PubMed Health⌕ Search

Biomedical subjects

Thomas W Jeffries

Publications and source records attributed to Thomas W Jeffries.

13 recordsLinked to original sources
PubMed2006

Engineering yeasts for xylose metabolism.

Technologies for the production of alternative fuels are receiving increased attention owing to concerns over the rising cost of petrol and global warming. One such technology under development is the use of yeasts for the commercial fermentation of xylose to ethanol. Several approaches have been employed to engineer xylose metabolism. These involve modeling, flux analysis, and expression analysis followed by the targeted deletion or altered expression of key genes. Expression analysis is increasingly being used to target rate-limiting steps. Quantitative metabolic models have also proved extremely useful: they can be calculated from stoichiometric balances or inferred from the labeling of intermediate metabolites. The recent determination of the genome sequence for P. stipitis is important, as its genome characteristics and regulatory patterns could serve as guides for further development in this natural xylose-fermenting yeast or in engineered Saccharomyces cerevisiae. Lastly, strain selection through mutagenesis, adaptive evolution or from nature can also be employed to further improve activity.

Adaptation, Biological↗
PubMed2005

Xylitol production by a Pichia stipitis D-xylulokinase mutant.

Xylitol production by Pichia stipitis FPL-YS30, a xyl3-delta1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions. Under both culture conditions, FPL-YS30 (xyl3-delta1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g xylitol/g xylose). However, xylose consumption increased five-fold under aerobic compared to oxygen-limited conditions. This suggests that the efficiency of the alternative route of xylose assimilation is affected by respiration. As a result, the FPL-YS30 strain produced 26 g/l of xylitol, and exhibited a higher volumetric productivity (0.22 g xylitol l(-1) h(-1)) under aerobic conditions.

Fermentation↗
PubMed2004

Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae.

Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast traditionally used in ethanol production from hexose. However, recombinant S. cerevisiae created in several laboratories have used xylose oxidatively rather than in the fermentative manner that this yeast metabolizes glucose. To understand the differences between glucose and engineered xylose metabolic networks, we performed a flux balance analysis (FBA) and calculated extreme pathways using a stoichiometric model that describes the biochemistry of yeast cell growth. FBA predicted that the ethanol yield from xylose exhibits a maximum under oxygen-limited conditions, and a fermentation experiment confirmed this finding. Fermentation results were largely consistent with in silico phenotypes based on calculated extreme pathways, which displayed several phases of metabolic phenotype with respect to oxygen availability from anaerobic to aerobic conditions. However, in contrast to the model prediction, xylitol production continued even after the optimum aeration level for ethanol production was attained. These results suggest that oxygen (or some other electron accepting system) is required to resolve the redox imbalance caused by cofactor difference between xylose reductase and xylitol dehydrogenase, and that other factors limit glycolytic flux when xylose is the sole carbon source.

Cell Proliferation↗
PubMed2004

Saccharomyces cerevisiae engineered for xylose metabolism exhibits a respiratory response.

Native strains of Saccharomyces cerevisiae do not assimilate xylose. S. cerevisiae engineered for d-xylose utilization through the heterologous expression of genes for aldose reductase (XYL1), xylitol dehydrogenase (XYL2), and d-xylulokinase (XYL3 or XKS1) produce only limited amounts of ethanol in xylose medium. In recombinant S. cerevisiae expressing XYL1, XYL2, and XYL3, mRNA transcript levels for glycolytic, fermentative, and pentose phosphate enzymes did not change significantly on glucose or xylose under aeration or oxygen limitation. However, expression of genes encoding the tricarboxylic acid cycle, respiration enzymes (HXK1, ADH2, COX13, NDI1, and NDE1), and regulatory proteins (HAP4 and MTH1) increased significantly when cells were cultivated on xylose, and the genes for respiration were even more elevated under oxygen limitation. These results suggest that recombinant S. cerevisiae does not recognize xylose as a fermentable carbon source and that respiratory proteins are induced in response to cytosolic redox imbalance; however, lower sugar uptake and growth rates on xylose might also induce transcripts for respiration. A petite respiration-deficient mutant (rho degrees ) of the engineered strain produced more ethanol and accumulated less xylitol from xylose. It formed characteristic colonies on glucose, but it did not grow on xylose. These results are consistent with the higher respiratory activity of recombinant S. cerevisiae when growing on xylose and with its inability to grow on xylose under anaerobic conditions.

Culture Media↗
PubMed2003

Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening.

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-beta-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml(-1). The assay is linear for sugar concentrations from 0 to 86 microg ml(-1) and can also be used to assay protein concentrations (0 to 143 microg ml(-1)) on the same plate. A variety of temperatures and pH conditions can be used and, after incubation, the assay requires only one detection reagent and one heating step.

Carbohydrates↗
PubMed2003

Optimal growth and ethanol production from xylose by recombinant Saccharomyces cerevisiae require moderate D-xylulokinase activity.

D-Xylulokinase (XK) is essential for the metabolism of D-xylose in yeasts. However, overexpression of genes for XK, such as the Pichia stipitis XYL3 gene and the Saccharomyces cerevisiae XKS gene, can inhibit growth of S. cerevisiae on xylose. We varied the copy number and promoter strength of XYL3 or XKS1 to see how XK activity can affect xylose metabolism in S. cerevisiae. The S. cerevisiae genetic background included single integrated copies of P. stipitis XYL1 and XYL2 driven by the S. cerevisiae TDH1 promoter. Multicopy and single-copy constructs with either XYL3 or XKS1, likewise under control of the TDH1 promoter, or with the native P. stipitis promoter were introduced into the recombinant S. cerevisiae. In vitro enzymatic activity of XK increased with copy number and promoter strength. Overexpression of XYL3 and XKS1 inhibited growth on xylose but did not affect growth on glucose even though XK activities were three times higher in glucose-grown cells. Growth inhibition increased and ethanol yields from xylose decreased with increasing XK activity. Uncontrolled XK expression in recombinant S. cerevisiae is inhibitory in a manner analogous to the substrate-accelerated cell death observed with an S. cerevisiae tps1 mutant during glucose metabolism. To bypass this effect, we transformed cells with a tunable expression vector containing XYL3 under the control of its native promoter into the FPL-YS1020 strain and screened the transformants for growth on, and ethanol production from, xylose. The selected transformant had approximately four copies of XYL3 per haploid genome and had moderate XK activity. It converted xylose into ethanol efficiently.

Culture Media↗
PubMed2003

Molecular characterization of a gene for aldose reductase ( CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae.

Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.

Aldehyde Reductase↗
PubMed2003

Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae.

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes (XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into the chromosome or by transforming cells with XYL2 in a multicopy vector. Genes in all three constructs were under control of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Enzymatic activity of XR and XDH in the recombinant strains increased with the copy number of XYL1 and XYL2. XR activity was not detected in the parent but was present at a nearly constant level in all of the transformants. XDH activity increased 12-fold when XYL2 was on a multicopy vector compared with when it was present in an integrated single copy. Product formation during xylose fermentation was affected by XDH activity and by aeration in recombinant S. cerevisiae. Higher XDH activity and more aeration resulted in less xylitol and more xylulose accumulation during xylose fermentation. Secretion of xylulose by strains with multicopy XYL2 and elevated XDH supports the hypothesis that D-xylulokinase limits metabolic flux in recombinant S. cerevisiae.

Aldehyde Reductase↗
PubMed2002

Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase.

Glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required forthe catabolism of methanol as a carbon source and certain primary amines, such as methylamine as nitrogen sources in methylotrophic yeasts. Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha. Unlike the recently described Pichia pastoris homologue, the H. polymorpha gene does not contain an intron. The predicted FLD1 product (Fld1p) is a protein of 380 amino acids (ca. 41 kDa) with 82% identity to P. pastoris Fld1p, 76% identity to the FLD protein sequence from n-alkane-assimilating yeast Candida maltosa and 63-64% identity to dehydrogenase class III enzymes from humans and other higher eukaryotes. The expression of FLD1 is strictly regulated and can be controlled at two expression levels by manipulation of the growth conditions. The gene is strongly induced under methylotrophic growth conditions; moderate expression is obtained under conditions in which a primary amine, e.g. methylamine, is used as nitrogen source. These properties render the FLD1 promoter of high interest for heterologous gene expression. The availability of the H. polymorpha FLD1 promoter provides an attractive alternative for expression of foreign genes besides the commonly used alcohol oxidase promoter.

Aldehyde Oxidoreductases↗
PubMed2002

SHAM-sensitive alternative respiration in the xylose-metabolizing yeast Pichia stipitis.

SHAM-sensitive (STO) alternative respiration is present in the xylose-metabolizing, Crabtree-negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood. We cloned PsSTO1, which encodes the SHAM-sensitive terminal oxidase (PsSto1p), by genome walking from wild-type CBS 6054 and subsequently deleted PsSTO1 by targeted gene disruption. The resulting sto1-delta deletion mutant, FPL-Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum. Levels of cytochromes b, c, c(1) and a.a(3) did not change in the sto1-delta mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool. Interestingly, the sto1-delta deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose. Heterologous expression of PsSTO1 in Saccharomyces cerevisiae increased its total oxygen consumption rate and imparted cyanide-resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis. We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild-type CBS 6054 but was bypassed in the cells during xylose metabolism. Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source. We propose that the non-proton-translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose-metabolizing cells and that this non-ATP-generating route serves a regulatory function in the complex redox network of P. stipitis.

Amino Acid Sequence↗
PubMed2002

Molecular cloning of XYL3 (D-xylulokinase) from Pichia stipitis and characterization of its physiological function.

XYL3, which encodes a D-xylulokinase (EC 2.7.1.17), was isolated from Pichia stipitis CBS 6054 genomic DNA by using primers designed against conserved motifs. Disruption of XYL3 eliminated D-xylulokinase activity, but D-ribulokinase activity was still present. Southern analysis of P. stipitis genomic DNA with XYL3 as a probe confirmed the disruption and did not reveal additional related genes. Disruption of XYL3 stopped ethanol production from xylose, but the resulting mutant still assimilated xylose slowly and formed xylitol and arabinitol. These results indicate that XYL3 is critical for ethanol production from xylose but that P. stipitis has another pathway for xylose assimilation. Expression of XYL3 using its P. stipitis promoter increased Saccharomyces cerevisiae D-xylulose consumption threefold and enabled the transformants to produce ethanol from a mixture of xylose and xylulose, whereas the parental strain only accumulated xylitol. In vitro, D-xylulokinase activity in recombinant S. cerevisiae was sixfold higher with a multicopy than with a single-copy XYL3 plasmid, but ethanol production decreased with increased copy number. These results confirmed the function of XYL3 in S. cerevisiae.

Amino Acid Sequence↗