Hemicelluloses for fuel ethanol: A review.

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden-Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient anaerobic fermentation of this pentose. L: -Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under 'academic' conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.

/pdf/alcoholic-fermentation-of-carbon-sources-in-biomass-2g5e4jm4xd.pdf

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae : current status

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g−1 sugar consumed, so commercialization seems feasible for some applications.

https://www.fpl.fs.fed.us/documnts/pdf2004/fpl_2004_jeffries001.pdf

Metabolic engineering for improved fermentation of pentoses by yeasts

Publisher Summary The response of a fungus to osmotic stress involves the integrated function of many components of cell metabolism. The dehydration stress is countered by an important mechanism that entails accumulation of polyols, primarily glycerol, to achieve an internal environment that is conducive for enzyme function and growth under water stress. The changes in the composition of the cytoplasm are controlled by systems for biosynthesis of polyols and for transport of inorganic ions. The intracellular retention of glycerol is controlled at the level of glycerol efflux and by systems for glycerol uptake. The osmoregulatory processes require energy to drive transport and carbon supply for polyol formation. The capacity for substrate uptake under osmotic stress and the efficiency operating the osmoregulatory processes are important in setting the limits for growth at low water potentials. The fungal response to changes in the external water potential must involve sensing as well as transduction of the received signal. Combined genetic and physiological analysis is required for a deeper understanding of fungus-water relations. Analysis at this level has revealed sequential induction of osmotically controlled genes in enteric bacteria and given exciting insights in signal transduction and regulation of the process.

Physiology of osmotolerance in fungi

Magnaporthe grisea produces an infection structure called an appressorium, which is used to breach the plant cuticle by mechanical force. Appressoria generate hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control and biochemical mechanism for turgor generation, we assayed glycerol biosynthetic enzymes during appressorium development, and the movement of storage reserves was monitored in developmental mutants. Enzymatic activities for glycerol generation from carbohydrate sources were present in appressoria but did not increase during development. In contrast, triacylglycerol lipase activity increased during appressorium maturation. Rapid glycogen degradation occurred during conidial germination, followed by accumulation in incipient appressoria and dissolution before turgor generation. Lipid droplets also moved to the incipient appressorium and coalesced into a central vacuole before degrading at the onset of turgor generation. Glycogen and lipid mobilization did not occur in a Δpmk1 mutant, which lacked the mitogen-activated protein kinase (MAPK) required for appressorium differentiation, and was retarded markedly in a ΔcpkA mutant, which lacks the catalytic subunit of cAMP-dependent protein kinase A (PKA). Glycogen and lipid degradation were very rapid in a Δmac1 sum1-99 mutant, which carries a mutation in the regulatory subunit of PKA, occurring before appressorium morphogenesis was complete. Mass transfer of storage carbohydrate and lipid reserves to the appressorium therefore occurs under control of the PMK1 MAPK pathway. Turgor generation then proceeds by compartmentalization and rapid degradation of lipid and glycogen reserves under control of the CPKA/SUM1-encoded PKA holoenzyme.

MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea.

SUMMARY: A mutant of Aspergillus niger unable to grow on d-xylose and l-arabinose has been isolated. Genetic analysis revealed that the mutation is located on linkage group IV. Enzymic analysis revealed a deficiency in d-xylulose kinase activity. After transfer of growing mycelium to a medium containing either d-xylose or l-arabinose, the mutant accumulates large amounts of arabitol and xylitol, as shown by 13C NMR spectroscopy. These data and an analysis of enzyme activities induced by d-xylose and l-arabinose in the wild-type strain led to the following catabolic pathway for d-xylose: d-xylose - xylitol - d-xylulose - d-xylulose 5-phosphate; and for l-arabinose: l-arabinose - l-arabitol - l-xylulose - xylitol - d-xylulose - d-xylulose 5-phosphate. The reduction steps of the sugars to the corresponding polyols are all NADPH dependent. The oxidation steps of the polyols to the sugars are all NAD+ dependent. Fractionation of cell-free extracts gave information about the specificity of the enzymes and showed that all the reactions are catalysed by different enzymes.

L-arabinose and D-xylose catabolism in Aspergillus niger.

An NADPH-dependent L-xylulose reductase [xylitol:NADP 4-oxidoreductase; EC 1.1.1.10 (LXDH)] from Aspergillus niger was purified and characterized. It is an octamer with a monomeric molecular mass of 32 kDa, showing high specificity for L-xylulose, xylitol and NADP(H). The K
m values for L-xylulose and xylitol are relatively high (16.5 and 925 mM, respectively). An NAD+-dependent xylitol dehydrogenase [xylitol:NAD+ 2-oxidoreductase; EC 1.1.1.9 (DXDH)] was partially purified from the same fungus. It has a monomeric molecular mass of 40 kDa and shows a high specificity for D-xylulose, xylitol and NAD(H). The K
m values for D-xylulose and xylitol are relatively low (approximately 4 and 50 mM, respectively). The reactivity towards xylitol, the product or substrate these enzymes have in common, confirms their role in the L-arabinose catabolic pathway.

Isolation and characterization of two xylitol dehydrogenases from Aspergillus niger.

Glycerol is catabolized in Aspergillus nidulans by glycerol kinase and a mitochondrial FAD-dependent sn-glycerol-3-phosphate dehydrogenase. The levels of both enzymes are controlled by carbon catabolite repression and by specific induction, Biochemical and genetical analyses show that dihydroxyacetone and d-glyceraldehyde are converted into glycerol and then catabolized by the same pathway, d-Glyceraldehyde can be reduced by NADP+-dependent glycerol dehydrogenase or by alcohol dehydrogenase I, while dihydroxyacetone is only reduced by the first enzyme. Three new glycerol non-utilizing mutants have been found. These three mutations define three hitherto unknown loci, glcE, glcF and glcG. The mutation in glcG leads to a greatly decreased sn-glycerol-3-phosphate dehydrogenase activity.

/pdf/glycerol-catabolism-in-aspergillus-nidulans-5e0i58r8ew.pdf

Glycerol catabolism in Aspergillus nidulans.

Summary: Glycerol dehydrogenase, NADP+-specific (EC 1.1.1.72), was purified from mycelium of Aspergillus nidulans and Aspergillus niger using different purification procedures. Both enzymes had an M
r of approximately 38000 and were immunologically cross-reactive, but had different amino acid compositions and isoelectric points. For both enzymes, the substrate specificity was limited to glycerol and erythritol for the oxidative reaction and to dihydroxyacetone (DHA), diacetyl, methylglyoxal, erythrose and D-glyceraldehyde for the reductive reaction. The A. nidulans enzyme had a turnover number twice that of the A. niger enzyme at pH 6.0, whereas inhibition by NADP+ was less (K
i = 45 μM vs 13 μM). It is proposed that both enzymes catalyse in vivo the reduction of DHA to glycerol and that they are regulated by the anabolic reduction charge.

R. Busink

Papers

L-arabinose and D-xylose catabolism in Aspergillus niger.

Isolation and characterization of two xylitol dehydrogenases from Aspergillus niger.

Glycerol catabolism in Aspergillus nidulans.

Purification and properties of NADP(+)-dependent glycerol dehydrogenases from Aspergillus nidulans and A. niger.

A permeabilized cell assay system for studying enzyme regulation and localisation in Aspergillus niger.