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K T Shanmugam - One of the best experts on this subject based on the ideXlab platform.

  • amino acid substitutions at glutamate 354 in dihydrolipoamide dehydrogenase of escherichia coli lower the sensitivity of pyruvate dehydrogenase to nadh
    Microbiology, 2012
    Co-Authors: Lonnie O Ingram, Zhentao Sun, Mun Su Rhee, Lakshmanan Govindasamy, Qingzhao Wang, K T Shanmugam
    Abstract:

    Pyruvate dehydrogenase (PDH) of Escherichia coli is inhibited by NADH. This inhibition is partially reversed by mutational alteration of the dihydrolipoamide dehydrogenase (LPD) component of the PDH complex (E354K or H322Y). Such a mutation in lpd led to a PDH complex that was functional in an Anaerobic culture as seen by restoration of Anaerobic Growth of a pflB, ldhA double mutant of E. coli utilizing a PDH- and alcohol dehydrogenase-dependent homoethanol fermentation pathway. The glutamate at position 354 in LPD was systematically changed to all of the other natural amino acids to evaluate the physiological consequences. These amino acid replacements did not affect the PDH-dependent aerobic Growth. With the exception of E354M, all changes also restored PDH-dependent Anaerobic Growth of and fermentation by an ldhA, pflB double mutant. The PDH complex with an LPD alteration E354G, E354P or E354W had an approximately 20-fold increase in the apparent K i for NADH compared with the native complex. The apparent K m for pyruvate or NAD+ for the mutated forms of PDH was not significantly different from that of the native enzyme. A structural model of LPD suggests that the amino acid at position 354 could influence movement of NADH from its binding site to the surface. These results indicate that glutamate at position 354 plays a structural role in establishing the NADH sensitivity of LPD and the PDH complex by restricting movement of the product/substrate NADH, although this amino acid is not directly associated with NAD(H) binding.

  • dihydrolipoamide dehydrogenase mutation alters the nadh sensitivity of pyruvate dehydrogenase complex of escherichia coli k 12
    Journal of Bacteriology, 2008
    Co-Authors: Youngnyun Kim, Lonnie O Ingram, K T Shanmugam
    Abstract:

    Under Anaerobic Growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to Growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported Anaerobic Growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher Ki for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in Anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the Growth and physiology of the organisms in a similar fashion.

  • pyruvate formate lyase and acetate kinase are essential for Anaerobic Growth of escherichia coli on xylose
    Journal of Bacteriology, 2004
    Co-Authors: Adnan Hasona, Frank Healy, L O Ingram, K T Shanmugam
    Abstract:

    During Anaerobic Growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell Growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative Growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under Anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic Growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under Anaerobic conditions, confirming the need for the ATP produced by acetate kinase for Anaerobic Growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew Anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the Growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support Growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for Anaerobic Growth of E. coli on xylose due to energetic constraints.

Pronk J.t. - One of the best experts on this subject based on the ideXlab platform.

  • A squalene-hopene cyclase in Schizosaccharomyces japonicus represents a eukaryotic adaptation to sterol-limited Anaerobic environments
    'Proceedings of the National Academy of Sciences', 2021
    Co-Authors: Bouwknegt J., Wiersma S.j., Ortiz Merino R.a., Doornenbal E.s.r., Buitenhuis Petrik, Giera Martin, Müller Christoph, Pronk J.t.
    Abstract:

    Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requiresmolecular oxygen. Anaerobic protists and deep-branching Anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent Growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353-358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing Anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of Anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop- 17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated Anaerobic Growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to Anaerobic Growth. The fast Anaerobic Growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in Anaerobic industrial processes.BT/Industrial MicrobiologyEconomics of Technology and InnovationBT/Biotechnolog

  • Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for Anaerobic Growth
    'Elsevier BV', 2021
    Co-Authors: Dekker W.j.c., Mooiman C., Ortiz Merino R.a., Kaljouw Astrid, Battjes Julius, Wiering, Frank W., De La Torre, P., Pronk J.t.
    Abstract:

    Current large-scale, Anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew Anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for Anaerobic industrial applications.BT/Industrial MicrobiologyBT/Bioprocess EngineeringBT/Biotechnolog

  • Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for Anaerobic Growth
    'Elsevier BV', 2021
    Co-Authors: Dekker W.j.c., Mooiman C., Ortiz Merino R.a., Kaljouw Astrid, Battjes Julius, Wiering, Frank W., De La Torre, P., Pronk J.t.
    Abstract:

    Current large-scale, Anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew Anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for Anaerobic industrial applications.

  • Anaerobic Growth of Saccharomyces cerevisiae CEN.PK113-7D does not depend on synthesis or supplementation of unsaturated fatty acids
    'Oxford University Press (OUP)', 2019
    Co-Authors: Dekker W.j.c., Wiersma S.j., Bouwknegt J., Mooiman C., Pronk J.t.
    Abstract:

    In Saccharomyces cerevisiae, acyl-coenzyme A desaturation by Ole1 requires molecular oxygen. Tween 80, a poly-ethoxylated sorbitan-oleate ester, is therefore routinely included in Anaerobic Growth media as a source of unsaturated fatty acids (UFAs). During optimization of protocols for Anaerobic bioreactor cultivation of this yeast, we consistently observed Growth of the laboratory strain S. cerevisiae CEN.PK113-7D in media that contained the Anaerobic Growth factor ergosterol, but lacked UFAs. To minimize oxygen contamination, additional experiments were performed in an Anaerobic chamber. After Anaerobic precultivation without ergosterol and Tween 80, strain CEN.PK113-7D and a congenic ole1Δ strain both grew during three consecutive batch-cultivation cycles on medium that contained ergosterol, but not Tween 80. During these three cycles, no UFAs were detected in biomass of cultures grown without Tween 80, while contents of C10 to C14 saturated fatty acids were higher than in biomass from Tween 80-supplemented cultures. In contrast to its UFA-independent Anaerobic Growth, aerobic Growth of the ole1Δ strain strictly depended on Tween 80 supplementation. This study shows that the requirement of Anaerobic cultures of S. cerevisiae for UFA supplementation is not absolute and provides a basis for further research on the effects of lipid composition on yeast viability and robustness.

  • Anaerobic Growth of Saccharomyces cerevisiae CEN.PK113-7D does not depend on synthesis or supplementation of unsaturated fatty acids
    'Oxford University Press (OUP)', 2019
    Co-Authors: Dekker W.j.c., Wiersma S.j., Bouwknegt J., Mooiman C., Pronk J.t.
    Abstract:

    In Saccharomyces cerevisiae, acyl-coenzyme A desaturation by Ole1 requires molecular oxygen. Tween 80, a poly-ethoxylated sorbitan-oleate ester, is therefore routinely included in Anaerobic Growth media as a source of unsaturated fatty acids (UFAs). During optimization of protocols for Anaerobic bioreactor cultivation of this yeast, we consistently observed Growth of the laboratory strain S. cerevisiae CEN.PK113-7D in media that contained the Anaerobic Growth factor ergosterol, but lacked UFAs. To minimize oxygen contamination, additional experiments were performed in an Anaerobic chamber. After Anaerobic precultivation without ergosterol and Tween 80, strain CEN.PK113-7D and a congenic ole1Δ strain both grew during three consecutive batch-cultivation cycles on medium that contained ergosterol, but not Tween 80. During these three cycles, no UFAs were detected in biomass of cultures grown without Tween 80, while contents of C10 to C14 saturated fatty acids were higher than in biomass from Tween 80-supplemented cultures. In contrast to its UFA-independent Anaerobic Growth, aerobic Growth of the ole1Δ strain strictly depended on Tween 80 supplementation. This study shows that the requirement of Anaerobic cultures of S. cerevisiae for UFA supplementation is not absolute and provides a basis for further research on the effects of lipid composition on yeast viability and robustness.BT/Industrial MicrobiologyBT/Biotechnolog

Joel H Weiner - One of the best experts on this subject based on the ideXlab platform.

  • the escherichia coli ynfefghi operon encodes polypeptides which are paralogues of dimethyl sulfoxide reductase dmsabc
    Archives of Biochemistry and Biophysics, 2003
    Co-Authors: Shannon P Lubitz, Joel H Weiner
    Abstract:

    Abstract The ynfEFGHI operon is a paralogue of the Escherichia coli dmsABC operon. ynfE and ynfF are paralogues of dmsA. ynfG and ynfH are paralogues of dmsB and dmsC, respectively. YnfI (dmsD) has no dms paralogue. YnfE/F and YnfG could be detected by immunoblotting with anti-DmsAB antibodies when expressed under the control of a tac or dms promoter. Cells harbouring ynfFGH on a multicopy plasmid supported Anaerobic Growth with dimethyl sulfoxide (DMSO) as respiratory oxidant in a dmsABC deletion, suggesting that YnfFGH forms a heterotimeric enzyme complex similar to DmsABC. Exchange of DmsC by YnfH (DmsAB-YnfH) resulted in membrane localization, Anaerobic Growth on DMSO, and binding of 2-n-heptyl 4-hydroxyquinoline-N-oxide, indicating that YnfH was a competent anchor. YnfG can also replace DmsB as the electron transfer subunit and assembled [Fe–S] clusters as judged by electron paramagnetic resonance spectroscopy. YnfE and/or YnfF could not form a functional complex with DmsBC and expression of YnfE prevented the accumulation of YnfFGH.

  • multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of escherichia coli
    Journal of Biological Chemistry, 2000
    Co-Authors: Damaraju Sambasivarao, Joanne Simalagrant, Raymond J Turner, Gillian Shaw, Joel H Weiner
    Abstract:

    Dimethyl sulfoxide (Me(2)SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under Anaerobic Growth conditions and is required for Anaerobic Growth with Me(2)SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support Growth on glycerol-Me(2)SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membrane-bound DmsABC with greatly reduced enzyme activity and inefficient Anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.

  • kinetic analysis and substrate specificity of escherichia coli dimethyl sulfoxide reductase
    Microbiology, 1996
    Co-Authors: Joanne Simalagrant, Joel H Weiner
    Abstract:

    We have characterized the substrate specificity of dimethyl sulfoxide reductase (DmsABC) of Escherichia coli by determining Km and Kcat values for 22 different substrates. The enzyme has a very broad substrate specificity. The Km values varied 470-fold, while Kcat values varied only 20-fold, implicating Km as the major determinant of Kcat/Km values. Sulfoxides and pyridine N-oxide exhibited the lowest Km values, followed by aliphatic N-oxides. The Kcat values for these compounds also followed the same pattern. Substitution at the 2 or 3 position of the pyridine N-oxide ring had little effect on Km while substitution at the 4 position had a greater effect, and increased Km. Negatively charged substrates were poorly accepted. A few compounds that are not S- or N-oxides were also reduced by the enzyme. Most compounds reduced by DmsABC were not toxic to E. coli under Anaerobic Growth conditions, and E. coli was able to use many of these compounds Anaerobically as terminal electron acceptors in the presence of glycerol. Anaerobic Growth on sulfoxides is solely due to DmsABC expression. However, there appears to be another as yet unidentified terminal reductase capable of using pyridine N-oxides as terminal electron acceptors.

  • multiple pathways of electron transfer in dimethyl sulfoxide reductase of escherichia coli
    Journal of Biological Chemistry, 1994
    Co-Authors: Catharine A Trieber, Richard A. Rothery, Joel H Weiner
    Abstract:

    The catalytic subunit of dimethyl sulfoxide (Me2SO) reductase, DmsA, contains six blocks of sequence that are homologous to other members of the superfamily of prokaryotic molybdoenzymes. The amino-terminal block contains 5 conserved residues (Cys38, Cys42, Cys75, Lys28, and Arg77). Site-directed mutagenesis of these residues did not alter membrane localization but in some cases less enzyme accumulated. The activity of Me2SO reductase was monitored by measuring Me2SO-dependent Anaerobic Growth, benzyl viologen, or dimethylnaphthoquinol oxidase activity, and using a quinol pool-coupling assay. Only Cys75 and Lys28 mutant enzymes were able to support Anaerobic Growth with Me2SO suggesting a critical role for Cys38, Cys42, and Arg77. Benzyl viologen oxidase activity was retained in the mutants although with reduced efficiency in Cys42-Ser. Electron transport with dimethylnaphthoquinol was reduced in Cys38-Ser, Cys42-Ser, and Cys75-Ser and almost totally eliminated in the Arg77-Ser mutant. Cys38-Ser, Cys42-Ser, and Arg77-Ser were unable to support quinol oxidation although electron transfer from the quinol pool to the [Fe-S] centers in DmsB was normal. These results indicate that the amino-terminal region is involved in functional electron transfer from the quinol pool to Me2SO and that electrons from benzyl viologen, dimethylnaphthoquinol, and menaquinol may follow different paths within the catalytic subunit.

Marc Strous - One of the best experts on this subject based on the ideXlab platform.

  • an improved medium for the Anaerobic Growth of paracoccus denitrificans pd1222
    Frontiers in Microbiology, 2014
    Co-Authors: Stefanie M Hahnke, Philipp Moosmann, Tobias J Erb, Marc Strous
    Abstract:

    Paracoccus denitrificans is a well studied model organism with respect to its aerobic and Anaerobic respiratory enzymes. However, until now, the Growth medium for this organism has not been optimized for Anaerobic Growth. In particular, the requirements of P. denitrificans for trace elements are not well known. In the present study we aimed to improve Growth rates of P. denitrificans Pd1222 on a defined medium under anoxic conditions. We designed media containing different combinations of trace elements at various concentrations, and tested their performance against previously reported media. Our results suggest that Growth rate and yield depend on the availability and concentration of trace elements in the medium. A chelated trace element solution was more suitable than an acidified trace element solution. Highest Growth rates were achieved with medium comprising the trace elements iron, manganese, molybdenum, copper and zinc ranging from 0.1 to 9 µM. On this medium, P. denitrificans Pd1222 grew with a generation time of 4.4 hours under anoxic conditions and 2.8 hours under oxic conditions. Diauxic Growth was clearly shown with respect to nitrate and nitrite reduction under anoxic conditions.

Dekker W.j.c. - One of the best experts on this subject based on the ideXlab platform.

  • Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for Anaerobic Growth
    'Elsevier BV', 2021
    Co-Authors: Dekker W.j.c., Mooiman C., Ortiz Merino R.a., Kaljouw Astrid, Battjes Julius, Wiering, Frank W., De La Torre, P., Pronk J.t.
    Abstract:

    Current large-scale, Anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew Anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for Anaerobic industrial applications.BT/Industrial MicrobiologyBT/Bioprocess EngineeringBT/Biotechnolog

  • Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for Anaerobic Growth
    'Elsevier BV', 2021
    Co-Authors: Dekker W.j.c., Mooiman C., Ortiz Merino R.a., Kaljouw Astrid, Battjes Julius, Wiering, Frank W., De La Torre, P., Pronk J.t.
    Abstract:

    Current large-scale, Anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew Anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for Anaerobic industrial applications.

  • Anaerobic Growth of Saccharomyces cerevisiae CEN.PK113-7D does not depend on synthesis or supplementation of unsaturated fatty acids
    'Oxford University Press (OUP)', 2019
    Co-Authors: Dekker W.j.c., Wiersma S.j., Bouwknegt J., Mooiman C., Pronk J.t.
    Abstract:

    In Saccharomyces cerevisiae, acyl-coenzyme A desaturation by Ole1 requires molecular oxygen. Tween 80, a poly-ethoxylated sorbitan-oleate ester, is therefore routinely included in Anaerobic Growth media as a source of unsaturated fatty acids (UFAs). During optimization of protocols for Anaerobic bioreactor cultivation of this yeast, we consistently observed Growth of the laboratory strain S. cerevisiae CEN.PK113-7D in media that contained the Anaerobic Growth factor ergosterol, but lacked UFAs. To minimize oxygen contamination, additional experiments were performed in an Anaerobic chamber. After Anaerobic precultivation without ergosterol and Tween 80, strain CEN.PK113-7D and a congenic ole1Δ strain both grew during three consecutive batch-cultivation cycles on medium that contained ergosterol, but not Tween 80. During these three cycles, no UFAs were detected in biomass of cultures grown without Tween 80, while contents of C10 to C14 saturated fatty acids were higher than in biomass from Tween 80-supplemented cultures. In contrast to its UFA-independent Anaerobic Growth, aerobic Growth of the ole1Δ strain strictly depended on Tween 80 supplementation. This study shows that the requirement of Anaerobic cultures of S. cerevisiae for UFA supplementation is not absolute and provides a basis for further research on the effects of lipid composition on yeast viability and robustness.

  • Anaerobic Growth of Saccharomyces cerevisiae CEN.PK113-7D does not depend on synthesis or supplementation of unsaturated fatty acids
    'Oxford University Press (OUP)', 2019
    Co-Authors: Dekker W.j.c., Wiersma S.j., Bouwknegt J., Mooiman C., Pronk J.t.
    Abstract:

    In Saccharomyces cerevisiae, acyl-coenzyme A desaturation by Ole1 requires molecular oxygen. Tween 80, a poly-ethoxylated sorbitan-oleate ester, is therefore routinely included in Anaerobic Growth media as a source of unsaturated fatty acids (UFAs). During optimization of protocols for Anaerobic bioreactor cultivation of this yeast, we consistently observed Growth of the laboratory strain S. cerevisiae CEN.PK113-7D in media that contained the Anaerobic Growth factor ergosterol, but lacked UFAs. To minimize oxygen contamination, additional experiments were performed in an Anaerobic chamber. After Anaerobic precultivation without ergosterol and Tween 80, strain CEN.PK113-7D and a congenic ole1Δ strain both grew during three consecutive batch-cultivation cycles on medium that contained ergosterol, but not Tween 80. During these three cycles, no UFAs were detected in biomass of cultures grown without Tween 80, while contents of C10 to C14 saturated fatty acids were higher than in biomass from Tween 80-supplemented cultures. In contrast to its UFA-independent Anaerobic Growth, aerobic Growth of the ole1Δ strain strictly depended on Tween 80 supplementation. This study shows that the requirement of Anaerobic cultures of S. cerevisiae for UFA supplementation is not absolute and provides a basis for further research on the effects of lipid composition on yeast viability and robustness.BT/Industrial MicrobiologyBT/Biotechnolog

  • Reassessment of requirements for Anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains
    'Oxford University Press (OUP)', 2018
    Co-Authors: Bracher J.m., Dekker W.j.c., Van Maris A.j.a., Martinez-rodriguez, Oscar A., Pronk J.t.
    Abstract:

    Expression of a heterologous xylose isomerase, deletion of the GRE3 aldose-reductase gene and overexpression of genes encoding xylulokinase (XKS1) and non-oxidative pentose-phosphate-pathway enzymes (RKI1, RPE1, TAL1, TKL1) enables aerobic Growth of Saccharomyces cerevisiae on d-xylose. However, literature reports differ on whether Anaerobic Growth on d-xylose requires additional mutations. Here, CRISPR-Cas9-assisted reconstruction and physiological analysis confirmed an early report that this basic set of genetic modifications suffices to enable Anaerobic Growth on d-xylose in the CEN.PK genetic background. Strains that additionally carried overexpression cassettes for the transaldolase and transketolase paralogs NQM1 and TKL2 only exhibited Anaerobic Growth on d-xylose after a 7-10 day lag phase. This extended lag phase was eliminated by increasing inoculum concentrations from 0.02 to 0.2 g biomass L-1. Alternatively, a long lag phase could be prevented by sparging low-inoculum-density bioreactor cultures with a CO2/N2-mixture, thus mimicking initial CO2 concentrations in high-inoculum-density, nitrogen-sparged cultures, or by using l-aspartate instead of ammonium as nitrogen source. This study resolves apparent contradictions in the literature on the genetic interventions required for Anaerobic Growth of CEN.PK-derived strains on d-xylose. Additionally, it indicates the potential relevance of CO2 availability and anaplerotic carboxylation reactions for Anaerobic Growth of engineered S. cerevisiae strains on d-xylose.BT/Industrial Microbiolog