Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s−1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae).

BackgroundSaccharomyces cerevisiae strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-xylose fermentation.ResultsFour XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for D-xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strain’s ability to grow in D-xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24, was further adapted for aerobic and fermentative growth by serial transfers of D-xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h-1 on D-xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment D-xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a Vmax of 0.81 μmole/min/mg protein and a Km of 34 mM.ConclusionThis study identifies a new xylose isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal D-xylose isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment D-xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-xylose fermenting strains, including industrial yeast genetic backgrounds.

Recombinant Arabidopsis NADH:nitrate reductase was expressed in Pichia pastoris using fermentation. Large enzyme quantities were purified for pre-steady-state kinetic analysis, which had not been done before with any eukaryotic nitrate reductase. Basic biochemical properties of recombinant nitrate reductase were similar to natural enzyme forms. Molybdenum content was lower than expected, which was compensated for by activity calculation on molybdenum basis. Stopped-flow rapid-scan spectrophotometry showed that the enzyme FAD and heme were rapidly reduced by NADH with and without nitrate present. NADPH reduced FAD at less than one-tenth of NADH rate. Reaction of NADH-reduced enzyme with nitrate yielded rapid initial oxidation of heme with slower oxidation of flavin. Rapid-reaction freeze-quench EPR spectra revealed molybdenum was maintained in a partially reduced state during turnover. Rapid-reaction chemical quench for quantifying nitrite production showed that the rate of nitrate reduction was initially greater than the steady-state rate, but rapidly decreased to near steady-state turnover rate. However, rates of internal electron transfer and nitrate reduction were similar in magnitude with no one step in the catalytic process appearing to be much slower than the others. This leads to the conclusion that the catalytic rate is determined by a combination of rates with no overall rate-limiting individual process.Nitrate reductase (NR, 1 EC 1.6.6.1-3) is a molybdenumcontaining enzyme, which catalyzes the pyridine nucleotide-dependent reduction of nitrate to nitrite in plants, fungi, and algae (1, 2). NR contains an internal electron transfer system consisting of an FAD, heme-iron, and molybdenum-molybdopterin (Mo-MPT). The internal redox cofactors, which have a stoichiometry of 1:1:1 for FAD:heme:Mo-MPT, are bound to independently folding domains of the ϳ100-kDa polypeptide, which forms homodimers and -tetramers in the active enzyme (3). NR has two active sites: one for NAD(P)H to donate electrons to the FAD, which is connected via a cyt b domain to the Mo-MPT domain that possesses the nitrate reducing activity. The structure of the recombinant cyt b reductase fragment of corn NADH:NR, representing the C-terminal 270 residues containing the FAD and NADH binding sites, has been determined (4, 5). The structure of the cyt b reductase fragment demonstrates that this portion of the enzyme is related to the ferredoxin NADP ϩ reductase family of enzymes (6). The N-terminal region of NR, containing the Mo-MPT binding site and nitratereducing active site, is related by sequence similarity to sulfite oxidase, another Mo-MPT-containing enzyme, for which a structure was recently determined (7). The bridge between these parts of NR is the cyt b domain, which is related by sequence similarity to mammalian cyt b 5 and to sulfite oxidase's cyt b domain (1, 7). Thus, a model for holo-NR has been generated by combining atom replacement models of the Nterminal Mo-MPT and cyt b domains with the cyt b reductase structure (1, 5). The...

No abstract