Abstract
Proteins with putative erythrose reductase activity have been identified in the filamentous fungi Trichoderma reesei, Aspergillus niger, and Fusarium graminearum by in silico analysis. The proteins found in T. reesei and A. niger had earlier been characterized as glycerol dehydrogenase and aldehyde reductase, respectively. Corresponding genes from all three fungi were cloned, heterologously expressed in Escherichia coli, and purified. Subsequently, they were used to establish optimal enzyme assay conditions. All three enzymes strictly require NADPH as cofactor, whereas with NADH no activity could be observed. The enzymatic characterization of the three enzymes using ten substrates revealed high substrate specificity and activity with D-erythrose and D-threose. The enzymes from T. reesei and A. niger herein showed comparable activities, whereas the one from F. graminearum reached only about a tenth of it for all tested substrates. In order to proof in vivo the proposed enzyme function, we overexpressed the erythrose reductase-encoding gene in T. reesei. An increased production of erythritol by the recombinant strain compared to the parental strain could be detected.
Highlights
Erythritol is a four-carbon sugar alcohol, which is applied as flavour enhancer, formulation aid, humectants, stabilizer, thickener, and as low-calorie sweetener, of which the latter is the main utilization
The following proteins were found in these organisms: for T. reesei the NADPdependent glycerol dehydrogenase (GLD1) (NCBI accession number ABD83952.1, query coverage 98%, max. ident. 50%, E-value 5e-87); for A. niger the aldehyde reductase 1 (Alr1) CBS 513.88 (NCBI accession number XP_001394119.2, query coverage 98%, max. ident. 49%, Evalue 8e-88); and for F. graminearum a hypothetical protein FG04223.1 (NCBI accession number XP_384399.1, query coverage 98%, max. ident. 48%, E-value 1e-98)
Query results are given relative to ER3, which showed a slightly better match with the protein found for T. reesei than ER1 and ER2
Summary
Erythritol is a four-carbon sugar alcohol, which is applied as flavour enhancer, formulation aid, humectants, stabilizer, thickener, and as low-calorie sweetener, of which the latter is the main utilization. It has a natural occurrence in several foods including beer, sake, wine, soy sauce, water melon, pear and grape (O’Donnell and Kearsley 2012; Sreenath and Venkatesh 2008) and is well tolerated by the human body (Munro et al 1998). Erythritol can be chemically synthesized from dialdehyde starch with a nickel catalyst at high temperatures, but this process is not stereospecific and low in yield, and not industrialized (Moon et al 2010). As substrate a highly concentrated glucose solution (typically 40% (w/v)) is applied, which is
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