| 1. |
- 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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| 2. |
- Liu, Can, et al.
(författare)
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Immobilized Crosslinked Pectinase Preparation on Porous ZSM-5 Zeolites as Reusable Biocatalysts for Ultra-Efficient Hydrolysis of beta-Glycosidic Bonds
- 2021
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Ingår i: Frontiers in Chemistry. - : Frontiers Media SA. - 2296-2646. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we immobilized pectinase preparation on porous zeolite ZSM-5 as an enzyme carrier. We realized this immobilized enzyme catalyst, pectinase preparation@ZSM-5, via a simple combined strategy involving the van der Waals adsorption of pectinase preparation followed by crosslinking of the adsorbed pectinase preparation with glutaraldehyde over ZSM-5. Conformal pectinase preparation coverage of various ZSM-5 supports was achieved for the as-prepared pectinase preparation@ZSM-5. The porous pectinase preparation@ZSM-5 catalyst exhibited ultra-efficient biocatalytic activity for hydrolyzing the beta-glycosidic bonds in the model substrate 4-nitrophenyl beta-D-glucopyranoside, with a broad operating temperature range, high thermal stability, and excellent reusability. The relative activity of pectinase preparation@ZSM-5 at a high temperature (70 degrees C) was nine times higher than that of free pectinase preparation. Using thermal inactivation kinetic analysis based on the Arrhenius law, pectinase preparation@ZSM-5 showed higher activation energy for denaturation (315 kJ mol(-1)) and a longer half-life (62 min(-1)) than free pectinase preparation. Moreover, a Michaelis-Menten enzyme kinetic analysis indicated a higher maximal reaction velocity for pectinase preparation@ZSM-5 (0.22 mu mol mg(-1) min(-1)). This enhanced reactivity was attributed to the microstructure of the immobilized pectinase preparation@ZSM-5, which offered a heterogeneous reaction system that decreased the substrate-pectinase preparation binding affinity and modulated the kinetic characteristics of the enzyme. Additionally, pectinase preparation@ZSM-5 showed the best ethanol tolerance among all the reported pectinase preparation-immobilized catalysts, and an activity 247% higher than that of free pectinase preparation at a 10% (v/v) ethanol concentration was measured. Furthermore, pectinase preparation@ZSM-5 exhibited potential for practical engineering applications, promoting the hydrolysis of beta-glycosidic bonds in baicalin to convert it into baicalein. This was achieved with a 98% conversion rate, i.e., 320% higher than that of the free enzyme.
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| 3. |
- He, Huihui, et al.
(författare)
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Sex-related differences in the hypertriglyceridemic-waist phenotype in association with hyperuricemia : a longitudinal cohort study
- 2023
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Ingår i: Lipids in Health and Disease. - : Springer Nature. - 1476-511X. ; 22
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is limited longitudinal evidence supporting the association between the hypertriglyceridemic-waist (HTGW) phenotype and hyperuricemia. This study aimed to examine the longitudinal relationship between hyperuricemia and the HTGW phenotype among males and females.Methods: A total of 5562 hyperuricemia-free participants aged 45 or over from the China Health and Retirement Longitudinal Study (mean age: 59.0) were followed for 4 years. The HTGW phenotype was defined as having elevated triglyceride levels and enlarged waist circumference (cutoffs for males: 2.0 mmol/L and 90 cm; females: 1.5 mmol/L and 85 cm). Hyperuricemia was determined by uric acid cutoffs (males: 7 mg/dl; females: 6 mg/dl. Multivariate logistic regression models were used to assess the association between the HTGW phenotype and hyperuricemia. The joint effect of the HTGW phenotype and sex on hyperuricemia was quantified, and the multiplicative interaction was assessed.Results: During the four-year follow-up, 549 (9.9%) incident hyperuricemia cases were ascertained. Compared with those with normal levels of triglycerides and waist circumference, participants with the HTGW phenotype had the highest risk of hyperuricemia (OR: 2.67; 95% CI: 1.95 to 3.66), followed by an OR of 1.96 (95% CI: 1.40 to 2.74) for only higher triglyceride levels and 1.39 (95% CI: 1.03 to 1.86) for only greater waist circumference. The association between HTGW and hyperuricemia was more prominent among females (OR = 2.36; 95% CI: 1.77 to 3.15) than males (OR = 1.29; 95% CI: 0.82 to 2.04), with evidence of a multiplicative interaction (P = 0.006).Conclusions: Middle-aged and older females with the HTGW phenotype may at the highest risk of hyperuricemia. Future hyperuricemia prevention interventions should be primarily targeted for females with the HTGW phenotype.
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| 4. |
- Hong, Wonbin, et al.
(författare)
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Guest Editorial Antenna-in-Package, Antenna-on-Chip, Antenna-IC Interface: Joint Design and Cointegration
- 2019
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Ingår i: IEEE Antennas and Wireless Propagation Letters. - 1548-5757 .- 1536-1225. ; 18:11, s. 2345-2350
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The twenty peer-reviewed letters in this special section examine the design and cointegration of antenna-in-package (AiP), antenna-on-chip (AoC), and antenna ICs (AIC). The letters are categorized in the four distinctive categories: 1) Fabrication technologies (four); 2) Measurement strategies; 3) Applications; and 4) New design and integration strategies. Fruition of major thrusts such as 5G/6G, high-resolution radar and imaging, autonomous driving, and space technology are highly intertwined with the advance of applied electromagnetics. Miniaturization and seamless integration of microwave components and radio systems can enable superior performance, form factor, and cost efficiencies leading to enhanced proliferation of such applications. Historically, radio frequency front ends, antennas, and microwave components have separately evolved using distinct fabrication and measurement technologies.
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| 5. |
- Liu, Zihe, 1984, et al.
(författare)
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Synthetic Biology of Yeast
- 2019
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 58:11, s. 1511-1520
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Forskningsöversikt (refereegranskat)abstract
- With the rapid development of DNA synthesis and next-generation sequencing, synthetic biology that aims to standardize, modularize, and innovate cellular functions, has achieved vast progress. Here we review key advances in synthetic biology of the yeast Saccharomyces cerevisiae, which serves as an important eukaryal model organism and widely applied cell factory. This covers the development of new building blocks, i.e., promoters, terminators and enzymes, pathway engineering, tools developments, and gene circuits utilization. We will also summarize impacts of synthetic biology on both basic and applied biology, and end with further directions for advancing synthetic biology in yeast.
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| 6. |
- 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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| 7. |
- 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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| 8. |
- Zhang, Yueping, et al.
(författare)
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Engineering yeast metabolism for production of terpenoids for use as perfume ingredients, pharmaceuticals and biofuels
- 2017
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 17:8
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Terpenoids represent a large class of natural products with significant commercial applications. These chemicals are currently mainly obtained through extraction from plants and microbes or through chemical synthesis. However, these sources often face challenges of unsustainability and low productivity. In order to address these issues, Escherichia coli and yeast have been metabolic engineered to produce non-native terpenoids. With recent reports of engineering yeast metabolism to produce several terpenoids at high yields, it has become possible to establish commercial yeast production of terpenoids that find applications as perfume ingredients, pharmaceuticals and advanced biofuels. In this review, we describe the strategies to rewire the yeast pathway for terpenoid biosynthesis. Recent advances will be discussed together with challenges and perspectives of yeast as a cell factory to produce different terpenoids.
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| 9. |
- Zhao, Yijin, et al.
(författare)
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Production of β-carotene in Saccharomyces cerevisiae through altering yeast lipid metabolism
- 2021
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 118:5, s. 2043-2052
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals. However, as a non-oleaginous yeast, S. cerevisiae has a limited production capacity for lipophilic compounds, such as β-carotene. To increase its accumulation of β-carotene, we engineered different lipid metabolic pathways in a β-carotene producing strain and investigated the relationship between lipid components and the accumulation of β-carotene. We found that overexpression of sterol ester synthesis genes ARE1 and ARE2 increased β-carotene yield by 1.5-fold. Deletion of phosphatidate phosphatase (PAP) genes (PAH1, DPP1, and LPP1) also increased β-carotene yield by twofold. Combining these two strategies resulted in a 2.4-fold improvement in β-carotene production compared with the starting strain. These results demonstrated that regulating lipid metabolism pathways is important for β-carotene accumulation in S. cerevisiae, and may also shed insights to the accumulation of other lipophilic compounds in yeast.
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| 10. |
- Zhao, Yijin, et al.
(författare)
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Protein engineering of invertase for enhancing yeast dough fermentation under high-sucrose conditions
- 2023
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Ingår i: Folia Microbiologica. - : Springer Science and Business Media LLC. - 0015-5632 .- 1874-9356. ; 68:2, s. 207-217
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Tidskriftsartikel (refereegranskat)abstract
- During yeast dough fermentation, such as the high-sucrose bread-making process, the yeast cells are subjected to considerable osmotic stress, resulting in poor outcomes. Invertase is important for catalyzing the irreversible hydrolysis of sucrose to free glucose and fructose, and decreasing the catalytic activity of the invertase may reduce the glucose osmotic stress on the yeast. In this study, we performed structural design and site-directed mutagenesis (SDM) on the Saccharomyces cerevisiae invertase (ScInV) in an Escherichia coli expression system to study the catalytic activity of ScInV mutants in vitro. In addition, we generated the same mutation sites in the yeast endogenous genome and tested their invertase activity in yeast and dough fermentation ability. Our results indicated that appropriately reduced invertase activity of yeast ScInV can enhance dough fermentation activity under high-sucrose conditions by 52%. Our systems have greatly accelerated the engineering of yeast endogenous enzymes both in vitro and in yeast, and shed light on future metabolic engineering of yeast.
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