FunsTF05 is a synthetic transcription factor (sTF). FunsTF05 should be able to interact with its inducer, furfural, and this interaction will facilitate transcription from 6x(LexO)-Pmin. This design of sTF is a possible sensing part of a biosensor.

FunsTF05 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand binding domain from Hmox1, the transactivation of domain VP16 and the nuclear localization signal (NLS) SV40. The linker between the LexA domain and the Hmox1 domain is a longer linker (Ottoz et. al (2014)) compared to the linker used in sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)). FunsTF05 was codon optimised to A. niger.

LexA is a repressor that regulates the SOS response in E. coli (Radman (1975)). LexA binds to a specific DNA motif, namely LexO sites (Erill. et. al (2003)). Hmox1 is the human heme oxygenase 1, which is the enzyme that initiates cleavage of heme (Tenhunen et. al (1969)). This enzyme was, despite the seemingly unrelated context, computationally shown to bind furfural (Santhakumar et. al (2021))

Viral Protein 16 (VP16) from Herpes simplex virus type 1 is a transcription factor with a transactivation domain that recruits RNA polymerase II (Hirai et. al (2010)).The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport of the protein to the nucleus (Garcia-Bustos et. al (1991)).

In short, FunsTF05 should be able to interact with furfural due to the domain from Hmox1, it should be able to bind to LexO site through the LexA domain and, and it should be able to activate transcription of a minimal promoter like 6xLexO-Pmin (BBa_K4129115) through the interaction between the transactivation domain of VP16 and the 6xLexO-Pmin.

In this fungal synthetic expression system (SES) FunsTF05 was constitutively expressed from PgpdA. The FunsTF05, which contains a LexA DNA-binding domain, would be able to bind to the LexO binding sites for the minimal promoter fused with LexO binding sites (6xLexO-Pmin). The efficiency of the FunsTF05 activation was measured by the reporter gene mCherry, which is expressed from the 6xLexO-Pmin.

The promoter gpdA from A. nidulans was minimised to the minimum amount of nucleotides (180 bp) for transcription and was named Pmin. The transcription from Pmin was minor, but can still assemble the RNA polymerase if that process is initiated by a transcription factor. This system was used to make a tetracycline inducible system, TetOn, (Wanka et. al (2016)).

LexO is the name of the binding motif for LexA. LexA is the main regulator for the SOS response in E. coli (Radman (1975)). It has been shown in A. niger, that repeating LexO sites increase binding affinity and expression of a minimal promoter. The consensus number of LexO binding sites are six, 6xLexO (Rantasalo et. al (2018)).

FunsTF02 is a synthetic transcription factor (sTF) based on a sensor of benzoic acid derivatives (sBAD), which is a sTF in S. cerevisiae (Castaño-Cerezo et. al (2020)). FunsTF02 deviates from sBAD, in that it has a nuclear localization signal (NLS) and is codon optimised to A. niger. FunsTF02 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from HbaR, transactivation domain B112 and the nuclear localization signal (NLS) SV40.

The designed function of FunsTF02 is to be used as a transcription factor that can initiate transcription from the 6xLexO minimal promoter (BBa_K4129115). This sTF is designed to be the sensing part of a biosensor.

LexA is a repressor that regulates the SOS response in E. coli (Radman. 1975). LexA binds to a specific DNA motif, namely LexO sites (Erill. et al (2003)). HbaR is a transcription factor from Rhodopseudomonas palustris that initiates transcription in the presence of benzoic acid (Egland. et al (2000) or in the presence of benzoic acid derivatives (Castaño-Cerezo et. al (2020)).

The transactivation domain B112 is from E. coli, and it was experimentally proven to initiate transcription of a synthetic promoter in S. cerevisiae (Ottoz et. al (2014)). The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport og the protein to the nucleus (Garcia-Bustos et. al (1991)).

This composite part was used to characterise the synthetic transcription factor (sTF) FunsTF02. The reporter cassette consists of the synthetic 6xLexO-Pmin (BBa_K4129120) that transcribe mCherry.

FunsTF04 is a synthetic transcription factor (sTF). FunsTF04 is designed to function as a transcription factor that can initiate transcription from the 6xLexO minimal promoter (BBa_K4129115). This sTF is designed to be the sensing part of a biosensor.

FunsTF04 is a fusion protein consisting of the DNA-binding domain from LexA, the ligand sensing domain from Hmox1, transactivation domain B112 and the nuclear localization signal (NLS) SV40. The linker between LexA and Hmox1 is a longer version linker (Ottoz et. al (2014) compared to sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)). FunsTF05 was codon optimised to A. niger.

LexA is a repressor that regulates the SOS response in E. coli (Radman. 1975). LexA binds to a specific DNA motif, namely LexO sites (Erill. et al (2003)), and it is the DNA binding domain, which is interacting with LexO, that is used in FunsTF04. Hmox1 is the human heme oxygenase 1, which is the enzyme that initiates cleavage of heme (Tenhunen et al. (1969)). This enzyme was, despite the seemingly unrelated context, computationally shown to bind furfural (Santhakumar et al (2021)).

The transactivation domain B112 is from E. coli, and was experimentally proven to initiate transcription of a synthetic promoter in S. cerevisiae (Ottoz et. al (2014)). The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport og the protein to the nucleus (Garcia-Bustos et. al (1991)).

This composite part was used to characterise the synthetic transcription factor (sTF) FunsTF04. The reporter cassette consists of the synthetic 6xLexO-Pmin (BBa_K4129120) that transcribe mCherry.

FunsTF18 is a synthetic transcription factor (sTF) based on sensor of benzoic acid derivatives (sBAD), which is a sTF in S. cerevisiae (Castaño-Cerezo et. al (2020)). FunsTF18 differ from sBAD, because it has an nuclear localization signal (NLS) and is codon optimised to A. niger. FunsTF18 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain HbaR12, transactivation domain B112 and the nuclear localization signal (NLS) SV40.

LexA is a repressor that regulates the SOS response in E. coli (Radman. (1975)). LexA binds to a specific DNA motif, namely LexO sites (Erill et. al (2003)). HbaR is a transcription factor from Rhodopseudomonas palustris that initiates transcription in the presence of benzoic acid (Egland et. al (2000) or in the presence of benzoic acid derivatives (Castaño-Cerezo et. al (2020)). We created 16 mutants of HbaR and FunsTF18 carry mutant 12 of HbaR, which had the following mutations: A45S, L64I, F85M, A86G, A88Y and Y96S.

The transactivation domain B112 is from E. coli, which were experimentally proven to initiate transcription of a synthetic promoter in S. cerevisiae (Ottoz et. al (2014)). The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport of the protein to the nucleus (Garcia-Bustos et. al (1991)).

This composite part was used to characterise the synthetic transcription factor (sTF) FunsTF18. The reporter cassette consists of the synthetic 6xLexO-Pmin (BBa_K4129120) that transcribe mCherry.

FunsTF70 is a synthetic transcription factor (sTF). FunsTF70 should function as a transcription factor that can initiate transcription of the 6xLexO minimal promoter (BBa_K4129115). This sTF will be the sensing part of the biosensor.

FunsTF70 is a fusion protein consisting of the DNA-binding domain LexA, the ligand sensing domain HbaR16, the transactivation domain VP16 and the nuclear localization signal (NLS) SV40. The linker between LexA and HbaR16 was a longer version (Ottoz et. al (2014) compared to sBAD, which was the reference sTF (Castaño-Cerezo et. al (2020)).

LexA is a repressor that regulates the SOS response in E. coli (Radman. 1975). LexA binds to a specific DNA motif, LexO (Erill. et al (2003)). HbaR is a transcription factor from Rhodopseudomonas palustris that initiates transcription in the presence of benzoic acid (Egland. et al (2000)) or in the presence of benzoic acid derivatives (Castaño-Cerezo et. al (2020)). We created 16 mutants of HbaR and FunsTF70 carried mutant 16 of HbaR, which had the following mutations: L64I, F85H, A86G, A90Y and L97G.

Viral Protein 16 (VP16) from Herpes simplex virus type 1 is a transcription factor that uses a transactivation domain to recruit the RNA polymerase II (Hirai et al. (2010)).The NLS SV40 is a small peptide sequence of PKKKRKV that enables transport of the protein to the nucleus (Garcia-Bustos et. al (1991)).

This composite part was used to characterise the synthetic transcription factor (sTF) FunsTF70. The reporter cassette consists of the synthetic 6xLexO-Pmin (BBa_K4129120) that transcribe mCherry.

This is a plasmid for transient expression in A. niger containing an E. coli ori, an ampicillin selection marker, a pyrG auxotrophyc selection marker and the AMA1 sequence. Inserts can be integrated into the plasmid by using a PacI/Nt.BbvCI USER assembly casette.

Transient expression platform for A. niger

The part consists of the 1000 bp upstream of Formate Dehydrogenase (FDH) CDS from A. niger. Since FDH genes have been shown to be upregulated in response to furfural in Saccharomyces cerevisiae, the part was utilised to test if anFDH promoter is also responsive to furfural. Quantification by qPCR of the FDH transcript and mCherry transcript from a transiently expressed BBa_K4129005 (BBa_K4129002 regulating mCherry reporter), showed that BBa_K4129002 is induced by furfural both in its native regulatory enviroment and when used in syntetic expression cassettes.