| 1. |
- Pereira, Rui, 1986, et al.
(författare)
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Metabolic Engineering of Yeast
- 2021
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Ingår i: Metabolic Engineering: Concepts and Applications: Volume 13a and 13b. - : Wiley. ; 13, s. 689-733
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- This chapter focuses on a few examples that can serve as illustrations of how powerful yeast metabolic engineering stands today. Yeast, especially S. cerevisiae, plays an essential role in bioethanol production. Rapid ethanol production by yeast cells makes the fermentation process less susceptible to contamination. Higher alcohols are attractive due to some advantages compared with bioethanol, such as higher energy density, better blending into gasoline, higher octane value, lower hygroscopicity, and less corrosivity. The ethanol production process in the industry is mainly achieved through simultaneous saccharification and fermentation. Production of insulin, by volume the largest pharmaceutical protein produced, has paved the way for a wide use of S. cerevisiae for production of recombinant proteins. Virus like particles are proteins of virus capsid, which are produced by recombinant DNA technology and are important for the development of viral vaccines as they can self-assemble and display similar immunogenic properties as native viruses.
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| 2. |
- Li, Xiaowei, 1986, et al.
(författare)
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Metabolic network remodelling enhances yeast’s fitness on xylose using aerobic glycolysis
- 2021
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Ingår i: Nature Catalysis. - : Springer Science and Business Media LLC. - 2520-1158. ; 4:9, s. 783-796
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Tidskriftsartikel (refereegranskat)abstract
- The reprogramming of metabolism in response to switching the carbon source from glucose to non-preferred carbon sources is well-studied for yeast. However, understanding how metabolic networks respond to utilize a non-natural carbon source such as xylose is limited due to the incomplete knowledge of cellular response mechanisms. Here we applied a combination of metabolic engineering, systems biology and adaptive laboratory evolution to gain insights into how yeast can perform a global rewiring of cellular processes to efficiently accompany metabolic transitions. Through metabolic engineering, we substantially enhanced the cell growth on xylose after the growth on glucose. Transcriptome analysis of the engineered strains demonstrated that multiple pathways were involved in the cellular reprogramming. Through genome resequencing of the evolved strains and reverse engineering, we further identified that SWI/SNF chromatin remodelling, osmotic response and aldehyde reductase were responsible for the improved growth. Combined, our analysis showed that glycerol-3-phosphate and xylitol serve as two key metabolites that affect cellular adaptation to growth on xylose. [Figure not available: see fulltext.].
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| 3. |
- Pereira, Rui, 1986, et al.
(författare)
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Adaptive laboratory evolution of tolerance to dicarboxylic acids in Saccharomyces cerevisiae
- 2019
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 56, s. 130-141
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Tidskriftsartikel (refereegranskat)abstract
- Improving the growth phenotypes of microbes in high product concentrations is an essential design objective in the development of robust cell factories. However, the limited knowledge regarding tolerance mechanisms makes rational design of such traits complicated. Here, adaptive laboratory evolution was used to explore the tolerance mechanisms that Saccharomyces cerevisiae can evolve in the presence of inhibiting concentrations of three dicarboxylic acids: glutaric acid, adipic acid and pimelic acid. Whole-genome sequencing of tolerant mutants enabled the discovery of the genetic changes behind tolerance and most mutations could be linked to the up-regulation of multidrug resistance transporters. The amplification of QDR3, in particular, was shown to confer tolerance not only to the three dicarboxylic acids investigated, but also towards muconic acid and glutaconic acid. In addition to increased acid tolerance, QDR3 overexpression also improved the production of muconic acid in the context of a strain engineered for producing this compound.
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| 4. |
- 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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| 5. |
- Yu, Tao, 1986, et al.
(författare)
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Reprogramming Yeast Metabolism from Alcoholic Fermentation to Lipogenesis
- 2018
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Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 174:6, s. 1549-1572
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Tidskriftsartikel (refereegranskat)abstract
- Engineering microorganisms for production of fuels and chemicals often requires major re-programming of metabolism to ensure high flux toward the product of interest. This is challenging, as millions of years of evolution have resulted in establishment of tight regulation of metabolism for optimal growth in the organism's natural habitat. Here, we show through metabolic engineering that it is possible to alter the metabolism of Saccharomyces cerevisiae from traditional ethanol fermentation to a pure lipogenesis metabolism, resulting in high-level production of free fatty acids. Through metabolic engineering and process design, we altered subcellular metabolic trafficking, fine tuned NADPH and ATP supply, and decreased carbon flux to biomass, enabling production of 33.4 g/L extracellular free fatty acids. We further demonstrate that lipogenesis metabolism can replace ethanol fermentation by deletion of pyruvate decarboxylase enzymes followed by adaptive laboratory evolution. Genome sequencing of evolved strains showed that pyruvate kinase mutations were essential for this phenotype.
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| 6. |
- Doughty, Tyler, 1987, et al.
(författare)
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Stress-induced expression is enriched for evolutionarily young genes in diverse budding yeasts
- 2020
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology.
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| 7. |
- Ferreira, Sofia, et al.
(författare)
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Discovery and implementation of a novel pathway for n-butanol production via 2-oxoglutarate
- 2019
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Background : One of the European Union directives indicates that 10% of all fuels must be bio-synthesized by 2020. In this regard, biobutanol - natively produced by clostridial strains - poses as a promising alternative biofuel. One possible approach to overcome the difficulties of the industrial exploration of the native producers is the expression of more suitable pathways in robust microorganisms such as Escherichia coli. The enumeration of novel pathways is a powerful tool, allowing to identify non-obvious combinations of enzymes to produce a target compound. Results : This work describes the in silico driven design of E. coli strains able to produce butanol via 2-oxoglutarate by a novel pathway. This butanol pathway was generated by a hypergraph algorithm and selected from an initial set of 105,954 different routes by successively applying different filters, such as stoichiometric feasibility, size and novelty. The implementation of this pathway involved seven catalytic steps and required the insertion of nine heterologous genes from various sources in E. coli distributed in three plasmids. Expressing butanol genes in E. coli K12 and cultivation in High-Density Medium formulation seem to favor butanol accumulation via the 2-oxoglutarate pathway. The maximum butanol titer obtained was 85 ± 1 mg L-1 by cultivating the cells in bioreactors. Conclusions : In this work, we were able to successfully translate the computational analysis into in vivo applications, designing novel strains of E. coli able to produce n-butanol via an innovative pathway. Our results demonstrate that enumeration algorithms can broad the spectrum of butanol producing pathways. This validation encourages further research to other target compounds.
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| 8. |
- Ferreira, Sofia, et al.
(författare)
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Metabolic engineering strategies for butanol production in Escherichia coli
- 2020
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:8, s. 2571-2587
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Forskningsöversikt (refereegranskat)abstract
- The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.
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| 9. |
- Gibson, B., et al.
(författare)
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Adaptive Laboratory Evolution of Ale and Lager Yeasts for Improved Brewing Efficiency and Beer Quality
- 2020
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Ingår i: Annual review of food science and technology. - : Annual Reviews. - 1941-1421 .- 1941-1413. ; 11, s. 23-44
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Forskningsöversikt (refereegranskat)abstract
- Yeasts directly impact the efficiency of brewery fermentations as well as the character of the beers produced. In recent years, there has been renewed interest in yeast selection and development inspired by the demand to utilize resources more efficiently and the need to differentiate beers in a competitive market. Reviewed here are the different, non-genetically modified (GM) approaches that have been considered, including bioprospecting, hybridization, and adaptive laboratory evolution (ALE). Particular emphasis is placed on the latter, which represents an extension of the processes that have led to the domestication of strains already used in commercial breweries. ALE can be used to accentuate the positive traits of brewing yeast as well as temper some of the traits that are less desirable from a modern brewer's perspective. This method has the added advantage of being non-GM and therefore suitable for food and beverage production.
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| 10. |
- Jenjaroenpun, Piroon, et al.
(författare)
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Complete genomic and transcriptional landscape analysis using third-generation sequencing: a case study of Saccharomyces cerevisiae CEN.PK113-7D
- 2018
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 46:7
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Tidskriftsartikel (refereegranskat)abstract
- Completion of eukaryal genomes can be difficult task with the highly repetitive sequences along the chromosomes and short read lengths of secondgeneration sequencing. Saccharomyces cerevisiae strain CEN. PK113-7D, widely used as a model organism and a cell factory, was selected for this study to demonstrate the superior capability of very long sequence reads for de novo genome assembly. We generated long reads using two common third-generation sequencing technologies (Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio)) and used short reads obtained using Illumina sequencing for error correction. Assembly of the reads derived from all three technologies resulted in complete sequences for all 16 yeast chromosomes, as well as themitochondrial chromosome, in one step. Further, we identified three types of DNA methylation (5mC, 4mC and 6mA). Comparison between the reference strain S288C and strain CEN. PK113-7D identified chromosomal rearrangements against a background of similar gene content between the two strains. We identified full-length transcripts through ONT direct RNA sequencing technology. This allows for the identification of transcriptional landscapes, including untranslated regions (UTRs) (5' UTR and 3' UTR) as well as differential gene expression quantification. About 91% of the predicted transcripts could be consistently detected across biological replicates grown either on glucose or ethanol. Direct RNA sequencing identified many polyadenylated non-coding RNAs, rRNAs, telomere-RNA, long non-coding RNA and antisense RNA. This work demonstrates a strategy to obtain complete genome sequences and transcriptional landscapes that can be applied to other eukaryal organisms.
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