In this study, we constructed single deletion mutants to confirm in vivo the function of the first three genes of the L-rhamnose catabolic pathway in A. niger (ΔlraA, ΔlraB and ΔlraC). These strains, together with the ΔrhaR mutant, were also used to investigate L-rhamnose-induced transcriptional up-regulation by RhaR in A. niger to elucidate the inducer molecule. Comparative growth analysis of the 3 L-rhamnose catabolic mutants showed that individual deletion of lraA, lraB and lraC results in an inability to use L-rhamnose as a sole carbon source. This indicates that there are no other enzymes capable of replacing the function of LraA, LraB and LraC, at least at a level that can rescue the growth phenotype on L-rhamnose. These results are in line with a previous study in which a double deletion mutant of lraA and lraC in A. niger was unable to grow on L-rhamnose [17, 27].
The second aim of our study was to search for lraD candidate genes in the A. niger genome and identify this function by gene deletion. L-rhamnose catabolism genes (RHA1, LRA2, LRA3 and LRA4) have been previously found in a chromosomal gene cluster in S. stipitis [8, 13]. In A. niger, the orthologs of the S. stipitis RHA1, LRA2 and LRA3 genes, the lraA, lraB and lraC genes, are clustered with rhaR on chromosome II, but the cluster does not contain an LRA4 homolog (lraD) [15]. In this study we selected three candidate genes for lraD that were specifically up-regulated in L-rhamnose and which all have similar PFAM and InterPro domains to those found in LRA4 of S. stipitis. However, deletion of these genes did not reduce growth on L-rhamnose, suggesting that neither of them encodes an L-KDR specific aldolase with a key role in L-rhamnose metabolism. Because the five remaining genes in the A. niger genome with a dihydrodipicolinate synthetase family domain (PF00701) were not induced on L-rhamnose or not expressed in any condition (Additional file 4: Table S4), it is very unlikely that these genes are involved in L-rhamnose metabolism in A. niger. A possibility is that the real lraD gene of A. niger belongs to a different aldolase family than the LRA4 of S. stipitis. Also, we cannot exclude that an alternative enzyme could convert L-KDR in the L-rhamnose pathway of A. niger. In Sphingomonas sp., a gene cluster consisting of LRA1–3, LRA5 and LRA6 has been found. LRA5 and LRA6 were assigned as new enzymes, L-KDR-4-dehydrogenase (KDRDH) and 2, 4-diketo-3-deoxy-L-rhamnonate hydrolase (DDRH), respectively [11]. LRA5 (KDRDH) was identified as an NAD+ specific enzyme belonging to the short-chain dehydrogenase/reductase (SDR) superfamily [11] and has been shown to convert L-KDR to 2, 4-diketo-3-deoxy-L-rhamnonate. Interestingly, another KDRDH belonging to the medium chain dehydrogenase reductase (MDR) superfamily has been biochemically characterized in Sulfobacillus thermosulfidooxidans and was found to catalyze the same metabolic reaction [10]. We analysed and compared the PFAM domains in the protein sequences of LRA5 of both species. The KDRDH from Sphingomonas sp. contains an enoyl-(Acyl carrier protein) reductase domain (PF13561) and the KDRDH from the acidophile S. thermosulfidooxidans contains an alcohol dehydrogenase GroES-like domain (PF08240) and a zinc-binding dehydrogenase domain (PF00107). In A. niger, a putative alcohol dehydrogenase (NRRL3_01492) is present in the lraA-C cluster (Fig. 1c), which is 33-fold up-regulated in the micro-array data of A. niger wild-type in L-rhamnose compared to D-glucose [15]. This putative alcohol dehydrogenase contained the same PFAM domains as KDRDH from S. thermosulfidooxidans. Interestingly, this putative alcohol dehydrogenase is well conserved within the genomes of the other Aspergilli [15]. This putative alcohol dehydrogenase might be a likely candidate in A. niger, to convert L-KDR into 2, 4-diketo-3-deoxy-L-rhamnonate.
The third aim was to study the transcript profiles of the L-rhamnose-induced genes in the metabolic deletion mutants to identify the inducer of RhaR in A. niger. In our RNA-seq analysis, genes involved in the L-rhamnose catabolism and in RG-I degradation were significantly lower expressed in ΔlraA, ΔlraB and ΔlraC mutants compared to the reference strain. Our results revealed that 12 of the 23 RG-I related pectinolytic genes were >1.5 fold down-regulated in all the deletion strains compared to the reference strain on L-rhamnose. In the D-galacturonic acid pathway in A. niger, deletion of gaaA and gaaB resulted in reduced expression profiles of pectinolytic genes on D-galacturonic-acid compared to the reference [21], while deletion of gaaC resulted in up-regulation of these genes whereas no difference was observed for the deletion of gaaD. This study also demonstrated that the inducer of the galacturonic acid degradation route is 2-keto-3-deoxy-L-galactonate, which is the substrate for GaaC, indicating that accumulation of an intermediate due to deletion of a pathway gene allows the identification of the inducer of the pathway. Since the expression of L-rhamnose-responsive genes was reduced in the strains in which lraA, lraB or lraC were deleted, this indicates that none of these deletions results in accumulation of the inducer of the pathway regulator (RhaR). The expression did not reduce to zero for all the known rhamnose catabolic genes in all the strains, suggesting that there is either a basal non-regulated expression of these genes or alternatively that they are also under control of other regulatory systems. Based on an alignment between TRC1 in S. stipitis and RhaR in A. niger, regions of the DNA Binding domain are conserved. This correlates with the phylogenetic analysis performed in Koivistoinen et al.,2012. Therefor RhaR appears to be an orthologue of TRC1. Also, since the transcription factor RhaR is conserved within the genomes of the other Aspergilli, and is also conserved in fungal species, we postulate that the product of the LraC reaction, L-KDR, is the inducer of the system. In S. stipitis another transcription factor than TRC1 has been found in the L-rhamnose cluster, FST14. This transcription factor was more up-regulated than TRC1 on L-rhamnose and it might be co-regulating the pectinolytic genes together with TRC1 [13]. This could also be the case in A. niger as previously suggested [15], even though we could not find an orthologue for this regulator in A. niger.
The L-rhamnose transporter encoding gene and the metabolic genes are L-rhamnose-induced and in the qPCR and RNA-seq analysis they have a similar gene expression profile in the metabolic and rhaR deletion mutants. These results and those of Sloothaak et al., 2016 [6] indicate that L-rhamnose is predominantly transported via the RhtA transporter and then converted through the L-rhamnose metabolic pathway.
Previously it was shown that only a small concentration of L-rhamnose is enough to induce the system [6], as also observed for the D-xylose regulatory system in A. niger [30]. The very low expression levels of lraA, lraB, rhaR, rglB, rgxA, rgaeA obtained in ΔlraC in the presence of L-rhamnonate showed that the third step is indeed necessary to generate the inducer. In the ΔlraA and ΔlraB strains the inability to generate the inducer from L-rhamnose can be overcome by supplying L-rhamnonate instead, which is the product of LraB. This then also explains why ΔlraA and ΔlraB can still grow on L-rhamnonate, while ΔlraC cannot. RNA-seq combined with the qPCR results of the L-rhamnose metabolic mutants demonstrated that L-rhamnose, L-rhamnono-γ-lactone and L-rhamnonate are not the inducers of RhaR. Interestingly and in contrast to transfer to L-rhamnose, upon transfer to L-rhamnonate the reference, ΔlraA and ΔlraB strains showed similar expression levels in all the genes tested. L-rhamnonate is unlikely to use the L-rhamnose transporter RhtA, due to its different chemical nature. The higher levels of expression observed in the reference, ΔlraA and ΔlraB strains suggest that by using L-rhamnonate as carbon source which avoids the first 2 steps in the L-rhamnose metabolic pathway, its metabolism still leads to induction of the L-rhamnose pathway. But induction requires a functional LraC since induction is severely affected in the lraC deletion strain.
Because the gene or genes involved in the conversion of L-KDR remain unknown in A. niger, studying metabolic gene deletion mutants further downstream of the LraC step is required to unambiguously establish the identity of the inducer. At this point in time we do not know whether L-KDR is converted by a yet unknown aldolase into L-lactaldehyde and pyruvate or, alternatively by LRA5 and LRA6 homologs to L-lactate and pyruvate. The putative alcohol dehydrogenase present in the cluster, which on the basis of the PFAM domains found and its rhamnose-responsive expression, makes it more likely that the L-rhamnonate degradation pathway involves formation of 2, 4-diketo-3-deoxy-L-rhamnonate from L-KDR. The final C3 metabolites formed (pyruvate and L-lactaldehyde or L-lactate) via these two non-phoshorylating pathways have to be further metabolized via gluconeogenesis. These metabolites are already part of central metabolism and are not expected to play any role in the induction of the rhamnose pathway itself. Lactaldehyde dehydrogenase is involved in the conversion of L-lactaldehyde to L-lactate. Several putative lactaldehyde dehydrogenases (AldA) based on a characterized AldA in E.coli were found in A. niger [31]. The first putative gene (NRRL3_11302) shares 33.3% identity with AldA from E. coli and it is significantly up-regulated in the reference strain compared to all the deletion mutants. Also, one putative lactate dehydrogenase (LDH; NRRL3_07593) that catalyzes the reaction from lactate to pyruvate, shares 37.4% identity with a characterized LDH from Rhizopus oryzae [32]. RNA-seq results showed that this putative gene is not induced after 2 h of transfer. This potentially leaves us with two options for the identity of the inducer, viz. L-KDR or 2, 4-diketo-3-deoxy-L-rhamnonate, and further work will be required to discriminate between these two options.