are positioned downstream of the bacteriophage T7 late promoter on medium copy number plasmids. However, this system has high background expression (8, 10), which causes inconsistency in the expression level and instability of the clones. The lac repressor-operator control system and T7 RNA lysozyme have been used to reduce background expression (4, 20). Despite the success in reducing leaky expression from the T7 system, the inducer, IPTG, is very expensive and toxic and could easily contaminate the protein products due to its indigestibility by cells (2, 5). Thus, the use of IPTG for production of human therapeutic proteins is undesirable (1, 5). These problems can be circumvented by utilizing lactose as an inferior inducer or by using a thermoinducible T7 system (18). However, the use of thermosensitive variants of the LacI repressor protein gave low soluble protein yield, equal to 13% of the total cell proteins (24), because a temperature upshift can cause protein aggregation (12, 19). Moreover, partial induction cannot be achieved, and the induction method is neither simple nor cost-effective. Recently, we developed an E. coli expression system based on the Salmonella enterica (S. enterica) prpBCDE promoter and prpR that has tight regulation of gene expression in LB medium. In addition, the nontoxic inducer, propionate, is 1000- fold less expensive than IPTG (15). The system is known to be subject to catabolite repression in LB medium containing glucose (15). Here, we tested the system in minimal medium containing either glucose or glycerol as a sole carbon source to examine the effect of catabolite repression on expression from pPro. Both glucose and glycerol have been widely used for large-scale production of recombinant proteins in E. coli. We compared the strength, uninduced expression, and induced expression of Salmonella P prpB to those of the T7 promoter using gfp in E. coli. Materials and Methods Bacterial Strains and Media. The bacterial strains and plasmids used in this study are listed in Table 1. Cultures were grown in Luria-Bertani (LB) medium or M9 medium with glucose (w/v) or glycerol (w/v) at 37 °C. Cell growth was monitored as the optical density at a wavelength of 600 nm (OD 600 ). Media were supplemented with ampicillin (100 µg/ mL). Either isopropyl--D-thiogalactopyranoside (IPTG) or sodium propionate (pH 8.0) was used as the inducer. Determination of in Vivo Promoter Activities. Promoter activities were tested by using GFP as an indirect, quantitative measurement of the transcriptional properties of gfp (3, 27). Cells grown overnight in LB medium at 37 °C were inoculated (1:100) into M9 medium with either glucose or glycerol (5 mL in culture tube) and grown at 37 °C in a shaking incubator for * Corresponding author. Telephone: 510-495-2620. Fax: 510-495-2630. E-mail: keasling@berkeley.edu. ² Department of Chemical Engineering. ‡ Department of Bioengineering. § Synthetic Biology Department. 1547 Biotechnol. Prog. 2006, 22, 1547-1551 10.1021/bp060193f CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 10/25/2006