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Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these [alpha]-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize altodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-[alpha]-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of 1-glucan substrates by P. furiosus.

In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive alpha-amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic alpha-amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant-produced alpha-amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant-made enzyme retained 85% of its initial activity after 3 h incubation at 100 degrees C, whereas the E. coli-produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca(2+) or plant cell extracts from tobacco and sweetpotato to the E. coli-produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium-rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available.

Bioinformatics analysis and transcriptional response information for Pyrococcus furiosus grown on alpha-glucans led to the identification of a novel isomaltase (PF0132) representing a new glycoside hydrolase (GH) family, a novel GH57 beta-amylase (PF0870), and an extracellular starch-binding protein (1,141 amino acids; PF1109-PF1110), in addition to several other putative alpha-glucan-processing enzymes.