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(12) United States Patent (10) Patent No.: US 7476,532 B2 Schneider Et Al

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(12) United States Patent (10) Patent No.: US 7476,532 B2 Schneider Et Al USOO7476532B2 (12) United States Patent (10) Patent No.: US 7476,532 B2 Schneider et al. (45) Date of Patent: Jan. 13, 2009 (54) MANNITOL INDUCED PROMOTER Makrides, S.C., "Strategies for achieving high-level expression of SYSTEMIS IN BACTERAL, HOST CELLS genes in Escherichia coli,” Microbiol. Rev. 60(3):512-538 (Sep. 1996). (75) Inventors: J. Carrie Schneider, San Diego, CA Sánchez-Romero, J., and De Lorenzo, V., "Genetic engineering of nonpathogenic Pseudomonas strains as biocatalysts for industrial (US); Bettina Rosner, San Diego, CA and environmental process.” in Manual of Industrial Microbiology (US) and Biotechnology, Demain, A, and Davies, J., eds. (ASM Press, Washington, D.C., 1999), pp. 460-474. (73) Assignee: Dow Global Technologies Inc., Schneider J.C., et al., “Auxotrophic markers pyrF and proC can Midland, MI (US) replace antibiotic markers on protein production plasmids in high cell-density Pseudomonas fluorescens fermentation.” Biotechnol. (*) Notice: Subject to any disclaimer, the term of this Prog., 21(2):343-8 (Mar.-Apr. 2005). patent is extended or adjusted under 35 Schweizer, H.P.. "Vectors to express foreign genes and techniques to U.S.C. 154(b) by 0 days. monitor gene expression in Pseudomonads. Curr: Opin. Biotechnol., 12(5):439-445 (Oct. 2001). (21) Appl. No.: 11/447,553 Slater, R., and Williams, R. “The expression of foreign DNA in bacteria.” in Molecular Biology and Biotechnology, Walker, J., and (22) Filed: Jun. 6, 2006 Rapley, R., eds. (The Royal Society of Chemistry, Cambridge, UK, 2000), pp. 125-154. (65) Prior Publication Data Stevens, R.C., “Design of high-throughput methods of protein pro duction for structural biology.” Structure, 8(9):R177-R185 (Sep. 15. US 2006/O292671 A1 Dec. 28, 2006 2000). Related U.S. Application Data Primary Examiner Nancy Vogel (60) Provisional application No. 60/687,763, filed on Jun. (74) Attorney, Agent, or Firm—Alston & Bird LLP 6, 2005. (57) ABSTRACT (51) Int. Cl. CI2N I/2 (2006.01) The present invention provides methods for producing CI2N 15/09 (2006.01) recombinant peptides in a bacterial host utilizing a mannitol, C7H 2L/04 (2006.01) arabitol, glucitol, or glycerol-inducible promoter, wherein the (52) U.S. Cl. ................ 435/252.34; 435/69.1:536/23.1; host bacterial cell that produces the peptide has been rendered 536/24.1 incapable of degrading or metabolizing mannitol, arabitol, or (58) Field of Classification Search ....................... None glucitol, or derivatives or analogues thereof. The present See application file for complete search history. invention provides bacterial cells that have been genetically altered to inhibit the metabolism or degradation of mannitol, (56) References Cited glucitol, or arabitol, or derivatives or analogues thereof. The OTHER PUBLICATIONS present invention utilizes mannitol, arabitol, glucitol, or glyc erol to induce expression of a target polypeptide from an Brinker, P. et al., “Structure and function of the genes involved in inducible promoter, allowing for the use of an inexpensive mannitol, arabitol and glucitol utilization from Pseudomonas and stable carbon Source inducer in the fermentation pro fluorescens DSM50106.” Gene, 206(1): 117-26 (Jan. 5, 1998). Hannig. G., and Makrides, S.C., "Strategies for optimizing cesses for the production of recombinant peptides. heterologous protein expression in Escherichia coli,' Trends Biotechnol., 16(2):54-60 (Feb. 1998). 12 Claims, 17 Drawing Sheets U.S. Patent Jan. 13, 2009 Sheet 1 of 17 US 7.476,532 B2 Mannitol, glucitol, arabitol Outside Mannitol Or Arabitol inside inside l mtD (NAD+) fructOSe Xylulose rtZ rtY fructose-6-phosphate xylulose-5-phosphate glycolysis pentose phosphate cycle FIGURE 1 U.S. Patent Jan. 13, 2009 Sheet 2 of 17 US 7476,532 B2 2S 00‘ON‘GI"OESv9VV99VLOOOOOLOVOLOOLLOL(?u0?)IVV10L(?u/L)VOL?11 --><= ZCHRI[10][H U.S. Patent Jan. 13, 2009 Sheet 5 Of 17 US 7476,532 B2 uo?eu?uuue),STEIou6 g-d-19-doO uo?60iOL uo?60IGº S(HIRIQ?IH I-G99INWOG]d oud U.S. Patent Jan. 13, 2009 Sheet 6 of 17 US 7476,532 B2 | N. O.) N. O CY (Y) V. V. S 3 O O a ?a O. O. U.S. Patent Jan. 13, 2009 Sheet 7 of 17 US 7476,532 B2 800 700 600 500 400 300 200 100 O 10 20 30 40 50 time after induction (h) FIGURE 7 U.S. Patent Jan. 13, 2009 Sheet 8 of 17 US 7476,532 B2 -8-2% glycerol not induced CD -- 2% glycerol induced -V-5% glycerol not induced -v- 5% glycerol induced -e-9.5% glycerol not induced -0-9.5% glycerol induced 10 20 30 40 50 time after induction (h) FIGURE 8 U.S. Patent Jan. 13, 2009 Sheet 9 Of 17 US 7476,532 B2 downstream of mtDYZ upstream of mtl RXFO5464 pyrf pDOW1373-3 tetR tetA FIGURE 9 U.S. Patent Jan. 13, 2009 Sheet 10 of 17 US 7476,532 B2 11 10 8 g -O-OD glc -- ODmt 5 2 -e-glo g/L 4 Amt g/L 3 to 2 S 1 O time (h) after inoculation FIGURE 10 U.S. Patent Jan. 13, 2009 Sheet 11 of 17 US 7476,532 B2 3500 -H2% gol No inductionAU 530 --5% gol No induction $250 -0-95% gol No induction 2000 --2%gol Induction at 24hr EFT 1so -A-5% gol Induction at 24 hr EFT s -e-9.5% gol Induction at 24h EFT 100 500 O Z O 10 20 30 40 50 60 time (h) after induction FIGURE 11 U.S. Patent Jan. 13, 2009 Sheet 12 of 17 US 7476,532 B2 a 2 4000 --5% gol mutant 3 3000 --5% gol wildtype 3 -A-9.5% gol mutant 9 - A - 9.5% gol wildtype R 2000 C CD N 1000 O 2 o O 10 20 30 40 50 60 time after induction (h) -o- 10%glo mutant -e-10%glc wildtype O 10 20 30 40 50 60 time after induction (h) FIGURE 12 U.S. Patent Jan. 13, 2009 Sheet 13 of 17 US 7476,532 B2 Fluorescence at 21-24 hr post induction 4000 -B-12.5% glucose-grown cells 3000 -V-9.5% glycerol-grown cells 2000 1000 O -0.25 0.00 0.25 0.50 0.75 1.00 1.25 % mannitol added at induction FIGURE 13 U.S. Patent Jan. 13, 2009 Sheet 14 of 17 US 7476,532 B2 O V O O O O O O O. O. O. f) O D O O CY Of CN CN V v. e3ue3SeOnly peZeu.JON U.S. Patent US 7476,532 B2 [106]Smo?JeA 3,01130sauDin? #0.0, SICHRIQOIH 30'u3Osalon? U.S. Patent Jan. 13, 2009 Sheet 16 of 17 US 7476,532 B2 Optical density 350 S C D CO O r C CD C s .9 5. O O 2O 40 60 80 EFT (h) -- corn syrup -- glycerol Fluorescence -20 - 10 0 10 20 30 40 50 60 Time after induction (h) -o- Corn Syrup -- glycerol FIGURE 16 US 7,476,532 B2 1. 2 MANNITOL INDUCED PROMOTER induction; stability of the inducer in the cell culture medium SYSTEMS IN BACTERAL, HOST CELLS so that the inducer need only be added once during the peptide accumulation phase of a fermentation process; easy passage CROSS-REFERENCE TO RELATED of the inducer of the promoter through the cell membrane so APPLICATIONS that the external concentrations of the inducer are directly related to the internal concentration within the cell, and lin This application claims priority to U.S. provisional patent early related to peptide production; and ability to be used in application No. 60/687,763 filed Jun. 6, 2005. tandem with other promoters. Such an ideal promoter allows recombinant peptides to be induced at high levels efficiently FIELD OF THE INVENTION 10 and inexpensively. The present invention relates to the field of recombinant One regulatable promoter that has found widespread use in peptide production. In particular, the present invention pro bacterial fermentation process for the production of recom vides for the improved production of recombinant peptides binant peptides is the lac promoter, and its derivatives, espe utilizing a mannitol, glucitol, or arabitol-inducible promoter, 15 cially the tac and trc promoters. In commercial fermentation wherein the host cell expressing the peptide from the pro systems using a lac-type promoter, the inducer isopropyl-3- moter is rendered deficient in its ability to metabolize or D-1-thiogalactopyranoside (“IPTG’) is almost universally degrade mannitol, glucitol, or arabitol, which is utilized to employed. IPTG is, however, expensive and must be carefully induce the promoter. In addition, the invention provides for controlled since it is significantly toxic to biological systems. improved bacterial host cells for the production of recombi Standard IPTG preparations are currently available at about nant peptides, wherein the bacterial cell has been rendered USD $18 per gram or about USD $125 per 10 grams. In deficient in its ability to metabolize or degrade mannitol, addition, standard IPTG preparations may contain dioxane, a glucitol, or arabitol, or analogues or derivatives thereof. toxin. Dioxane-free IPTG is available on the market, but costs roughly twice the price of standard IPTG. Furthermore, envi BACKGROUND OF THE INVENTION 25 ronmental and health regulatory issues arise in regard to the presence of IPTG in the fermentation, or in the protein puri The use of bacterial cells to produce recombinant peptides fied from the fermentation, given the IPTG toxicity risks to is increasing in commercial importance. One of the goals in humans, animals, and other biological organisms. developing a bacterial expression system is the production of Because of the toxicities and costs associated with IPTG, high quality target polypeptides quickly, efficiently, and 30 alternative inducible promoter systems have been proposed abundantly.
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