Table 1 Representative examples and design principles of engineering metabolite-responsive transcriptional factors (TFs) in eukaryotes
Host | TF and source | Effector (small metabolite) | Reporter | Transcriptional effect | Characteristic/architecture |
|---|---|---|---|---|---|
Mammalian cells | |||||
Hela cells | TetR-VP16 | Doxycycline | Transcription activator | TetR blocks the transcription of tetA, which encoding for the tetracycline efflux pump. When tetracycline binds to TetR, tetA would be expressed and functioning as tetracycline export pump [23] | |
COS-1 cells | FapR-VP16 | Malonyl-CoA | Luciferase or a destabilized short half-life GFP | Transcriptional activator | Bacillus subtilis FapR as a transcriptional repressor inhibits most of genes involved in fatty acid biosynthesis. FapR undergoes a conformation shift when malonyl-CoA binding to it and thus releases operators of many fatty acid synthesis genes. While VP16 is a herpes virus transcriptional activator. In frame fusion of FapR with VP16 converts FapR into a transcriptional activator in the absence of malonyl-CoA. Nucleus localization signal (NLS) is used [24] |
Human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells | E.coli PhlF, or PhlF-VP16 | 2,4-Diacetylphloroglucinol | YFP | Transcriptional repressor (PhlF) or Transcriptional activator (PhlF-VP16) | PhlF is equipped with eukaryotic specific nuclear localization signal (NLS). And multiple operator sites are integrated into responsive promoters [25] |
Human K562 cells | VP16 | Digoxin or progesterone | yEGFP | Transcriptional activator | The same biosensor as used in yeast [26] |
Human K562 cells | none | none | EGFP | CRISPR/Cas9 genome editing | Ligand biding domain DIG3 and PRO1 were fused upstream of a non-functional EGF variant with a premature stop codon. A gRNA was designed to target premature stop codon and to restore the EGFP activity [26] |
Yeast | |||||
Saccharomyces cerevisiae | LysR-type transcriptional regulator BenM from Acinetobacter sp. ADP1 | Cis,cis-muconic acid (CCM) | Green fluorescence protein (GFP) | Transcription activator | DNA-binding site of BenM (BenO) was inserted into a truncated CYC1 promoter [27] |
S. cerevisiae | FdeR from Herbaspirillum seropedicae | Naringenin | GFP | Transcription activator | DNA-binding site of BenM (BenO) was inserted into a truncated CYC1 promoter [27] |
S. cerevisiae | PcaQ from Sinorhizobium meliloti | Protocatechuic acid | GFP | Transcription activator | DNA-binding site of BenM (BenO) was inserted into a truncated CYC1 promoter [27] |
S. cerevisiae | ArgP from Escherichia coli | l-Arginine | GFP | Transcription activator | DNA-binding site of BenM (BenO) was inserted into a truncated CYC1 promoter [27] |
S. cerevisiae | MdcR from Klebsiella pneumonia | Malonic acid | GFP | Transcription activator | DNA-binding site of BenM (BenO) was inserted into a truncated CYC1 promoter [27] |
S. cerevisiae | Tetracycline-responsive TetR | Tetracycline | Transcription repressor | Hybrid TetO-CYC promoter [28] | |
S. cerevisiae | FadR from Escherichia coli and Vibrio cholerae | Fatty acid or fatty acyl-CoA | yEGFP | Transcription activator | bacterial FadR transcriptional repressors and yeast synthetic promoters containing varying number of FadR‐binding operators [29] |
S. cerevisiae | MetJ-B42 | S-adenosyl-methionine | Venus, HIS3 | Transcriptional activator | Transcription factor domain B42 is fused with E. coli MetJ [30] |
S. cerevisiae | XylR from Tetragenococcus halophile, Clostridium difficile, and Lactobacillus pentosus | Xylose sugars | yEGFP | Transcription repressor | Constitutive expression of heterologous XylR under a synthetic promoter with XylR operator-binding sites [31] |
S. cerevisiae | Gal4-Ada | Methyl phosphotriester adduct | GFP | Transcriptional activation | Fusing the N-terminal domain of E. coli Ada protein, which can detect methylating compounds, to the Gal4 transcriptional activator [32] |
S. cerevisiae | FapR from B. subtilis | Malonyl-CoA | GFP | Transcription activator | Malonyl-CoA reductase (MCRCa) from Chloroflexus aurantiacus is under the control of FapR, to create a self-regulatory system [33] |
S. cerevisiae | Yap1 from S. cerevisiae | Diamide | GFP | Transcription activator | Yap1 target promoter TRX2 with an extra yap responsive stie, or TRX2 promoter is fused with 1-5 upstream activating sequence [34] |
S. cerevisiae | The herpes virus protein VP16 or VP64 | Digoxin | yEGFP | Transcription activator | Computationally-designed ligand binding domain DIG0 or PRO0 was inserted between N-terminal DNA binding domain and C-terminal transcriptional activation domain [26] |
S. cerevisiae | LexA | Digoxin | luciferase | Transcription repressor | Replace the Gal4 DNA binding sites in GAL1 promoter with LexA binding sites [26] |
Plant | |||||
Arabidopsis thaliana | The herpes virus protein VP16 | Digoxin or digaxigenin | Luciferase | Transcription activator | A degron MATα2 from Arabidopsis with VP16 transcriptional activation domain were inserted downstream of a Gal4-activated plant promoter [26] |