| 1. |
- Qin, Ning, 1990, et al.
(author)
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Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast
- 2023
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In: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 186:4, s. 748-763.e15
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Journal article (peer-reviewed)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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| 2. |
- Qin, Ning, et al.
(author)
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Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production
- 2020
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In: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 9:12, s. 3236-3244
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Journal article (peer-reviewed)abstract
- The central carbon metabolite acetyl-CoA and the cofactor NADPH are important for the synthesis of a wide array of biobased products. Here, we constructed a platform yeast strain for improved provision of acetyl-CoA and NADPH, and used the production of 3-hydroxypropionic acid (3-HP) as a case study. We first demonstrated that the integration of phosphoketolase and phosphotransacetylase improved 3-HP production by 41.9% and decreased glycerol production by 48.1% compared with that of the control strain. Then, to direct more carbon flux toward the pentose phosphate pathway, we reduced the expression of phosphoglucose isomerase by replacing its native promoter with a weaker promoter, and increased the expression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase by replacing their native promoters with stronger promoters. This further improved 3-HP production by 26.4%. Furthermore, to increase the NADPH supply we overexpressed cytosolic aldehyde dehydrogenase, and improved 3-HP production by another 10.5%. Together with optimizing enzyme expression of acetyl-CoA carboxylase and malonyl-CoA reductase, the final strain is able to produce 3-HP with a final titer of 864.5 mg/L, which is a more than 24-fold improvement compared with that of the starting strain. Our strategy combines the PK pathway with the oxidative pentose phosphate pathway for the efficient provision of acetyl-CoA and NADPH, which provides both a higher theoretical yield and overall yield than the reported yeast-based 3-HP production strategies via the malonyl-CoA reductase-dependent pathway and sheds light on the construction of efficient platform cell factories for other products.
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| 3. |
- Wang, ZJ., et al.
(author)
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Metabolic flux analysis of the central carbon metabolism of the industrial vitamin B-12 producing strain Pseudomonas denitrificans using C-13-labeled glucose
- 2012
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In: Journal of the Taiwan Institute of Chemical Engineers. - : Elsevier BV. - 1876-1070. ; 43:2, s. 181-187
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Journal article (peer-reviewed)abstract
- The network topology and metabolic fluxes of central carbon metabolism in the industrial vitamin B-12 producing strain Pseudomonas denitrificans were characterized under oxygen limiting levels. Cultivations were carried out with 100% [1-C-13] or 20% [U-C-13] glucose as substrates under different oxygen supply conditions. The labeling patterns of the proteinogenic amino acids of exponentially growing cells were used to accurately estimate the fluxes in the central carbon metabolism of P. denitrificans. Metabolic flux analysis showed that glucose was mostly catabolized by the Entner-Doudoroff and pentose phosphate pathways. Up to 33% of glucose was consumed via the PP pathway under high specific oxygen uptake rate (SOUR) conditions. This amount was 77.9% higher than that under low oxygen uptake conditions. Quantitative evidence was also found for reversible serine hydroxymethyl transferase and threonine aldolase activities. Metabolic flux and cofactor analyses further showed that higher SOUR accelerated the supply of precursors and methyl groups. SOUR also provided more NADPH for higher vitamin B12 production under the same glucose consumption.
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| 4. |
- Zhang, Yiming, 1986, et al.
(author)
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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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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14
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Journal article (peer-reviewed)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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| 5. |
- Zhang, Yiming, 1986, et al.
(author)
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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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In: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:4
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Journal article (peer-reviewed)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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| 6. |
- Chen, Yun, 1978, et al.
(author)
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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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In: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:3, s. 1-8
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Journal article (peer-reviewed)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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| 7. |
- Krivoruchko, Anastasia, 1984, et al.
(author)
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Microbial acetyl-CoA metabolism and metabolic engineering
- 2015
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In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 28, s. 28-42
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Journal article (other academic/artistic)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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| 8. |
- Liu, Lifang, 1979, et al.
(author)
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Improving heterologous protein secretion at aerobic conditions by activating hypoxia-induced genes in Saccharomyces cerevisiae
- 2015
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In: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:7, s. 10-
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Journal article (peer-reviewed)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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| 9. |
- Zhang, Yiming, 1986
(author)
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Engineering cytosolic acetyl-CoA metabolism in Saccharomyces cerevisiae
- 2015
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Doctoral thesis (other academic/artistic)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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| 10. |
- Zhang, Yiming, 1986, et al.
(author)
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Engineering yeast mitochondrial metabolism for 3-hydroxypropionate production
- 2023
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In: Biotechnology for Biofuels and Bioproducts. - 2731-3654. ; 16:1
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Journal article (peer-reviewed)abstract
- Background: With unique physiochemical environments in subcellular organelles, there has been growing interest in harnessing yeast organelles for bioproduct synthesis. Among these organelles, the yeast mitochondrion has been found to be an attractive compartment for production of terpenoids and branched-chain alcohols, which could be credited to the abundant supply of acetyl-CoA, ATP and cofactors. In this study we explored the mitochondrial potential for production of 3-hydroxypropionate (3-HP) and performed the cofactor engineering and flux control at the acetyl-CoA node to maximize 3-HP synthesis. Results: Metabolic modeling suggested that the mitochondrion serves as a more suitable compartment for 3-HP synthesis via the malonyl-CoA pathway than the cytosol, due to the opportunity to obtain a higher maximum yield and a lower oxygen consumption. With the malonyl-CoA reductase (MCR) targeted into the mitochondria, the 3-HP production increased to 0.27 g/L compared with 0.09 g/L with MCR expressed in the cytosol. With enhanced expression of dissected MCR enzymes, the titer reached to 4.42 g/L, comparable to the highest titer achieved in the cytosol so far. Then, the mitochondrial NADPH supply was optimized by overexpressing POS5 and IDP1, which resulted in an increase in the 3-HP titer to 5.11 g/L. Furthermore, with induced expression of an ACC1 mutant in the mitochondria, the final 3-HP production reached 6.16 g/L in shake flask fermentations. The constructed strain was then evaluated in fed-batch fermentations, and produced 71.09 g/L 3-HP with a productivity of 0.71 g/L/h and a yield on glucose of 0.23 g/g. Conclusions: In this study, the yeast mitochondrion is reported as an attractive compartment for 3-HP production. The final 3-HP titer of 71.09 g/L with a productivity of 0.71 g/L/h was achieved in fed-batch fermentations, representing the highest titer reported for Saccharomyces cerevisiae so far, that demonstrated the potential of recruiting the yeast mitochondria for further development of cell factories.
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