Phosphoketolase Pathway for Xylose Catabolism in Clostridium acetobutylicum Revealed by ¹³ C Metabolic Flux Analysis

Abstract

b Solvent-producing clostridia are capable of utilizing pentose sugars, including xylose and arabinose; however, little is known about how pentose sugars are catabolized through the metabolic pathways in clostridia. In this study, we identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-13 C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter ؊1 . To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation. Substrate cost is a major factor impacting the economics of fermentative solvent production by clostridia (7, 21). To reduce the substrate cost, abundant and inexpensive lignocellulosic materials could be used, and one of their major components is pentose-rich hemicellulose (15). Solventogenic clostridia, including Clostridium acetobutylicum and Clostridium beijerinckii, are capable of utilizing the hemicellulosic pentoses xylose and arabinose (25). However, knowledge of clostridial pentose metabolism is very limited.Recently, our group has characterized two key enzymes in the xylose utilization pathway of C. acetobutylicum, xylose isomerase and xylulokinase, which convert xylose to xylulose-5-P (14). In many bacteria such as Escherichia coli, xylulose-5-P is further catabolized to form the central intermediate glyceraldehyde-3-P by the pentose phosphate pathway enzymes, including transketolase and transaldolase. Another xylose catabolic pathway through the phosphoketolase is found to be present in heterofermentative and facultative homofermentative lactic acid bacteria (19, 37). The thiamine diphosphate-dependent phosphoketolases (EC 4.1.2.9) cleave xylulose-5-P into acetyl-P and glyceraldehyde-3-P. In bifidobacteria, phosphoketolases are key enzymes of the fructose-6-P shunt to convert fructose-6-P to acetyl-P and erythrose-4-P (12, 34).Little is known about how xylose is metabolized through the metabolic pathways in solventogenic clostridia. Recent studies have shown that genes of the pentose phosphate pathway and a putative phosphoketolase-encoding gene in C. acetobutylicum are induced by pentose sugars (13, 33). However, the contribution of individual pathways to clostridial xylose catabolism has not been analyzed. To manipulate clostridia for efficient xylose utilization, it is important to gain insight into the responses of xylose catabolic pathways to environmental an...

Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

“…Flux distributions under different sugar media were calculated from the following steady-state 13 C-labeling experiments: [ We constructed a carbon mapping model to calculate the fluxes through PKP, PPP, and upper glycolysis from the labeling patterns of ribose-5-phosphate (R5P), Xu5P, glucose-6-phosphate (G6P), fructose-1,6-bisphosphate (FBP), dihydroxyacetone-phosphate (DHAP), and 3-phosphoglycerate (3-PG) (see Fig. S2 in the supplemental material).…”

“…Historically, it was assumed that carbons from Xyl and Arab pass from the PPP through glycolysis to supply acidogenesis and solventogenesis. However, two recent studies (5,13) reported that acids and solvents can be produced from these sugars from a glycolysis-independent pathway, the phosphoketolase pathway (PKP) (Fig. 1B).…”

“…1B). Using 13 C metabolic flux analysis, Liu et al (13) calculated that Xyl catabolic flux through the PKP in C. acetobutylicum increases with increasing Xyl concentration. Furthermore, due to both higher ratios of excreted acetate and a more than 100-fold increase in the mRNA expression of the phosphoketolase enzyme in the presence of Arab, Servinsky et al (5) proposed that the PKP is more significant when the cells are grown with Arab as the C source than with either Gluc or Xyl.…”

“…The PKP involves cleavage of xylulose-5-phosphate (Xu5P) into glyceraldehyde-3-phosphate (GAP) and acetyl-phosphate (Ac-P) and, to a lesser extent, cleavage of fructose-6-phosphate (F6P) into erythrose-4-phosphate (E4P) and Ac-P (Fig. 1B) (5,13). The metabolite Ac-P is a direct precursor to either acetate or acetyl coenzyme A (acetyl-CoA), which are both involved in clostridial production of acids and solvents (Fig.…”

Enhancement of Simultaneous Xylose and Glucose Utilization by Regulating ZWF1 and PGI1 in Saccharomyces Cerevisiae

“…Xylose-specific transporters and high-affinity transporters of xylose were developed by evolution of xylose-utilizing yeast, directed evolution or rational design of glucose transporters [23][24][25][26][27]. Except for transporter engineering, reconstitution of carbohydrate metabolic networks, such as the phosphoketolase pathway, is another approach to improve xylose utilization [15,28]. Recently, it has been reported that disruption of some genes in glycolysis and pentose phosphate (PP) pathway resulted in simultaneous xylose and glucose utilization [5,29].…”