Exploring the limits of the xylS/Pm expression cassette for recombinant protein production in Escherichia coli:

Recombinant protein production is applied for a wide range of applications, however, so far no universal system exists which allows for production of sufficient amounts of any protein of interest. Thus improvement of existing systems and better understanding of their molecular function is still as important as the discovery and development of new expression tools. Particularly expression systems which allow a certain degree of flexibility and adaptation concerning growth conditions, expression levels, and choice of expression host appear promising. The work described in this thesis concentrates on vectors with these characteristics. They combine the positively regulated XylS/Pm promoter system with the broad-host-range RK2 mini-replicon. They are well established and have been shown to be capable of industrial-level production of several medically important proteins. Intensive studies, especially with bla (coding for β-lactamase) as reporter gene, and mutagenesis of several of the control elements in the applied system have resulted in identification of variants that increase expression levels also for other reporter genes tested. In this thesis it was aimed to assess, whether there was potential for further improvements of the vectors, and indeed, a combination of several high-expression variants in one cassette resulted in higher expression levels than those achieved by single variants. With the reporter gene bla about 75 times as much protein as with the wild type system could be produced when variants for Pm, the DNA-region corresponding to the 5’-untranslated region of mRNA (5′-UTR) upstream of the reporter gene, and the gene of the positive regulator of the system, XylS, were combined. As a result of this combination corresponding transcript levels could also be increased up to 63-fold. Testing of these new vectors with several other reporter genes demonstrated that expression of them in general could be increased, however, only within the limits of gene- and protein-specific boundaries. This was particularly investigated in the case of celB (coding for phosphoglucomutase), and a new role for the plasmid copy number was discovered: an increase in plasmid copy number does not only lead to higher expression levels due to increased production of transcript, but also facilitates better exploitation of the resources due to the larger distribution of mRNA across the cell. The transcription factor XylS is of central importance for the here applied vectors and a study of its expression revealed new insights into limitations and mechanisms by which it activates Pm. Its expression could not be increased by codon optimization, which was partly, but not only, caused by secondary structure formation in its translation initiation region. However, XylS expression could be improved either by exchange of its 5’-UTR and/or the promoter from which it was expressed. The choice of the positively regulated promoter system ChnR/Pb for transcription of xylS allowed variation of XylS expression levels with concurrent investigation of its mechanisms of activation of Pm. A synthetic operon construct permitted to detect XylS at physiological concentrations. This revealed the existence of a roof for induced expression from Pm depending on XylS expression levels, which probably is caused by oligomerization of the transcription factor at high levels inside the cell. This indicates that it is redundant to increase XylS expression levels beyond the saturating concentration, and the maximum induction ratio at Pm with the wild type system was found to be around 700-fold, with bla as reporter gene. Regulation of XylS expression was also demonstrated to be useful in reduction of background expression from Pm, which often is increased, when induced expression is improved. This might provide benefits for expressing host-toxic proteins. Also background levels of the vectors which combine several high-expression variants, could be reduced by XylS regulation. Another way of background reduction and enhancement of the induction ratio was demonstrated to be the usage of low-expression 5′-UTR variants, which turned out to be a promising approach for metabolic engineering purposes. This also revealed Rho-dependent termination of celB-transcription at low expression levels. The combination of high-expression variants turned out to be useful in reduction of the metabolic load, which is imposed onto cells by recombinant protein production. The high expression levels which were achieved by this approach allowed expression from one single copy on the chromosome at levels higher than that of the wild type plasmid system. The findings described here demonstrate that there is potential to further improve expression from vectors that combine the XylS/Pm expression system with the RK2 mini-replicon. In addition there already exist variants that cause varying phenotypes for several of the elements in these vectors, and it offers a large toolkit with many possibilities for combination of these features. Thus the system probably can be adapted to expression requirements of many proteins. Some of the reported findings can also be seen in a broader context and potentially adapted to other systems, as for example the combination of variants with defined phenotype. Low-expression variants might be helpful to reduce background levels of strong promoters like in the largely applied T7-system. The discovery of the new role in plasmid copy number might be of importance for every expression system and the mechanisms which were revealed for XylS may potentially also exist for other and related transcription factors

Regulation of the expression level of transcription factor XylS reveals new functional insight into its induction mechanism at the Pm promoter

Background XylS is the positive regulator of the inducible Pm promoter, originating from Pseudomonas putida, where the system controls a biochemical pathway involved in degradation of aromatic hydrocarbons, which also act as inducers. The XylS/Pm positive regulator/promoter system is used for recombinant gene expression and the output from Pm is known to be sensitive to the intracellular XylS concentration. Results By constructing a synthetic operon consisting of xylS and luc, the gene encoding luciferase, relative XylS expression levels could be monitored indirectly at physiological concentrations. Expression of XylS from inducible promoters allowed control over a more than 800-fold range, however, the corresponding output from Pm covered only an about five-fold range. The maximum output from Pm could not be increased by introducing more copies of the promoter in the cells. Interestingly, a previously reported XylS variant (StEP-13), known to strongly stimulate expression from Pm, caused the same maximum activity from Pm as wild-type XylS at high XylS expression levels. Under uninduced conditions expression from Pm also increased as a function of XylS expression levels, and at very high concentrations the maximum activity from Pm was the same as in the presence of inducer. Conclusion According to our proposed model, which is in agreement with current knowledge, the regulator, XylS, can exist in three states: monomers, dimers, and aggregates. Only the dimers are active and able to induce expression from Pm. Their maximum intracellular concentration and the corresponding output from Pm are limited by the concentration-dependent conversion into inactive aggregates. Maximization of the induction ratio at Pm can be obtained by expression of XylS at the level where aggregation occurs, which might be exploited for recombinant gene expression. The results described here also indicate that there might exist variants of XylS which can exist at higher active dimer concentrations and thus lead to increased expression levels from Pm

Strong stimulation of recombinant protein production in <it>Escherichia coli </it>by combining stimulatory control elements in an expression cassette

<p>Abstract</p> <p>Background</p> <p>The XylS/<it>Pm</it> expression system has been used to produce recombinant proteins at industrial levels in <it>Escherichia coli</it>. Activation of transcription from the <it>Pm</it> promoter takes place in the presence of benzoic acid or derivatives of it. Previous mutagenesis studies resulted in identification of several variants of the expression control elements <it>xylS</it> (X), <it>Pm</it> (P) and the 5'-untranslated region (U) that individually gave rise to strongly stimulated expression. The goal of this study was to test if combination of such stimulatory mutations in the same expression vectors would lead to further increase of expression levels.</p> <p>Results</p> <p>We combined X, P and U variants that were originally identified due to their ability to strongly stimulate expression of the reporter gene <it>bla</it> (resistance to penicillin). Combination of optimized elements stimulated <it>bla</it> expression up to 75-fold (X, P and U combined) relative to the wild-type system, while accumulated transcript levels increased about 50-fold. This is much more than for the elements individually. We also tested combination of the variant elements on two other and unrelated genes, <it>celB</it> (encoding phosphoglucomutase) and the human growth factor gene <it>gm-csf</it>. Protein production from these genes is much more efficient than from <it>bla</it> in the wild-type system, but expression was still significantly stimulated by the combination of X, P and U variants, although not to the same extent as for <it>bla</it>.</p> <p>We also integrated a single copy of the expression cassette with each gene into the <it>E. coli</it> chromosome and found that the expression level from this single copy was higher for <it>bla</it> than for the wild-type plasmid system, while it was lower for <it>celB</it> and <it>gm-csf</it>.</p> <p>Conclusion</p> <p>Our results show that combination of stimulatory expression control elements can be used to further increase production of different proteins in <it>E. coli</it>. For one reporter gene (<it>bla</it>) this allowed for more protein production from a single gene copy integrated on the chromosome, compared to the wild-type plasmid system. The approach described here should in principle be applicable for improvement of any expression cassette.</p

PLL-Stabilized Optical Communications in Millimeter-Wave RoF Systems

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Sideband stabilization in the presence of LO phase noise: analysis and system demonstrator

International audienceWe present a technique allowing the stabilization and tuning of a modulation sideband in the presence of high carrier frequency jitter and increased carrier phase noise. This technique is of particular interest in communication systems where oscillators providing the carrier signal cannot be stabilized by a conventional phase-locked loop, such as systems relying on low-cost optical LO generation techniques. The results obtained in simulation are validated by measurements carried out on a modular system demonstrator. Index Terms-frequency jitter, phase noise, phase-locked loop, radio-over-fiber systems, mode-locked lasers

Modulation Techniques in Radio-over-Fiber Systems

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