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Enumeration	Reference	Cover	Find
1.	Almquist, Joachim, 1980, et al. (author) Kinetic models in industrial biotechnology - Improving cell factory performance 2014 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 24, s. 38-60 Journal article (peer-reviewed)abstract An increasing number of industrial bioprocesses capitalize on living cells by using them as cell factories that convert sugars into chemicals. These processes range from the production of bulk chemicals in yeasts and bacteria to the synthesis of therapeutic proteins in mammalian cell lines. One of the tools in the continuous search for improved performance of such production systems is the development and application of mathematical models. To be of value for industrial biotechnology, mathematical models should be able to assist in the rational design of cell factory properties or in the production processes in which they are utilized. Kinetic models are particularly suitable towards this end because they are capable of representing the complex biochemistry of cells in a more complete way compared to most other types of models. They can, at least in principle, be used to in detail understand, predict, and evaluate the effects of adding, removing, or modifying molecular components of a cell factory and for supporting the design of the bioreactor or fermentation process. However, several challenges still remain before kinetic modeling will reach the degree of maturity required for routine application in industry. Here we review the current status of kinetic cell factory modeling. Emphasis is on modeling methodology concepts, including model network structure, kinetic rate expressions, parameter estimation, optimization methods, identifiability analysis, model reduction, and model validation, but several applications of kinetic models for the improvement of cell factories are also discussed.
2.	Asadollahi, M. A., et al. (author) Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering 2009 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 11:6, s. 328-334 Journal article (peer-reviewed)abstract A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using Opt Gene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function. Deletion of NADPH-dependent glutamate dehydrogenase encoded by GDH1 was identified as the best target gene for the improvement of sesquiterpene biosynthesis in yeast. Deletion of this gene enhances the available NADPH in the cytosol for other NADPH requiring enzymes, including HMG-CoA reductase. However, since disruption of GDH1 impairs the ammonia utilization, simultaneous over-expression of the NADH-dependent glutamate dehydrogenase en coded by GDH2 was also considered in this study. Deletion of GDH1 led to an approximately 85% increase in the final cubebol titer. However, deletion of this gene also caused a significant decrease in the maximum specific growth rate. Over-expression of GDH2 did not show a further effect on the final cubebol titer but this alteration significantly improved the growth rate compared to the GDH1 deleted strain. (C) 2009 Elsevier Inc. All rights reserved.
3.	Aslan, Selcuk, et al. (author) Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana 2014 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 25, s. 103-112 Journal article (peer-reviewed)abstract In a future bin based economy, renewable sources for lipid compounds at attractive cost are needed for applications where today petrochemical derivatives are dominating. Wax esters and fatty alcohols provide diverse industrial uses, such as in lubricant and surfactant production. In this study, chloroplast metabolism was engineered to divert intermediates from de nova fatty acid biosynthesis to wax ester synthesis. To accomplish this, chloroplast targeted fatty acyl recluctases (EAR) and wax ester synthases (WS) were transiently expressed in Nic"onana benthamiuna loaves. Wax esters of different qualities and quantities were produced providing insights to the properties and interaction of the individual enzymes used. In particular, a phytyl ester synthase was found to be a premium candidate for medium chain wax ester synthesis. Catalytic activities of FAR and WS were also expressed as a fusion protein and determined functionally equivalent to the expression of individual enzymes for wax ester synthesis in chloroplasts. (C) 2014 The Authors. Published by Elsevier Inc. On behalf of International Metabolic Engineering Society.
4.	Asplund-Samuelsson, Johannes, et al. (author) Thermodynamic analysis of computed pathways integrated into the metabolic networks of E. coli and Synechocystis reveals contrasting expansion potential 2018 In: Metabolic engineering. - : Academic Press Inc.. - 1096-7176 .- 1096-7184. ; 45, s. 223-236 Journal article (peer-reviewed)abstract Introducing biosynthetic pathways into an organism is both reliant on and challenged by endogenous biochemistry. Here we compared the expansion potential of the metabolic network in the photoautotroph Synechocystis with that of the heterotroph E. coli using the novel workflow POPPY (Prospecting Optimal Pathways with PYthon). First, E. coli and Synechocystis metabolomic and fluxomic data were combined with metabolic models to identify thermodynamic constraints on metabolite concentrations (NET analysis). Then, thousands of automatically constructed pathways were placed within each network and subjected to a network-embedded variant of the max-min driving force analysis (NEM). We found that the networks had different capabilities for imparting thermodynamic driving forces toward certain compounds. Key metabolites were constrained differently in Synechocystis due to opposing flux directions in glycolysis and carbon fixation, the forked tri-carboxylic acid cycle, and photorespiration. Furthermore, the lysine biosynthesis pathway in Synechocystis was identified as thermodynamically constrained, impacting both endogenous and heterologous reactions through low 2-oxoglutarate levels. Our study also identified important yet poorly covered areas in existing metabolomics data and provides a reference for future thermodynamics-based engineering in Synechocystis and beyond. The POPPY methodology represents a step in making optimal pathway-host matches, which is likely to become important as the practical range of host organisms is diversified.
5.	Baebprasert, Wipawee, et al. (author) Increased H(2) production in the cyanobacterium Synechocystis sp strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway 2011 In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 13:5, s. 610-616 Journal article (peer-reviewed)abstract The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 contains a single bidirectional NiFe-Hox-hydrogenase, which evolves hydrogen under certain environmental conditions. The nitrate assimilation pathway is a potential competing pathway that may reduce the electron flow to the hydrogenase and thereby limit hydrogen production. To improve H(2) production, the nitrate assimilation pathway was disrupted by genetic engineering to redirect the electron flow towards the Hox-hydrogenase. Mutant strains disrupted in either nitrate reductase (Delta narB) or nitrite reductase (Delta nirA) or both nitrate reductase and nitrite reductase (Delta narB:Delta nirA) were constructed and tested for their ability to produce hydrogen. H(2) production and Hox-hydrogenase activities in all the mutant strains were higher than those in wild-type. Highest H(2) production was observed in the Delta narB:Delta nirA strain. Small changes were observed for Hox-hydrogenase enzyme activities and only minor changes in transcript levels of hoxH and hoxY were not correlated with H(2) production. The results suggest that the high rate of H(2) production observed in the Delta narB:Delta nirA strain of the cyanobacterium Synechocystis sp. strain PCC 6803 is the result of redirecting the electron supply from the nitrate assimilation pathway, through genetic engineering, towards the Hox-hydrogenase.
6.	Borodina, I., et al. (author) Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via beta-alanine 2015 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 27, s. 57-64 Journal article (peer-reviewed)abstract Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharolnyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the beta-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of beta-alanine and its subsequent conversion into 3HP using a novel beta-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7 +/- 0.3 g L-1 with a 0.14 +/- 0.0 C-mol C-mol(-1) yield on glucose in 80 h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.
7.	Cao, Xuan, et al. (author) Engineering yeast for high-level production of diterpenoid sclareol 2023 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 75, s. 19-28 Journal article (peer-reviewed)abstract The diterpenoid sclareol is an industrially important precursor for alternative sustainable supply of ambergris. However, its current production from plant extraction is neither economical nor environmental-friendly, since it requires laborious and cost-intensive purification procedures and plants cultivation is susceptible to environmental factors. Engineering cell factories for bio-manufacturing can enable sustainable production of natural products. However, stringent metabolic regulation poses challenges to rewire cellular metabolism for overproduction of compounds of interest. Here we used a modular approach to globally rewire the cellular metabolism for improving sclareol production to 11.4 g/L in budding yeast Saccharomyces cerevisiae, the highest reported diterpenoid titer in microbes. Metabolic flux analysis showed that modular balanced metabolism drove the metabolic flux toward the biosynthesis of targeted molecules, and transcriptomic analysis revealed that the expression of central metabolism genes was shaped for a new balanced metabolism, which laid a foundation in extensive metabolic engineering of other microbial species for sustainable bio-production.
8.	Chen, Que, et al. (author) Combining retinal-based and chlorophyll-based (oxygenic) photosynthesis : Proteorhodopsin expression increases growth rate and fitness of a Delta PSI strain of Synechocystis sp. PCC6803 2019 In: Metabolic engineering. - : Elsevier. - 1096-7176 .- 1096-7184. ; 52, s. 68-76 Journal article (peer-reviewed)abstract To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (Delta PSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the Delta PSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of Delta PSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (Delta PSI/Delta SynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing Delta PSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP(+)-reduction.
9.	Chen, Xin, 1980, et al. (author) Suppressors of amyloid-β toxicity improve recombinant protein production in yeast by reducing oxidative stress and tuning cellular metabolism 2022 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 311-324 Journal article (peer-reviewed)abstract High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer's disease (AD) model overexpression of amyloid-β peptides (Aβ42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aβ42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins.
10.	Chen, Yun, 1978, et al. (author) Coupled incremental precursor and co-factor supply improves 3-hydroxypropionic acid production in Saccharomyces cerevisiae 2014 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 22, s. 104-109 Journal article (peer-reviewed)abstract 3-Hydroxypropionic acid (3-HP) is an attractive platform chemical, which can be used to produce a variety of commodity chemicals, such as acrylic acid and acrylamide. For enabling a sustainable alternative to petrochemicals as the feedstock for these commercially important chemicals, fermentative production of 3-HP is widely investigated and is centered on bacterial systems in most cases. However, bacteria present certain drawbacks for large-scale organic acid production. In this study, we have evaluated the production of 3-HP in the budding yeast Saccharomyces cerevisiae through a route from malonyl-CoA, because this allows performing the fermentation at low pH thus making the overall process cheaper. We have further engineered the host strain by increasing availability of the precursor malonyl-CoA and by coupling the production with increased NADPH supply we were able to substantially improve 3-HP production by five-fold, up to a final titer of 463 mg l(-1). Our work thus led to a demonstration of 3-HP production in yeast via the malonyl-CoA pathway, and this opens for the use of yeast as a cell factory for production of bio-based 3-HP and derived acrylates in the future. (C) 2014 International Metabolic Engineering Society.
11.	Chen, Yun, 1978, et al. (author) Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism 2013 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 15:1, s. 48-54 Journal article (peer-reviewed)abstract Production of fuels and chemicals by industrial biotechnology requires efficient, safe and flexible cell factory platforms that can be used for production of a wide range of compounds. Here we developed a platform yeast cell factory for efficient provision of acetyl-CoA that serves as precursor metabolite for a wide range of industrially interesting products. We demonstrate that the platform cell factory can be used to improve the production of alpha-santalene, a plant sesquiterpene that can be used as a perfume by four-fold. This strain would be a useful tool to produce a wide range of acetyl-CoA-derived products.
12.	Choi, BoHyun, 1986, et al. (author) Engineering of Saccharomyces cerevisiae for enhanced metabolic robustness and L-lactic acid production from lignocellulosic biomass 2024 In: Metabolic Engineering. - 1096-7176 .- 1096-7184. ; 84, s. 23-33 Journal article (peer-reviewed)abstract Metabolic engineering for high productivity and increased robustness is needed to enable sustainable biomanufacturing of lactic acid from lignocellulosic biomass. Lactic acid is an important commodity chemical used for instance as a monomer for production of polylactic acid, a biodegradable polymer. Here, rational and model-based optimization was used to engineer a diploid, xylose fermenting Saccharomyces cerevisiae strain to produce L-lactic acid. The metabolic flux was steered towards lactic acid through the introduction of multiple lactate dehydrogenase encoding genes while deleting ERF2, GPD1, and CYB2. A production of 93 g/L of lactic acid with a yield of 0.84 g/g was achieved using xylose as the carbon source. To increase xylose utilization and reduce acetic acid synthesis, PHO13 and ALD6 were also deleted from the strain. Finally, CDC19 encoding a pyruvate kinase was overexpressed, resulting in a yield of 0.75 g lactic acid/g sugars consumed, when the substrate used was a synthetic lignocellulosic hydrolysate medium, containing hexoses, pentoses and inhibitors such as acetate and furfural. Notably, modeling also provided leads for understanding the influence of oxygen in lactic acid production. High lactic acid production from xylose, at oxygen-limitation could be explained by a reduced flux through the oxidative phosphorylation pathway. On the contrast, higher oxygen levels were beneficial for lactic acid production with the synthetic hydrolysate medium, likely as higher ATP concentrations are needed for tolerating the inhibitors therein. The work highlights the potential of S. cerevisiae for industrial production of lactic acid from lignocellulosic biomass.
13.	Demski, Kamil, et al. (author) Manufacturing specialized wax esters in plants 2022 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 391-402 Journal article (peer-reviewed)abstract Biologically produced wax esters can fulfil different industrial purposes. These functionalities almost drove the sperm whale to extinction from hunting. After the ban on hunting, there is a niche in the global market for biolubricants with properties similar to spermaceti. Wax esters can also serve as a mechanism for producing insect sex pheromone fatty alcohols. Pheromone-based mating disruption strategies are in high demand to replace the toxic pesticides in agriculture and manage insect plagues threatening our food and fiber reserves. In this study we set out to investigate the possibilities of in planta assembly of wax esters, for specific applications, through transient expression of various mix-and-match combinations of genes in Nicotiana benthamiana leaves. Our synthetic biology designs were outlined in order to pivot plant lipid metabolism into producing wax esters with targeted fatty acyl and fatty alcohols moieties. Through this approach we managed to obtain industrially important spermaceti-like wax esters enriched in medium-chain fatty acyl and/or fatty alcohol moieties of wax esters. Via employment of plant codon-optimized moth acyl-CoA desaturases we also managed to capture unusual, unsaturated fatty alcohol and fatty acyl moieties, structurally similar to moth pheromone compounds, in plant-accumulated wax esters. Comparison between outcomes of different experimental designs identified targets for stable transformation to accumulate specialized wax esters and helped us to recognize possible bottlenecks of such accumulation.
14.	Englund, Elias, et al. (author) Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound 2018 In: Metabolic engineering. - : Academic Press Inc.. - 1096-7176 .- 1096-7184. ; 49, s. 164-177 Journal article (peer-reviewed)abstract Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain.
15.	Fletcher, Eugene, 1986, et al. (author) Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments 2017 In: Metabolic engineering. - : Academic Press. - 1096-7176 .- 1096-7184. ; 39, s. 19-28 Journal article (peer-reviewed)abstract Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3 M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest.
16.	Gatto, Francesco, 1987, et al. (author) Pan-cancer analysis of the metabolic reaction network 2020 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 57, s. 51-62 Journal article (peer-reviewed)abstract Metabolic reprogramming is considered a hallmark of malignant transformation. However, it is not clear whether the network of metabolic reactions expressed by cancers of different origin differ from each other or from normal human tissues. In this study, we reconstructed functional and connected genome-scale metabolic models for 917 primary tumor samples across 13 types based on the probability of expression for 3765 reference metabolic genes in the sample. This network-centric approach revealed that tumor metabolic networks are largely similar in terms of accounted reactions, despite diversity in the expression of the associated genes. On average, each network contained 4721 reactions, of which 74% were core reactions (present in >95% of all models). Whilst 99.3% of the core reactions were classified as housekeeping also in normal tissues, we identified reactions catalyzed by ARG2, RHAG, SLC6 and SLC16 family gene members, and PTGS1 and PTGS2 as core exclusively in cancer. These findings were subsequently replicated in an independent validation set of 3388 genome-scale metabolic models. The remaining 26% of the reactions were contextual reactions. Their inclusion was dependent in one case (GLS2) on the absence of TP53 mutations and in 94.6% of cases on differences in cancer types. This dependency largely resembled differences in expression patterns in the corresponding normal tissues, with some exceptions like the presence of the NANP-encoded reaction in tumors not from the female reproductive system or of the SLC5A9-encoded reaction in kidney-pancreatic-colorectal tumors. In conclusion, tumors expressed a metabolic network virtually overlapping the matched normal tissues, raising the possibility that metabolic reprogramming simply reflects cancer cell plasticity to adapt to varying conditions thanks to redundancy and complexity of the underlying metabolic networks. At the same time, the here uncovered exceptions represent a resource to identify selective liabilities of tumor metabolism.
17.	Grotkjær, Thomas, et al. (author) Comparative metabolic network analysis of two xylose fermenting recombinant Saccharomyces cerevisiae strains 2005 In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 7:5-6, s. 437-444 Journal article (peer-reviewed)abstract The recombinant xylose fermenting strain Saccharomyces cerevisiae TMB3001 can grow on xylose, but the xylose utilisation rate is low. One important reason for the inefficient fermentation of xylose to ethanol is believed to be the imbalance of redox co-factors. In the present study, a metabolic flux model was constructed for two recombinant S. cerevisiae strains: TMB3001 and CPB.CR4 which in addition to xylose metabolism have a modulated redox metabolism, i.e. ammonia assimilation was shifted from being NADPH to NADH dependent by deletion of gdh1 and over-expression of GDH2. The intracellular fluxes were estimated for both strains in anaerobic continuous cultivations when the growth limiting feed consisted of glucose (2.5 g L−1) and xylose (13 g L−1). The metabolic network analysis with 13C labelled glucose showed that there was a shift in the specific xylose reductase activity towards use of NADH as co-factor rather than NADPH. This shift is beneficial for solving the redox imbalance and it can therefore partly explain the 25% increase in the ethanol yield observed for CPB.CR4. Furthermore, the analysis indicated that the glyoxylate cycle was activated in CPB.CR4.
18.	Headman Van Vleet, Jennifer, et al. (author) Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in recombinant Saccharomyces cerevisiae improves growth and ethanol production on D-xylose 2008 In: Metabolic Engineering. - 1096-7176 .- 1096-7184. ; :10, s. 360-369 Journal article (peer-reviewed)abstract Overexpression of D-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled bioreactors using engineered S. cerevisiae CEN.PK. Growth on glucose was not significantly affected in pho13∆ mutants, but acetate production increased by 75%. Cell growth, ethanol production, and xylose consumption all increased markedly in pho13∆ mutants. The specific growth rate and rate of specific xylose uptake were approximately 1.5 times higher in the deletion strain than in the parental strain when growing on glucose-xylose mixtures and up to 10-fold higher when growing on xylose alone. In addition to showing higher acetate levels, pho13∆ mutants also produced less glycerol on xylose, suggesting that deletion of Pho13p could improve growth by altering redox levels when cells are grown on xylose.
19.	Hellgren, John, 1991, et al. (author) Promiscuous phosphoketolase and metabolic rewiring enables novel non-oxidative glycolysis in yeast for high-yield production of acetyl-CoA derived products 2020 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 62, s. 150-160 Journal article (peer-reviewed)abstract Carbon-conserving pathways have the potential of increasing product yields in biotechnological processes. The aim of this project was to investigate the functionality of a novel carbon-conserving pathway that produces 3 mol of acetyl-CoA from fructose-6-phosphate without carbon loss in the yeast Saccharomyces cerevisiae. This cyclic pathway relies on a generalist phosphoketolase (Xfspk), which can convert xylulose-5-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate (S7P) to acetyl phosphate. This cycle is proposed to overcome bottlenecks from the previously reported non-oxidative glycolysis (NOG) cycle. Here, in silico simulations showed accumulation of S7P in the NOG cycle, which was resolved by blocking the non-oxidative pentose phosphate pathway and introducing Xfspk and part of the riboneogenesis pathway. To implement this, a transketolase and transaldolase deficient S. cerevisiae was generated and a cyclic pathway, the Glycolysis AlTernative High Carbon Yield Cycle (GATHCYC), was enabled through xfspk expression and sedoheptulose bisphosphatase (SHB17) overexpression. Flux through the GATHCYC was demonstrated in vitro with a phosphoketolase assay on crude cell free extracts, and in vivo by constructing a strain that was dependent on a functional pathway to survive. Finally, we showed that introducing the GATHCYC as a carbon-conserving route for 3-hydroxypropionic acid (3-HP) production resulted in a 109% increase in 3-HP titers when the glucose was exhausted compared to the phosphoketolase route only.
20.	Hing, Nathaphon Yu King, et al. (author) Combining isotopically non-stationary metabolic flux analysis with proteomics to unravel the regulation of the Calvin-Benson-Bassham cycle in Synechocystis sp. PCC 6803 2019 In: Metabolic engineering. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1096-7176 .- 1096-7184. ; 56, s. 77-84 Journal article (peer-reviewed)abstract Photosynthetic microorganisms are increasingly being investigated as a sustainable alternative to existing bio-industrial processes, converting CO2 into desirable end products without the use of carbohydrate feedstock. The Calvin-Benson-Bassham (CBB) cycle is the main pathway of carbon fixation metabolism in photosynthetic organisms. In this study, we analyzed the metabolic fluxes in two strains of the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) that overexpressed fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) and transketolase (TK), respectively. These two potential carbon flux control enzymes in the CBB cycle had previously been shown to improve biomass accumulation when overexpressed under air and low light (15 mu mol m(-2) s(-1)) conditions (Liang and Lindblad, 2016). We measured the growth rates of Synechocystis under atmospheric and high (3% v/v) CO2 conditions at 80 mu mol m(-2) s(-1). Surprisingly, the cells overexpressing transketolase (tktA) demonstrated no significant increase in growth rates when CO2 was increased, suggesting an altered carbon flux distribution and a potential metabolic bottleneck in carbon fixation. Moreover, the tktA strain had an increased susceptibility to oxidative stress under high light as revealed by its chlorotic phenotype under high light conditions. In contrast, the fructose-1,6/sedoheptulose-1,7-bisphosphatase (70glpX) and wildtype cells demonstrated increases in growth rates as expected. To investigate the disparate phenotypical responses of these different Synechocystis strains, isotopically non-stationary metabolic flux analysis (INST-MFA) was used to estimate the carbon flux distribution of tktA, 70glpX, and a kanamycin-resistant control (Km), under atmospheric conditions. In addition, untargeted label-free proteomics, which can detect changes in relative enzymatic abundance, was employed to study the possible effects caused by overexpressing each enzyme. Fluxomic and proteomic results indicated a decrease in oxidative pentose phosphate pathway activity when either FBP/SBPase or TK were overexpressed, resulting in increased carbon fixation efficiency. These results are an example of the integration of multiple omic-level experimental techniques and can be used to guide future metabolic engineering efforts to improve performances and efficiencies.
21.	Hofmann, G., et al. (author) Recombinant bacterial hemoglobin alters metabolism of Aspergillus niger 2009 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 11:1, s. 8-12 Journal article (peer-reviewed)abstract The filamentous fungus Aspergillus niger is used extensively for the production of enzymes and organic acids. A major problem in industrial fermentations with this fungus is to ensure sufficient supply of oxygen required for respiratory metabolism of the fungus. In case of oxygen limitation, the fungus will produce various by-products like organic acids and polyols. In order to circumvent this problem we here study the effects of the expression of a bacterial hemoglobin protein on the metabolism of A. niger. We integrated the vgb gene from Vitreoscilla sp. into the genome at the pyrA locus behind the strong gpdA promoter from Aspergillus nidulans. Analysis of secreted metabolites, oxygen uptake, CO2 evolution and biomass formation points towards a relief of stress in the mutant expressing VHB when it is exposed to oxygen limitation. Our findings therefore point to an interesting strategy to attenuate unwanted side effects resulting from oxygen limitation during industrial fermentations with A. niger. (C) 2008 Elsevier Inc. All rights reserved.
22.	Hong, Kuk-ki, 1976, et al. (author) Adaptively evolved yeast mutants on galactose show trade-offs in carbon utilization on glucose 2013 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 16:1, s. 78-86 Journal article (peer-reviewed)abstract Adaptive evolution offers many opportunities in metabolic engineering; however, several constraints still exist as evolutionary trade-offs may impose collateral cost to obtain new traits. The application of adaptive evolution for strains development could be further improved by elucidating the molecular mechanisms. In this study, adaptively evolved yeast mutants with improved galactose utilization ability showed impaired glucose utilization. The molecular genetic basis of this trade-off was investigated using a systems biology approach. Transcriptional and metabolic changes resulting from the improvement of galactose utilization were found maintained during growth on glucose. Moreover, glucose repression related genes showed conserved expression patterns during growth on both sugars. Mutations in the RAS2 gene that were identified as beneficial for galactose utilization in evolved mutants exhibited significant correlation with attenuation of glucose utilization. These results indicate that antagonistic pleiotropy is the dominant mechanism in the observed trade-off, and it is likely realized by changes in glucose signaling.
23.	Hou, Jin, 1982, et al. (author) Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae 2012 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 14:2, s. 120-127 Journal article (peer-reviewed)abstract The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Secl/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and alpha-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases alpha-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae.
24.	Hou, J., et al. (author) Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae 2009 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 11:4-5, s. 253-261 Journal article (peer-reviewed)abstract Redox cofactors play a pivotal role in coupling catabolism with anabolism and energy generation during metabolism. There exists a delicate balance in the intracellular level of these cofactors to ascertain an optimal metabolic output. Therefore, cofactors are emerging to be attractive targets to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol production, while decreasing mitochondrial NADH lowered ethanol production. However, when these reactions were coupled with NADPH production, the metabolic changes were more moderated. The direct consequence of these perturbations could be seen in the shift of the intracellular concentrations of the cofactors. The changes in product profile and intracellular metabolite levels were closely linked to the ATP requirement for biomass synthesis and the efficiency of oxidative phosphorylation, as estimated from a simple stoichiometric model. The results presented here will provide valuable insights for a quantitative understanding and prediction of cellular response to redox-based perturbations for metabolic engineering applications. (C) 2009 Elsevier Inc. All rights reserved.
25.	Hou, Jianshen, et al. (author) Rewiring carbon flux in Escherichia coli using a bifunctional molecular switch 2020 In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 61, s. 47-57 Journal article (peer-reviewed)abstract The unbalanced distribution of carbon flux in microbial cell factories can lead to inefficient production and poor cell growth. Uncoupling cell growth and chemical synthesis can therefore improve microbial cell factory efficiency. Such uncoupling, which requires precise manipulation of carbon fluxes, can be achieved by up-regulating or down-regulating the expression of enzymes of various pathways. In this study, a dynamic turn-off switch (dTFS) and a dynamic turn-on switch (dTNS) were constructed using growth phase-dependent promoters and degrons. By combining the dTFS and dTNS, a bifunctional molecular switch that could orthogonally regulate two target proteins was introduced. This bifunctional molecular switch was used to uncouple cell growth from shikimic acid and D-glucaric acid synthesis, resulting in the production of 14.33 g/L shikimic acid and the highest reported productivity of D-glucaric acid (0.0325 g/L/h) in Escherichia coli MG1655. This proved that the bifunctional molecular switch could rewire carbon fluxes by controlling target protein abundance.

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