Engineering Filamentous Fungi for Conversion of d -Galacturonic Acid to l -Galactonic Acid

Abstract

ABSTRACT d -Galacturonic acid, the main monomer of pectin, is an attractive substrate for bioconversions, since pectin-rich biomass is abundantly available and pectin is easily hydrolyzed. l -Galactonic acid is an intermediate in the eukaryotic pathway for d -galacturonic acid catabolism, but extracellular accumulation of l -galactonic acid has not been reported. By deleting the gene encoding l -galactonic acid dehydratase ( lgd1 or gaaB ) in two filamentous fungi, strains were obtained that converted d -galacturonic acid to l -galactonic acid. Both Trichoderma reesei Δ lgd1 and Aspergillus niger Δ gaaB strains produced l -galactonate at yields of 0.6 to 0.9 g per g of substrate consumed. Although T. reesei Δ lgd1 could produce l -galactonate at pH 5.5, a lower pH was necessary for A. niger Δ gaaB . Provision of a cosubstrate improved the production rate and titer in both strains. Intracellular accumulation of l -galactonate (40 to 70 mg g biomass −1 ) suggested that export may be limiting. Deletion of the l -galactonate dehydratase from A. niger was found to delay induction of d -galacturonate reductase and overexpression of the reductase improved initial production rates. Deletion of the l -galactonate dehydratase from A. niger also delayed or prevented induction of the putative d -galacturonate transporter An14g04280. In addition, A. niger Δ gaaB produced l -galactonate from polygalacturonate as efficiently as from the monomer.