Hydrolytic and phosphorolytic metabolism of cellobiose by the marine aerobic bacterium Saccharophagus degradans 2-40T

Abstract

Saccharophagus degradans 2-40 is a marine gamma proteobacterium that can produce polyhydroxyalkanoates from lignocellulosic biomass using a complex cellulolytic system. This bacterium has been annotated to express three surface-associated β-glucosidases (Bgl3C, Ced3A, and Ced3B), two cytoplasmic β-glucosidases (Bgl1A and Bgl1B), and unusual for an aerobic bacterium, two cytoplasmic cellobiose/cellodextrin phosphorylases (Cep94A and Cep94B). Expression of the genes for each of the above enzymes was induced when cells were transferred into a medium containing Avicel as the major carbon source except for Bgl1B. Both hydrolytic and phosphorolytic degradation of cellobiose by crude cell lysates obtained from cellulose-grown cells were demonstrated and all of these activities were cell-associated. With the exception of Cep94B, each purified enzyme exhibited their annotated activity upon cloning and expression in E. coli. The five β-glucosidases hydrolyzed a variety of glucose derivatives containing β-1, (2, 4, or 6) linkages but did not act on any α-linked glucose derivatives. All but one β-glucosidases exhibited transglycosylation activity consistent with the formation of an enzyme-substrate intermediate. The biochemistry and expression of these cellobiases indicate that external hydrolysis by surface-associated β-glucosidases coupled with internal hydrolysis and phosphorolysis are all involved in the metabolism of cellobiose by this bacterium.

Engineering Escherichia coli Cells for Cellobiose Assimilation through a Phosphorolytic Mechanism

“…Because the phosphorylation uses inorganic phosphate as a donor, the phosphorolytic mechanism is an ATP-saving mechanism more often associated with some cellulolytic bacteria (5,12,15,20). All known cellobiose phosphorylases are cytoplasmic, as they lack signal peptides, and experimental data support this notion (1,19). Thus, cellobiose assimilation through phosphorolytic mechanism requires a transporter that delivers the unmodified disaccharide to the cytoplasm.…”

“…The concentration of NADPH formed was determined by measuring the change in absorbance at 340 nm. Glucose-1-phosphate formation was estimated assuming molar equivalence to the NADPH produced (19). The beta-glucosidase activity was determined using pNP-beta-glucoside (Sigma-Aldrich, St. Louis, MO) as the substrate, and released p-nitrophenol was measured spectrophotometrically.…”

“…Each subcellular sample was analyzed for cellobiose phosphorylase and beta-glucosidase activity. The phosphorylase activity of recombinant CepA was determined by measuring the amount of glucose-1-phosphate generated from cellobiose and phosphate (19). The reaction mixture in a total volume of 200 l consisted of 50 mM PIPES [piperazine-N,N=-bis(2-ethanesulfonic acid)] buffer, 8 mM phosphate at pH 7.0, 5 mM MgSO 4 , 0.5 mM NADP, 4 U of phosphoglucomutase, 2 U of glucose-6-phosphate dehydrogenase (Sigma-Aldrich, St. Louis, MO), 250 M pure cellobiose, and 50 l of enzyme solution.…”

Complex Expression of the Cellulolytic Transcriptome of Saccharophagus degradans

“…S. degradans is a pleomorphic, Gram-negative, aerobic, motile gammaproteobacterium that was isolated from decaying Spartina alterniflora in the Chesapeake Bay (1,4,8). Twenty-two enzymes have been shown to be components of the unusual cellulolytic system in this bacterium (17,20,23). The system was found to lack cellobiohydrolases but instead appears to use processive endoglucanases to degrade cellulose.…”

“…S. degradans expresses little glucanase activity when it grows on glucose as the primary carbon source (5), but cellulase activity in general and several specific glucanases, such as Cel5F, Cel5H, and Cel5I, as well as their mRNAs, are detected when the bacterium is grown on Avicel (5,17,20,23). This suggests that these genes are under the transcriptional control of one or more regulatory systems that are responsive to cellulose.…”