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

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 ⌬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...

Conversion of orange peel to L-galactonic acid in a consolidated process using engineered strains of Aspergillus niger

“…This value is relatively close to the initial productivities of 46–64 and 54–70 mg l −1  h −1 that were obtained with Δ gaaB and Δ gaaB - gaaA , respectively, in SmF of a defined medium supplemented with 10 g l −1 of D-galacturonic acid and 2 g l −1 of D-xylose (Kuivanen et al [2012]). The product yields (% of theoretical maximum, i.e.…”

“…Since this strain produces pectinases, it was able to produce L-galactonate from polygalacturonate in a submerged fermentation (Kuivanen et al [2012]). The aim of the current work was to determine whether this strain could be used to hydrolyse the pectin in the CPW and convert the resulting D-galacturonic acid to L-galactonic acid in a single fermentation process.…”

The interaction of induction and repression mechanisms in the regulation of galacturonic acid-induced genes in Aspergillus niger

“…The genes encoding these enzymes (gaaA, gaaB, gaaC, and gaaD have been identified and the biochemical properties of the enzymes have been determined (Kuorelahti et al, 2005Hilditch et al, 2007;Mojzita et al, 2010;Wiebe et al, 2010;Liepins et al, 2006;Zhang et al, 2011;Kuivanen et al, 2012). Specific sugar transporters that are able to transport GA over the plasma membrane have recently been identified and characterised in Neurospora crassa (Benz et al, 2014) as well as in A. niger (Sloothaak et al, 2014) and Botrytis cinerea (Zhang et al, 2014).…”

Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans

“…library.cornell.edu/usda/current/CropProdSu/CropProdSu-01-10-2014.pdf). Pectins are currently used primarily as animal feed or discarded; the use as livestock feed involves drying the pulp to preclude spoilage, with an attendant energy requirement 30-40% of the total energy input for beet processing [1,2], while disposal incurs added expense, and it is thus becoming economically and environmentally desirable to develop value-added products from these bio-resources. Structurally, pectins comprise a family of heterogeneous polysaccharides from plant primary cell walls wherein d-galacturonic acid (GalUA) occurs in abundance, and they can be grouped as homogalacturonan (HG), xylogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II [3].…”