By measuring the levels of RNA in A.
niger, changes in gene expression at the transcriptional level can be seen. From the results, there is a clear induction of certain genes as a response from A.niger being starved. The presence of wheat straw is able to show if certain genes were under the control of XInR and if gene expression was mediated under CreA. It has been previously shown that CAzy gene transcription takes up 3% of the total mRNA after 48 hours of growth in 1% glucose. CAzy genes refer to cbhA, cbhB, eglA and glaA.Controls (yefC, cbhA, cbhB) A.
niger cbhA contains the XInR binding site within its promoter sequence and is therefore under complete control of the transcription factor, XInR . In the presence of a carbon source (which contains hemicellulose) xylan degradation occurs so XInR induces cbhA transcription at 9 hours (figure 1A). Catabolite repression of glucose can be seen at the S+G time point as CreA represses the level of cbhA expression.When comparing the level of expression in between the two timelines, a basal level of expression of cbhA is seen without a carbon source suggesting that cbhA is not being transcribed (figure 1A and 4A). When there is no xylose or carbon source, XInR is not activated because there is no complex polysaccharide to degrade therefore, genes under the control of XInR are not induced (figure 4A). Figure 1A and 4A shows bands present at the 475bp. This shows that A.
niger is able to degrade wheat straw if the solution it is contained in has no CreA stimulants such as glucose.Figure 1B and 4B show that cbhB expression is only partially under the control of XInR. XInR induces hemicellulolytic genes in the presence of a carbon source hence the positive effect on the transcriptional level of cbhB seen in figure 1B at the 6 hour time point. However, if XInR was completely controlled cbhB, there would be no expression shown in figure 4B.
This reaffirms previous studies showing that XInR only partially controls cbhB. The results are not able to show this but cbhB is under the control of CreA.yefC is a known housekeeping gene which encodes for a translational elongation factor 3 (An07g02650) [12, 52].
Housekeeping genes are involved in maintenance metabolism and are required to remain on, regardless of environmental conditions to A.niger. Expression of yefC is not affected under stressful condition for A.
niger as level of expression in both timelines remains constant (figure 1C and 4C).eglA encodes for endoglucanase A (EglA), a 440 amino acid polypeptide that is part of CBM Family 1 (from CAzy database) and GH Family 12. Endoglucanase are involved in the glucan degradation to glucose and EglA in particular, is highly specific to beta-glucan.
EglA has, as its family name suggests, a CBD mediated by three aromatic residues and therefore is involved in hydrolysing O-glycosyl compounds. CBM Family 1 modules are exclusive to fungi and have approximately 40 residues. In addition, EglA also has cellulase and cation binding activity. In A.niger, eglA is expressed when grown on sugarcane bagasse  as well as in response to wheat straw as shown in this study. XInR activates the expression of eglA and eglB in the presence of xylose, not sophorose [28, 42].Both figures 2 and 5 show a band present 200bp representing the PCR product of eglA at 201bp; with introns, the gene size is 253bp. The transcription pattern of both eglA and cbhA are very similar; induction at nine hours and repression after glucose is added into the media ( figure 1A and 2) .
However, without wheat straw, expression of eglA is turned on earlier at 6 hours and stayed highly induced till 24 hours (figure 5) suggesting that expression of eglA must be induced by starvation. In figure 5, there is an anomaly in the band pattern because the band at the 24 hour time point should still show eglA being highly induced. The cause of this anomaly is most likely due to not enough cDNA being synthesised.
The glaA gene encodes for a 640 amino acid glucoamylase which has two alternate names; 1,4-alpha- D-glucan glucohydrolase and glucan 1,4-alpha-glucosidase and is responsible for 65% of CAzy gene transcription. GlaA is involved in starch and sucrose metabolism to release glucose from the terminal ends of ?-1,4-glucan subsequently glaA is expressed in high levels of starch[56, 57]. In terms of the CAzy database, glaA is placed in CBM20 and GH15 families. Specifically, glaA has a starch-binding domain unlike cbhB or eglA which have CBMs specific for cellulose.
Expression of glaA is under the control of the promoter, PglaA, the transcriptional factor AmyR and CreA[53, 59]. Furthermore, It has been shown that overexpression of glaA gene results in an increase in glaA mRNA and in extracellular production of GLA. Finally, in the food industry, A.niger glucoamylase plays an essential role for the modification of starch.Figure 3 and 6 both show that the level of glaA expression is not affected by the presence of wheat straw and is constantly on. As glaA expression is very similar to yefC (figure 1C and 4C), it would be easy to assume that glaA is also a housekeeping gene.
However, glaA is not under the control of XInR but is AmyR-dependant which has been shown to be induced by D-maltose and D-glucose and repressed by xylose[56, 59]. Induction of glaA expression by glucose is stronger than that of xylose therefore, despite the wheat straw degradation seen at the carbon timeline, it must be noted that glucose is still a component of wheat straw and the xylose concentration was very low, consequently, glaA transcription remains constantly on (figure 3).ConclusionThis study has shown that expression of CAzy genes can be affected by wheat straw at a transcriptional level. A.niger can be used as a model organism for the degradation of lignocelluloses as it is able to release specific plant cell-wall degrading enzymes. It is still unclear the precise mechanism by XInR and CreA modulate their expression therefore, experimentation in mutated strains of ?xInR and ?creA in 1% wheat straw timeline would show how they affect CAzy gene expression. Most of the CAzy genes (cbhA, cbhB and eglA) are XInR-dependant but glaA is under the control AmyR. In the ?xInR mutants, expression levels of glaA would still be the same as in wild type, cbhA expression will not be seen and cbhB may be partially seen.
In ?creA mutants, expression of all the CAzy genes will be induced and not turned off.An additional experiment would be to do a low xylose (0.01%) timeline. This would provide further evidence of xylose induction of CAzy genes. Xylose induces of cbhA, cbhB and eglA expression but represses glaA.
Both cbhB and glaA are induced by starvation therefore, induction should be seen at 6 and 9 hours respectively. With regards to glaA, there is contradicting research of xylose repression of glaA. An experiment with increasing xylose concentration would confirm if xylose has a repressive effect via CreA on the gene as well as confirm the precise concentration at which xylose repression is in effect. This experiment can also be applied to the other CAzy genes to further confirm the precise concentration for when CreA is active.