Abstract
A novel inducible gene expression system using p-isopropyl benzoate (cumate) as an inducer was developed for the industrial production hosts, Bacillus subtilis and Bacillus megaterium. Cumate is non-toxic to the host, inexpensive, and carbon source-independent inducer which provides an economical option for large-scale production of valuable proteins and chemicals from Bacillus strains. The synthetic cumate-inducible system was constructed by combining the strong constitutive Bacillus promoter Pveg with regulatory elements of the Pseudomonas putida, CymR repressor, and its operator sequence CuO. The designed expression cassette containing a sfGFP reporter under the cumate-inducible promoter was assembled into a Bacillus-E. coli shuttle and gene expression investigated in the two Bacillus strains. Characterization of gene expression levels, expression kinetics, and dose-response to cumate inducer concentration confirmed high-level, but tightly controlled GFP reporter expression in tunable, cumate concentration-dependent manner. Unexpectedly, this expression system works equally well for Escherichia coli, resulting in a platform that can be used both in gram-positive and gram-negative expression host. Its tight regulation and controllable expression makes this system useful for metabolic engineering, synthetic biology studies as well industrial protein production.
Similar content being viewed by others
References
Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2005) Tuning genetic control through promoter engineering. Proc Natl Acad Sci U S A 102(36):12678–12683. https://doi.org/10.1073/pnas.0504604102
Bernhard K, Schrempf H, Goebel W (1978) Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. J Bacteriol 133(2):897–903
Biedendieck R, Gamer M, Jaensch L, Meyer S, Rohde M, Deckwer WD, Jahn D (2007) A sucrose-inducible promoter system for the intra- and extracellular protein production in Bacillus megaterium. J Biotechnol 132(4):426–430. https://doi.org/10.1016/j.jbiotec.2007.07.494
Biedendieck R, Malten M, Barg H, Bunk B, Martens JH, Deery E, Leech H, Warren MJ, Jahn D (2010) Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium. Microb Biotechnol 3(1):24–37. https://doi.org/10.1111/j.1751-7915.2009.00125.x
Borriss R, Danchin A, Harwood CR, Medigue C, Rocha EPC, Sekowska A, Vallenet D (2018) Bacillus subtilis, the model gram-positive bacterium: 20 years of annotation refinement. Microb Biotechnol 11(1):3–17. https://doi.org/10.1111/1751-7915.13043
Borujeni AE, Channarasappa AS, Salis HM (2014) Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res 42(4):2646–2659. https://doi.org/10.1093/nar/gkt1139
Chen PT, Shaw JF, Chao YP, David Ho TH, Yu SM (2010) Construction of chromosomally located T7 expression system for production of heterologous secreted proteins in Bacillus subtilis. J Agric Food Chem 58(9):5392–5399. https://doi.org/10.1021/jf100445a
Choi YJ, Morel L, Bourque D, Mullick A, Massie B, Miguez CB (2006) Bestowing inducibility on the cloned methanol dehydrogenase promoter (PmxaF) of Methylobacterium extorquens by applying regulatory elements of Pseudomonas putida F1. Appl Environ Microbiol 72(12):7723–7729. https://doi.org/10.1128/AEM.02002-06
Choi YJ, Morel L, Le Francois T, Bourque D, Bourget L, Groleau D, Massie B, Miguez CB (2010) Novel, versatile, and tightly regulated expression system for Escherichia coli strains. Appl Environ Microbiol 76(15):5058–5066. https://doi.org/10.1128/AEM.00413-10
Eaton RW (1996) p-Cumate catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA carrying the cmt operon. J Bacteriol 178(5):1351–1362. https://doi.org/10.1128/jb.178.5.1351-1362.1996
Engstrom MD, Pfleger BF (2017) Transcription control engineering and applications in synthetic biology. Synth Syst Biotechnol 2(3):176–191. https://doi.org/10.1016/j.synbio.2017.09.003
Eppinger M, Bunk B, Johns MA, Edirisinghe JN, Kutumbaka KK, Koenig SS, Creasy HH, Rosovitz MJ, Riley DR, Daugherty S, Martin M, Elbourne LD, Paulsen I, Biedendieck R, Braun C, Grayburn S, Dhingra S, Lukyanchuk V, Ball B, Ul-Qamar R, Seibel J, Bremer E, Jahn D, Ravel J, Vary PS (2011) Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319. J Bacteriol 193(16):4199–4213. https://doi.org/10.1128/JB.00449-11
Gamer M, Starnmen S, Biedendieck R, Frode D, Yang Y, Jahn D (2007) Bacillus megaterium - an alternative expression system. J Biotechnol 131(2):S220–S220. https://doi.org/10.1016/j.jbiotec.2007.07.396
Gamer M, Frode D, Biedendieck R, Stammen S, Jahn D (2009) A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium. Appl Microbiol Biotechnol 82(6):1195–1203. https://doi.org/10.1007/s00253-009-1952-5
Guiziou S, Sauveplane V, Chang HJ, Clerte C, Declerck N, Jules M, Bonnet J (2016) A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Res 44(15):7495–7508. https://doi.org/10.1093/nar/gkw624
Harwood CR, Cutting SM (1990) Molecular biological methods for Bacillus. Wiley, Chichester
Harwood CR, Pohl S, Smith W, Wipat A (2013) Bacillus subtilis: model gram-positive synthetic biology chassis. Microbial Synthetic Biology 40:87–117. https://doi.org/10.1016/B978-0-12-417029-2.00004-2
Horbal L, Fedorenko V, Luzhetskyy A (2014) Novel and tightly regulated resorcinol and cumate-inducible expression systems for Streptomyces and other actinobacteria. Appl Microbiol Biotechnol 98(20):8641–8655. https://doi.org/10.1007/s00253-014-5918-x
Kaczmarczyk A, Vorholt JA, Francez-Charlot A (2013) Cumate-inducible gene expression system for sphingomonads and other Alphaproteobacteria. Appl Environ Microbiol 79(21):6795–6802. https://doi.org/10.1128/AEM.02296-13
Keasling JD (1999) Gene-expression tools for the metabolic engineering of bacteria. Trends Biotechnol 17(11):452–460
Ming YM, Wei ZW, Lin CY, Sheng GY (2010) Development of a Bacillus subtilis expression system using the improved Pglv promoter. Microb Cell Factories 9. Artn 55. https://doi.org/10.1186/1475-2859-9-55
Moore SJ, Lawrence AD, Biedendieck R, Deery E, Frank S, Howard MJ, Rigby SE, Warren MJ (2013) Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12). Proc Natl Acad Sci U S A 110(37):14906–14911. https://doi.org/10.1073/pnas.1308098110
Mullick A, Xu Y, Warren R, Koutroumanis M, Guilbault C, Broussau S, Malenfant F, Bourget L, Lamoureux L, Lo R, Caron AW, Pilotte A, Massie B (2006) The cumate gene-switch: a system for regulated expression in mammalian cells. BMC Biotechnol 6:43. https://doi.org/10.1186/1472-6750-6-43
Ohta Y, Maeda M, Kudo T (2001) Pseudomonas putida CE2010 can degrade biphenyl by a mosaic pathway encoded by the tod operon and cmtE, which are identical to those of P. putida F1 except for a single base difference in the operator-promoter region of the cmt operon. Microbiol-SGM 147:31–41. https://doi.org/10.1099/00221287-147-1-31
Overkamp W, Beilharz K, Weme RDO, Solopova A, Karsens H, Kovacs AT, Kok J, Kuipers OP, Veening JW (2013) Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl Environ Microbiol 79(20):6481–6490. https://doi.org/10.1128/Aem.02033-13
Phan TT, Tran LT, Schumann W, Nguyen HD (2015) Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microb Cell Factories 14:72. https://doi.org/10.1186/s12934-015-0255-z
Ren Q, Henes B, Fairhead M, Thony-Meyer L (2013) High level production of tyrosinase in recombinant Escherichia coli. BMC Biotechnol 13:18. https://doi.org/10.1186/1472-6750-13-18
Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27(10):946–U112. https://doi.org/10.1038/nbt.1568
Schumann W (2007) Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol 62(62):137–189. https://doi.org/10.1016/S0065-2164(07)62006-1
Selinger LB, McGregor NF, Khachatourians GG, Hynes MF (1990) Mobilization of closely related plasmids pUB110 and pBC16 by Bacillus plasmid pXO503 requires trans-acting open reading frame beta. J Bacteriol 172(6):3290–3297
Stammen S, Muller BK, Korneli C, Biedendieck R, Gamer M, Franco-Lara E, Jahn D (2010) High-yield intra- and extracellular protein production using Bacillus megaterium. Appl Environ Microbiol 76(12):4037–4046. https://doi.org/10.1128/AEM.00431-10
Vary PS, Biedendieck R, Fuerch T, Meinhardt F, Rohde M, Deckwer WD, Jahn D (2007) Bacillus megaterium--from simple soil bacterium to industrial protein production host. Appl Microbiol Biotechnol 76(5):957–967. https://doi.org/10.1007/s00253-007-1089-3
Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta Mol Cell Res 1694(1–3):299–310. https://doi.org/10.1016/j.bbammcr.2004.02.011
Zhang G, Quin MB, Schmidt-Dannert C (2018) Self-assembling protein scaffold system for easy in vitro coimmobilization of biocatalytic cascade enzymes. ACS Catal 8(6):5611–5620. https://doi.org/10.1021/acscatal.8b00986
Acknowledgments
The authors thank Dr. Maureen Quin for helpful discussion about the project, and Dr. Antony Dean and Dr. Xiao Yi at the University of Minnesota for flow cytometer use.
Funding
This work was supported by funding from the Defense Advanced Research Projects Agency (DARPA) #HR0011-17-2-0038.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no competing interests.
Electronic supplementary material
ESM 1
(PDF 1074 kb)
Rights and permissions
About this article
Cite this article
Seo, SO., Schmidt-Dannert, C. Development of a synthetic cumate-inducible gene expression system for Bacillus. Appl Microbiol Biotechnol 103, 303–313 (2019). https://doi.org/10.1007/s00253-018-9485-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-018-9485-4