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Yun Chen - One of the best experts on this subject based on the ideXlab platform.
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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs Sugar Phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
Microbial Cell Factories, 2019Co-Authors: Alexandra Bergman, John Hellgren, Thomas Moritz, Verena Siewers, Jens Nielsen, Yun ChenAbstract: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 CO_2. 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 CO_2. 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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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs Sugar Phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
Microbial Cell Factories, 2019Co-Authors: Alexandra Bergman, John Hellgren, Thomas Moritz, Verena Siewers, Jens Nielsen, Yun ChenAbstract: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. 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. 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.
Alexandra Bergman - One of the best experts on this subject based on the ideXlab platform.
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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs Sugar Phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
Microbial Cell Factories, 2019Co-Authors: Alexandra Bergman, John Hellgren, Thomas Moritz, Verena Siewers, Jens Nielsen, Yun ChenAbstract: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 CO_2. 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 CO_2. 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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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs Sugar Phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
Microbial Cell Factories, 2019Co-Authors: Alexandra Bergman, John Hellgren, Thomas Moritz, Verena Siewers, Jens Nielsen, Yun ChenAbstract: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. 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. 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.
Taifo Mahmud - One of the best experts on this subject based on the ideXlab platform.
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Structure of a Sedoheptulose 7‑Phosphate Cyclase: ValA from Streptomyces hygroscopicus
2016Co-Authors: Kelsey M Kean, Shumpei Asamizu, Taifo Mahmud, Sara J Codding, Andrew P KarplusAbstract:encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the Sugar Phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all Sugar Phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no Sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparison
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structure of a sedoheptulose 7 Phosphate cyclase vala from streptomyces hygroscopicus
Biochemistry, 2014Co-Authors: Kelsey M Kean, Shumpei Asamizu, Taifo Mahmud, Sara J Codding, Andrew P KarplusAbstract:Sedoheptulose 7-Phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 A resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the Sugar Phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all Sugar Phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no Sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site diff...
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Evolutionary Divergence of Sedoheptulose 7-Phosphate Cyclases Leads to Several Distinct Cyclic Products
2012Co-Authors: Shumpei Asamizu, Patricia M Flatt, Pengfei Xie, Corey J. Brumsted, Taifo MahmudAbstract:Sedoheptulose 7-Phosphate cyclases are enzymes that utilize the pentose Phosphate pathway intermediate, sedoheptulose 7-Phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway and are part of the more recently recognized superfamily of Sugar Phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes catalyze the conversion of sedoheptulose 7-Phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like Sugar Phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules
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a comparative analysis of the Sugar Phosphate cyclase superfamily involved in primary and secondary metabolism
ChemBioChem, 2007Co-Authors: Patricia M Flatt, Oliver Schlorke, Axel Zeeck, Tohru Dairi, Taifo MahmudAbstract:Sugar Phosphate Cyclases (SPCs) catalyze the cyclization of Sugar Phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, e.g., aromatic amino acids, and clinically relevant secondary metabolites, e.g., aminocyclitol/aminoglycoside and ansamycin antibiotics. Feeding experiments with isotopically-labeled cyclitols revealed that cetoniacytone A, a unique C7N-aminocyclitol antibiotic isolated from an insect endophytic Actinomyces sp., is derived from 2-epi-5-epi-valiolone, a product of SPC. Using heterologous probes from the 2-epi-5-epi-valiolone synthase class of SPCs, an SPC homolog gene, cetA, was isolated from the cetoniacytone producer. CetA is closely related to BE-orf9 found in the BE-40644 biosynthetic gene cluster from Actinoplanes sp. strain {"type":"entrez-protein","attrs":{"text":"A40644","term_id":"421200","term_text":"pir||A40644"}}A40644. Recombinant expression of cetA and BE-orf9 and biochemical characterization of the gene products confirmed their function as 2-epi-5-epi-valiolone synthases. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs that may regulate the biosynthesis of a novel set of secondary metabolites.
Peter C Maloney - One of the best experts on this subject based on the ideXlab platform.
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altered oxyanion selectivity in mutants of uhpt the pi linked Sugar Phosphate carrier of escherichia coli
Journal of Biological Chemistry, 2005Co-Authors: Jason A Hall, Peter C MaloneyAbstract:Abstract In Escherichia coli, the UhpT transporter catalyzes the electroneutral accumulation of Sugar 6-Phosphate by exchange with internal inorganic Phosphate (Pi). The substrate specificity of UhpT is regulated at least in part by constituents of an Asp388-Lys391 intrahelical salt bridge, and mutations that remove one but not both of these residues alter UhpT preference for organoPhosphate substrates. Using site-directed mutagenesis, we examined the role played by these two positions in the selection of the oxyanion countersubstrate. We show that derivatives having aliphatic or polar residues at positions 388 and 391 are gain-of-function mutants capable of transporting SO4 as well as Pi. These oxyanions share similar structures but differ significantly in the presence of a proton(s) on Pi. Our findings therefore lead us to suggest that the Asp388-Lys391 ion pair acts normally as a filter that prevents substrates lacking a proton that can be donated from occupying the UhpT active site.
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altered substrate selectivity in a mutant of an intrahelical salt bridge in uhpt the Sugar Phosphate carrier of escherichia coli
Journal of Biological Chemistry, 1999Co-Authors: Jason A Hall, Mon Chou Fann, Peter C MaloneyAbstract:Abstract Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the Sugar Phosphate carrier ofEscherichia coli. Glucose 6-Phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced. This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of32Pi by homologous Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Piexchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP.
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functional symmetry of uhpt the Sugar Phosphate transporter of escherichia coli
Journal of Biological Chemistry, 1998Co-Authors: Mon Chou Fann, Peter C MaloneyAbstract:UhpT, the Sugar Phosphate transporter ofEscherichia coli, acts to exchange internal inorganic Phosphate for external hexose 6-Phosphate. Because of this operational asymmetry, we studied variants in which right-side-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His10K291C/K294N, in which the sole external tryptic cleavage site (Lys294) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His10K291C/K294N led to loss of about 50% of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His10V284C derivative, in which a newly inserted external cysteine (Cys284) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N-maleimidylpropionyl)biocytin treatment of proteoliposomes containing His10V284C gave about a 60% loss of activity, and immunodetection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (His10K291C/K294N) or ISO orientation (His10V284C) with respect to the kinetics of glucose 6-Phosphate transport by Phosphate-loaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-Phosphate, mannose 6-Phosphate, galactose 6-Phosphate, fructose 6-Phosphate, and inorganic Phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.
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identification of two essential arginine residues in uhpt the Sugar Phosphate antiporter of escherichia coli
The Journal of Membrane Biology, 1998Co-Authors: Mon Chou Fann, A H Davies, Atul Varadhachary, T Kuroda, Carolyn S Sevier, Tomofusa Tsuchiya, Peter C MaloneyAbstract:Three lines of evidence indicate that arginine-46 (R46) and arginine-275 (R275) are essential to the function of UhpT, the Pi-linked antiport protein of Escherichia coli. A role for arginine was initially suggested by the sensitivity of UhpT to inhibition by 2,3-butanedione, an arginine-directed probe. Since the presence of substrate protected against this inhibition, this work further suggested that arginine(s) may lie at or near the UhpT active site. In other work, each UhpT arginine was examined individually by using site-directed mutagenesis to generate a cysteine or a lysine derivative. With two exceptions (R46, R275), all arginines could be replaced by either cysteine (10 of 14 residues) or lysine (12 of 14) without loss of function, implicating R46 and R275 as essential to UhpT function. This idea was strengthened by examining a multiple alignment of the eleven known UhpT-related proteins (≥30% identity). That alignment showed R46 and R275 were two of the only three arginines strongly conserved in this group of proteins. Considered together, these different approaches lead us to conclude that UhpT and its relatives have only two arginine residues (R46, R275) whose presence is essential to function. Prior biochemical work had placed R275 at the external entrance to the translocation pathway, and a symmetry argument emerging from the multiple alignment suggests a similar position for R46. Accordingly, by virtue of their locations at the entrance to this pathway, we speculate that R46 and R275 function in establishing substrate specificity.
John Hellgren - One of the best experts on this subject based on the ideXlab platform.
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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs Sugar Phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
Microbial Cell Factories, 2019Co-Authors: Alexandra Bergman, John Hellgren, Thomas Moritz, Verena Siewers, Jens Nielsen, Yun ChenAbstract: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 CO_2. 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 CO_2. 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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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs Sugar Phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
Microbial Cell Factories, 2019Co-Authors: Alexandra Bergman, John Hellgren, Thomas Moritz, Verena Siewers, Jens Nielsen, Yun ChenAbstract: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. 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. 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.
