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Bärbel Hahn-Hägerdal

Publications and source records attributed to Bärbel Hahn-Hägerdal.

At least 19 recordsLinked to original sources
PubMed2006

High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae.

Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high xylose reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the xylose consumption rate. The results indicate that ethanolic xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which xylose is introduced in the central metabolism.

Aldehyde Reductase↗
PubMed2006

Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400.

The two main sugars in the agricultural by-product corn stover are glucose and xylose. Co-fermentation of glucose and xylose at high content of water-insoluble solids (WIS) without detoxification is a prerequisite to obtain high ethanol concentration and to reduce production costs. A recombinant strain of Saccharomyces cerevisiae, TMB3400, was used in simultaneous saccharification and fermentation (SSF) of whole pretreated slurry of corn stover at high WIS. TMB3400 co-fermented glucose and xylose with relatively high ethanol yields giving high final ethanol concentration. The ethanol productivity increased with increasing concentration of pretreatment hydrolysate in the yeast production medium and when SSF was performed in a fed-batch mode.

Biomass↗
PubMed2006

A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance.

The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol production is inhibited by 5-hydroxymethyl furfural (HMF), a furan derivative which is formed during the hydrolysis of lignocellulosic materials. The inhibition can be avoided if the yeast strain used in the fermentation has the ability to reduce HMF to 5-hydroxymethylfurfuryl alcohol. To enable the identification of enzyme(s) responsible for HMF conversion in S. cerevisiae, microarray analyses of two strains with different abilities to convert HMF were performed. Based on the expression data, a subset of 15 reductase genes was chosen to be further examined using an overexpression strain collection. Three candidate genes were cloned from two different strains, TMB3000 and the laboratory strain CEN.PK 113-5D, and overexpressed using a strong promoter in the strain CEN.PK 113-5D. Strains overexpressing ADH6 had increased HMF conversion activity in cell-free crude extracts with both NADPH and NADH as co-factors. In vitro activities were recorded of 8 mU/mg with NADH as co-factor and as high as 1200 mU/mg for the NADPH-coupled reduction. Yeast strains overexpressing ADH6 also had a substantially higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures, showing that the overexpression indeed conveyed the desired increased reduction capacity.

Aerobiosis↗
PubMed2006

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains.

BACKGROUND: Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. RESULTS: We describe the engineering of laboratory and industrial S. cerevisiae strains to co-ferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. CONCLUSION: Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production.

Journal Article↗
PubMed2006

The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae.

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.

Aldehyde Reductase↗
PubMed2005

Role of cultivation media in the development of yeast strains for large scale industrial use.

The composition of cultivation media in relation to strain development for industrial application is reviewed. Heterologous protein production and pentose utilization by Saccharomyces cerevisiae are used to illustrate the influence of media composition at different stages of strain construction and strain development. The effects of complex, defined and industrial media are compared. Auxotrophic strains and strain stability are discussed. Media for heterologous protein production and for bulk bio-commodity production are summarized.

Journal Article↗
PubMed2005

Construction of cellobiose-growing and fermenting Saccharomyces cerevisiae strains.

Beta-glucosidase genes of fungal origins were isolated and heterologously expressed in Saccharomyces cerevisiae to enable growth on the disaccharide, cellobiose. To promote secretion of the beta-glucosidases, the genes were fused to the secretion signal of the Trichoderma reesei xyn2 gene and constitutively expressed from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The two most promising enzymes, BGL1 from Saccharomycopsis fibuligera and BglA from Aspergillus kawachii, were anchored to the yeast cell surface by fusing the mature proteins to the alpha-agglutinin (AGalpha1) or cell wall protein 2 (Cwp2) peptides. The maximum specific growth rates (mu(max)) of the recombinant S. cerevisiae strains were determined in batch cultivation. S. cerevisiae secreting the recombinant S. fibuligera BGL1 enzyme sustained growth aerobically and anaerobically, in minimal medium containing 5g L(-1) cellobiose at 0.23 h(-1) (compared to 0.29 h(-1) on glucose) and 0.18 h(-1) (compared to 0.25 h(-1) on glucose), respectively. Substrate consumption and product formation were determined to evaluate product yields in glucose and cellobiose.

Agglutinins↗
PubMed2005

Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering.

A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae.

Aldehyde Reductase↗
PubMed2005

Ethanolic fermentation of acid pre-treated starch industry effluents by recombinant Saccharomyces cerevisiae strains.

Two industrial effluents, a pre-fermentation effluent and a post-fermentation effluent from a wheat starch production plant, were used as substrates for fuel ethanol production in anaerobic batch cultures using minimal nutritional amendment. The performances of three metabolically engineered xylose-utilizing Saccharomyces cerevisiae strains: TMB 3001 expressing XYL1, XYL2 and XKS1, redox metabolism modulated CPB.CR1 and glucose de-repressed CPB.CR2, as well as a reference strain CEN.PK 113-7D not fermenting xylose, were evaluated. For the recombinant strains a glucose consumption phase preceded the xylose consumption phase. In both effluents, biomass and ethanol production occurred predominantly during the glucose consumption phase, whereas xylitol and glycerol formation were predominant in the xylose consumption phase. Total specific ethanol productivities on glucose were 6-fold higher than on xylose in the pre-fermentation effluent and 15-fold higher than on xylose in the post-fermentation effluent. CPB.CR1 showed impaired growth compared to the two other xylose-utilizing strains, but displayed 18% increased ethanol yield in the post-fermentation effluent.

Biomass↗
PubMed2005

Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium.

Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated. The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions. The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production. Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate. The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism.

Amino Acids↗
PubMed2005

Amino acid supplementation, controlled oxygen limitation and sequential double induction improves heterologous xylanase production by Pichia stipitis.

Heterologous endo-beta-1,4-xylanase was produced by Pichia stipitis under control of the hypoxia-inducible PsADH2-promoter in a high-cell-density culture. After promoter induction by a shift to oxygen limitation, different aeration rates (oxygen transfer rates) were applied while maintaining oxygen-limitation. Initially, enzyme production was higher in oxygen-limited cultures with high rates of oxygen transfer, although the maximum xylanase activity was not significantly influenced. Amino acid supplementation increased the production of the heterologous endo-beta-1,4-xylanase significantly in highly aerated oxygen-limited cultures, until glucose was depleted. A slight second induction of the promoter was observed in all cultures after the glucose had been consumed. The second induction was most obvious in amino acid-supplemented cultures with higher oxygen transfer rates during oxygen limitation. When such oxygen-limited cultures were shifted back to fully aerobic conditions, a significant re-induction of heterologous endo-beta-1,4-xylanase production was observed. Re-induction was accompanied by ethanol consumption. A similar protein production pattern was observed when cultures were first grown on ethanol as sole carbon source and subsequently glucose and oxygen limitation were applied. Thus, we present the first expression system in yeast with a sequential double-inducible promoter.

Aerobiosis↗
PubMed2005

Cofactor dependence in furan reduction by Saccharomyces cerevisiae in fermentation of acid-hydrolyzed lignocellulose.

A decreased fermentation rate due to inhibition is a significant problem for economic conversion of acid-pretreated lignocellulose hydrolysates to ethanol, since the inhibition gives rise to a requirement for separate detoxification steps. Together with acetic acid, the sugar degradation products furfural and 5-hydroxymethyl furfural are the inhibiting compounds found at the highest concentrations in hydrolysates. These aldehydes have been shown to affect both the specific growth rate and the rate of fermentation by yeast. Two strains of Saccharomyces cerevisiae with different abilities to ferment inhibiting hydrolysates were evaluated in fermentations of a dilute acid hydrolysate from spruce, and the reducing activities for furfural and 5-hydroxymethyl furfural were determined. Crude cell extracts of a hydrolysate-tolerant strain (TMB3000) converted both furfural and 5-hydroxymethyl furfural to the corresponding alcohol at a rate that was severalfold higher than the rate observed for cell extracts of a less tolerant strain (CBS 8066), thereby confirming that there is a correlation between the fermentation rate in a lignocellulosic hydrolysate and the bioconversion capacity of a strain. The in vitro NADH-dependent furfural reduction capacity of TMB3000 was three times higher than that of CBS 8066 (1,200 mU/mg protein and 370 mU/mg protein, respectively) in fed-batch experiments. Furthermore, the inhibitor-tolerant strain TMB3000 displayed a previously unknown NADH-dependent reducing activity for 5-hydroxymethyl furfural (400 mU/mg protein during fed-batch fermentation of hydrolysates). No corresponding activity was found in strain CBS 8066 (<2 mU/mg). The ability to reduce 5-hydroxymethyl furfural is an important characteristic for the development of yeast strains with increased tolerance to lignocellulosic hydrolysates.

Cellulose↗
PubMed2004

Comparison of three expression systems for heterologous xylanase production by S. cerevisiae in defined medium.

The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated.

Amino Acids↗
PubMed2004

Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains.

Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation performance and robustness of eight recombinant strains and two evolved populations on glucose/xylose mixtures in defined and lignocellulose hydrolysate-containing medium. Generally, the polyploid industrial strains depleted xylose faster and were more resistant to the hydrolysate than the laboratory strains. The industrial strains accumulated, however, up to 30% more xylitol and therefore produced less ethanol than the haploid strains. The three most attractive strains were the mutated and selected, extremely rapid xylose consumer TMB3400, the evolved C5 strain with the highest achieved ethanol titer, and the engineered industrial F12 strain with by far the highest robustness to the lignocellulosic hydrolysate.

Cellulose↗
PubMed2004

Acidic proteome of growing and resting Lactococcus lactis metabolizing maltose.

The acidic proteome of Lactococcus lactis grown anaerobically was compared for three different growth conditions: cells growing on maltose, resting cells metabolizing maltose, and cells growing on glucose. In maltose metabolizing cells several proteins were up-regulated compared with glucose metabolizing cells, however only some of the up-regulated proteins had apparent relation to maltose metabolism. Cells growing on maltose produced formate, acetate and ethanol in addition to lactate, whereas resting cells metabolizing maltose and cells growing on glucose produced only lactate. Increased levels of alcohol-acetaldehyde dehydrogenase (ADH) and phosphate acetyltransferase (PTA) in maltose-growing cells compared with glucose-growing cells coincided with formation of mixed acids in maltose-growing cells. The resting cells did not grow due to lack of an amino acid source and fermented maltose with lactate as the sole product, although ADH and PTA were present at high levels. The maltose consumption rate was approximately three times lower in resting cells than in exponentially growing cells. However, the enzyme levels in resting and growing cells metabolizing maltose were similar, which indicates that the difference in product formation in this case is due to regulation at the enzyme level. The levels of 30S ribosomal proteins S1 and S2 increased with increasing growth rate for resting cells metabolizing maltose, maltose-growing cells and glucose-growing cells. A modified form of HPr was synthesized under amino acid starvation. This is suggested to be due to alanine misincorporation for valine, which L. lactis is auxotrophic for. L. lactis conserves the protein profile to a high extent, even after prolonged amino acid starvation, so that the protein expression profile of the bacterium remains almost invariant.

Acetates↗
PubMed2004

Reappraisal of the regulation of lactococcal L-lactate dehydrogenase.

Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed.

Fructosediphosphates↗
PubMed2004

Molecular basis for anaerobic growth of Saccharomyces cerevisiae on xylose, investigated by global gene expression and metabolic flux analysis.

Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a Saccharomyces cerevisiae strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production.

Adenosine Triphosphate↗