Gene Expression

We have constructed a series of broad-host-range vectors useful for multiple aspects of recombinant protein expression. The vectors are based on the replication elements (oriV + trfA) of the broad-host range RK2 plasmid and they harbor two alternative promoter/regulator systems (Pu/xylR or Pm/xylS) for expression of cloned genes. The Pu and Pm promoters have many natural inducers that wander passively into gram-negative cells, and expression levels of cloned genes are affected by the type of inducer and its concentration. The vector copy-numbers can be set to any level between 5 and 100 per genome in the host cell, which is useful for numerous purposes. We have applied these vectors for metabolic engineering experiments in different bacterial species to control the biosynthesis of biopolymers such as xhanthan, alginate, and amylose. We are also using these vectors for industrial production of various human proteins in high-cell-denisty cultures of Escherichia coli. To further study this expression system, we are producing mutant banks for the -10 region and the XylS binding sites of Pm, and also for the untranslated leader mRNA region, and the xylS gene. The aim is to identify mutants with modified expression properties (e.g. elevated expression level, low uinduced expression level), and finally to obtain better control and understanding of this expression system.

Key Papers

Berg, L., Lale, R., Bakke, I., Burroughs, N. and Valla, S. (2009). The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5'-untranslated part of mRNA. Microb. Biotechnol. 2(3), 379-389.

Bakke, I., Berg, L., Aune, T. E. V., Brautaset, T., Sletta, H., Tøndervik, A. and Valla, S. (2009). Random mutagenesis of the Pm promoter as a powerful strategy for improvement of recombinant-gene expression. Appl. Environ. Microbiol. 75(7), 2002–2011.

Brautaset, T., Lale, R., and Valla, S. (2008). Positively regulated bacterial expression systems. Microb. Biotechnol. 2(1), 15-30

Sletta, H., Tøndervik, A., Hakvåg, S., Aune, T. E. V., Nedal, A., Aune, R., Evensen, G., Valla, S., Ellingsen, T. E. and Brautaset, T. (2007). The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of Escherichia coli . Appl. Environ. Microbiol. 73 ,(3), 906–912.

Sletta H, Nedal A, Aune TE, Hellebust H, Hakvag S, Aune R, Ellingsen TE, Valla S, Brautaset T. (2004). Broad-Host-Range Plasmid pJB658 Can Be Used for Industrial-Level Production of a Secreted Host-Toxic Single-Chain Antibody Fragment in Escherichia coli . Appl Environ Microbiol . Dec;70(12):7033-9.

Winther-Larsen, H. C., Josefsen, K. D., Brautaset, T., and Valla, S. (2000). Parameters affecting gene expression from the Pm promoter in gram negative bacteria. Metabolic Engineering, 2(2): 79-91.

Blatny, J.M., Brautaset, T., Winther-Larsen, H.C., Karunakaran, P., and Valla, S. (1997). Improved broad-host-range vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid 38, 35-51.

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Biopolymers
Bioprospecting of marine microorganisms
Biosyntesis of L-Lysin from Methanol
Engineered antibiotic synthesis
Osmoregulation
Metabolic Engineering