Abstract
Bacterial expression systems play an indispensable role in the biosynthesis of recombinant proteins. Different proteins and the tasks associated with them may require different systems. The purpose of this work is to make an expression vector that allows switching on and off the expression of the target gene during cell incubation. Several expression vectors for use in Escherichia coli cells were developed using elements of the luxR/luxI type quorum sensing system of psychrophilic bacterium Aliivibrio logei. These vectors contain A. logei luxR2 and (optionally) luxI genes and LuxR2-regulated promoter, under the control of which a target gene is intended to be inserted. The synthesis of the target protein depends directly on the temperature: gene expression starts when the temperature drops to 22 °C and stops when it rises to 37 °C, which makes it possible to fix the desired amount of the target protein in the cell. At the same time, the expression of the target gene at a low temperature depends on the concentration of the autoinducer (L-homoserine N-(3-oxohexanoyl)-lactone, AI) in the culture medium in a wide range from 1 nM to 10 μM, which makes it possible to smoothly regulate the rate of target protein synthesis. Presence of luxI in the vector provides the possibility of autoinduction. Constructed expression vectors were tested with gfp, ardA, and ardB genes. At maximum, we obtained the target protein in an amount of up to 33% of the total cellular protein.
Key points
• A. logei quorum sensing system elements were applied in new expression vectors
• Expression of target gene is inducible at 22 °C and it is switched off at 37 °C
• Target gene expression at 22 °C is tunable by use different AI concentrations
Graphical Abstract
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Data availability
Constructed plasmids and their nucleotide sequences are available at https://nbp.biophystech.ru/. The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.
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Funding
Expression vector constructing and testing with gfp by SVB and ESS were supported of the Ministry of Science and Higher Education of the Russian Federation (grant MK-1164.2022.1.4). Theoretical assessment of applicability in biotechnology, literature review, and revision of the text of the publication, conducted by IVM, was supported by the Ministry of Science and Higher Education of the Russian Federation (project 1022060200069–0-1.6.2;1.6.4;1.6.5;1.6.10;1.6.19, “Development of technology for rational and highly productive use of agro- and bioresources, their efficient processing and obtaining safe and high-quality sources of food and non-food products”). Cloning and expression of ardA and ardB antirestriction genes, conducted by AAU and AAK, was supported by the Russian Science Foundation (project No. 22–74-00027).
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SVB and IVM conceived and designed research. SVB, ESS, RAE, AAU, and AAK conducted experiments. SVB and IVM analyzed data and wrote the original draft. SVB, ESS, AAU, AAK, and IVM reviewed and edited manuscript. All authors read and approved the manuscript.
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Bazhenov, S.V., Scheglova, E.S., Utkina, A.A. et al. New temperature-switchable acyl homoserine lactone-regulated expression vector. Appl Microbiol Biotechnol 107, 807–818 (2023). https://doi.org/10.1007/s00253-022-12341-y
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DOI: https://doi.org/10.1007/s00253-022-12341-y