Synthetische Naturstoffbiosensor - Design, Konstruktion und Anwendung
Mikrobielle Ökologie und Angewandte Mikrobiologie
Zusammenfassung der Projektergebnisse
The rapid development of synthetic biology tools in recent decades has resulted in significant progress in the metabolic engineering of microbes. Despite that, the evaluation of phenotypes in engineering cycles remains a major bottleneck. Through our work aimed at improving the production of the antimycobacterial compound pamamycin in S. alboniger, we have developed the first design recommendations for the construction and tuning of biosensors to achieve better performance. We found that our G0 biosensors, consisting of a promoter (pamW) regulated by a native, pamamycin-responsive transcription factor, were poorly suited for downstream applications. We suggest that the limited operating and dynamic ranges of the sensor are due to the fact that the native receptor protein evolved to act over intracellular concentrations of ligand, rather than concentrations that are useful for applications of synthetic biology and biotechnology. This constraint was partially overcome by switching from selection (Neo) to screening (gusA) procedure, but this provided only a marginal increase in throughput of the procedure when compared to the conventional methods, like activity-based screening. Manipulating the regulatory (promoter strength, operators number and location) and signal output modules (translation efficiency) of the sensor influenced the basal level of output signal and dynamic range, but had no major effect on the operating range. To optimize the regulatory module of a sensor, detailed knowledge of the functional properties of the regulatory protein was required. Tuning the biosensor using promoters of different strengths led to lower basal signal and improved related dynamic range. The location of operator sequences had no significant effect, although care should be taken with placement of operators between -10 and -35, as this can affect overall promoter activity. Amplification of the output signal (by moderating the translation initiation rate through the alteration of the start codon of the reporter gene – gusAATG vs gusACTG in our case) should also be performed carefully, because of the high probability of increasing the system noise. From our experience it became obvious that the use of a strong promoter is preferred when tuning is aimed at increasing the dynamic range of the biosensor and a weak promoter when higher sensitivity is desired. A reporter with a high rate of translation initiation might be used as signal output module when a rapid response is aimed. We have used the developed design rules to generate several other (e.g. cheocardine) biosensors in order to show ist broad applicability. The development of biosensors for use in screening for antibiotic producers is more straightforward than the development of biosensors designed to screen for producers of other bacterial metabolites. The TetR family is the most abundant group of transcription factors in actinobacteriaand many members of that family are associated with the secondary metabolite biosynthetic gene clusters responsible for producing their ligands. The design principles described here will aid in the development and tuning of these biosensors.
Projektbezogene Publikationen (Auswahl)
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(2018) A set of synthetic versatile genetic control elements for the efficient expression of genes in Actinobacteria. Sci Rep. 8(1):491
Horbal L., Siegl T., Luzhetskyy A.
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(2018) Design, development and application of whole-cell based antibiotic-specific biosensor. Metab Eng. 47: 263-270
Rebets Y., Schmelz S., Gromyko O., Tistechok S., Petzke L., Scrima A., Luzhetskyy A.
