Compartmentalization and metabolic channeling for multienzymatic biosynthesis: practical strategies and modeling approaches.

: The construction of efficient enzyme complexes for multienzymatic biosynthesis is of increasing interest in order to achieve maximum yield and to minimize the interference due to shortcomings that are typical for straightforward one-pot multienzyme catalysis. These include product or intermediate feedback inhibition, degeneration, and diffusive losses of reaction intermediates, consumption of co-factors, and others. The main mechanisms in nature to tackle these effects in transient or stable protein associations are the formation of metabolic channeling and microcompartments, processes that are desirable also for multienzymatic biosynthesis in vitro. This chapter provides an overview over two main aspects. First, numerous recent strategies for establishing compartmentalized multienzyme associations and constructed synthetic enzyme complexes are reviewed. Second, the computational methods at hand to investigate and optimize such associations systematically, especially with focus on large multienzyme complexes and metabolic channeling, are discussed. Perspectives on future studies of multienzymatic biosynthesis concerning compartmentalization and metabolic channeling are presented.

Evaluations of cellulose accessibilities of lignocelluloses by solute exclusion and protein adsorption techniques.

Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by drying the never dried pretreated sample and a set of differently pretreated lodgepople pine substrates, were evaluated using solute exclusion and protein adsorption methods. Direct measurements of cellulase adsorption onto cellulose surface of the set of pretreated substrates were also carried out using an in situ UV-Vis spectrophotometric technique. The cellulose accessibilities measured by the solute exclusion and a cellulose-binding module (CBM)-containing green fluorescent protein (TGC) adsorption methods correlate well for both sets of samples. The substrate enzymatic digestibilities (SEDs) of the hornified substrates are proportional to the measured cellulose accessibilities. Approximately over 90% of the SED was contributed by the accessible pore surfaces of the hornified substrates, suggesting that the substrate external surface plays a minor role contributing to cellulose accessibility and SED. The cellulose accessibilities of the pretreated substrates correlated well with the amounts of cellulase adsorbed. The SEDs of these substrates directly correlated with the amounts of adsorbed cellulase.

Glycoside hydrolase family 9 processive endoglucanase from Clostridium phytofermentans: heterologous expression, characterization, and synergy with family 48 cellobiohydrolase.

The glycoside hydrolase family 9 cellulase (Cel9) from Clostridium phytofermentans has a multi-modular structure and is essential for cellulose hydrolysis. In order to facilitate production and purification of Cel9, recombinant Cel9 was functionally expressed in Escherichia coli. Cel9 exhibited maximum activity at pH 6.5 and 65 degrees C on carboxymethyl cellulose in a 10-min reaction period. The hydrolysis products on regenerated amorphous cellulose (RAC) were cellotetraose (a major product), cellotriose, cellobiose and glucose, and 71-80% of the reducing sugars produced by Cel9 were in soluble form, suggesting that Cel9 was a processive endoglucanase. The highest synergy between C. phytofermentans Cel9 and C. phytofermentans cellobiohydrolase Cel48 on Avicel was about 1.8 at a ratio of about 1:5. Cel9 alone was sufficient to solublize filter paper while Cel48 was not; however, it enhanced the solublization process along with Cel9 synergistically. This study provided useful information for understanding of the cellulose hydrolysis mechanism of this cellulolytic bacterium with potential industrial importance.

A highly active phosphoglucomutase from Clostridium thermocellum: cloning, purification, characterization and enhanced thermostability.

AIMS: Discovery and utilization of highly active and thermostable phosphoglucomutase (PGM) would be vital for biocatalysis mediated by multiple enzymes, for example, high-yield production of enzymatic hydrogen. METHODS AND RESULTS: The thermophilic cellulolytic bacterium Clostridium thermocellum was hypothesized to have a very active PGM because of its key role in microbial cellulose utilization. The Cl. thermocellum ORF Cthe1265 encoding a putative PGM was cloned and expressed in Escherichia coli. The purified enzyme appeared to be a monomer with an estimated molecular weight of 64.9 kDa. This enzyme was found to be a dual-specificity enzyme - PGM/phosphomannomutase (PMM). Mg(2+) and Mn(2+) were activators. Ser144 was identified as an essential catalytic residue through site-directed mutagenesis. The k(cat) and K(m) of PGM were 190 s(-1) and 0.41 mmol l(-1) on glucose-1-phosphate and 59 s(-1) and 0.44 mmol l(-1) on mannose-1-phosphate, respectively, at 60 degrees C. Thermostability of PGM at a low concentration (2 nmol l(-1), 100 U l(-1)) was enhanced by 12-fold (i.e. t(1/2) = 72 h) at 60 degrees C with addition of bovine serum albumin, Triton X-100, Mg(2+)and Mn(2+). CONCLUSIONS: The ORF Cthe1265 was confirmed to encode a PGM with PMM activity. This enzyme was the most active PGM reported. SIGNIFICANCE AND IMPACT OF THE STUDY: This highly active PGM with enhanced thermostability would be an important building block for in vitro synthetic biology projects (complicated biotransformation mediated by multiple enzymes in one pot).

Biosynthesis of radiolabeled cellodextrins by the Clostridium thermocellum cellobiose and cellodextrin phosphorylases for measurement of intracellular sugars.

The Clostridium thermocellum cellobiose and cellodextrin phosphorylases (glucosyl transferases) in the cell extract were used to synthesize radiolabeled cellodextrins with a degree of polymerization (DP=2-6) from nonradioactive glucose-1-phosphate and radioactive glucose. Chain lengths of synthesized cellodextrin were controlled by the absence or presence of dithiothreitol and by reaction conditions. All cellodextrins have the sole radioactive glucose unit located at the reducing ends. Mixed cellodextrins (G2-G6) were separated efficiently by size-exclusion chromatography or less efficiently by thin-layer chromatography. A new rapid sampling device was developed using disposable syringes containing an ultracold methanol-quenching buffer. It was simple, less costly, and especially convenient for anaerobic fermentation. After an impulse feed of radiolabeled cellobiose, the intracellular sugar levels were measured after a series of operations-sampling, extracting, concentrating, separating, and reading. Results showed that the largest amount of radioactivity was cellobiose with lesser amounts of glucose, cellotriose, and cellotetraose, and an average DP of intracellular cellodextrins was ca. 2.