Production of Galactose Oxidase Inside the Fusarium fujikuroi Species Complex and Recombinant Expression and Characterization of the Galactose Oxidase GaoA Protein from Fusarium subglutinans

Faria, Carla Bertechini; Castro, Fausto Fernandes de; Martim, Damaris Batistão; Abe, Camila Agnes Lumi; Prates, Kelly Valério; Oliveira, Marco Aurélio Schüler de; Barbosa-Tessmann, Ione Parra

doi:10.1007/s12033-019-00190-6

Cited by 8 publications

(11 citation statements)

References 66 publications

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“…Building upon our previous discovery of unique substrate specificities among Colletotrichum AA5_2 homologs [ 18 , 19 ], we were keen to explore alternative homologs of AA5_2 members from Fusarium species, from which a number of galactose oxidases have been described [ 53 , 54 , 56 ], including the archetypal Fgr GalOx [ 17 , 25 , 57 , 58 ]. A molecular phylogeny was generated to guide target selection, using 45 AA5_2 catalytic modules selected from the CAZy database [ 13 ], including previously characterized homologs from Fusarium and Colletotrichum (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…numbers also listed. Data from previous studies comprise AAO95371 ( Fgr GalOx) [ 17 , 57 , 58 ], AHA90705.1 ( Fox GalOx) [ 54 ], AIR07394.1 ( Fsa GalOx) [ 53 ], AJE27923 ( Fsu GalOx) [ 56 ], ADG08188.1 and ADG08187.1 ( Fve AA5 and Fsu AA5) [ 59 ], XP_011322138 and XP_018246910 ( Fgr AAO and Fox AAO, this study), EFQ27661.1 ( Cgr AAO) [ 19 ], EFQ30446 and ELA25906.1 ( Cgr AlcOx and Cgl AlcOx) [ 18 ], EFQ36699.1 ( Cgr RafOx) [ 55 ], OBR05259.1 ( Chi AlcOx) and XP_003719369.1 ( Po AlcOx) [ 26 ] …”

Section: Resultsmentioning

confidence: 99%

“…Fgr AAO and Fox AAO have ca . 60% pairwise identity versus the AA5 archetype Fgr GalOx [ 17 , 61 ] and with characterized GalOx from F. oxysporum, F. sambucinum and F. subglutinans [ 53 , 54 , 56 ] (Fig. 1 and Additional file 1 : Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…In summary, a detailed biochemical characterization of two orthologous CROs from Fusarium graminearum and Fusarium oxysporum , which segregate into a previously uncharacterized clade with AA5_2, revealed a distinct substrate specificity profile versus known GalOx [ 17 , 25 , 53 – 56 ], AlcOx [ 18 , 26 ], and AAO [ 19 ] within the family. Despite strong conservation of active-site architecture compared to the archetypal Fgr GalOx, both Fgr AAO and Fox AAO have a novel native preference for mannose and fructose over galactose or galactosides, and a clear preference for aryl alcohols, specifically 3-methoxybenzyl alcohol and veratryl alcohol.…”

Section: Discussionmentioning

confidence: 99%

“…The diversity of CROs from AA5 remains largely unexplored, but features a breadth of sequences from fungal sources that can be mined for potentially new industrial applications. Of the hundreds of publicly available putative AA5 sequences currently in the CAZy database [ 13 ], only ten AA5_2 members from this subfamily have been biochemically characterized, with the majority (five) characterized as galactose or galactoside oxidases (four from Fusarium species) [ 53 – 56 ]. In this context, we have recently revealed novel non-carbohydrate-active CROs in AA5_2 from the phytopathogenic fungus Colletotrichum graminicola : Cgr AlcOx displays high activity on primary aliphatic, benzyl, and other unsaturated alcohols [ 18 ], while Cgr AAO is most active on a range of benzyl alcohols and is also active on hydroxymethylfurfural (HMF, EC 1.1.3.47) [ 19 ].…”

Section: Introductionmentioning

confidence: 99%

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Two Fusarium copper radical oxidases with high activity on aryl alcohols

Cleveland

Lafond

Xia

et al. 2021

Biotechnol Biofuels

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Background Biomass valorization has been suggested as a sustainable alternative to petroleum-based energy and commodities. In this context, the copper radical oxidases (CROs) from Auxiliary Activity Family 5/Subfamily 2 (AA5_2) are attractive biocatalysts for the selective oxidation of primary alcohols to aldehydes. Originally defined by the archetypal galactose 6-oxidase from Fusarium graminearum, fungal AA5_2 members have recently been shown to comprise a wide range of specificities for aromatic, aliphatic and furan-based alcohols. This suggests a broader substrate scope of native CROs for applications. However, only 10% of the annotated AA5_2 members have been characterized to date. Results Here, we define two homologues from the filamentous fungi Fusarium graminearum and F. oxysporum as predominant aryl alcohol oxidases (AAOs) through recombinant production in Pichia pastoris, detailed kinetic characterization, and enzyme product analysis. Despite possessing generally similar active-site architectures to the archetypal FgrGalOx, FgrAAO and FoxAAO have weak activity on carbohydrates, but instead efficiently oxidize specific aryl alcohols. Notably, both FgrAAO and FoxAAO oxidize hydroxymethyl furfural (HMF) directly to 5-formyl-2-furoic acid (FFCA), and desymmetrize the bioproduct glycerol to the uncommon L-isomer of glyceraldehyde. Conclusions This work expands understanding of the catalytic diversity of CRO from AA5_2 to include unique representatives from Fusarium species that depart from the well-known galactose 6-oxidase activity of this family. Detailed enzymological analysis highlights the potential biotechnological applications of these orthologs in the production of renewable plastic polymer precursors and other chemicals.

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