Loss of AA13 LPMOs impairs degradation of resistant starch and reduces the growth of Aspergillus nidulans

Abstract

Background Lytic polysaccharide monooxygenases (LPMOs) are often studied in simple models involving activity measurements of a single LPMO or a blend thereof with hydrolytic enzymes towards an insoluble substrate. However, the contribution of LPMOs to polysaccharide breakdown in complex cocktails of hydrolytic and oxidative enzymes, similar to fungal secretomes, remains elusive. Typically, two starch-specific AA13 LPMOs are encoded by mainly Ascomycota genomes. Here, we investigate the impact of LPMO loss on the growth and degradation of starches of varying resistance to amylolytic hydrolases by Aspergillus nidulans . Results Deletion of the genes encoding An AA13A that possesses a CBM20 starch-binding module, An AA13B (lacking a CBM20) or both AA13 genes resulted in reduced growth on solid media with resistant, but not soluble processed potato starch. Larger size and amount of residual starch granules were observed for the AA13-deficient strains as compared to the reference and the impairment of starch degradation was more severe for the strain lacking An AA13A based on a microscopic analysis. After 5 days of growth on raw potato starch in liquid media, the mount of residual starch was about fivefold higher for the AA13 gene deletion strains compared to the reference, which underscores the importance of LPMOs for degradation of especially resistant starches. Proteomic analyses revealed substantial changes in the secretomes of the double AA13 gene deletion, followed by the An AA13A-deficient strain, whereas only a single protein was significantly different in the proteome of the An AA13B-deficient strain as compared to the reference. Conclusions This study shows that the loss of AA13, especially the starch-binding An AA13A, impairs degradation of resistant potato starch, but has limited impact on less-resistant wheat starch and no impact on processed solubilized starch. The effects of LPMO loss are more pronounced at the later stages of fungal growth, likely due to the accumulation of the less-accessible regions of the substrate. The striking impairment in granular starch degradation due to the loss of a single LPMO from the secretome offers insight into the crucial role played by AA13 in the breakdown of resistant starch and presents a methodological framework to analyse the contribution of distinct LPMOs towards semi-crystalline polysaccharides under in vivo conditions.

Discovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs

“…T he involvement of oxidative processes in polysaccharide degradation by fungi has been proposed by the pioneering work of Eriksson et al in 1974 1 . This notion has gained strong support by the recent discovery of lytic polysaccharide monooxygenases (LPMOs) that uniquely catalyse the oxidative cleavage of glycosidic bonds in (semi)crystalline polysaccharides such as starch [2][3][4] , chitin 5 , cellulose [6][7][8] and cellulose-bound hemicelluloses, e.g., xyloglucan 9 and xylan 10 . Besides LPMOs, filamentous fungi co-secrete an impressing diversity of carbohydrate-specific oxidoreductases 11 .…”

Combination of genetic engineering and random mutagenesis for improving production of raw-starch-degrading enzymes in Penicillium oxalicum

“…AA13 catalyses oxidative cleavage of insoluble starch. Deletion of AA13 in Aspergillus nidulans seriously impaired the degradation of resistant starch, but showed no effects against soluble starch [ 20 ]. Unexpectedly, the transcriptional abundance of two α-glucosidase genes ( POX_f08248 and POX_e06687 ), 1,4-α-glucan-branching enzyme gene POX_d05520 , and α-amylase gene POX_b03245 were downregulated in GXUR001 (Fig.…”

A seven-transmembrane methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases

“…C8V530, gene ID AN10419, is classified as a member of Auxiliary Activity Family 9 (AA9 LPMO) of the CAZy class of glycoside hydrolases by InterPro 17 and Pfam 18 (Table S1). Q5B1W7 (AN5463) plays a crucial role in starch degradation 19 , and similarly contains a predicted glycoside hydrolase domain followed by a predicted carbohydrate-binding module by Pfam (Table S2). Q5AU55 (AN4702) is structurally predicted to be most similar to an AA11-type LPMO from A. oryzae.…”