Fungal Synthetic Transcription Factor

Almost all functions in a cell are carried out by proteins. These proteins are synthesised from genes, where some genes are under control of transcription factors (TFs). TFs are a diverse group of proteins with widely different mechanisms, but they still share some common features. Some of these features are facilitating transcription which combines the ability to bind DNA, the ability to bind a ligand of interest, and further initiate transcription. The transcription initiation is specific to the system, since eukaryotes and prokaryotes have a different initiation of transcription, and this is reflected in the TF.

An hypothesis was made; can a prokaryotic TF be reprogrammed to function as an eukaryotic TF? The challenge with reprogramming a prokaryotic TF is transcription initiation and the answer is found in nature. Namely, vira that hijack the replication and transcription machinery of an eukaryotic, they have TFs that initiate transcription. One of these vira was the herpes simplex virus type 1, which encodes the transcription factor Viral Protein 16 (VP16) (Hirai. et al (2010)). The hypothesis was tested using the TetR from the tet operon of bacterial origin. TetR is a repressor in the original system in bacteria, but it was remodelled to be an activator in eukaryotes known as TetOn. Specifically, the binding site of TetR was moved in order to eliminate the steric hindrance and, subsequently, the transactivation domain of VP16 was fused to the repressor. This allowed the TetOn regulator to facilitate transcription in the presence of tetracycline, its inducer (Gossen et al (1995), Wanka et. al (2016)).

The idea proved to work in the TetOn system and it was applied to different known transcription factors, giving rise to a new type of transcription factor given many names. The most common are chimaera transcription factor, artificial transcription factor and synthetic transcription factor. We will refer to these transcription factors as synthetic transcription factors (sTFs). The primary organism where these sTF have been tested is in Saccharomyces cerevisiae and one of these sTFs, Z3EV, utilised the ligand binding domain from the human oestrogen receptor (hER) (McIsaac, et al. (2014)) and VP16 as transactivation domain, which made this yeast inducible by beta-estradiol. Another major point of interest was the DNA binding domain, since we prefer specific binding to our promoter with as few off binding sites as possible. Z3EV used the zinc finger repeating Zif268 as DNA binding domain (McIsaac, et al. (2014)).

This is a proof of concept that these sTF work in S. cerevisiae, but an estradiol biosensor is far from a furfural biosensor. A compound that is closer to furfural is benzoic acid, which has a known transcription factor from Rhodopseudomonas palustris, namely HbaR (Egland et. al (2000)). HbaR has already been shown to work in the framework of a sTF in S. cerevisiae with sensor a of benzoic acid derivatives (sBAD) (Castaño-Cerezo et. al (2020)) . Notably sBAD uses a different DNA binding domain and activation domain than Z3EV, but sBAD was constructed from a derivative of Z3EV. The DNA binding domain is LexA from bacterial origin and the transactivation domain was B112, which also originates from a group of bacterial activators (Ottoz et al. (2014)).

This brings us to our work, and our sTFs that were designed as a modular mix-and-match system. The standardised modules were a DNA binding domain, ligand sensing domains, and transactivation domains. The DNA binding domain used was LexA. There were two ligand sensing domains; HbaR and hmox1. The transactivation domains used in the system were B112 and VP16. In addition to the three modular domains, the linker between the DNA-binding domain and ligand sensing domain also had two variants: a short linker and a longer linker. The total number of combinations were thus eight sTF and of these eight possible sTF, six sTF were created.

16 variants of the HbaR domain were designed using rational computational design, to increase the binding affinity of the HbaR domain to furfural.

Figure 1: To the left is an overview of the modules used to construct our fungal synthetic transcription factor. To the right is a shortened list of the tested FunsTFs. An extended figure can be downloaded here.

Promoter

The modular sTF needs a specific promoter in order to facilitate transcription. The promoter was designed by fusing six LexO binding sites from an in-house and a minimal promoter of gdpA used in the TetOn system. The promoter LexO-Pmin would drive transcription of a mCherry (BBa_J06504) as a reporter gene. The sTF should bind to the LexO binding sites where the transactivation domain can facilitate transcription of mCherry, allowing a measurable output in response to the ligand.

In theory, the synthetic transcription factor should work as follows. The sTF starts being constitutively expressed from PgpdA (BBa_K4129024) and terminated by TcgrA (BBa_K3669007). The sTF contains a SV40 NLS in the C-terminus of the sTF so it did not interfere with the LexA domain. The SV40 NLS localises the sTF to the nucleus and it is hypothesised that the sTF will interact with the compound of interest here, namely furfural or benzoic acid, to induce conformational change, ultimately enabling the transactivation domain to facilitate transcription of mCherry.

Figure 2: SES, with modular sTF gene upstream of sTF regulated promoter and mCherry.

References

Castaño-Cerezo S, Fournié M, Urban P, Faulon JL, Truan G. Development of a Biosensor for Detection of Benzoic Acid Derivatives in Saccharomyces cerevisiae. Front Bioeng Biotechnol. 7, 372. (2020)

Egland PG, Harwood CS. HbaR, a 4-hydroxybenzoate sensor and FNR-CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation by Rhodopseudomonas palustris. J Bacteriol. 182, 100-1066. (2000)

Gossen M, Freundlieb S, Bender G, Müller G, Hillen W, Bujard H. Transcriptional activation by tetracyclines in mammalian cells. Science. 23, 1766-1769, (1995)

Hirai H, Tani T, Kikyo N. Structure and functions of powerful transactivators: VP16, MyoD and FoxA. Int J Dev Biol. 54, 1589-1596 (2010)

McIsaac, et al. Synthetic biology tools for programmaing gene expression without nutritional perturbations in Saccharomyces cerevisiae. Nucleic Acids Research, 42, pages. e48 (2014)

Ottoz DS, Rudolf F, Stelling J. Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae. Nucleic Acids Res. 42(2014

Wanka F, Cairns T, Boecker S, Berens C, Happel A, Zheng X, Sun J, Krappmann S, Meyer V. Tet-on, or Tet-off, that is the question: Advanced conditional gene expression in Aspergillus. Fungal Genet Biol. 89, 72-83 (2016)