| 1. |
- Bergenholm, David, 1987, et al.
(författare)
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Construction of mini-chemostats for high-throughput strain characterization
- 2019
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 116:5, s. 1029-1038
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Tidskriftsartikel (refereegranskat)abstract
- To achieve large-scale, high-throughput experiments for systems biology research of microorganisms, reliable data from robust cultivation systems are needed. Chemostats are such systems, ensuring reproducibility and quality by providing a stable, well-controlled environment for the cells. However, many of the available chemostat systems require large amounts of media and are complex to set up and expensive to purchase and maintain. To address these concerns, we developed a mini-chemostat (MC) system with 16 reactors, each at a working volume of 40 ml. Sensors measure dissolved oxygen in the reactor, while OD600 is measured in the outflow. We further developed a CO 2 and pH sensor array that can be plugged into the outflow of the reactors. The system was used to characterize yeast physiology at four metabolically different conditions: limitations of glucose, both aerobic and anaerobic, nitrogen, and ethanol. The physiology of yeast cells grown at the four different conditions in the MC system was compared with the yeast cells grown in a DASGIP 1 L system using RNAseq analysis. The results show that the MC system provides the same environmental conditions as the DASGIP system and that the MC system is reproducible between different runs. The system is built to be easily scalable with more reactors and to include more sensors, if available. Our study shows that a robust, reproducible chemostat system for high-throughput and large-scale experiments can be built at low costs.
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| 2. |
- Bergenholm, David, 1987, et al.
(författare)
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Modulation of saturation and chain length of fatty acids in Saccharomyces cerevisiae for production of cocoa butter-like lipids
- 2018
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 115:4, s. 932-942
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Tidskriftsartikel (refereegranskat)abstract
- Chain length and degree of saturation plays an important role for the characteristics of various products derived from fatty acids, such as fuels, cosmetics, and food dditives. The seeds of Theobroma cacao are the source of cocoa butter, a natural lipid of high interest for the food and cosmetics industry. Cocoa butter is rich in saturated fatty acids that are stored in the form of triacylglycerides (TAGs). One of the major TAG species of cocoa butter, consisting of two stearic acid molecules and one oleic acid molecule (stearic acid-oleic acid-stearic acid, sn-SOS), is particularly rare in nature as the saturated fatty acid stearic acid is typically found only in low abundance. Demand for cocoa butter is increasing, yet T. cacao can only be cultivated in some parts of the tropics. Alternative means of production of cocoa butter lipids (CBLs) are, therefore, sought after. Yeasts also store fatty acids in the form of TAGs, but these are typically not rich in saturated fatty acids. To make yeast an attractive host for microbial production of CBLs, its fatty acid composition needs to be optimized. We engineered Saccharomyces cerevisiae yeast strains toward a modified fatty acid synthesis. Analysis of the fatty acid profile of the modified strains showed that the fatty acid content as well as the titers of saturated fatty acids and the titers of TAGs were increased. The relative content of potential CBLs in the TAG pool reached up to 22% in our engineered strains, which is a 5.8-fold increase over the wild-type. SOS content reached a level of 9.8% in our engineered strains, which is a 48-fold increase over the wild type.
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| 3. |
- Buijs, Nicolaas, 1985, et al.
(författare)
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Long-chain Alkane Production by the Yeast Saccharomyces cerevisiae
- 2015
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 112:6, s. 1275-1279
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Tidskriftsartikel (refereegranskat)abstract
- In the past decade industrial-scale production of renewable transportation biofuels has been developed as an alternative to fossil fuels, with ethanol as the most prominent biofuel and yeast as the production organism of choice. However, ethanol is a less efficient substitute fuel for heavy-duty and maritime transportation as well as aviation due to its low energy density. Therefore, new types of biofuels, such as alkanes, are being developed that can be used as drop-in fuels and can substitute gasoline, diesel, and kerosene. Here, we describe for the first time the heterologous biosynthesis of long-chain alkanes by the yeast Saccharomyces cerevisiae. We show that elimination of the hexadecenal dehydrogenase Hfdl and expression of a redox system are essential for alkane biosynthesis in yeast. Deletion of HFDI together with expression of an alkane biosynthesis pathway resulted in the production of the alkanes tridecane, pentadecane, and heptadecane. Our study provides a proof of principle for producing long-chain alkanes in the industrial workhorse S. cerevisiae, which was so far limited to bacteria. We anticipate that these findings will be a key factor for further yeast engineering to enable industrial production of alkane based drop-in biofuels, which can allow the biofuel industry to diversify beyond bioethanol.
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| 4. |
- Cámara, Elena, 1985, et al.
(författare)
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Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
- 2019
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 3 February:https://doi.org/10.1002/bit.26947
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Tidskriftsartikel (refereegranskat)abstract
- The methanol-regulated alcohol oxidase promoter (P AOX1 ) of Pichia pastoris (syn. Komagataella spp.) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures.
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| 5. |
- Clark, Douglas S., et al.
(författare)
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Klaus Mosbach Tribute
- 2015
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 112:4, s. 645-647
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 6. |
- Feldman, Hannah, et al.
(författare)
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Model-based analysis and optimization of a full-scale industrial high-rate anaerobic bioreactor
- 2018
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; , s. 2726-2739
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this paper is to present the model-based optimization results of an anaerobic granular sludge internal circulation reactor. The International Water Association Anaerobic Digestion Model No. 1 extended with phosphorus (P), sulfur (S), and ethanol is used to describe the main biological and physico-chemical processes. The high-rate conditions within the reactor are simulated using a flow + reactor model comprised of a series of continuous stirred tank reactors followed by an ideal total suspended solids separation unit. Following parameter estimation by least squares on the measured data, the model had a relative mean error of 13 and 15% for data set #1 and data set #2, respectively. Response surfaces show that the reactor performance index (a metric combining energy recovery in the form of heat and electricity, as well as chemicals needed for pH control) could be improved by 45% when reactor pH is reduced down to 6.8. Model-based results reveal that influent S does not impose sufficient negative impacts on energy recovery (+5.7%, in MWh/day,+0.20 M€/year when influent S is removed) to warrant the cost of its removal (3.58 M€/year). In fact, the process could handle even higher S loads (ensuring the same degree of conversion) as long as the pH is maintained above 6.8. Nevertheless, a higher S load substantially increases the amount of added NaOH to maintain the desired operational pH (>25%) due to the acidic behavior of HS −. CO 2 stripping decreases the buffer capacity of the system and hence use of chemicals for pH control. Finally, the paper discusses the possibilities and limitations of the proposed approach, and how the results of this study will be put into practice.
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| 7. |
- Fletcher, Eugene, 1986, et al.
(författare)
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Industrial systems biology and its impact on synthetic biology of yeast cell factories
- 2016
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 113:6, s. 1164-1170
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Forskningsöversikt (refereegranskat)abstract
- Engineering industrial cell factories to effectively yield a desired product while dealing with industrially relevant stresses is usually the most challenging step in the development of industrial production of chemicals using microbial fermentation processes. Using synthetic biology tools, microbial cell factories such as Saccharomyces cerevisiae can be engineered to express synthetic pathways for the production of fuels, biopharmaceuticals, fragrances, and food flavors. However, directing fluxes through these synthetic pathways towards the desired product can be demanding due to complex regulation or poor gene expression. Systems biology, which applies computational tools and mathematical modeling to understand complex biological networks, can be used to guide synthetic biology design. Here, we present our perspective on how systems biology can impact synthetic biology towards the goal of developing improved yeast cell factories. (C) 2015 Wiley Periodicals, Inc.
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| 8. |
- Irani, Z. A., et al.
(författare)
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Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins
- 2016
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 113:5, s. 961-969
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Tidskriftsartikel (refereegranskat)abstract
- Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes.
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| 9. |
- Lehmann, L., et al.
(författare)
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Linking hydrolysis performance to Trichoderma reesei cellulolytic enzyme profile
- 2016
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 113:5, s. 1001-1010
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Tidskriftsartikel (refereegranskat)abstract
- Trichoderma reesei expresses a large number of enzymes involved in lignocellulose hydrolysis and the mechanism of how these enzymes work together is too complex to study by traditional methods, for example, by spiking with single enzymes and monitoring hydrolysis performance. In this study, a multivariate approach, partial least squares regression, was used to see whether it could help explain the correlation between enzyme profile and hydrolysis performance. Diverse enzyme mixtures were produced by T. reesei Rut-C30 by exploiting various fermentation conditions and used for hydrolysis of washed pretreated corn stover as a measure of enzyme performance. In addition, the enzyme mixtures were analyzed by liquid chromatography-tandem mass spectrometry to identify and quantify the different proteins. A multivariate model was applied for the prediction of enzyme performance based on the combination of different proteins present in an enzyme mixture. The multivariate model was used for identification of candidate proteins that are correlated to enzyme performance on pretreated corn stover. A very large variation in hydrolysis performance was observed and this was clearly caused by the difference in fermentation conditions. Besides β-glucosidase, the multivariate model identified several xylanases, Cip1 and Cip2, as relevant proteins to study further.
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| 10. |
- Lindahl, Lina, 1984, et al.
(författare)
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Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii
- 2016
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 113:4, s. 744-753
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Tidskriftsartikel (refereegranskat)abstract
- Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition, and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic-acid-tolerant yeast Zygosaccharomyces bailii was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in Z. bailii was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane. Z. bailii cultured with the drug myriocin, known to decrease cellular sphingolipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin-treated cells than in control cells. This indicates a higher inflow rate of acetic acid, and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid, was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of Z. bailii to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stress
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