Elucidation of the Molecular Weight Control Mechanism ofFungal Cell Wall Polysaccharide Nigeran and Establishment of aNigeran Fermentation Production SystemKeiko UECHI
Faculty of Agriculture, University of the Ryukyus
Research objective
Filamentous fungi of the genus Aspergillus belonging to the Nigri section, including
Aspergillus luchuensis, produce the fungal cell wall component, nigeran, an unbranched Dglucan with alternating α-1,3- and α-1,4-glucoside linkages, under nitrogen starvation
conditions. We previously identified the A. luchuensis nigeran synthase gene, nisA1).
Furthermore, we reported that the disruption of two genes adjacent to nisA, αglucanotransferase gene (agtC) and a gene of unknown function (gnsA), affected nigeran
production and its molecular weight. In this study, we expressed both agtC and gnsA as
recombinant enzymes and examined their effects on the molecular weight of nigeran.
In recent years, biomass-based plastics made from polysaccharides have attracted
considerable attention as substitutes for plastics. We investigated whether nigeran producedby the A. oryzae nisA-overexpressing strain could be used as a biomass-based plastic material2).
We attempted to obtain strains with high nigeran production that constitutively expressed nisA.
Methods
1. Characterization of recombinant AgtC and GnsA
The α-glucanotransferase gene (agtC) was amplified using polymerase chain reaction(PCR) and expressed as a recombinant enzyme using the A. oryzae expression system. The
gene of unknown function (gnsA) was also amplified using PCR and expressed as arecombinant enzyme using the Escherichia coli expression system. rAgtC and rGnsA werereacted with α-glucans, such as nigeran, starch, and α-1,3-glucan, and the molecular weightsof α-glucans were analyzed using size exclusion chromatography. Furthermore, we analyzed
the structures of the reaction products of rAgtC and nigeran oligosaccharides prepared via acidhydrolysis.
2. Construction of a nigeran fermentation production system
We replaced the A. luchuensis nisA promoter with an improved enolase promoter (PenoA142), which is highly expressed in the glycolytic system of A. oryzae, and obtained atransformant that constitutively expressed nisA. A. oryzae was transformed with a plasmid inwhich the high-expression vector pNEN142 and nisA were ligated. The hosts used fortransformation included a strain (AGΔ), in which all three α-1,3-glucan synthase (AG) genes
were disrupted, and another strain, in which the galactosaminogalactan (GAG) synthase andAG genes were disrupted (AG-GAGΔ). The yield and molecular weight of nigeran obtained
from nisA-overexpressing strains were analyzed.
Results
1. Characterization of recombinant AgtC and GnsA
Purified rAgtC and rGnsA were reacted with nigeran, and the change in their molecularweights was analyzed using size exclusion chromatography. We found that the molecularweight of nigeran was reduced by rAgtC treatment. In contrast, rGnsA did not affect themolecular weight of nigeran. When rAgtC and rGnsA were combined and reacted with nigeran,the molecular weight of nigeran decreased, as in the case of rAgtC alone. Reacting both rAgtCand rGnsA with nigeran resulted in a similar reduction in the molecular weight of nigeran
compared to rAgtC alone. In a previous study, both an increase and decrease in nigeran
molecular weight were observed in the A. luchuensis ΔgnsA strain (AgtC functions). Incontrast, the molecular weight of nigeran did not change in the ΔagtC strain, in which GnsAfunctioned. This is consistent with the results obtained in this study that rAgtC reduces themolecular weight of nigeran, whereas rGnsA does not affect its molecular weight. In addition,
starch or α-1,3-glucan reacted with rAgtC and rGnsA, but the molecular weights of theseglucans did not change, suggesting that rAgtC acts specifically in nigeran.
Fungal α-glucanotransferases (Agt) act mainly on α-1,4-glucosidic bonds in α-1,4-glucan
and maltooligosaccharides3). To clarify whether rAgtC acts on α-1,4- or α-1,3-glucosidic
bonds in nigeran, a nigeran oligosaccharide mixture prepared via the acid hydrolysis of nigeran
was reacted with rAgtC. We found that rAgtC did not act on nigeran oligosaccharides with anestimated degree of polymerization (DP) of less than 6, and only hydrolyzed the nigeran
oligosaccharides with an estimated DP of more than 6. The obtained product was fractionated,and its structural analysis is underway.
2. Construction of a nigeran fermentation production system
A comparison of nigeran production by the A. luchuensis parental strain and nisAoverexpressing strain with the promoter replaced revealed that the nisA-overexpressing strain
produced approximately twice as much nigeran as the parental strain. Compared to the nisAoverexpressing strain generated from A. oryzae niaD300, the nisA-overexpressing strains
generated from AGΔ and AG-GAGΔ strains produced 2–3 times more nigeran. Based on thehigh homology between nisA and fungal α-1,3-glucan synthase genes, we suggest that thesynthetic substrates of nigeran and α-1,3-glucan are the same. Because the A. oryzae AGΔstrain does not synthesize α-1,3-glucan, it is presumed that its nigeran production was higher
than that of the A. oryzae niaD300 strain, which synthesizes α-1,3-glucan.
The molecular weight of nigeran was determined using size-exclusion chromatography. The
nigeran molecular weight of the nisA-overexpressing strain generated from the A. oryzae AGGAGΔ strain was the highest, followed by the A. oryzae niaD300, A. oryzae AGΔ, A.
luchuensis nisA-overexpressing, and A. luchuensis parent strains.
Conclusion
The mechanism of nigeran synthesis is unclear compared to that of other fungal cell wall
polysaccharides. The characterization of nigeran synthesis-related enzymes in this study has
provided fundamental knowledge leading to an understanding of nigeran synthesis
mechanisms. To clarify the significance of nigeran production in fungi, we will continue toconduct extensive research on the enzymes and genes involved in nigeran synthesis.
Here, we found that the A. oryzae nisA-overexpressing strain produced more nigeran in ashorter culture period than the wild-type A. luchuensis strain. Furthermore, we obtained a nisAoverexpressing strain that produced higher-molecular-weight nigeran. Therefore, the resultsof this study can contribute to the future research on biomass-based plastics using nigeran.
References
1) Uechi, K., Yaguchi, H., Tokashiki, J., Taira, T., and Mizutani, O. (2021) Identification ofgenes involved in the synthesis of the fungal cell wall component nigeran and regulationof its polymerization in Aspergillus luchuensis. Appl. Environ. Microbiol. 87(21):e0114421.
2) Togo, A., Uechi, K., Mizutani, O., Kimura, S., and Iwata T. (2021) Synthesis andcharacterization of α-1,3-alt-α-1,4-glucan (nigeran) ester derivatives. Polymer 214:123343.
3) Kaaij, R. M. van der et al., (2007) Two novel, putatively cell wall-associated, andglycosylphosphatidylinositol-anchored α-glucanotransferase enzymes of Aspergillus
niger. Eukaryot. Cell 6(7): 1178-1188.