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Transcriptional and secretome analysis of Rasamsonia emersonii lytic polysaccharide mono-oxygenases

ABSTRACT: The study is first to describe the temporal and differential transcriptional expression of two lytic polysaccharide monooxygenase (LPMO) genes of Rasamsonia emersonii in response to various carbon sources. The mass spectrometry based expression of carbohydrate active enzymes (CAZymes) on different carbon sources showed varying levels of LPMOs (AA9), AA3, AA7, catalase, and superoxide dismutase pointing to the redox interplay between the LPMOs and auxiliary enzymes. Moreover, it was observed that cello-oligosaccharides have a negative impact on the expression of LPMOs, which has not been highlighted in any previous research. The LPMO1 (30 kDa) and LPMO2 (47 kDa), cloned and expressed in Pichia pastoris were catalytically active with (Kcat/Km) of 6.6 × 10-2 mg-1 ml min-1 and 1.8 × 10-2 mg-1 ml min-1 against Avicel, respectively. The mass spectrometry of hydrolysis products of Avicel/CMC showed presence of C1/C4 oxidized oligosaccharides indicating them to be type 3 LPMOs. The 3D structural analysis of LPMO1 and LPMO2 revealed distinct arrangement of conserved catalytic residues. Furthermore, the developed enzyme cocktails consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant LPMO1/LPMO2 resulted in significantly enhanced saccharification of steam/acid pretreated unwashed rice straw slurry from PRAJ industries (Pune, India). The current work indicates that LPMO1 and LPMO2 are catalytically efficient and have a high degree of thermostability, emphasising their usefulness in improving benchmark enzyme cocktail performance.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Fungi Rasamsonia Emersonii

TISSUE(S): Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Marcos Di Falco

LAB HEAD: Bhupinder Singh Chadha

PROVIDER: PXD046292 | Pride | 2024-10-17

REPOSITORIES: Pride

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Transcriptional and secretome analysis of Rasamsonia emersonii lytic polysaccharide mono-oxygenases.

Raheja Yashika Y Singh Varinder V Kumar Nitish N Agrawal Dhruv D Sharma Gaurav G Di Falco Marcos M Tsang Adrian A Chadha Bhupinder Singh BS

Applied microbiology and biotechnology 20240821 1

The current study is the first to describe the temporal and differential transcriptional expression of two lytic polysaccharide monooxygenase (LPMO) genes of Rasamsonia emersonii in response to various carbon sources. The mass spectrometry based secretome analysis of carbohydrate active enzymes (CAZymes) expression in response to different carbon sources showed varying levels of LPMOs (AA9), AA3, AA7, catalase, and superoxide dismutase enzymes pointing toward the redox-interplay between the LPMO ...[more]

PMID: 39167166

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Project description:Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes found in viruses, archaea, bacteria as well as eukaryotes, such as fungi, algae and insects, actively contributing to the degradation of different polysaccharides. In Aspergillus nidulans, LPMOs from family AA9 (AnLPMO9s), along with an AA3 cellobiose dehydrogenase (AnCDH1), are co-secreted upon growth on crystalline cellulose and lignocellulosic substrates, indicating their role in the degradation of plant cell wall components. Functional analysis revealed that the three main secreted LPMO9s (AnLPMO9C, AnLPMO9F and AnLPMO9G) correspond to cellulose- active enzymes with distinct regioselectivity. Deletion and overexpression studies confirmed that the abundantly secreted AnLPMO9F is a major component of the extracellular cellulolytic system, while AnLPMO9G, less abundant in the secretome, has an important role by oxidizing crystalline fractions of cellulose. Single or double deletion of these AnLPMO9s partially impair fungal growth on sugarcane straw but not on crystalline cellulose, demonstrating the importance of LPMO9s for the saprophytic fungal lifestyle in the degradation of complex lignocellulosic substrates. Although the deletion of AnCDH1 slightly reduced the cellulolytic activity, it did not affect fungal growth indicating the existence of other electron donors to LPMOs. Additionally, double or triple knockouts of these enzymes had no accumulative deleterious effect on the cellulolytic activity nor on fungal growth, regardless of the deleted gene. The extracelllular proteomes of single deleted mutants growing in Avicel was analysed on a Q-TOF mass spectrometer and revealed an overall reduction in cellulase secretion, and some specifically some changes in the secretion of some enzymes, suggesting an adaptive mechanism adopted to compensate LPMO9s absence during cellulose degradation. Overexpression of AnLPMO9s in a cellulose-induced secretome background confirmed the importance and applicability of AnLPMO9G to improve lignocellulose saccharification.

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Dataset Information

Transcriptional and secretome analysis of Rasamsonia emersonii lytic polysaccharide mono-oxygenases

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Transcriptional and secretome analysis of Rasamsonia emersonii lytic polysaccharide mono-oxygenases.

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