By guest contributor, Konstantinos Vavitsas
One thing absent in a heterologous pathway is substrate regulation: a molecular sensor that adjusts the abundance of the respective enzymes according to substrate availability. A research group from the California Institute of Technology addressed this issue by synthesizing a transcription regulation system that responds to vanillin.
Bacteria adjust to different environmental conditions mainly by modulating transcription. Internal and external stimuli affect regulatory elements, all of which formulate the complex transcriptional profile of an organism at any given moment. In synthetic biology, the use of synthetic genetic circuits and metabolic pathways, which are often unresponsive to the aforementioned native regulation, is a common procedure. Such behavior can be advantageous, as it allows the organism to remain unaffected by unpredictable perturbations. In many cases, however, the lack of interaction with the internal control leads to undesired effects: metabolic intermediate accumulation, reduced fitness, and decreased product yields. This sets the framework of a recent research paper from the groups of Stephen Mayo and Richard Murray, where they describe a de novo transcription factor that is regulated by vanillin.
Vanillin is a byproduct of lignin degradation and an important substrate for the flavor industry. It is a phenolic compound with cytotoxic effects. In this article, the researchers modified qacR, a tetR-family repressor to bind vanillin, which binds to DNA via a helix-turn-helix domain. In the absence of the effector molecule, qacR physically inhibits RNA polymerase from transcribing the region downstream the binding site (see figure). The inducer causes conformational changes that prevent this binding, thus activating the gene. The procedure for qacR engineering consists of three steps:
(i) computational protein design. The researchers superimposed vanillin with the qacR crystal structure, enabling them to identify the potential binding conformations and the crucial aminoacids. They subsequently came up with a number of protein mutants that could form the correct interactions with vanillin and did not have steric clashes.
(ii) cell-free initial screening. The proteins resulting from the previous step were tested in an in vitro transcription/translation system. This methodology let the authors of the paper validate the repression qacR imposes on a reporter gene (GFP), while screening the functionality of the engineered proteins. Since all of the initial modified proteins failed to be activated by vanillin, the researchers went back to step (i) and designed more modifications. This time, two mutants showed the desired phenotype.
(iii) in vivo validation. As a last step, the two proteins from step (ii) were tested in E.coli. Both were able to suppress GFP expression in the lack of activator. One of them responded positively to increasing concentrations of vanillin, resulting in increased fluorescence.
While comprehensive, the authors – to their credit – acknowledge and extensively discuss the problems with this study. In the case of qacR, there are high-resolution crystal structures available for both DNA-binding and non-binding states; that reduces the scope of this approach and restricts the potential applications to the better-studied protein targets. The limitations of the computational design became apparent during the in vitro screening. The binding model was probably wrong, hence the derived proteins were not responsive to the substrate. Finally, the in vitro system did not heed any warnings on the toxicity observed for both the engineered qacR and vanillin.
Nevertheless, the paper narrates a nice story: the aim of the study was achieved, the rationale is clear, and the thinking behind this synthetic system can be expanded and utilized in a more generic manner. Computer-aided design can pinpoint targets of interest and provide a starting point when none is discernible. A high-throughput system is vital for the validation of the (hopefully many) potential solutions the computational work resulted in. The implementation within the target organism is the final step that hides additional challenges and more unknowns.
The design process in synthetic biology has been addressed in a previous blog, the take-home message of which I will try to rephrase and apply in this work: plan rationally, but be prepared to embrace the chaos.
Disclaimer: Any views and opinions belong to the author and do not necessarily reflect the PLOS Synbio community.
Konstantinos Vavitsas is a PhD student at the Copenhagen Plant Science Centre, University of Copenhagen, working on the photosynthetic production of high-value compounds (Plant Power project). Find him on LinkedIn or follow him on Twitter.