Date Published: April 25, 2019
Publisher: Public Library of Science
Author(s): Natalia V. Geraskina, Elena V. Sycheva, Valery V. Samsonov, Natalia S. Eremina, Christine D. Hook, Vsevolod A. Serebrianyi, Nataliya V. Stoynova, Zhiqiang Wen.
Artificial metabolically regulated inducible expression systems are often used for the production of essential compounds. In most cases, the application of such systems enables regulating the expression of an entire group of genes in response to any internal signal such as an aerobic/anaerobic switch, a transition to stationary phase, or the exhausting of essential compounds. In this work, we demonstrate an example of another type of artificial autoinducible module, denoted a positive feedback module. This positive feedback module generates an inducer molecule that in turn enhances its own synthesis, promoting an activation signal. Due to the use of acetolactate, an intermediate of the L-valine biosynthetic pathway, as a specific inducer molecule, we realized a positive feedback loop in the biosynthetic pathway of branched chain amino acids. Such positive feedback was demonstrated to improve the production of a target compound.
At the present time, many useful substances, e.g., L-amino acids, biofuels, and fine chemicals, are produced at a large scale by microbiological fermentation [1–5]. For this purpose, specific strain-producers are required. The basic approach for developing a producing strain is activating a biosynthetic pathway making the desired substance by enhancing key enzyme gene expression. For this goal, many specific regulatory elements, native or artificial, and metabolic toggle switches are widely used for metabolic flux redirection [6–9]. For example, the gene of interest may be placed under control of a well-characterized regulatory region, such as the lac promoter, trp promoter, PR or PL promoters of λ phage, and tac promoter . These promoters have different strengths and, in the absence of corresponding repressors, provide high constitutive expression of the target genes. There are also inducer-free expression systems based on growth phase- or stress-specific promoters, such as the promoter of the rpsF operon or the promoter of the pst operon in gram-positive bacteria [11, 12].
In bacterial cells, many regulatory mechanisms are involved in negative feedback circuits that control the biosynthesis of metabolites, thereby preventing their excessive production, which is undesirable under certain conditions. Meanwhile, the activation of gene expression occurs in response to environmental or intracellular signals that indicate a necessity for adaptation to changing conditions (catabolism or transport of compounds, coordinated synthesis of separate structural elements in common biosynthetic pathways, stress defense, etc). In a native prokaryotic cell, examples of positive feedback circuits are rather rare and practically limited by signal transmission, such as “quorum sensing” [49–51]. In contrast, in artificial biological systems aimed to overproduce a target compound, such a strategy can be realized.