Propionate-Regulated High-Yield Protein Production in Escherichia coli Sung Kuk Lee, Jay D. Keasling Department of Chemical Engineering and Bioengineering, University of California, Synthetic Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720; telephone: 510-495-2620; fax: 510-495-2630; e-mail: keasling@berkeley.edu Received 20 July 2005; accepted 24 October 2005 Published online 6 December 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20784 Abstract: A new expression system containing the Sal- monella enterica prpBCDE promoter (P prpB ) responsible for expression of the propionate catabolic genes (prp BCDE) and prpR encoding the positive regulator of this promoter has been developed and tested. The main feat- ures of the expression system compared to those based on the bacteriophage T7 promoter are low background expression and high induced expression in Escherichia coli strains BL21, BL21(DE3), MG1655, and W3110. In addition, propionate is an inexpensive, simple-to-use, nontoxic inducer that is attractive for large-scale protein production. Hence, this new system is highly comple- mentary to the widely used T7 promoter-driven expres- sion systems. ß 2005 Wiley Periodicals, Inc. Keywords: expression system; prpBCDE promoter; prpR; Salmonella enterica; T7 promoter INTRODUCTION The T7-based expression systems have been widely used for overexpression of recombinant proteins in bacterial cells (Studier and Moffatt, 1986; Tabor and Richardson, 1992). In order to use the T7 system in Escherichia coli, the host must carry a chromosomal copy of the T7 gene 1 encoding T7 RNA polymerase, which specifically interacts with a T7 promoter (P T7 ), under control of an inducible promoter such as the lacUV5 promoter, P lacUV5 . A common host strain is E. coli BL21(DE3) (Studier and Moffatt, 1986; Studier et al., 1990). Upon induction by isopropyl b-D-thiogalactopyrano- side (IPTG), the gene fused to P T7 on the plasmid in E. coli BL21(DE3) becomes highly expressed. In particular, the T7 RNA polymerase exhibited superior processivity compared to E. coli housekeeping RNA polymerase (Studier and Moffatt, 1986). Despite great success in using the T7 system for protein overproduction, it has considerable problems. Overexpres- sion systems with IPTG-inducible promoters, such as P tac , P trc , or P T7 , require IPTG as an inducer; this inducer is costly, limiting the usefulness of these systems to small-scale production (Baneyx, 1999; Figge et al., 1988; Neubauer et al., 1992). In addition, IPTG could easily contaminate the products due to its indigestibility by cells (Figge et al., 1988). Moreover, the use of IPTG for production of human thera- peutic proteins is undesirable because of its toxicity (Baneyx, 1999; Figge et al., 1988). The serious issue of IPTG toxicity can be circumvented by utilizing lactose as an inferior inducer or by using a thermoinducible T7 system (Neubauer et al., 1992). More recently, a thermoinducible T7 system was developed by making use of thermosensitive variants of the LacI repressor protein that allow thermal induction of recombinant protein synthesis (Chao et al., 2002a,b; Wang et al., 2004); however, this system gave low soluble protein production yield equal to 13% of the total cell protein (Wang et al., 2004) because the use of thermoregulatable promoters that are inducible by a temperature upshift can cause protein aggregation (Kiefhaber et al., 1991; Rinas, 1996). Other problems afflicting the T7 expression system are inconsistency in expression level and instability of the clones due to leaky expression of the T7 RNA polymerase (Hattman et al., 1985; Hoang et al., 1999; Jeong and Lee, 1999), even though the gene was placed under control of the inducible P lacUV5 (Doherty et al., 1993). To reduce the leaky expres- sion, the lac repressor-operator control system is often used to block transcription from P T7 (Dubendorff and Studier, 1991), or T7 RNA lysozyme is expressed from plasmids pLysS or pLysL to inhibit the activity of the T7 RNA polymerase in the un-induced state (Moffatt and Studier, 1987; Studier, 1991). However, lacI-regulated genes are not efficiently repressed even in the absence of IPTG (Figs. 1B and 2A), and the T7 lysozyme causes cell fragility and sometimes reduces protein production (Hattman et al., 1985; Studier and Moffatt, 1986). Recently, the cluster of genes required for the catabolism of propionate was identified in both E. coli and Salmonella enterica (S. enterica) serovar Typhimurium LT2 and characterized for S. enterica (Horswill and Escalante- Semerena, 1997, 1999a,b; Textor et al., 1997). The expres- sion of prpBCDE genes encoding the propionate-degrading enzymes essential for propionate metabolism is dependent on ß 2005 Wiley Periodicals, Inc. Correspondence to: J.D. Keasling Contract grant sponsor: National Institutes of Health Contract grant number: GM070763-01