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.

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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

Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.

High‐level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?

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.

Summary: A glycerol-kinase-deficient mutant of Aspergillus niger was isolated. Genetic analysis revealed that the mutation is located on linkage group VI. The phenotype of this mutant differed from that of a glycerol kinase mutant of Aspergillus nidulans in its ability to utilize dihydroxyacetone (DHA). The weak growth on glycerol of the A. niger glycerol kinase mutant showed that glycerol phosphorylation is an important step in glycerol catabolism. The mutant could still grow normally on DHA because of the presence of a DHA kinase. This enzyme, probably in combination with an NAD+-dependent glycerol dehydrogenase, present only in the mutant, is responsible for the weak growth of the mutant on glycerol. Enzymic analysis of both the mutant and the parental strain showed that at least three different glycerol dehydrogenases were formed under different physiological conditions: the NAD+-dependent enzyme described above, a constitutive NADP+-dependent enzyme and a d-glyceraldehyde-specific enzyme induced on d-galacturonate. The glycerol kinase mutant showed impaired growth on d-galacturonate.

Characterization of a glycerol kinase mutant of Aspergillus niger.

~~ ~~ A new class of glycerol non-utilizing mutants, designated glcC, has been isolated. The glcC gene was mapped in linkage group VI and mutants were found to complement the reference strains glcA2 (linkage group V) and glcB33 (linkage group I) in diploids. The new mutants were unable to grow on glycerol. However, in contrast to theglcA andglcB phenotype these mutants did grow well on dihydroxyacetone and D-galacturonate. By in vivo 3C NMR spectroscopy it was shown that the glcC mutant did not take up glycerol but did take up dihydroxyacetone. The latter substrate was converted intracellularly into glycerol which was then catabolized as normal.

/pdf/glycerol-uptake-mutants-of-the-hyphal-fungus-aspergillus-4eyvzi118p.pdf

C. Dijkema

Papers

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

Characterization of a glycerol kinase mutant of Aspergillus niger.

Glycerol uptake mutants of the hyphal fungus Aspergillus nidulans.