| 1. |
- Zhang, Yiming, 1986, et al.
(författare)
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Adaptive mutations in sugar metabolism restore growth on glucose in a pyruvate decarboxylase negative yeast strain
- 2015
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase (PDC) genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for the production of pyruvate derived biochemicals. However, it cannot grow on glucose as the sole carbon source, and requires supplementation of C2 compounds to the medium in order to meet the requirement for cytosolic acetyl-CoA for biosynthesis of fatty acids and ergosterol. Results: In this study, a Pdc negative strain was adaptively evolved for improved growth in glucose medium via serial transfer, resulting in three independently evolved strains, which were able to grow in minimal medium containing glucose as the sole carbon source at the maximum specific rates of 0.138, 0.148, 0.141 h(-1), respectively. Several genetic changes were identified in the evolved Pdc negative strains by genomic DNA sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1 encoding a negative regulator of the glucose-sensing signal transduction pathway, HXT2 encoding a hexose transporter, CIT1 encoding a mitochondrial citrate synthase, and RPD3 encoding a histone deacetylase. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH1(81D) allele restored its growth on glucose at a maximum specific rate of 0.053 h(-1) in minimal medium with 2% glucose, and the CIT1 deletion in the reverse engineered strain further increased the maximum specific growth rate to 0.069 h(-1). Conclusions: In this study, possible evolving mechanisms of Pdc negative strains on glucose were investigated by genome sequencing and reverse engineering. The non-synonymous mutations in MTH1 alleviated the glucose repression by repressing expression of several hexose transporter genes. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain.
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| 2. |
- Zhang, Yiming, 1986, et al.
(författare)
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Functional pyruvate formate lyase pathway expressed with two different electron donors in Saccharomyces cerevisiae at aerobic growth
- 2015
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:4
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Tidskriftsartikel (refereegranskat)abstract
- Pyruvate formate lyase (PFL) is characterized as an enzyme functional at anaerobic conditions, since the radical in the enzyme's active form is sensitive to oxygen. In this study, PFL and its activating enzyme from Escherichia coli were expressed in a Saccharomyces cerevisiae strain lacking pyruvate decarboxylase and having a reduced glucose uptake rate due to a mutation in the transcriptional regulator Mth1, IMI076 (Pdc-MTH1-Delta T ura3-52). PFL was expressed with two different electron donors, reduced ferredoxin or reduced flavodoxin, respectively, and it was found that the coexpression of either of these electron donors had a positive effect on growth under aerobic conditions, indicating increased activity of PFL. The positive effect on growth was manifested as a higher final biomass concentration and a significant increase in transcription of formate dehydrogenases. Among the two electron donors reduced flavodoxin was found to be a better electron donor than reduced ferredoxin.
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| 3. |
- Chen, Yun, 1978, et al.
(författare)
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Ach1 is involved in shuttling mitochondrial acetyl units for cytosolic C2 provision in Saccharomyces cerevisiae lacking pyruvate decarboxylase
- 2015
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:3, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- Acetyl-coenzyme A (acetyl-CoA) is not only an essential intermediate in central carbon metabolism, but also an important precursor metabolite for native or engineered pathways that can produce many products of commercial interest such as pharmaceuticals, chemicals or biofuels. In the yeast Saccharomyces cerevisiae, acetyl-CoA is compartmentalized in the cytosol, mitochondrion, peroxisome and nucleus, and cannot be directly transported between these compartments. With the acetyl-carnitine or glyoxylate shuttle, acetyl-CoA produced in peroxisomes or the cytoplasm can be transported into the cytoplasm or the mitochondria. However, whether acetyl-CoA generated in the mitochondria can be exported to the cytoplasm is still unclear. Here, we investigated whether the transfer of acetyl-CoA from the mitochondria to the cytoplasm can occur using a pyruvate decarboxylase negative, non-fermentative yeast strain. We found that mitochondrial Ach1 can convert acetyl-CoA in this compartment into acetate, which crosses the mitochondrial membrane before being converted into acetyl-CoA in the cytosol. Based on our finding we propose a model in which acetate can be used to exchange acetyl units between mitochondria and the cytosol. These results will increase our fundamental understanding of intracellular transport of acetyl units, and also help to develop microbial cell factories for many kinds of acetyl-CoA derived products.
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| 4. |
- Krivoruchko, Anastasia, 1984, et al.
(författare)
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Microbial acetyl-CoA metabolism and metabolic engineering
- 2015
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 28, s. 28-42
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Recent concerns over the sustainability of petrochemical-based processes for production of desired chemicals have fueled research into alternative modes of production. Metabolic engineering of microbial cell factories such as Saccharomyces cerevisiae and Escherichia coli offers a sustainable and flexible alternative for the production of various molecules. Acetyl-CoA is a key molecule in microbial central carbon metabolism and is involved in a variety of cellular processes. In addition, it functions as a precursor for many molecules of biotechnological relevance. Therefore, much interest exists in engineering the metabolism around the acetyl-CoA pools in cells in order to increase product titers. Here we provide an overview of the acetyl-CoA metabolism in eukaryotic and prokaryotic microbes (with a focus on S. cerevisiae and E. coli), with an emphasis on reactions involved in the production and consumption of acetyl-CoA. In addition, we review various strategies that have been used to increase acetyl-CoA production in these microbes.
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| 5. |
- Liu, Lifang, 1979, et al.
(författare)
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Improving heterologous protein secretion at aerobic conditions by activating hypoxia-induced genes in Saccharomyces cerevisiae
- 2015
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:7, s. 10-
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Tidskriftsartikel (refereegranskat)abstract
- Oxygen is important for normal aerobic metabolism, as well as for protein production where it is needed for oxidative protein folding. However, several studies have reported that anaerobic conditions seem to be more favorable in terms of recombinant protein production. We were interested in increasing recombinant protein production under aerobic conditions so we focused on Rox1p regulation. Rox1p is a transcriptional regulator, which in oxidative conditions represses genes induced in hypoxia. We deleted ROX1 and studied the effects on the production of recombinant proteins in Saccharomyces cerevisiae. Intriguingly, we found a 100% increase in the recombinant fungal alpha-amylase yield, as well as productivity. Varied levels of improvements were also observed for the productions of the human insulin precursor and the yeast endogenous enzyme invertase. Based on the genome-wide transcriptional response, we specifically focused on the effect of UPC2 upregulation on protein production and suggested a possible mechanistic explanation.
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| 6. |
- Zhang, Yiming, 1986
(författare)
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Engineering cytosolic acetyl-CoA metabolism in Saccharomyces cerevisiae
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for biochemical production. However, it cannot grow on glucose as the sole carbon source due to the lack of cytosolic acetyl-CoA for lipid biosynthesis. Its growth inability on glucose could be restored through directed evolution, which was explained by an in-frame internal deletion in MTH1 (MTH1-∆T). The MTH1-∆T allele resulted in reduced glucose uptake, which may attenuate the repression of respiratory metabolism. However, it was not clear what mechanism could provide the cells with sufficient precursors for cytosolic acetyl-CoA. Here we investigated this using a Pdc negative strain with MTH1-∆T, IMI076. Our results identified a route relying on Ach1 that could transfer acetyl units from mitochondria to the cytoplasm. Based on the results a new model was proposed, in which acetyl units are shuttled from the mitochondria to the cytoplasm in the form of acetate. In addition, a collection of Pdc negative strains was constructed and one of them was adaptively evolved on glucose via serial transfer. Three independently evolved strains were obtained, which can grow on glucose as the sole carbon source at maximum specific rates of 0.138 h-1, 0.148 h-1, 0.141 h-1, respectively. Several genetic changes were identified in the evolved Pdc negative strains by genome sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1, HXT2, CIT1, and RPD3. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH181D allele restored its growth on glucose at a maximum specific rate of 0.05 h-1 in minimal medium with 2% glucose. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain. In connection with biobased chemical production, it is necessary to engineer the metabolism of cell factories such that the raw material, typically sugars, can be efficiently converted to the product of interest. Although IMI076 could grow on glucose, it was still inefficient at conversion of pyruvate to cytosolic acetyl-CoA. To increase cytosolic acetyl-CoA supply from pyruvate, pyruvate formate lyase and its activating enzyme from Escherichia coli were expressed with two different cofactors, ferredoxin or flavodoxin, and their reductase, respectively, and it was found that the co-expression of either of these cofactors had a positive effect on growth under aerobic conditions, indicating increased activity of PFL. The positive effect on growth was manifested as a higher final biomass concentration and a significant increase in transcription of formate dehydrogenase genes (FDHs). Among the two cofactors reduced flavodoxin was found to be a better electron donor than reduced ferredoxin.
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| 7. |
- Zhang, Yiming, 1986, et al.
(författare)
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Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid–derived hydrocarbons
- 2018
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 115:9, s. 2139-2147
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Forskningsöversikt (refereegranskat)abstract
- Fatty acid–derived hydrocarbons attract increasing attention as biofuels due to their immiscibility with water, high-energy content, low freezing point, and high compatibility with existing refineries and end-user infrastructures. Yeast Saccharomyces cerevisiae has advantages for production of fatty acid–derived hydrocarbons as its native routes toward fatty acid synthesis involve only a few reactions that allow more efficient conversion of carbon substrates. Here we describe major biosynthetic pathways of fatty acid–derived hydrocarbons in yeast, and summarize key metabolic engineering strategies, including enhancing precursor supply, eliminating competing pathways, and expressing heterologous pathways. With recent advances in yeast production of fatty acid–derived hydrocarbons, our review identifies key research challenges and opportunities for future optimization, and concludes with perspectives and outlooks for further research directions.
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