Introduction

Two different mutants were obtained, each with a different furfural converting enzyme (FCE). These will be annotated as Arz and hmfH and are transformed with the following plasmids BBa_K4129018 and BBa_K4129020, respectively. These were then subsequently tested in a BioLector alongside WT A. niger.

Results

We have not been able to experimentally show the FCE working in A. niger. However, we have shown that the expression of the FCEs have a detrimental effect on both growth rate and final biomass gain, suggesting they are a metabolic burden to the fungus under stressful environments. This also suggests that A. niger has a better way to handle furfural in the media than our proposed pathways enabled by the FCEs, granted they work in A. niger.

Characterising furfurals effect on the growth of A. niger

With WT A. niger inoculum, wells with differing concentrations of furfural were inoculated. After just seven hours it was clear that furfural in the media had a detrimental effect on the growth rate of WT A. niger. This is greatly illustrated on figure 1. But what was also discovered was that WT A. niger does not reach the same biomass gain when growing in the presence of furfural as it does without the presence of furfural. This agrees with the literature, and sets the basis for reducing the effect of furfural on fungal growth by giving WT A. niger another pathway to deal with furfural.

Figure 1: Cultivation of WT A. niger in YPD media with differing concentrations of furfural. Lines are mean values of triplicates and shadow marks +/- standard deviation of those. Left) linear plot, Right) semilog plot. As can be seen on both subfigures, increasing concentration of furfural has a detrimental effect on the growth rate of WT A. niger .

Characterising the effect of furfural on the growth of A. niger mutants

This cultivation was intervention styled: Spores were inoculated in minimal media (MM) with no furfural. After germination and when the cultures entered the exponential growth phase, furfural was added to a final concentration of: 0.0 g/l, 0.6 g/l, 1.2 g/l and 3.0 g/l. As can be seen in figure 2, hmfH mutant outgrew WT, while Arz mutant had similar growth curve until 60 hours of cultivation. However, increasing the concentrations of furfural impacted both the growth rate and final biomass gain of the mutants comapred to WT.

Figure 2: WT, Arz and hmfH mutants grown in MM with differing concentrations of furfural. Lines are mean values of triplicates and shadow is +/- the standard deviation of those. With no furfural, hmfH and to a certain extend Arz mutants grew better than WT, but with furfural added WT grew quite a bit better.

After this cultivation, samples were processed and sent to our high-performance liquid chromatography (HPLC) lab for furfural quantification. With this, we could hopefully describe what could not be seen on the growth curves. However, it was not possible to identify the furfural peak in the data from HPLC and subsequent analysis was thus not performed.

Characterization of furfurals effect on the germination of A. niger spores

With the intervention styled experiment done, we wanted to see the effect of furfural on the germination of spores. Spores were added to MM with differing concentrations of furfural. After about 15 hour, WT spores in MM with no furfural began to germinate and Arz mutant spores germinated similarly compared to WT. Arz mutant spores only germinated slower than WT in low concentration of furfural (0.6 g/l). hmfH mutant spores were in general quite dealyed compared to WT.

Figure 3: WT, Arz and hmfH mutants grown in MM with differing concentrations of furfural. Lines are mean values of triplicates and shadow is +/- the standard deviation of those. Arz mutant germinated either similarly or a bit delayed depending on furfural concentration. hmfH mutant germination was also quite delayed compared to WT.

Introduction

Engineered A. niger strains transiently expressing mCherry under the regulation of the fungal promoters scFDH1, BBa_K4129004; anFDH, BBa_K4129005; or scFDH2, BBa_K4129006 were used to investigate the activity of the promoters and their response to furfural. Strains that carried only the empty vector (BBa_K4129025) or mCherry cassette with a prokaryotic promoter (BBa_K3046009) were used as negative controls, while strains carrying a constitutively expressed mCherry cassette (BBa_K3046004) either on a plasmid or as a genomic insertion were selected as positive controls. The strains were grown in liquid minimal media. Half of the samples were induced with furfural either 20 or 70 hours after inoculation. Fluorescence and growth were monitored with a plate reader to evaluate promoter activity. The anFDH promoter was further investigated by qPCR. The strain transiently expressing BBa_K4129005 and a WT were grown on liquid minimal media, after 24 hours half the samples were induced with furfural. The FDH and mCherry transcripts were quantified at 1.5 hours following induction.

Results

The strains utilising the investigated fungal promoters did not show a significantly higher fluorescence per biomass compared to the negative controls (Kruskal – Wallis, α = 0.05) (Figure 1), suggesting that, in A. niger, these promoters are neither constitutively active nor inducible in response to furfural. 1.5 hours after induction (Figure 1 D), many samples containing furfural showed an increase in fluorescence; however, this was also observed in the negative controls, suggesting that the increased fluorescence is due to a perturbation caused by furfural rather than a change in promoter activity.

Figure 1. Comparison of the samples with or without furfural using fluorescence per biomass as a proxy for promoter activity. The bars indicate the mean fluorescence per biomass, the error bars correspond to +/-1 standard error. A-C shows the fluorescence per biomass of the samples induced at 20 hours after inoculation, as measured at 71.5, 94, or 118 hours, respectively. D-F shows the fluorescence per biomass of the samples induced at 70 hours after inoculation, as measured at 71, 94, or 118 hours, respectively.

The qPCR results show that the FDH gene is strongly upregulated in response to furfural (Mann-Whitney test, α = 0.05) (Fig. 2 A). A similar trend was observed for the mCherry mRNA level (Fig. 2 B), however this difference was not supported by our statistical analysis (Mann-Whitney test, p-value = 0.1). We suspect that this discrepancy is caused by the small sample size at our disposal for the mCherry transcript, as the data from the WT strain could not be included in the mCherry qPCR, while both WT and BBa_K4129005-expressing strains were included in the FDH mRNA quantification. This should be studied further, but until new data are collected we will simply suggest that BBa_K4129005 is likely induced by furfural when transiently expressed in A. niger, and clearly upregulated when genomically integrated..

Figure 2. Investigation of anFDH promoter activity in response to furfural. The plots show the mRNA levels relative to actin mRNA of FDH (A), and mCherry transiently expressed from BBa_K4129005 (B), as measured by qPCR.

While the anFDH promoter shows strong induction both from the FDH locus and the plasmid vector, the absolute expression is clearly higher when measuring the FDH transcript. At the FDH locus, the promoter is integrated in a chromosome and in its native regulatory environment. While in the mCherry reporter, it is limited to 1000 bp and found on a vector for transient expression. We see two possible explanations for the observed difference in expression: either the anFDH regulatory regulatory region contains an enhancer that is not included in the 1000 bp we amplified, or the transient expression system is less efficient than the genomic locus. The latter is more likely; transient expression systems in filamentous fungi are unreliable, as can be observed in the fluorescence experiment where the positive control BBa_K3046004 showed higher and more consistent fluorescence when genomically integrated rather than expressed transiently (Fig.1). To date,the anFDH promoter is the only A. niger promoter to be shown to be responsive to furfural. At the FDH locus the promoter has a considerable dynamic range and high activity, making it ideal for engineering a biosensor. Once genomically integrated, BBa_K4129005 will be the first furfural biosensor ever built.

Introduction

The fungal synthetic expression system (SES) constructed in this project consists of our synthetic transcription factor (sTF), synthetic minimal promoter (6xLexO-Pmin) and mCherry. The sTF is constitutively expressed from PgpdA and it is expected that the sTF promotes transcription of the mCherry.

We successfully assembled 27 SESs each with their unique sTF, and 12 of these had their functionality tested in A. niger, both grown on solid media and in liquid media, by measuring fluorescence. The solid media experiments were carried out in two batches and there were minor differences in execution of the assessment. The liquid media experiments were measured with the same gain across multiple plates in order to compare fluorescence.

The 12 SESs were tested and a few SESs were highlighted to give a quick overview of some results. The sTF of the SESs were FunsTF02, FunsTF04, FunsTF05, FunsTF18, FunsTF18, FunsTF57 and FunsTF70. All of the sTF used lexA as a DNA-binding domain. FunsTF04 and FunsTF05 have Hmox1 as ligand-sensing domain, while FunsTF02 has HbaR. FunsTF18, FunsTF57 and FunsTF70 have mutated variants of HbaR. The transactivation domain B112 is used in FunsTF02, FunsTF04 and FunsTF18, while FunsTF05, FunsTF57 and FunsTF70 have VP16.

Table 1: An overview of floursences results of the following highlighted sTFs. They are FunsTF02, FunsTF04, FunsTF05, FunsTF18, FunsTF57 and FunsTF70. The sTFs were tested in minimal media, MM with 2 mM benzoic acid and MM with 0.6 g/L furfural. The results are illustrated relative to the best performing SES of FunsTF05.

Results

This chain of experiments did not supply us with the groundbreaking results we had hoped for, but will provide a good starting point for further work, since there are only a handful of synthetic systems that function in filamentous fungi. All SESs documented to work in filamentous fungi until now are constitutive systems, making this SES the first documented inducible SES in filamentous fungi. sThose systems do not have the possibility to become inducible. They were created to be constitutive.

Characterization on solid media

This chain of experiments did not supply us with the groundbreaking results we had hoped for, but will provide a good starting point for further work, since there are only a handful of synthetic systems that function in filamentous fungi. All SESs documented to work in filamentous fungi until now are constitutive systems, making this SES the first documented inducible SES in filamentous fungi. Those systems do not have the possibility to become inducible like our systems.

Figure 1: Pictures of fluorescence from A. niger, which carries either BBa_K4129025 or genomically integrated BBa_K3046004. The exposure time was set to 1.04 seconds. The A. niger is grown on plates containing minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.

The first iteration had an exposure time of 1.04 seconds and the genomic integrated BBa_K3046004 and BBa_K4129025 showed the expected outcome. BBa_K4129025 is an empty vector, thus it did not account for the potential expression leak from 6xLexA-Pmin (BBa_K4129115)

FunsTF04 and FunsTF05 have a similar structure but the transactivation domain was B112 and VP16 for FunsTF04 and FunsTF05, respectively.

FunsTF05 displays higher intensity than genomic integrated BBa_K3046004. A contributing factor, to this observation, was that FunsTF05 was carried on a high copy transient expression plasmid. The genomic integrated BBa_K3046004 was integrated as a single copy (figure 1). FunsTF04 displays only fluorescence at the perimeter of A. niger when grown on minimal media (figure 1). The fluorescence from FunsTF04 has a higher intensity than that of BBa_K4129025. It can be argued that the fluorescence was due to leaky expression from 6xLexA-Pmin (BBa_K4129115). The leaky expression does not fit because FunsTF04 on benzoic acid does not express, thus it indicates there is no leak or working sTF (figure 2). To summarise, The sTF of FunsTF05 promotes constitutive expression of mCherry. In addition, the activity of FunsTF04 was not at the same level as FunsTF05. Which indicates that VP16 was a more active transactivation domain than B112, in this specific combination of modules (LexA, Hmox1, and SV40).

Figure 2: Pictures of fluorescence from A. niger, which carries either FunsTF04 or genomically integrated FunsTF05. The exposure time was set to 1.04 seconds. The A. niger is grown on plates containing minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.

The SES with FunsTF02 (BBa_K4129101) also displays slight fluorescence at the perimeter of the colonies when grown on minimal media. This fluorescence was not present on any other MM plates with either benzoic acid or furfural (figure 3).FunsTF02 is interesting because it is the sTF, which is closest to sBAD (Castaño-Cerezo et. al (2020)). A sTF that was inducible in S. cerevisiae, is working in A. niger, but not it is not inducible yet. FunsTF57 did not display any fluorescence even when compared to BBa_K4129025 across all three different medias (figure 3).

Figure 3: Pictures of fluorescence from A. niger, which carries either FunsTF02or genomically integrated FunsTF57. The exposure time was set to 1.04 seconds. The A. niger is grown on plates containing minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.

The second iteration used an exposure time of 0.72 seconds. At this exposure time FunsTF18 and FunsTF70 were constitutively expressing mCherry, although it was with mid intensity. FunsTF18 had the modules as FunsTF02, but the HbaR domain of FunsTF18 was our mutation 12. FunsTF18 was observed to be constitutively, so another set of mutations to HbaR is required to improve inducibility.

Figure 4: Pictures of fluorescence from A. niger, which carries either FunsTF18or genomically integrated FunsTF70. The exposure time was set to 0.72 seconds. The A. niger is grown on plates containing minimal media, minimal media with 2 mM benzoic acid or minimal media with 0.6 g/L furfural. The fluorescent is depicted as grey-white intensity.

The remaining SESs did not show any fluorescence and all pictures can be found here (link to Download PDF with all pictures).

Characterization in liquid culture

The A. niger expressing our SESs were cultivated in a 48 well CellStar cultivation plate and 24 hours after inoculation the SES systems were induced. The inducers were 2 mM benzoic acid and 0.6 g/L furfural. The fluorescence and optical density at 600 nm (OD600) was measured at four time points: 2 hours, 26, hours, 47 hours and 70 hours after induction. The measurements were performed in a CLARIOstar microplate reader.

The genomic integrated BBa_K3046004 was used as positive control and pAC572 (BBa_K4129025 ) was used as a negative control. A clear inducibility of the sTFs was not observed, because the samples induced with 2 mM benzoic acid or 0.6 g/L furfural did not fluoresce more than the uninduced samples (figure 5).

Two hours after induction, the observed fluorescence for the sTFs were slightly higher than that of pAC572. This was supported by the sTFs are all significantly different than pAC572 (Kruskal - Wallis, α=0.05) FunsTF05, while uninduced, had the highest fluorescence of all the sTF at around 3300 RFU (figure xx). FunsTF02 induced with 2 mM benzoic acid did also have a high fluorescence a little under 3000 RFU, but it was not significantly different from pAC572 induced with 2 mM benzoic acid (Kruskal - Wallis, α=0.05).

The time points of 26 hours, 47 hours and 70 hours after induction displayed similar observations of fluorescence measurement, The main point of interest is that FunsTF05 has high activity throughout time and FunsTF18 ramps up slowly over time.

Figure 5: Barplot of log transformed fluorescence (RFU) with standard deviation displayed as arrows. The fluorescence measurements from A. niger carrying genomically integrated BBa_K3046004, BBa_K4129025, FunsTF02, FunsTF04, FunsTF05, FunsTF18, FunsTF57 and FunsTF70. The A. niger was grown in minimal media, where it was uninduced (blue), induced with 0.6 g/L furfural (red) or induced with 2 mM benzoic acid (green). The fluorescence was measured A) 2 hours, B) 26 hours, C) 47 hours, and D) 70 hours after induction.

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