FN Thomson Reuters Web of Knowledge
VR 1.0
PT J
AU Zelle, Rintze M.
   Harrison, Jacob C.
   Pronk, Jack T.
   van Maris, Antonius J. A.
TI Anaplerotic Role for Cytosolic Malic Enzyme in Engineered Saccharomyces
   cerevisiae Strains
SO APPLIED AND ENVIRONMENTAL MICROBIOLOGY
VL 77
IS 3
BP 732
EP 738
DI 10.1128/AEM.02132-10
PD FEB 2011
PY 2011
AB Malic enzyme catalyzes the reversible oxidative decarboxylation of
   malate to pyruvate and CO(2). The Saccharomyces cerevisiae MAE1 gene
   encodes a mitochondrial malic enzyme whose proposed physiological roles
   are related to the oxidative, malate-decarboxylating reaction. Hitherto,
   the inability of pyruvate carboxylase-negative (Pyc(-)) S. cerevisiae
   strains to grow on glucose suggested that Mae1p cannot act as a
   pyruvate-carboxylating, anaplerotic enzyme. In this study, relocation of
   malic enzyme to the cytosol and creation of thermodynamically favorable
   conditions for pyruvate carboxylation by metabolic engineering, process
   design, and adaptive evolution, enabled malic enzyme to act as the sole
   anaplerotic enzyme in S. cerevisiae. The Escherichia coli NADH-dependent
   sfcA malic enzyme was expressed in a Pyc(-) S. cerevisiae background.
   When PDC2, a transcriptional regulator of pyruvate decarboxylase genes,
   was deleted to increase intracellular pyruvate levels and cells were
   grown under a CO(2) atmosphere to favor carboxylation, adaptive
   evolution yielded a strain that grew on glucose (specific growth rate,
   0.06 +/- 0.01 h(-1)). Growth of the evolved strain was enabled by a
   single point mutation (Asp336Gly) that switched the cofactor preference
   of E. coli malic enzyme from NADH to NADPH. Consistently, cytosolic
   relocalization of the native Mae1p, which can use both NADH and NADPH,
   in a pyc1,2 Delta pdc2 Delta strain grown under a CO(2) atmosphere, also
   enabled slow-growth on glucose. Although growth rates of these strains
   are still low, the higher ATP efficiency of carboxylation via malic
   enzyme, compared to the pyruvate carboxylase pathway, may contribute to
   metabolic engineering of S. cerevisiae for anaerobic, high-yield
   C(4)-dicarboxylic acid production.
TC 0
Z9 0
SN 0099-2240
UT WOS:000286597100004
ER

PT J
AU Zelle, Rintze M.
   Trueheart, Josh
   Harrison, Jacob C.
   Pronk, Jack T.
   van Maris, Antonius J. A.
TI Phosphoenolpyruvate Carboxykinase as the Sole Anaplerotic Enzyme in
   Saccharomyces cerevisiae
SO APPLIED AND ENVIRONMENTAL MICROBIOLOGY
VL 76
IS 16
BP 5383
EP 5389
DI 10.1128/AEM.01077-10
PD AUG 2010
PY 2010
AB Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown
   cultures of wild-type Saccharomyces cerevisiae. Pyruvate
   carboxylase-negative (Pyc(-)) S. cerevisiae strains cannot grow on
   glucose unless media are supplemented with C(4) compounds, such as
   aspartic acid. In several succinate-producing prokaryotes,
   phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic
   role. However, the S. cerevisiae PEPCK encoded by PCK1 is repressed by
   glucose and is considered to have a purely decarboxylating and
   gluconeogenic function. This study investigates whether and under which
   conditions PEPCK can replace the anaplerotic function of pyruvate
   carboxylase in S. cerevisiae. Pyc(-) S. cerevisiae strains
   constitutively overexpressing the PEPCK either from S. cerevisiae or
   from Actinobacillus succinogenes did not grow on glucose as the sole
   carbon source. However, evolutionary engineering yielded mutants able to
   grow on glucose as the sole carbon source at a maximum specific growth
   rate of ca. 0.14 h(-1), one-half that of the (pyruvate
   carboxylase-positive) reference strain grown under the same conditions.
   Growth was dependent on high carbon dioxide concentrations, indicating
   that the reaction catalyzed by PEPCK operates near thermodynamic
   equilibrium. Analysis and reverse engineering of two independently
   evolved strains showed that single point mutations in pyruvate kinase,
   which competes with PEPCK for phosphoenolpyruvate, were sufficient to
   enable the use of PEPCK as the sole anaplerotic enzyme. The PEPCK
   reaction produces one ATP per carboxylation event, whereas the original
   route through pyruvate kinase and pyruvate carboxylase is ATP neutral.
   This increased ATP yield may prove crucial for engineering of efficient
   and low-cost anaerobic production of C(4) dicarboxylic acids in S.
   cerevisiae.
TC 1
Z9 1
SN 0099-2240
UT WOS:000280633400006
ER

PT J
AU Zelle, Rintze M.
   De Hulster, Erik
   Kloezen, Wendy
   Pronk, Jack T.
   van Maris, Antonius J. A.
TI Key Process Conditions for Production of C(4) Dicarboxylic Acids in
   Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae
   Strain
SO APPLIED AND ENVIRONMENTAL MICROBIOLOGY
VL 76
IS 3
BP 744
EP 750
DI 10.1128/AEM.02396-09
PD FEB 2010
PY 2010
AB A recent effort to improve malic acid production by Saccharomyces
   cerevisiae by means of metabolic engineering resulted in a strain that
   produced up to 59 g liter(-1) of malate at a yield of 0.42 mol (mol
   glucose)(-1) in calcium carbonate-buffered shake flask cultures. With
   shake flasks, process parameters that are important for scaling up this
   process cannot be controlled independently. In this study, growth and
   product formation by the engineered strain were studied in bioreactors
   in order to separately analyze the effects of pH, calcium, and carbon
   dioxide and oxygen availability. A near-neutral pH, which in shake
   flasks was achieved by adding CaCO(3), was required for efficient C(4)
   dicarboxylic acid production. Increased calcium concentrations, a side
   effect of CaCO(3) dissolution, had a small positive effect on malate
   formation. Carbon dioxide enrichment of the sparging gas (up to 15%
   [vol/vol]) improved production of both malate and succinate. At higher
   concentrations, succinate titers further increased, reaching 0.29 mol
   (mol glucose)(-1), whereas malate formation strongly decreased. Although
   fully aerobic conditions could be achieved, it was found that moderate
   oxygen limitation benefitted malate production. In conclusion, malic
   acid production with the engineered S. cerevisiae strain could be
   successfully transferred from shake flasks to 1-liter batch bioreactors
   by simultaneous optimization of four process parameters (pH and
   concentrations of CO(2), calcium, and O(2)). Under optimized conditions,
   a malate yield of 0.48 +/- 0.01 mol (mol glucose)(-1) was obtained in
   bioreactors, a 19% increase over yields in shake flask experiments.
TC 2
Z9 2
SN 0099-2240
UT WOS:000274017400015
ER

PT J
AU Abbott, Derek A.
   Zelle, Rintze M.
   Pronk, Jack T.
   van Maris, Antonius J. A.
TI Metabolic engineering of Saccharomyces cerevisiae for production of
   carboxylic acids: current status and challenges
SO FEMS YEAST RESEARCH
VL 9
IS 8
BP 1123
EP 1136
DI 10.1111/j.1567-1364.2009.00537.x
PD DEC 2009
PY 2009
AB To meet the demands of future generations for chemicals and energy and
   to reduce the environmental footprint of the chemical industry,
   alternatives for petrochemistry are required. Microbial conversion of
   renewable feedstocks has a huge potential for cleaner, sustainable
   industrial production of fuels and chemicals. Microbial production of
   organic acids is a promising approach for production of chemical
   building blocks that can replace their petrochemically derived
   equivalents. Although Saccharomyces cerevisiae does not naturally
   produce organic acids in large quantities, its robustness, pH tolerance,
   simple nutrient requirements and long history as an industrial workhorse
   make it an excellent candidate biocatalyst for such processes. Genetic
   engineering, along with evolution and selection, has been successfully
   used to divert carbon from ethanol, the natural endproduct of S.
   cerevisiae, to pyruvate. Further engineering, which included expression
   of heterologous enzymes and transporters, yielded strains capable of
   producing lactate and malate from pyruvate. Besides these metabolic
   engineering strategies, this review discusses the impact of transport
   and energetics as well as the tolerance towards these organic acids. In
   addition to recent progress in engineering S. cerevisiae for organic
   acid production, the key limitations and challenges are discussed in the
   context of sustainable industrial production of organic acids from
   renewable feedstocks.
TC 11
Z9 11
SN 1567-1356
UT WOS:000271264400001
ER

PT J
AU Zelle, Rintze M.
   de Hulster, Erik
   van Winden, WoUter A.
   de Waard, Pieter
   Dijkema, Cor
   Winkler, Aaron A.
   Geertman, Jan-Maarten A.
   van Dijken, Johannes P.
   Pronk, Jack T.
   van Maris, Antonius J. A.
TI Malic acid production by Saccharomyces cerevisiae: Engineering of
   pyruvate carboxylation, oxaloacetate reduction, and malate export
SO APPLIED AND ENVIRONMENTAL MICROBIOLOGY
VL 74
IS 9
BP 2766
EP 2777
DI 10.1128/AEM.02591-07
PD MAY 2008
PY 2008
AB Malic acid is a potential biomass-derivable "building block" for
   chemical synthesis. Since wild-type Saccharomyces cerevisiae strains
   produce only low levels of malate, metabolic engineering is required to
   achieve efficient malate production with this yeast. A promising pathway
   for malate production from glucose proceeds via carboxylation of
   pyruvate, followed by reduction of oxaloacetate to malate. This redox-
   and ATP-neutral, CO2-fixing pathway has a theoretical maximum yield of 2
   mol malate (mol glucose)(-1). A previously engineered glucose-tolerant,
   C-2-independent pyruvate decarboxylase-negative S. cerevisiae strain was
   used as the platform to evaluate the impact of individual and combined
   introduction of three genetic modifications: (i) overexpression of the
   native pyruvate carboxylase encoded by PYC2, (ii) high-level expression
   of an allele of the MDH3 gene, of which the encoded malate dehydrogenase
   was retargeted to the cytosol by deletion of the C-terminal peroxisomal
   targeting sequence, and (iii) functional expression of the
   Schizosaccharomyces pombe malate transporter gene SpMAE1. While single
   or double modifications improved malate production, the highest malate
   yields and titers were obtained with the simultaneous introduction of
   all three modifications. In glucose-grown batch cultures, the resulting
   engineered strain produced malate at titers of up to 59 g liter(-1) at a
   malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis
   showed that metabolite labeling patterns observed upon nuclear magnetic
   resonance analyses of cultures grown on C-13-labeled glucose were
   consistent with the envisaged nonoxidative, fermentative pathway for
   malate production. The engineered strains still produced substantial
   amounts of pyruvate, indicating that the pathway efficiency can be
   further improved.
TC 15
Z9 17
SN 0099-2240
UT WOS:000255567900024
ER

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