| 1. |
- Bergman, Alexandra Linda, 1985, et al.
(författare)
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Effects of overexpression of STB5 in Saccharomyces cerevisiae on fatty acid biosynthesis, physiology and transcriptome
- 2019
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 19:3
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Tidskriftsartikel (refereegranskat)abstract
- Microbial conversion of biomass to fatty acids (FA) and products derived thereof is an attractive alternative to the traditional oleochemical production route from animal and plant lipids. This study examined if NADPH-costly FA biosynthesis could be enhanced by overexpressing the transcription factor Stb5 in Saccharomyces cerevisiae. Stb5 activates expression of multiple genes encoding enzymes within the pentose phosphate pathway (PPP) and other NADPH-producing reactions. Overexpression of STB5 led to a decreased growth rate and an increased free fatty acid (FFA) production during growth on glucose. The improved FFA synthetic ability in the glucose phase was shown to be independent of flux through the oxidative PPP. RNAseq analysis revealed that STB5 overexpression had wide-ranging effects on the transcriptome in the batch phase, and appeared to cause a counterintuitive phenotype with reduced flux through the oxidative PPP. During glucose limitation, when an increased NADPH supply is likely less harmful, an overall induction of the proposed target genes of Stb5 (eg. GND1/2, TAL1, ALD6, YEF1) was observed. Taken together, the strategy of utilizing STB5 overexpression to increase NADPH supply for reductive biosynthesis is suggested to have potential in strains engineered to have strong ability to consume excess NADPH, alleviating a potential redox imbalance.
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| 2. |
- Bergman, Alexandra Linda, 1985, et al.
(författare)
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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
- 2019
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized. Results: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses. Conclusion: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.
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| 3. |
- Hellgren, John, 1991
(författare)
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Engineering central carbon metabolism with phosphoketolase pathways in Saccharomyces cerevisiae
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- We need more efficient biocatalysts to make sustainable microbial production of chemicals and fuels more profitable before they can replace petroleum-based sources. Rewiring the metabolic pathways in the biocatalysts to avoid the loss of carbon as CO2 can aid in improving product yields and thereby the profitability of the process. In this thesis, I investigated the use of phosphoketolase (PK) pathways in the yeast Saccharomyces cerevisiae to produce the precursor metabolite acetyl-CoA without loss of carbon as CO2. Firstly, we investigated the effect of acetyl-phosphate (AcP) accumulation from the linear PK pathway when downstream product formation is limited. Accumulated AcP was degraded to acetate, which limited the benefit of the PK pathway. Furthermore, we investigated a combinatorial strategy to supply acetyl-CoA and NADPH for fatty acid (FA) production. We combined the PK strategy with overexpression of the transcription factor Stb5 to activate NADPH generating pathways. This strategy increased the FA titer in the glucose phase, but with a counteractive response that possibly arose from the lack of an effective NADPH sink. Secondly, we expanded the linear PK pathway to a novel configuration of the cyclic non-oxidative glycolysis (NOG) that can recycle all the carbon from glucose into acetyl-CoA, thus potentially increasing product yields even further. We showed through kinetic modeling that the new configuration resolves potential bottlenecks in the previous configuration. We verified both in vitro and in vivo functionality of the cycle in S. cerevisiae . Furthermore, we demonstrated increased titers of an acetyl-CoA-derived product in the glucose phase compared to the linear PK pathway, indicating increased precursor supply from the cycle. Finally, we further characterized the S. cerevisiae strain with the cycle, using omics. Most notably, the cycle strain yielded respiro-fermentative growth in chemostat cultures with acetate as the main overflow metabolite. This points to a metabolic imbalance and extensive AcP degradation to acetate, which needs to be resolved before the cycle can be efficiently utilized. This thesis highlights the status of this novel NOG configuration and will aid in the further development of cell factories with high-yield production of acetyl-CoA-derived products.
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| 4. |
- Hellgren, John, 1991, et al.
(författare)
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Promiscuous phosphoketolase and metabolic rewiring enables novel non-oxidative glycolysis in yeast for high-yield production of acetyl-CoA derived products
- 2020
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 62, s. 150-160
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Tidskriftsartikel (refereegranskat)abstract
- Carbon-conserving pathways have the potential of increasing product yields in biotechnological processes. The aim of this project was to investigate the functionality of a novel carbon-conserving pathway that produces 3 mol of acetyl-CoA from fructose-6-phosphate without carbon loss in the yeast Saccharomyces cerevisiae. This cyclic pathway relies on a generalist phosphoketolase (Xfspk), which can convert xylulose-5-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate (S7P) to acetyl phosphate. This cycle is proposed to overcome bottlenecks from the previously reported non-oxidative glycolysis (NOG) cycle. Here, in silico simulations showed accumulation of S7P in the NOG cycle, which was resolved by blocking the non-oxidative pentose phosphate pathway and introducing Xfspk and part of the riboneogenesis pathway. To implement this, a transketolase and transaldolase deficient S. cerevisiae was generated and a cyclic pathway, the Glycolysis AlTernative High Carbon Yield Cycle (GATHCYC), was enabled through xfspk expression and sedoheptulose bisphosphatase (SHB17) overexpression. Flux through the GATHCYC was demonstrated in vitro with a phosphoketolase assay on crude cell free extracts, and in vivo by constructing a strain that was dependent on a functional pathway to survive. Finally, we showed that introducing the GATHCYC as a carbon-conserving route for 3-hydroxypropionic acid (3-HP) production resulted in a 109% increase in 3-HP titers when the glucose was exhausted compared to the phosphoketolase route only.
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