Metabolic Engineering of Fungal Strains for Conversion of d -Galacturonate to meso -Galactarate

Abstract

D-Galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of D-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical. In fungi D- D-Galacturonate is the main component of pectin, an abundant and cheap raw material. Sugar beet pulp and citrus peel are both rich in pectin residues. At present, these residues are mainly used as cattle feed. However, since energy-consuming drying and pelletizing of the residues is required to prevent them from rotting, it is not always economical to process the residues, and it is desirable to find alternative uses.Various microbes which live on decaying plant material have the ability to catabolize D-galacturonate using various, completely different pathways (19). Eukaryotic microorganisms use a reductive pathway in which D-galacturonate is first reduced to L-galactonate by an NAD(P)H-dependent reductase (12,17). In the following steps a dehydratase, aldolase, and reductase convert the L-galactonate to pyruvate and glycerol (9,11,14).In Hypocrea jecorina (anamorph Trichoderma reesei) the gar1 gene codes for a strictly NADPH-dependent D-galacturonate reductase. In Aspergillus niger a homologue gene sequence, gar2, exists; however, a different gene, gaaA, is upregulated during growth on D-galacturonate containing medium (16). The gaaA codes for a D-galacturonate reductase with different kinetic properties than the H. jecorina enzyme, having a higher affinity toward D-galacturonate and using either NADH or NADPH as cofactor. It is not known whether gar2 codes for an active protein.Some bacteria, such as Agrobacterium tumefaciens or Pseudomonas syringae, have an oxidative pathway for D-galacturonate catabolism. In this pathway D-galacturonate is first oxidized to meso-galactarate (mucate) by an NAD-utilizing D-galacturonate dehydrogenase. Galactarate is then converted in the following steps to ␣-ketoglutarate. This route is sometimes called the ␣-ketoglutarate pathway (20). Galactarate can also be catabolized through the glycerate pathway (20). The products of this pathway are pyruvate and D-glycerate. These pathways have been described in prokaryotes, and it is not certain whether similar pathways also exist in fungi, some of which are able to metabolize galactarate.D-Galacturonate dehydrogenase (EC 1.1.1.203) has been described in Agrobacterium tumefaciens and in Pseudomonas syringae, and the enzymes from these organisms have been purified and characterized (3,6,22). Recently, the corresponding genes were also identified (4, 24). Both enzymes are specific for NAD as a cofactor but are not specific for the substrate. They oxidize D-galacturonate and D-glucuronate to meso-galactarate (mucate) and D-glucarate (saccharate), respectively. The reaction product is probably the hexaro-lactone which spontaneously hydrolyzes. The reverse reaction can only be observed at acidic ...

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

“…1) that includes reactions catalyzed by D-galacturonic acid reductase (10), L-galactonate dehydratase (9), 2-keto-3-deoxy galactonate aldolase (8), and L-glyceraldehyde reductase (11); the intermediates are L-galactonate, 2-keto-3-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate), and L-glyceraldehyde, and the products of the pathway are pyruvate and glycerol. D-Galacturonic acid can induce pectinolytic and D-galacturonic acid catabolic genes in A. niger, regardless of whether D-galacturonic acid is metabolized or not (4,14).…”

“…Genetically modified bacteria have been used to produce ethanol from pectin-rich biomass (5,7). Using genetically modified fungi, D-galacturonic acid has been converted to galactaric acid (14) or to 2-keto-3-deoxy-L-galactonic acid (20).…”

“…1) that includes reactions catalyzed by D-galacturonic acid reductase (10), L-galactonate dehydratase (9), 2-keto-3-deoxy galactonate aldolase (8), and L-glyceraldehyde reductase (11); the intermediates are L-galactonate, 2-keto-3-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate), and L-glyceraldehyde, and the products of the pathway are pyruvate and glycerol. D-Galacturonic acid can induce pectinolytic and D-galacturonic acid catabolic genes in A. niger, regardless of whether D-galacturonic acid is metabolized or not (4,14).By disrupting the native D-galacturonic acid catabolic pathway, it is possible to engineer fungal strains for alternative D-galacturonic acid conversions (14,20). In the case of galactaric acid production, the gene encoding D-galacturonic acid reductase was deleted and a gene encoding a D-galacturonic acid dehydrogenase expressed (14).…”

“…By disrupting the native D-galacturonic acid catabolic pathway, it is possible to engineer fungal strains for alternative D-galacturonic acid conversions (14,20). In the case of galactaric acid production, the gene encoding D-galacturonic acid reductase was deleted and a gene encoding a D-galacturonic acid dehydrogenase expressed (14).…”

Genetic improvement of microorganisms for applications in biorefineries

“…Nowadays, strains that are able to produce a variety of chemical compounds in concentrations as high as above 90% m/m of the theoretical maximum are available (Table 1) (Figure 2). The strategies to increase product formation generally include a series of modifications in the microorganism metabolism, achieved by overexpression or knockout of enzymes in the producing pathway [13,32], changing redox balancing of the cell by redirecting carbon fluxes from NADPH-to NADH consuming reactions [33][34][35][36], engineering global transcription machinery [37] and others (Table 1). All these types of modification were employed, for instance, to obtain S. cerevisiae strains that are able to produce ethanol from sugars that are present in lignocellulosic hydrolysates [38].…”