Xylose Uptake Rate Research Articles

Metabolic flux analysis of xylose metabolism in recombinant Saccharomyces cerevisiae using continuous culture

This study focused on elucidating metabolism of xylose in a Saccharomyces cerevisiae strain that overexpresses xylose reductase and xylitol dehydrogenase from Pichia stipitis, as well as the endogenous xylulokinase. The influence of xylose on overall metabolism was examined supplemented with low glucose levels with emphasis on two potential bottlenecks; cofactor requirements and xylose uptake. Results of metabolic flux analysis in continuous cultivations show changes in central metabolism due to the cofactor imbalance imposed by the two-step oxidoreductase reaction of xylose to xylulose. A comparison between cultivations on 27:3 g/L xylose–glucose mixture and 10 g/L glucose revealed that the NADPH-generating flux from glucose-6-phosphate to ribulose-5-phosphate was almost tenfold higher on xylose–glucose mixture and due to the loss of carbon in that pathway the total flux to pyruvate was only around 60% of that on glucose. As a consequence also the fluxes in the citric acid cycle were reduced to around 60%. As the glucose level was decreased to 0.1 g/L the fluxes to pyruvate and in the citric acid cycle were further reduced to 30% and 20%, respectively. The results from in vitro and in vivo xylose uptake measurements showed that the specific xylose uptake rate was highest at the lowest glucose level, 0.1 g/L.

Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures.

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h(-1) g (dry weight) of cells(-1) (0.24 to 0.30 g h(-1) g [dry weight] of cells(-1)) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h(-1). The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h(-1) g (dry weight) of cells(-1) when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h(-1) g (dry weight) of cells(-1) when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.

Effects of initial pH on biological synthesis of xylitol using xylose-rich hydrolysate.

Sugarcane bagasse, an agricultural residue plentiful in Brazil, was utilized for xylitol production by a biotechnological process. A medium fermentation prepared with this xylose-rich biomass at an oxygen transfer volumetric coefficient of 10/h1 and different initial pH values was inoculated with cells of Candida guilliermondii FTI 20037. The maximum values of xylitol and cell volumetric productivities (Qp = 0.56 g/[L.h] and Qx = 0.11 g/[g.h]), xylitol yield factor (YP/S = 0.79 g/g), and xylose uptake rate (qs = 0.197 g/[g.h]) were attained at pH 7.0 without further pH control. The results show that the yeast performance was influenced by the pH, an important bioengineering parameter in this fermentation process.

Conversion of glucose/xylose mixtures by Pichia stipitis under oxygen-limited conditions

Oxygen has a large effect on the growth, substrate utilization, and product formation of yeasts. A good understanding of the influence of oxygen on the conversion of mixed substrates by Pichia stipitis is necessary for a good control of the process. A model was developed to describe the effect of the oxygen supply rate on the conversion of a mixture of glucose and xylose by P. stipitis. This model was validated by means of batch and continuous culture experiments under oxygen-limited conditions. The diauxic conversion of a glucose/xylose mixture by Pichia stipitis can be described by a competitive inhibition of one sugar on the uptake of the other sugar. The influence of xylose on the glucose conversion is negligible. Glucose, however, has a strong inhibitory effect on the xylose conversion. The xylose conversion is inhibited completely at glucose concentrations of 2.3 g l −1 and higher. The model can predict batch conversions of a glucose/xylose mixture. For extremely oxygen-limited conditions, the specific xylose uptake rate equals the anaerobic xylose uptake rate. The continuous fermentations are well described by the model for the oxygen-limited parts of these fermentations. Maximum biomass concentrations of 3.5-4 g l −1 were reached because of other limitations than oxygen. The simulations show that the ethanol yield is maximal at an oxygen uptake rate close to zero. For this situation, the volumetric ethanol production will be low as a result of the low biomass concentration. Therefore, the optimal conditions for ethanol production from hemicellulosic wastes with P. stipitis will be a compromise between the ethanol yield and the volumetric production rate.

Effect of Oxygenation on Xylose Fermentation by Pichia stipitis

The effect of oxygen limitation on xylose fermentation by Pichia stipitis (CBS 6054) was investigated in continuous culture. The maximum specific ethanol productivity (0.20 g of ethanol g dry weight h) and ethanol yield (0.48 g/g) was reached at an oxygen transfer rate below 1 mmol/liter per h. In the studied range of oxygenation, the xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were constant as well as the ratio between the NADPH and NADH activities of xylose reductase. No xylitol production was found. The pyruvate decarboxylase (EC 4.1.1.1) activity increased and the malate dehydrogenase (EC 1.1.1.37) activity decreased with decreasing oxygenation. With decreasing oxygenation, the intracellular intermediary metabolites sedoheptulose 7-phosphate, glucose 6-phosphate, fructose 1,6-diphosphate, and malate accumulated slightly while pyruvate decreased. The ratio of the xylose uptake rate under aerobic conditions, in contrast to that under anaerobic assay conditions, increased with increasing oxygenation in the culture. The results are discussed in relation to the energy level in the cell, the redox balance, and the mitochondrial function.

Xylose metabolism by Candida shehatae in continuous culture

Xylose metabolism by Candida shehatae in continuous culture was examined under both fully-aerobic and semi-aerobic conditions. Growth did not occur in the absence of respiration. Under fully-aerobic conditions, the cell yield was constant at 0.51 g/g and the specific respiration rate Qo2was linearly related to the specific growth rate μ with a slope of 15 mmol g-1 and an intercept of 1.2 mmol g-1 h-1. Under semi-aerobic conditions, QO2was proportional to μ with a slope consistent with a cell yield from oxygen YO2of 35±7 g mol-1. The specific xylose uptake rate was a constant independent of μ and equal to the maximum rate of xylose transport. Ethanol production was observed under semi-aerobic conditions only, and the specific fermentation rate was inversely related to μ.