Biomedical subjects

Gunnar Lidén

Publications and source records attributed to Gunnar Lidén.

15 recordsLinked to original sources

PubMed2007

Anaerobic glycerol production by Saccharomyces cerevisiae strains under hyperosmotic stress.

Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80-90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.

Aldehyde Dehydrogenase↗

PubMed2006

Fed-batch cultivation of Saccharomyces cerevisiae on lignocellulosic hydrolyzate.

Saccharomyces cerevisiae grows very poorly in dilute acid lignocellulosic hydrolyzate during the anaerobic fermentation for fuel ethanol production. However, yeast cells grown aerobically on the hydrolyzate have increased tolerance for the hydrolyzate. Cultivation of yeast on part of the hydrolyzate has therefore the potential of enabling increased ethanol productivity in the fermentation of the hydrolyzate. To evaluate the ability of the yeast to grow in the hydrolyzate, fed-batch cultivations were run using the ethanol concentration as input variable to control the feed-rate. The yeast then grew in an undetoxified hydrolyzate with a specific growth rate of 0.19 h(-1) by controlling the ethanol concentration at a low level during the cultivation. However, the biomass yield was lower for the cultivation on hydrolyzate compared to synthetic media: with an ethanol set-point of 0.25 g/l the yield was 0.46 g/g on the hydrolyzate, compared to 0.52 g/g for synthetic media. The main reason for the difference was not the ethanol production per se, but a significant production of glycerol at a high specific growth rate. The glycerol production may be attributed to an insufficient respiratory capacity.

Cellulose↗

PubMed2006

Transcriptome analysis of a shikimic acid producing strain of Escherichia coli W3110 grown under carbon- and phosphate-limited conditions.

Shikimic acid, which is produced in the aromatic amino acid pathway in plants and microorganisms, is an industrially interesting chiral starting material for the synthesis of many chemical substances, e.g. the influenza medicine Tamiflu. When produced by genetically modified Escherichia coli it has previously been found that carbon-rich conditions (e.g. phosphate-limitation) favors production of shikimic acid over shikimate pathway by-products, whereas the situation is the opposite at carbon-(glucose-) limited conditions. In the present study, gene expression patterns of the shikimate producing strain W3110.shik1 (W3110 with aroL deletion and plasmid-overexpressed aroF) and the wild type strain W3110 grown under carbon- and phosphate-limited (carbon-rich) chemostat conditions (D=0.23h(-1)) were analyzed. The study suggests that the by-product formation under carbon-limitation is explained by a set of upregulated genes coupled to the shikimate pathway. The genes, ydiB, aroD and ydiN, were strongly induced only in carbon-limited W3110.shik1. Compared to W3110 the lg(2)-fold changes were: 6.25 (ydiB); 3.93 (aroD) and 8.18 (ydiN). In addition, the transcriptome analysis revealed a large change in the gene expression when comparing phosphate- to carbon-limitation, which to a large part could be explained by anabolic-catabolic uncoupling, which is present under phosphate-limitation but not under carbon-limitation. Interestingly, there was also a larger difference between the two strains under carbon-limitation than under phosphate-limitation. The reason for this difference is interpreted in terms of starvation for aromatic amino acids under carbon-limitation which is relieved under phosphate-limitation due to an upregulation of aroK and aroA.

Bioreactors↗

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↗

PubMed2005

Shikimic acid production by a modified strain of E. coli (W3110.shik1) under phosphate-limited and carbon-limited conditions.

Shikimic acid is one of several industrially interesting chiral starting materials formed in the aromatic amino acid pathway of plants and microorganisms. In this study, the physiology of a shikimic acid producing strain of Escherichia coli (derived from W3110) deleted in aroL (shikimic acid kinase II gene), was compared to that of a corresponding control strain (W3110) under carbon- and phosphate-limited conditions. For the shikimic acid producing strain (referred to as W3110.shik1), phosphate limitation resulted in a higher yield of shikimic acid (0.059 +/- 0.012 vs. 0.024 +/- 0.005 c-mol/c-mol) and a lower yield of by-products from the shikimate pathway, when compared to carbon-limited condition. The yield of the by-product 3-dehydroshikimic acid (DHS) decreased from 0.076 +/- 0.028 to 0.022 +/- 0.001 c-mol/c-mol. Several other by-products were only detected under carbon-limited conditions. The latter group included 3-dehydroquinic acid (0.021 +/- 0.021 c-mol/c-mol), quinic acid (0.012 +/- 0.005 c-mol/c-mol), and gallic acid (0.002 +/- 0.001 c-mol/c-mol). For both strains, more acetate was produced under phosphate than the carbon-limited case. Considerable cell lysis was found for both strains but was higher for W3110.shik1, and increased for both strains under phosphate limitation. The advantages of the latter condition in terms of an increased shikimic acid yield was thus counteracted by an increased cell lysis, which may make downstream processing more difficult.

Bioreactors↗

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

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose.

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources-including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucose-grown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO2 evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO2 evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO2 evolution are analyzed and discussed herein.

Biomass↗

PubMed2004

Controlled fed-batch fermentations of dilute-acid hydrolysate in pilot development unit scale.

Inhibitors formed during wood hydrolysis constitute a major problem in fermenting dilute-acid hydrolysates. By applying a fed-batch technique, the levels of inhibitory compounds may be held low, enabling high ethanol productivity. In this study, a previously developed fed-batch strategy was modified and implemented for use in pilot development unit (PDU) scale. The rate of total gas formation, measured with a mass flow meter, was used as input variable in the control algorithm. The feed rate in the PDU-scale experiments could be properly controlled based on the gas evolution from the reactor. In fed-batch experiments utilizing TMB 3000, an inhibitor-tolerant strain of Saccharomyces cerevisiae, close to 100% of the hexoses in the hydrolysate was converted.

Acids↗

PubMed2003

Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae.

Saccharomyces cerevisiae TMB3001 has previously been engineered to utilize xylose by integrating the genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpressing the native xylulokinase (XK) gene. The resulting strain is able to metabolize xylose, but its xylose utilization rate is low compared to that of natural xylose utilizing yeasts, like Pichia stipitis or Candida shehatae. One difference between S. cerevisiae and the latter species is that these possess specific xylose transporters, while S. cerevisiae takes up xylose via the high-affinity hexose transporters. For this reason, in part, it has been suggested that xylose transport in S. cerevisiae may limit the xylose utilization. We investigated the control exercised by the transport over the specific xylose utilization rate in two recombinant S. cerevisiae strains, one with low XR activity, TMB3001, and one with high XR activity, TMB3260. The strains were grown in aerobic sugar-limited chemostat and the specific xylose uptake rate was modulated by changing the xylose concentration in the feed, which allowed determination of the flux response coefficients. Separate measurements of xylose transport kinetics allowed determination of the elasticity coefficients of transport with respect to extracellular xylose concentration. The flux control coefficient, C(J) (transp), for the xylose transport was calculated from the response and elasticity coefficients. The value of C(J) (transp) for both strains was found to be < 0.1 at extracellular xylose concentrations > 7.5 g L(-1). However, for strain TMB3260 the flux control coefficient was higher than 0.5 at xylose concentrations < 0.6 g L(-1), while C(J) (transp) stayed below 0.2 for strain TMB3001 irrespective of xylose concentration.

Aldehyde Reductase↗

PubMed2003

Engineering of the metabolism of Saccharomyces cerevisiae for anaerobic production of mannitol.

Under anaerobic conditions, Saccharomyces cerevisiae uses NADH-dependent glycerol-3-phosphate dehydrogenase (Gpd1p and Gpd2p) to re-oxidize excess NADH, yielding substantial amounts of glycerol. In a Deltagpd1 Deltagpd2 double-null mutant, the necessary NAD+ regeneration through glycerol production is no longer possible, and this mutant does not grow under anaerobic conditions. The excess NADH formed can potentially be used to drive other NADH-dependent reactions or pathways. To investigate this possibility, a double-null mutant was transformed with a heterologous gene (mtlD) from Escherichia coli, coding for NADH-dependent mannitol-1-phosphate dehydrogenase. Expression of this gene in S. cerevisiae should result in NADH oxidation by the NADH-requiring formation of mannitol-1-phosphate from fructose-6-phosphate. The strain was characterized using step-change experiments, in which, during the exponential growth phase, the inlet gas was changed from air to nitrogen. It was found that the mutant produced mannitol only under anaerobic conditions. However, anaerobic growth was not regained, which was probably due to the excessive accumulation of mannitol in the cells.

Anaerobiosis↗

PubMed2003

Effects of furfural on the respiratory metabolism of Saccharomyces cerevisiae in glucose-limited chemostats.

Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid. Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD+. At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred. Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred. Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.

Aerobiosis↗

PubMed2002

Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.

The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition.

Alcohol Dehydrogenase↗

PubMed1998

Distribution of 14C-labelled carbon from glucose and glutamate during anaerobic growth of Saccharomyces cerevisiae.

The distribution of carbon from glucose and glutamate was studied using anaerobically grown Saccharomyces cerevisiae. The yeast was grown on glucose (20 g l-1) as the carbon/energy source and glutamic acid (3.5 g l-1) as additional carbon and sole nitrogen source. The products formed were identified using labelled [U-14C]glucose or [U-14C]glutamic acid. A seldom-reported metabolite in S. cerevisiae, 2-hydroxyglutarate, was found in significant amounts. It is suggested that 2-hydroxyglutarate is formed from the reduction of 2-oxoglutarate in a reaction catalysed by a dehydrogenase. Succinate, 2-oxoglutarate and 2-hydroxyglutarate were found to be derived exclusively from glutamate. Based on radioactivity measurements, 55%, 17% and 14% of the labelled glutamate was converted to 2-oxoglutarate, succinate and 2-hydroxyglutarate, respectively, and 55%, 9% and 3% of the labelled glucose was converted to ethanol, glycerol and pyruvate, respectively. No labelled glucose was converted to 2-oxoglutarate, succinate or 2-hydroxyglutarate. Furthermore, very little of the evolved CO2 was derived from glutamate. Separation of the amino acids from biomass by paper chromatography revealed that the glutamate family of amino acids (glutamic acid, glutamine, proline, arginine and lysine) originated almost exclusively from the carbon skeleton of glutamic acid. It can be concluded that the carbon flow follows two separate paths, and that the only major reactions utilized in the tricarboxylic acid (TCA) cycle are those reactions involved in the conversion of 2-oxoglutarate to succinate.

Amino Acids↗

PubMedDate not supplied

MFA for overdetermined systems reviewed and compared with rna expression data to elucidate the difference in shikimate yield between carbon- and phosphate-limited continuous cultures of E. coli W3110.shik1.

The present contribution focuses on the mathematical techniques used to solve steady state metabolic models for the case of an overdetermined system. Even when parts of the system are underdetermined it is possible to solve the model partially and obtain statistically meaningful results. This is illustrated with data gathered from a set of E. coli W3110.shik1 phosphate- or carbon-limited continuous cultures. It is shown that the low yield in shikimate for C-limited cultures is not due to a lower flux going to the shikimate pathway but is caused by a high secretion of byproducts. Carbon-limited cultures could be better for shikimate production than carbon-abundant cultures provided the byproduct secretion is reduced. Finally, flux calculations are compared with RNA expression data.

Carbon↗

PubMedDate not supplied

A study of long-term effects on plasmid-containing Escherichia coli in carbon-limited chemostat using 2D-fluorescence spectrofluorimetry.

Strain stability of plasmid-containing recombinant organisms is clearly important for industrial applications. Stability is normally assessed by methods such as selective colony forming units or by simply measuring the recombinant product. These methods are typically performed off-line, are time-consuming, and do not give detailed information on the changes in the metabolism. In the current work, long-term stability of a plasmid-containing strain of Escherichia coli (W3110.shik1) capable of shikimic acid overproduction was studied by means of a 2D-fluorescence sensor (BioView) able to emit and detect light in ranges of 260-560 nm and 300-600 nm, respectively. Long-term carbon-limited chemostat experiments were made under both selective (tetracycline-containing medium) and nonselective conditions. It is shown that the fluorescence spectra provide information about metabolic changes at an earlier stage, thereby giving a noninvasive method for monitoring of strain stability. Further, the fluorescence measurements showed that (i) the metabolic changes in the strain W3110.shik1 with time were qualitatively different in selective and nonselective environment, (ii) plasmid recombination resulted primarily in increased biomass yield, and (iii) a change in metabolism probably involving FAD/FMN and pyridoxal-5-P occurred in all experiments. It was concluded that the strain was not stable in any growth condition for more than about 25 growth generations and even less if plasmid recombination took place.

Bacteriological Techniques↗