Fermenco: The Technology

Introduction to SPL technology

The SPL Technology is based on randomisation of spacer sequences in the vicinity of fixed consensus boxes of standard promoters to generate libraries of promoters covering very broad ranges of activities in small steps of activity change. The use of the SPL approach eases the work for the genetic engineer to obtain an accurate optimisation of the expression of genes in a cell. In combination with a PCR approach the method offers the possibility for easy, accurate and individual optimisation of the expression of numerous genes in a single cell, which is not really feasible with other expression systems. Moreover, the random approach of the technology allows for simultaneous modulation of several genes in a cell.

The SPL approach has been developed for a broad range of microorganisms including various bacteria, yeast and even mammalian cell lines (Jensen and Hammer, 1998a, b, c; Solem and Jensen, 2002; Jeppson et al., 2003; Tornoe et al., 2002) as described below.

SPL Technology for prokaryotes

In bacteria a majority of promoters possess consensus sequences known as the hexamer boxes -35 and -10 with which the s-factor make site-specific contact and facilitates the binding of the RNA polymerase, which then allows for transcription to occur. The length of the spacer region connecting the two hexamers is also conserved.

The strategy to develop synthetic promoter libraries for prokaryotes is based on randomisation of the DNA sequences (spacers) that separate the individual consensus sequences of promoters (Jensen and Hammer, 1998a, b, c). The sequences of the spacer areas are less important for the strength of a particular promoter than the consensus sequences. By randomising many base pairs simultaneously in the vicinity of the consensus sequences, it is therefore possible to change the DNA structure and the binding of transcription factors to the promoter sequences. The promoter libraries obtained by this approach contain promoters with virtually any activity. So far the promoters have been developed for Lactococcus lactis, Leuconostoc carnosum, Lactobacillus plantarum, Bacillus subtilis and Escherichia coli.

The approach to apply the SPL technology for modulation of gene expression is greatly facilitated by incorporation of the randomised promoter sequences in a primer used for PCR amplification of the gene of interest (Fig. 2A) (Solem and Jensen, 2002). In short, this strategy allows for generation of either a truncated or a full-length version of the gene behind synthetic promoters depending on whether or not an extra gene copy is to be introduced (Fig. 2B).

Fig. 2. An overview of the approach used for modulating enzyme activities.
(A) Shows the chromosome with an arbitrary gene ( geneX) and its upstream region ( upsX). The promoter primer (N can be any of the four nucleobases while R is 50% A and 50% G) and two reverse primers are shown. (B) Shows the two possible PCR products, the first one of which contains a truncated geneX while the second contains a full length geneX. (C) Shows the resulting plasmids obtained after cloning of the PCR fragments. If a chromosomal promoter is to be replaced by a synthetic one then Strategy I is followed. If the resulting plasmid is to be used in a simple integration event then Strategy Ib is followed. If the resulting plasmid is to take part of a double crossover event then Strategy Ia is followed. If the plan is to introduce an additional gene copy into the chromosome then Strategy II is followed (From Solem and Jensen, 2002).

A truncated version of the gene can be used directly for replacing the chromosomal promoter with a synthetic promoter through homologous recombination, while the full-length version can be used for introducing an extra gene copy, for instance on the chromosome or on a plasmid (Fig. 2C).

Examples of promoter libraries by using the SPL approach for different prokaryotic organisms are presented in Fig. 3.

Fig. 3. Examples on synthetic promoter libraries in Escherichia coli and Bacillus subtilis.

SPL Technology for yeast
The need for modulation and fine-tuning of steady state gene expression in yeast also initiated the development of synthetic promoter library for this organism as follows:

Synthetic constitutive promoters
A second synthetic promoter library for yeast has also been developed for yeast (Jeppsson et al., 2003). In contrast to prokaryotic promoters that have a relatively compact and constant consensus sequence, upon which a synthetic promoter can be modeled, Saccharomyces cerevisiae appears to have less strict consensus sequence for promoters. Constructions of synthetic promoters were made by piecing together a combination of structures from several S. cerevisiae promoters and separate the conserved structures by stretches of randomized DNA. The resulting promoter library covers some three orders of magnitude between the lowest and the highest activity. Importantly, the range of promoter activities is covered in small steps, facilitating the fine-tuning of gene expression.

Example on a synthetic promoter library in yeast is presented in Fig. 4.

Fig. 4. Example on a synthetic promoter library in the yeast Saccharomyces cerevisiae

Regulated synthetic promoters
Another approach for construction of synthetic regulated promoters for S. cerevisiae is based on the ARG8 promoter, which has the advantage that the individual promoter elements (consensus sequences) are located on a relatively small DNA fragment. Degenerated oligonucleotides containing elements are regulatory features from the ARG8 promoter, and randomised spacers were designed, and promoters of different strengths are obtained (Andersen et al., 1998). The library of synthetic ARG8 promoters consists of a series of promoters which differ in strength and which are i) down regulated by amino acids in the medium in general and ii) down regulated specifically by arginine. These promoters therefore have the best of both worlds: the can be activated upon demand which is important for many industrial applications, and they have different strength in the uninduced as well as in the induced state which allows for metabolic optimisation and control analysis (Jensen and Hammer, 1998b).

SPL Technology for mammalian cell lines – a technology for gene therapy vectors

The fundamental understanding of transcription initiation from mammalian promoters is a process significantly more complicated than transcription from prokaryotic promoters. The SPL approach for modulation of gene expression is based on designing a synthetic promoter, the JeT promoter, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human b-actin and polyubiquitin C (UbC) promoters (Tornoe et al., 2002). A library of synthetic JeT-derived promoters with incremental differences spanning a 10-fold range of transcriptional activity in cell culture was subsequently obtained by randomising the spacer sequences separating the transcription factor binding sites included in the JeT promoter (Fig. 5). These findings established modification of spacer nucleotide composition as a means for modulating transcriptional activity from mammalian promoters to attain fine-tuning of expression levels. Importantly, a number of synthetic JeT promoters delivered sustained high-level reporter gene expression in in vitro cell culture studies at levels comparable to the strong, naturally occurring CMV, UbC and SV40 promoters, although being considerably smaller in size (Tornoe et al., 2002). Promoters from the JeT promoter library are therefore attractive candidates for inclusion in gene therapy vectors where DNA sequence length is a limiting factor, e.g. viral vectors.

Fig. 5 Example on a synthetic promoter library in a mammalian cell line

References
Andersen, H. W., Schürmann, R., Madsen, K., Pedersen, M. B., Købmann, B. J., Piskur, J., Hammer, K., and Jensen, P. R. 1998. Synthetic promoters for experimental control analysis. In Proceedings of the 8^th International Meeting on BioThermoKinetics: BioThermoKinetics In the Post Genomic Era (Larsson, C., Påhlman, I., and Gustafsson, L. eds.), Chalmers Reproservice, Göteborg, Sweden, pp. 11-17.

Jensen, P. R., and Hammer, K. 1998a. Artificial promoters for metabolic optimization. Biotechnol Bioeng. 58:191-195. Review

Jensen, P. R., and Hammer, K. 1998b. The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl. Environ. Microbiol. 64:82-87.

Jensen, P.R. and Hammer, K. 1998c. Artificial promoter libraries for selected organisms and promoters derived from such libraries. International Publication Number, WO 98/07846
Jeppsson, M., Johansson, B., Jensen, P. R., Hahn-Hägerdal, B., and Gorwa-Grauslund, M.F. 2003. The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains. Yeast 20:1263-1275.

Solem, C., and Jensen, P. R. 2002. Modulation of gene expression made easy. Appl Environ Microbiol. 68:2397-2403.

Tornøe, J., Kusk, P., Johansen, T.E., and Jensen, P. R. 2002. Generation of a Synthetic Mammalian Promoter Library by modification of sequences spacing transcription factor binding sites. Gene, 297:21-32 .