Introduction
The Fun4al project can roughly be divided into two topics: 1) sensing furfural and 2) converting furfural to less toxic compounds. Very few putative furfural-converting enzymes have been reported in literature. To prove that we could make a furfural-converting strain of A. niger, we needed to test a selection of these enzymes expressed in A. niger. Designing a furfural-sensing system in A. niger turned out to be even more complex; we had to design this system almost from scratch with only a few tools available for A. niger compared to for example S. cerevisiae or E. coli. Additionally, A. niger is a slow-growing microorganism compared to S. cerevisiae and E. coli. For both of our topics, quantifying the furfural conversion and the functionality of our biosensors turned out to be a greater challenge than anticipated, mainly due to the slow and non-homogeneous growth of A. niger. Collectively, progressing through the DBTL engineering cycle when working with A. niger was an aggravating and time-consuming process, especially when factoring in the time constraints of the iGEM competition. Despite all these challenges, we managed to design, build, and test a functional synthetic expression system (SES) in A. niger, as well as a furfural inducible biosensor using a promoter native to A. niger. To the best of our knowledge, our SES is one out of very few published SES of this kind for filamentous fungi (Rantasalo et al 2018) and the first ever made in iGEM.
Integration and mechanisms of our SES in A. niger
We successfully designed and integrated a novel SES into A. niger composed of a synthetic transcription factor (sTF), FunsTF05, expressing the fluorescent marker gene, mCherry, from the synthetic promoter, 6xLexO-Pmin (Fig.1). Out of 12 tested sTFs, FunsTF05 gave the strongest fluorescent signal when grown on plates with minimal media as described in the Results page under "Synthetic transcription factors" section.
Figure 1. FunsTF05 mechanism of action: An example of a synthetic expression system.
FunsTF05 is a synthetic transcription factor mainly consisting of three domains from different bacteria. The LexA domain of FunsTF05 binds the 6xLexO-Pmin sites at the synthetic promoter binding site, inducing expression of the red fluorescent protein mCherry
We made this FunsTF05 with inspiration from the architecture of the sTF sBAD from yeast (Castaño-Cerezo et al. (2020)), but where we exchanged the sensing domain and the transactivation domain. We also added a nuclear localization signal (NLS) as well as a longer linker (LL) between the DNA binding domain and the sensing domain. Thus, FunsTF05 is composed of LexA as the DNA-binding domain, Hmox1 as the sensing domain, VP16 as the activation domain, and SV40 as the NLS, hence we named it LexA-LL-Hmox1-VP16-SV40.
The synthetic promoter, 6xLexO-Pmin, was designed by modifying the tetracycline inducible promoter, TetON, proven to work in A. niger (Wanka et al. (2016)). TetON is activated by the binding of the regulator TetR to the operator tetO. To make this promoter respond to our sTF instead of TetR, we simply replaced the tetO sequence with 6 copies of the LexO binding site sequence. Our final construct was therefore 6 LexO sequences fused to the minimal promoter (Pmin) from TetON.
Due to the complex non-homogeneous growth of A. niger in small volumes, we were not able to measure the induction of fluorescence by furfural in a plate reader. We thus tried to induce the production of the fluorescent marker with furfural on solid media, but the A. niger strains did not grow even though only low concentrations of furfural was added to the plates. Nevertheless, we regard the design, assembly, and successful demonstration of this highly modular SES in A. niger as an important proof of concept, and as a great foundation for further developing a furfural-indusible biosensor by exchanging the sensing domain. In the same manner, we believe our SES could be modified to detect other compounds by the use of different sensing domains. Evidently, this SES has the potential to provide the foundation for easy construction of various custom-made biosensors in filamentous fungi.
Regulation using native promoters
We predicted that anFDH would be upregulated in response to furfural, since it showed ~61% identity to scFDH1 in yeast which was highly upregulated in response to furfural see the project description. We constructed the furfural inducible biosensor, BBa_K4129006, consisting of the A. niger anFDH promoter in front of mCherry, as seen in figure 2.
Figure 2. Identification of an A. niger native promoter activated in response to furfural and construction of a furfural biosensor.
From a yeast transcriptome dataset, we identified scFDH1 as a highly upregulated gene in response to furfural.
The putative anFDH in A. niger shows a 61% identity to scFDH1 and the promoter in front of this gene was therefore used to construct the furfural biosensor, BBa_K412900.
To verify if anFDH was actually upregulated in response to furfural in A. niger, we measured its expression levels of the WT with qPCR when exposed to 1 g/L furfural for 1.5 hours see the results here. We also measured the expression levels of mCherry in our strain containing our biosensor using the native anFDH promoter.
The qPCR results clearly indicate that FDH in A. niger is upregulated. We are also confident that the mCherry gene is upregulated in the strain containing our biosensor BBa_K4129006. We strongly believe that the cause of the high p-value (Mann-Whitney test, p=0.1) is due to a small sample size. Nonetheless, we feel confident that repeating the experiment with more replicates would indefinitely confirm that we indeed generated a furfural inducible biosensor using a native promoter naturally upregulated in response to furfural.
With this BBa_K4129006, we demonstrated a straightforward and elegant way of designing a biosensor without the need for rational engineering and assembling synthetic transcription factors and promoters. We believe that it would be possible to identify more and maybe even better promoters activated by furfural, by using the script we generated to identify promoters in front of upregulated genes from transcriptome datasets Read our script here..
References
- Rantasalo, Anssi, et al. A universal gene expression system for fungi. Nucleic acids research 46.18 (2018).
- Castaño-Cerezo S. et al. Development of a biosensor for detection of benzoic acid derivatives in Saccharomyces cerevisiae. Frontiers in bioengineering and biotechnology 7 (2020), p. 372.
- Wanka F. et al. Tet-on, or Tet-off, that is the question: Advanced conditional gene expression in Aspergillus. Fungal Genetics and Biology 89, (2016) p. 72-83