| 1. |
- Ng, C, et al.
(författare)
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Phase stability and tensile properties of Co-free Al 0.5CrCuFeNi2 high-entropy alloys
- 2014
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Ingår i: Journal of Alloys and Compounds. - : Elsevier BV. - 0925-8388. ; 584, s. 530-537
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Tidskriftsartikel (refereegranskat)abstract
- High-entropy alloys (HEAs) are becoming new research frontiers in the metallic materials field. The phase stability of HEAs is of critical significance, but a convincing understanding on it has been somewhat held back by the slow diffusion kinetics, which prevents the completion of diffusion assisted phase transformations toward the equilibrium state. Here a unique methodology, combining both the thermomechanical treatments and thermodynamic calculations, was employed to reveal the phase stability of HEAs, exemplified using the newly developed Al0.5CrCuFeNi2 alloy. The metastable nature of the solid solution phases in this high-entropy alloy was uncovered through thermomechanical treatments induced phase transformations, and supported by the thermodynamic calculations. Meanwhile, the tensile properties for both the as-cast and thermomechanically treated alloys were measured, and correlated to their indentation behavior. © 2013 Published by Elsevier B.V.
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| 2. |
- de Jong, Bouke Wim, 1983, et al.
(författare)
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Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway
- 2014
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Due to an increasing demand of transportation fuels, a lower availability of cheap crude oil and a lack of sustainability of fossil fuels, a gradual shift from petroleum based fuels towards alternative and renewable fuel resources will be required in the near future. Fatty acid ethyl esters (FAEEs) have properties similar to current crude diesel and could therefore form an important contribution to the development of sustainable transportation fuels in future. It is important to develop novel cell factories for efficient production of FAEEs and their precursors. Results: Here, a Saccharomyces cerevisiae cell factory expressing a heterologous wax ester synthase (ws2) from Marinobacter hydrocarbonoclasticus was used to produce FAEEs from ethanol and acyl-coenzyme A (acyl-CoA). The production of acyl-CoA requires large amounts of NADPH and acetyl-CoA. Therefore, two metabolic engineering strategies for improved provision of NADPH and acetyl-CoA were evaluated. First, the ethanol degradation pathway was employed to re-channel carbon flow towards the synthesis of acetyl-CoA. Therefore, ADH2 and ALD6 encoding, respectively, alcohol dehydrogenase and acetaldehyde dehydrogenase were overexpressed together with the heterologous gene acs(SE)(L641P) encoding acetyl-CoA synthetase. The co-overexpression of ADH2, ALD6 and acs(SE)(L641P) with ws2 resulted in 408 +/- 270 mu g FAEE gCDW-1, a 3-fold improvement. Secondly, for the expression of the PHK pathway two genes, xpkA and ack, both descending from Aspergillus nidulans, were co-expressed together with ws2 to catalyze, respectively, the conversion of xylulose-5-phosphate to acetyl phosphate and glyceraldehyde3-phosphate and acetyl phosphate to acetate. Alternatively, ack was substituted with pta from Bacillus subtilis, encoding phosphotransacetylase for the conversion of acetyl phosphate to acetyl-CoA. Both PHK pathways were additionally expressed in a strain with multiple chromosomally integrated ws2 gene, which resulted in respectively 5100 +/- 509 and 4670 +/- 379 mu g FAEE gCDW(-1), an up to 1.7-fold improvement. Conclusion: Two different strategies for engineering of the central carbon metabolism for efficient provision of acetyl-CoA and NADPH required for fatty acid biosynthesis and hence FAEE production were evaluated and it was found that both the ethanol degradation pathway as well as the phosphoketolase pathway improve the yield of FAEEs.
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| 3. |
- Gong, Guiping, et al.
(författare)
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GTR 2.0: GRNA-tRNA Array and Cas9-NG Based Genome Disruption and Single-Nucleotide Conversion in Saccharomyces cerevisiae
- 2021
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:6, s. 1328-1337
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Tidskriftsartikel (refereegranskat)abstract
- Targeted genome disruptions and single-nucleotide conversions with the CRISPR/Cas system have greatly facilitated the development of gene therapy, basic biological research, and synthetic biology. With vast progress in this field, there are still aspects to be optimized, including the target range, the ability to multiplex, the mutation efficiency and specificity, as well as the requirement of adjusting protospacer adjacent motifs (PAMs). Here, we report the development of a highly efficient genome disruption and single-nucleotide conversion tool with a gRNA-tRNA array and SpCas9-NG (GTR 2.0). We performed gene disruptions in yeast cells covering all 16 possible NGN PAMs and all 12 possible single-nucleotide conversions (N to N) with near 100% efficiencies. Moreover, we applied GTR 2.0 for multiplexed single-nucleotide conversions, resulting in 66.67% mutation efficiency in simultaneous generation of 4 single-nucleotide conversions in one gene, as well as 100% mutation efficiency for simultaneously generating 2 single-nucleotide conversions in two different genes. GTR 2.0 will substantially expand the scope, efficiency, and capabilities of yeast genome editing, and will be a versatile and invaluable addition to the toolbox of synthetic biology and metabolic engineering.
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| 4. |
- Lin, Zhenquan, et al.
(författare)
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Characterization of cross-species transcription and splicing from Penicillium to Saccharomyces cerevisiae
- 2021
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Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 48:9-10
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Tidskriftsartikel (refereegranskat)abstract
- Heterologous expression of eukaryotic gene clusters in yeast has been widely used for producing high-value chemicals and bioactive secondary metabolites. However, eukaryotic transcription cis-elements are still undercharacterized, and the cross-species expression mechanism remains poorly understood. Here we used the whole expression unit (including original promoter, terminator, and open reading frame with introns) of orotidine 5'-monophosphate decarboxylases from 14 Penicillium species as a showcase, and analyzed their cross-species expression in Saccharomyces cerevisiae. We found that pyrG promoters from the Penicillium species could drive URA3 expression in yeast, and that inefficient cross-species splicing of Penicillium introns might result in weak cross-species expression. Thus, this study demonstrates cross-species expression from Penicillium to yeast, and sheds light on the opportunities and challenges of cross-species expression of fungi expression units and gene clusters in yeast without refactoring for novel natural product discovery.
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| 5. |
- Qin, Ning, 1990, et al.
(författare)
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Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast
- 2023
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Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 186:4, s. 748-763.e15
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Tidskriftsartikel (refereegranskat)abstract
- Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.
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| 6. |
- Qin, Ning, et al.
(författare)
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Increased CO 2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast
- 2024
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Ingår i: Nature Communications. - 2041-1723 .- 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- CO2 fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO2 fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO2, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO2 fixation strategies pave the way for CO2 being used as the sole carbon source.
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| 7. |
- Shi, Shuobo, 1981, et al.
(författare)
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Engineering of Chromosomal Wax Ester Synthase Integrated Saccharomyces Cerevisiae Mutants for Improved Biosynthesis of Fatty Acid Ethyl Esters
- 2014
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 111:9, s. 1740-1747
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast.
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| 8. |
- Shi, Shuobo, 1981, et al.
(författare)
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Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production
- 2012
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 5, s. Art. no. 7-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundWax ester synthases (WSs) can synthesize wax esters from alcohols and fatty acyl coenzyme A thioesters. The knowledge of the preferred substrates for each WS allows the use of yeast cells for the production of wax esters that are high-value materials and can be used in a variety of industrial applications. The products of WSs include fatty acid ethyl esters, which can be directly used as biodiesel.ResultsHere, heterologous WSs derived from five different organisms were successfully expressed and evaluated for their substrate preference in Saccharomyces cerevisiae. We investigated the potential of the different WSs for biodiesel (that is, fatty acid ethyl esters) production in S. cerevisiae. All investigated WSs, from Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6 and Psychrobacter arcticus 273-4, have different substrate specificities, but they can all lead to the formation of biodiesel. The best biodiesel producing strain was found to be the one expressing WS from M. hydrocarbonoclasticus DSM 8798 that resulted in a biodiesel titer of 6.3 mg/L. To further enhance biodiesel production, acetyl coenzyme A carboxylase was up-regulated, which resulted in a 30% increase in biodiesel production.ConclusionsFive WSs from different species were functionally expressed and their substrate preference characterized in S. cerevisiae, thus constructing cell factories for the production of specific kinds of wax ester. WS from M. hydrocarbonoclasticus showed the highest preference for ethanol compared to the other WSs, and could permit the engineered S. cerevisiae to produce biodiesel.
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| 9. |
- Shi, Shuobo, 1981, et al.
(författare)
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Improved production of fatty acids by Saccharomyces cerevisiae through screening a cDNA library from the oleaginous yeast Yarrowia lipolytica
- 2016
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae.
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| 10. |
- Shi, Shuobo, 1981, et al.
(författare)
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Microbial production of chemicals driven by CRISPR-Cas systems
- 2022
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Ingår i: Current Opinion in Biotechnology. - : Elsevier BV. - 0958-1669 .- 1879-0429. ; 73, s. 34-42
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Forskningsöversikt (refereegranskat)abstract
- Microorganisms have provided an attractive route for biosynthesis of various chemicals from renewable resources. CRISPR-Cas systems have served as powerful mechanisms for generating cell factories with desirable properties by manipulating nucleic acids quickly and efficiently. The CRISPR-Cas system provides a toolbox with excellent opportunities for identifying better biocatalysts, multiplexed fine-tuning of metabolic flux, efficient utilization of low-cost substrates, and improvement of metabolic robustness. The overall goal of this review highlights recent advances in the development of microbial cell factories for chemical production using various CRISPR-Cas systems. The perspectives for further development or applications of CRISPR-Cas systems for strain improvement are also discussed.
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| 11. |
- Shi, Shuobo, 1981, et al.
(författare)
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Prospects for microbial biodiesel production
- 2011
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 6:3, s. 277-285
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Tidskriftsartikel (refereegranskat)abstract
- As the demand for biofuels for transportation is increasing, it is necessary to develop technologies that will allow for low-cost production of biodiesel. Conventional biodiesel is mainly produced from vegetable oil by chemical transesterification. This production, however, has relatively low land-yield and is competing for agricultural land that can be used for food production. Therefore, there is an increasing interest in developing microbial fermentation processes for production of biodiesel as this will allow for the use of a wide range of raw-materials, including sugar cane, corn, and biomass. Production of biodiesel by microbial fermentation can be divided into two different approaches, (1) indirect biodiesel production from oleaginous microbes by in vitro transesterification, and (2) direct biodiesel production from redesigned cell factories. This work reviews both microbial approaches for renewable biodiesel production and evaluates the existing challenges in these two strategies.
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| 12. |
- Valle Rodriguez, Juan Octavio, 1984, et al.
(författare)
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Directed evolution of a wax ester synthase for production of fatty acid ethyl esters in Saccharomyces cerevisiae
- 2023
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 107:9, s. 2921-2932
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: Wax ester synthases (WSs) utilize a fatty alcohol and a fatty acyl-coenzyme A (activated fatty acid) to synthesize the corresponding wax ester. There is much interest in developing novel cell factories that can produce shorter esters, e.g., fatty acid ethyl esters (FAEEs), with properties similar to biodiesel in order to use these as transportation fuels. However, ethanol is a poor substrate for WSs, and this may limit the biosynthesis of FAEEs. Here, we implemented a random mutagenesis approach to enhance the catalytic efficiency of a WS from Marinobacter hydrocarbonoclasticus (MhWS2, encoded by the ws2 gene). Our selection system was based on FAEE formation serving as a detoxification mechanism for excessive oleate, where high WS activity was essential for a storage-lipid free yeast to survive. A random mutagenesis library of ws2 was used to transform the storage-lipid free yeast, and mutants could be selected by plating the transformants on oleate containing plates. The variants encoding WS with improved activity were sequenced, and an identified point mutation translated into the residue substitution at position A344 was discovered to substantially increase the selectivity of MhWS2 toward ethanol and other shorter alcohols. Structural modeling indicated that an A344T substitution might affect the alcohol selectivity due to change of both steric effects and polarity changes near the active site. This work not only provides a new WS variant with altered selectivity to shorter alcohols but also presents a new high-throughput selection system to isolate WSs with a desired selectivity. Key Points: • The work provides WS variants with altered substrate preference for shorter alcohols • A novel method was developed for directed evolution of WS of desired selectivity.
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| 13. |
- Valle Rodriguez, Juan Octavio, 1984, et al.
(författare)
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Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid ethyl esters, an advanced biofuel, by eliminating non-essential fatty acid utilization pathways
- 2014
-
Ingår i: Applied Energy. - : Elsevier BV. - 1872-9118 .- 0306-2619. ; 115, s. 226-232
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Tidskriftsartikel (refereegranskat)abstract
- Microbial production of fatty acid derived chemicals and fuels is currently of great interest due to the limited resources and increasing prices of petroleum and petroleum-based products. The development of Saccharomyces cerevisiae as a fatty acid ethyl ester (FAEE) cell factory would represent an opportunity for biodiesel production due to its successful history in the biotechnology area. However, fatty acid (FA) biosynthesis is highly regulated and usually not high enough for developing an efficient production process. In S. cerevisiae, FAs are degraded by beta-oxidation and a large fraction is utilized to synthesize steryl esters (SEs) and triacylglycerols (TAGs), which are not essential for the cell. Here, by eliminating nonessential FA utilization pathways, we developed a metabolic engineering strategy resulting in a S. cerevisiae strain that can overproduce FAs and in turn use these for producing FAEEs (biodiesel). Compared to the wild-type, there is an about 3-fold increase in free FA content in a strain devoid of both TAG and SE formation, a 4-fold increase in free FA content in a strain that is incapable of beta-oxidation, and a 5-fold increase of free FAs in a strain lacking all of these non-essential FA utilization pathways. It is also demonstrated that there are similar positive effects on FAEE production in these deletion strains. The highest production of FAEEs is 17.2 mg/l in the strain in which all these pathways were blocked. The results of this study serve as a basis for further strategies to improve the production of FA derivatives in S. cerevisiae. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
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| 14. |
- Zhang, Yueping, et al.
(författare)
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A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.
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