Abstract
Pectin-rich biomasses, such as citrus peel and sugar beet pulp, hold promise as inexpensive feedstocks for microbial fermentations as enzymatic hydrolysis of their component polysaccharides can be accomplished inexpensively to yield high concentrations of fermentable sugars and d-galacturonic acid (d-galUA). In this study, we tackle a number of challenges associated with engineering a microbial strain to convert pectin-rich hydrolysates into commodity and specialty chemicals. First, we engineer d-galUA utilization into yeast, Saccharomyces cerevisiae. Second, we identify that the mechanism of d-galUA uptake into yeast is mediated by hexose transporters and that consumption of d-galUA is inhibited by d-glucose. Third, we enable co-utilization of d-galUA and d-glucose by identifying and expressing a heterologous transporter, GatA, from Aspergillus niger. Last, we demonstrate the use of this transporter for production of the platform chemical, meso-galactaric acid, directly from industrial Navel orange peel waste.
Highlights
Pectin-rich biomasses, such as citrus peel and sugar beet pulp, hold promise as inexpensive feedstocks for microbial fermentations as enzymatic hydrolysis of their component polysaccharides can be accomplished inexpensively to yield high concentrations of fermentable sugars and D-galacturonic acid (D-galUA)
Since D-galUA is oxidized compared to neutral hexoses, pectin hydrolysates equilibrate at low pH due to the high concentration of free D-galUA
We found that D-galacturonate reductase from Trichoderma reesei (GAR1) and 2-keto-3-deoxy-L-galactonate aldolase from Aspergillus niger (GAAC) have activities near the rate-limiting specific activity of glycolysis: 0.1 μmol min−1 mg−1 protein (Supplementary Table 2) 32
Summary
Pectin-rich biomasses, such as citrus peel and sugar beet pulp, hold promise as inexpensive feedstocks for microbial fermentations as enzymatic hydrolysis of their component polysaccharides can be accomplished inexpensively to yield high concentrations of fermentable sugars and D-galacturonic acid (D-galUA). Buffering at high pH with titrants eliminates this hygiene benefit and results in byproduct formation (e.g., gypsum) Both filamentous fungi and yeast have been proposed as fermentation hosts for pectin-rich wastes due to their tolerance of acidic conditions and use in industrial processes. Filamentous fungi have been studied for their native D-galUA metabolism, transport, and secretion of pectinases for depolymerization of pectin cell walls[18] These efforts have culminated in the engineering of an Aspergillus niger strain that produced 3.1 g/ L meso-galactaric acid from citrus peel in a consolidated bioprocess by disruption of D-galUA catabolism and expression of uronate dehydrogenase (UDH)[19]. Co-transport of D-xylose and D-glucose in hemicellulose hydrolysates has remained a heavily investigated area of yeast engineering[30,31]
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