D-Galacturonic acid is the main constituent of pectin, a naturally abundant compound. Pectin-rich residues accumulate when sugar is extracted from sugar beet or juices are produced from citrus fruits. It is a cheap raw material but currently mainly used as animal feed. Pectin has the potential to be an important raw material for biotechnological conversions to fuels or chemicals. In this paper, we review the microbial pathways for the catabolism of D-galacturonic acid that would be relevant for the microbial conversion to useful products.

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 ...

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 ⌬lgd1 and Aspergillus niger ⌬gaaB strains produced L-galactonate at yields of 0.6 to 0.9 g per g of substrate consumed. Although T. reesei ⌬lgd1 could produce L-galactonate at pH 5.5, a lower pH was necessary for A. niger ⌬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 ⌬gaaB produced L-galactonate from polygalacturonate as efficiently as from the monomer. D-Galacturonic acid is the principal component of pectin, a major constituent of sugar beet pulp and citrus peel, which are abundant and inexpensive raw materials. The annual worldwide production of sugar beet and citrus fruit is about 250 ϫ 10 6 and 115 ϫ 10 6 metric tons, respectively. After beet processing, 5 to 10% of the sugar beet remains as dried sugar beet pulp. This pulp contains ca. 25% pectin (6). Citrus peel contains ca. 20% pectin on a dry mass basis. Sugar beet pulp and citrus peel are mainly used as cattle feed, or they are dumped. The use as cattle feed requires that the pulp and peel are dried since; otherwise, they rot rapidly. Disposal of the material is problematic because of the bad odor generated at the dumping sites. In the case of sugar beet pulp the energy consumption for drying and pelleting are 30 to 40% of the total energy used for beet processing (6). This process is only economical when done on a large scale and when energy costs are low. Other products, such as pectin and limonene, may be extracted from citrus peel. Pectin is used as a gelling agent in the food industry; limonene as a flavor compound. These are limited markets, and with increasing energy costs and alternative animal feed sources reducing the revenues from pectin-rich biomass for cattle feed sales, it is desirable to find new ways to convert this biomass to other useful products. This may be accomplished by microbial fermentation (16). 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...

BackgroundThe D-galacturonic acid derived from plant pectin can be converted into a variety of other chemicals which have potential use as chelators, clarifiers, preservatives and plastic precursors. Among these is the deoxy-keto acid derived from L-galactonic acid, keto-deoxy-L-galactonic acid or 3-deoxy-L-threo-hex-2-ulosonic acid. The keto-deoxy sugars have been found to be useful precursors for producing further derivatives. Keto-deoxy-L-galactonate is a natural intermediate in the fungal D-galacturonate metabolic pathway, and thus keto-deoxy-L-galactonate can be produced in a simple biological conversion.ResultsKeto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) accumulated in the culture supernatant when Trichoderma reesei Δlga1 and Aspergillus niger ΔgaaC were grown in the presence of D-galacturonate. Keto-deoxy-L-galactonate accumulated even if no metabolisable carbon source was present in the culture supernatant, but was enhanced when D-xylose was provided as a carbon and energy source. Up to 10.5 g keto-deoxy-L-galactonate l-1 was produced from 20 g D-galacturonate l-1 and A. niger ΔgaaC produced 15.0 g keto-deoxy-L-galactonate l-1 from 20 g polygalacturonate l-1, at yields of 0.4 to 1.0 g keto-deoxy-L-galactonate [g D-galacturonate consumed]-1. Keto-deoxy-L-galactonate accumulated to concentrations of 12 to 16 g l-1 intracellularly in both producing organisms. This intracellular concentration was sustained throughout production in A. niger ΔgaaC, but decreased in T. reesei.ConclusionsBioconversion of D-galacturonate to keto-deoxy-L-galactonate was achieved with both A. niger ΔgaaC and T. reesei Δlga1, although production (titre, volumetric and specific rates) was better with A. niger than T. reesei. A. niger was also able to produce keto-deoxy-L-galactonate directly from pectin or polygalacturonate demonstrating the feasibility of simultaneous hydrolysis and bioconversion. Although keto-deoxy-L-galactonate accumulated intracellularly, concentrations above ~12 g l-1 were exported to the culture supernatant. Lysis may have contributed to the release of keto-deoxy-L-galactonate from T. reesei mycelia.

There are two distinctly different pathways for the catabolism of l‐rhamnose in microorganisms. One pathway with phosphorylated intermediates was described in bacteria; here the enzymes and the corresponding gene sequences are known. The other pathway has no phosphorylated intermediates and has only been described in eukaryotic microorganisms. For this pathway, the enzyme activities have been described but not the corresponding gene sequences. The first enzyme in this catabolic pathway is the NAD‐utilizing l‐rhamnose 1‐dehydrogenase. The enzyme was purified from the yeast Pichia stipitis, and the mass of its tryptic peptides was determined using MALDI‐TOF MS. This enabled the identification of the corresponding gene, RHA1. It codes for a protein with 258 amino acids belonging to the protein family of short‐chain alcohol dehydrogenases. The ORF was expressed in Saccharomyces cerevisiae. As the gene contained a CUG codon that codes for serine in P. stipitis but for leucine in S. cerevisiae, this codon has changed so that the same amino acid was expressed in S. cerevisiae. The heterologous protein showed the highest activity and affinity with l‐rhamnose and a lower activity and affinity with l‐mannose and l‐lyxose. The enzyme was specific for NAD. A northern blot analysis revealed that transcription in P. stipitis is induced during growth on l‐rhamnose but not on other carbon sources.