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

Pitkänen, Juha-Pekka; Aristidou, Aristos; Salusjärvi, Laura; Ruohonen, Laura; Penttilä, Merja

doi:10.1016/s1096-7176(02)00012-5

Cited by 108 publications

(81 citation statements)

References 48 publications

(52 reference statements)

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“…In contrast to the expression in C1, the levels of expression of genes involved in glycolysis, redox metabolism, and ethanol or glycerol production were not altered in TMB3400, which may explain why this mutant cannot grow anaerobically on xylose alone (49). Our conclusion that increased cytosolic NADPH formation and NADH consumption are important for anaerobic growth of yeast on xylose is consistent with flux and proteome data obtained for recombinant, xylose-fermenting S. cerevisiae during a shift from glucose-containing medium to glucose-xylosecontaining medium (26,31). In particular, it was shown that strains overexpressing the preferentially NADPH-dependent xylose reductase from P. stipitis generate the required NADPH primarily through the oxidative pentose phosphate pathway (16).…”

Section: Discussionsupporting

confidence: 90%

Molecular Basis for Anaerobic Growth of Saccharomyces cerevisiae on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis

Sonderegger

Jeppsson

Hahn‐Hägerdal

et al. 2004

Appl Environ Microbiol

120

109

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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.

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Section: Discussionsupporting

confidence: 90%

Molecular Basis for Anaerobic Growth of Saccharomyces cerevisiae on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis

Sonderegger

Jeppsson

Hahn‐Hägerdal

et al. 2004

Appl Environ Microbiol

120

109

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“…NADH/NAD + and NADPH/NADP ratios, are important markers when considering strategies for metabolic engineering of an organism (Moreira dos Santos et al, 2003;Pitkanen et al, 2003;Thomas et al, 2007). In particular, increased redox potential is considered as an important factor in improving BDO production in (Zhuang et al, 2013).…”

Section: Redox Potential Considerationsmentioning

confidence: 99%

Identification of metabolic engineering targets for the enhancement of 1,4-butanediol production in recombinant E. coli using large-scale kinetic models

Andreozzi

Chakrabarti

Soh

et al. 2016

Metabolic Engineering

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Identification of metabolic engineering targets for the enhancement of 1,4-butanediol production in recombinant E. coli using largescale kinetic models, Metabolic Engineering, http://dx.doi.org/10. 1016/j.ymben.2016.01.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Entities) to analyze the physiology of recombinant E. coli producing 1,4-butanediol (BDO) and to identify potential strategies for improved production of BDO. The framework allowed us to integrate data across multiple levels and to construct a population of large-scale kinetic models despite the lack of available information about kinetic properties of every enzyme in the metabolic pathways. We analyzed these models and we found that the enzymes that primarily control the fluxes leading to BDO production are part of central glycolysis, the lower branch of tricarboxylic acid (TCA) cycle and the novel BDO production route. Interestingly, among the enzymes between the glucose uptake and the BDO pathway, the enzymes belonging to the lower branch of TCA cycle have been identified as the most important for improving BDO production and yield. We also quantified the effects of changes of the target enzymes on other intracellular states like energy charge, cofactor levels, redox state, cellular growth, and byproduct formation.Independent earlier experiments on this strain confirmed that the computationally 3 obtained conclusions are consistent with the experimentally tested designs, and the findings of the present studies can provide guidance for future work on strain improvement. Overall, these studies demonstrate the potential and effectiveness of ORACLE for the accelerated design of microbial cell factories.

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“…Thus, low, but non-zero, concentration of glucose enhances xylose metabolism. The effect has been attributed to improved co-factor generation [22] and the induction of genes for transporter systems [22] and glycolytic enzymes [23].…”

Section: Introductionmentioning

confidence: 99%

Sequential Targeting of Xylose and Glucose Conversion in Fed-Batch Simultaneous Saccharification and Co-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

et al. 2017

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Efficient conversion of both glucose and xylose in lignocellulosic biomass is necessary to make secondgeneration bioethanol from agricultural residues competitive with first-generation bioethanol and gasoline. Simultaneous saccharification and co-fermentation (SSCF) is a promising strategy for obtaining high ethanol yields. However, with this method, the xylose-fermenting capacity and viability of yeast tend to decline over time and restrict the xylose utilization. In this study, we examined the ethanol production from steampretreated wheat straw using an established SSCF strategy with substrate and enzyme feeding that was previously applied to steam-pretreated corn cobs. Based on our findings, we propose an alternative SSCF strategy to sustain the xylose-fermenting capacity and improve the ethanol yield. The xylose-rich hydrolyzate liquor was separated from the glucose-rich solids, and phases were co-fermented sequentially. By prefermentation of the hydrolyzate liquor followed fedbatch SSCF, xylose, and glucose conversion could be targeted in succession. Because the xylose-fermenting capacity declines over time, while glucose is still converted, it was advantageous to target xylose conversion upfront. With our strategy, an overall ethanol yield of 84% of the theoretical maximum based on both xylose and glucose was reached for a slurry with higher inhibitor concentrations, versus 92% for a slurry with lower inhibitor concentrations. Xylose utilization exceeded 90% after SSCF for both slurries. Sequential targeting of xylose and glucose conversion sustained xylose fermentation and improved xylose utilization and ethanol yield compared with fed-batch SSCF of whole slurry.

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

Cited by 108 publications

References 48 publications

Molecular Basis for Anaerobic Growth of Saccharomyces cerevisiae on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis

Molecular Basis for Anaerobic Growth of Saccharomyces cerevisiae on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis

Identification of metabolic engineering targets for the enhancement of 1,4-butanediol production in recombinant E. coli using large-scale kinetic models

Sequential Targeting of Xylose and Glucose Conversion in Fed-Batch Simultaneous Saccharification and Co-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

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