| 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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| 11. |
- Lopez, J., et al.
(författare)
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Production of beta-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae
- 2015
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14:1, s. Art. no. 84-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants-like raspberries and roses-by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid beta-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP. Results: Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of beta-ionone (0.073 +/- 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased beta-ionone concentration fivefold (0.34 +/- 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of beta-ionone concentration (0.63 +/- 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase. Conclusions: An efficient beta-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene-the highest beta-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.
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| 12. |
- Pereira, Rui, 1986, et al.
(författare)
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Elucidating aromatic acid tolerance at low pH in Saccharomyces cerevisiae using adaptive laboratory evolution
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:45, s. 27954-27961
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Tidskriftsartikel (refereegranskat)abstract
- Toxicity from the external presence or internal production of compounds can reduce the growth and viability of microbial cell factories and compromise productivity. Aromatic compounds are generally toxic for microorganisms, which makes their production in microbial hosts challenging. Here we use adaptive laboratory evolution to generate Saccharomyces cerevisiae mutants tolerant to two aromatic acids, coumaric acid and ferulic acid. The evolution experiments were performed at low pH (3.5) to reproduce conditions typical of industrial processes. Mutant strains tolerant to levels of aromatic acids near the solubility limit were then analyzed by whole genome sequencing, which revealed prevalent point mutations in a transcriptional activator (Aro80) that is responsible for regulating the use of aromatic amino acids as the nitrogen source. Among the genes regulated by Aro80, ESBP6 was found to be responsible for increasing tolerance to aromatic acids by exporting them out of the cell. Further examination of the native function of Esbp6 revealed that this transporter can excrete fusel acids (byproducts of aromatic amino acid catabolism) and this role is shared with at least one additional transporter native to S. cerevisiae (Pdr12). Besides conferring tolerance to aromatic acids, ESBP6 overexpression was also shown to significantly improve the secretion in coumaric acid production strains. Overall, we showed that regulating the activity of transporters is a major mechanism to improve tolerance to aromatic acids. These findings can be used to modulate the intracellular concentration of aromatic compounds to optimize the excretion of such products while keeping precursor molecules inside the cell.
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| 13. |
- Pereira, Rui, 1986, et al.
(författare)
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Improving the flux distributions simulated with genome-scale metabolic models of Saccharomyces cerevisiae
- 2016
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Ingår i: Metabolic Engineering Communications. - : Elsevier BV. - 2214-0301. ; 3, s. 153-163
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Tidskriftsartikel (refereegranskat)abstract
- Genome-scale metabolic models (GEMs) can be used to evaluate genotype-phenotype relationships and their application to microbial strain engineering is increasing in popularity. Some of the algorithms used to simulate the phenotypes of mutant strains require the determination of a wild-type flux distribution. However, the accuracy of this reference, when calculated with flux balance analysis, has not been studied in detail before.Here, the wild-type simulations of selected GEMs for Saccharomyces cerevisiae have been analysed and most of the models tested predicted erroneous fluxes in central pathways, especially in the pentose phosphate pathway. Since the problematic fluxes were mostly related to areas of the metabolism consuming or producing NADPH/NADH, we have manually curated all reactions including these cofactors by forcing the use of NADPH/NADP+ in anabolic reactions and NADH/NAD+ for catabolic reactions. The curated models predicted more accurate flux distributions and performed better in the simulation of mutant phenotypes.
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| 14. |
- Yu, R., et al.
(författare)
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Nitrogen limitation reveals large reserves in metabolic and translational capacities of yeast
- 2020
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Cells maintain reserves in their metabolic and translational capacities as a strategy to quickly respond to changing environments. Here we quantify these reserves by stepwisereducing nitrogen availability in yeast steady-state chemostat cultures, imposing severe restrictions on total cellular protein and transcript content. Combining multi-omics analysis with metabolic modeling, we find that seven metabolic superpathways maintain >50% metabolic capacity in reserve, with glucose metabolism maintaining >80% reserve capacity. Cells maintain >50% reserve in translational capacity for 2490 out of 3361 expressed genes (74%), with a disproportionately large reserve dedicated to translating metabolic proteins. Finally, ribosome reserves contain up to 30% sub-stoichiometric ribosomal proteins, with activation of reserve translational capacity associated with selective upregulation of 17 ribosomal proteins. Together, our dataset provides a quantitative link between yeast physiology and cellular economics, which could be leveraged in future cell engineering through targeted proteome streamlining. © 2020, The Author(s).
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| 15. |
- Zhiwei, Zhu, 1985, et al.
(författare)
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Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids
- 2020
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Ingår i: Nature Catalysis. - : Springer Science and Business Media LLC. - 2520-1158. ; 3:1, s. 64-74
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Tidskriftsartikel (refereegranskat)abstract
- Medium-chain fatty acids (MCFAs; C6–C12) are valuable molecules used for biofuel and oleochemical production; however, it is challenging to synthesize these fatty acids efficiently using microbial biocatalysts due to the cellular toxicity of MCFAs. In this study, both the endogenous fatty acid synthase (FAS) and an orthogonal bacterial type I FAS were engineered for MCFA production in the yeast Saccharomyces cerevisiae. To improve cellular tolerance to toxic MCFAs, we performed directed evolution of the membrane transporter Tpo1 and strain adaptive laboratory evolution, which elevated the MCFA production by 1.3 ± 0.3- and 1.7 ± 0.2-fold, respectively. We therefore further engineered the highly resistant strain to augment the metabolic flux towards MCFAs. This multidimensional engineering of the yeast at the single protein/enzyme level, the pathway level and the cellular level, combined with an optimized cultivation process, resulted in the production of >1 g l−1 extracellular MCFAs—a more than 250-fold improvement over the original strain.
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