scholarly journals Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures of Saccharomyces cerevisiae

2003 ◽  
Vol 69 (4) ◽  
pp. 2094-2099 ◽  
Author(s):  
Antonius J. A. van Maris ◽  
Marijke A. H. Luttik ◽  
Aaron A. Winkler ◽  
Johannes P. van Dijken ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alternative Pathway ◽  
Pyruvate Decarboxylase ◽  
Lysine Biosynthesis ◽  
Carbon Limitation ◽  
Growth Media ◽  
Nutritional Requirement ◽  
Acetyl Coa ◽  
Threonine Aldolase ◽  
Chemostat Cultures

ABSTRACT Pyruvate decarboxylase-negative (Pdc−) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C2 requirement of Pdc− S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of l-carnitine to growth media did not restore growth of a Pdc− strain on glucose, indicating that the C2 requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc− strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc− GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc− strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.

scholarly journals Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Harmen M. van Rossum ◽  
Barbara U. Kozak ◽  
Matthijs S. Niemeijer ◽  
James C. Dykstra ◽  
Marijke A. H. Luttik ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Lipoic Acid ◽  
Intracellular Transport ◽  
Pyruvate Dehydrogenase Complex ◽  
Constitutive Expression ◽  
Growth Media ◽  
Acetyl Coa ◽  
Mitochondrial Fatty Acid

ABSTRACTIn many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grownSaccharomyces cerevisiaecells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occurin vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineeredS. cerevisiaestrain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, wasl-carnitine dependent, indicating thatin vivoexport of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabledl-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enablingin vivoexport of mitochondrial acetyl units via the carnitine shuttle.IMPORTANCEThis study demonstrates, for the first time, thatSaccharomyces cerevisiaecan be engineered to employ the carnitine shuttle for export of acetyl moieties from the mitochondria and, thereby, to act as the sole source of cytosolic acetyl-CoA. Further optimization of this ATP-independent mechanism for cytosolic acetyl-CoA provision can contribute to efficient, yeast-based production of industrially relevant compounds derived from this precursor. The strains constructed in this study, whose growth on glucose depends on a functional carnitine shuttle, provide valuable models for further functional analysis and engineering of this shuttle in yeast and other eukaryotes.

scholarly journals Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae, Yielding a C2-Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast

2004 ◽  
Vol 70 (1) ◽  
pp. 159-166 ◽  
Author(s):  
Antonius J. A. van Maris ◽  
Jan-Maarten A. Geertman ◽  
Alexander Vermeulen ◽  
Matthijs K. Groothuizen ◽  
Aaron A. Winkler ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptome Analysis ◽  
Molecular Mechanisms ◽  
Synthetic Medium ◽  
Pyruvate Decarboxylase ◽  
Exponential Growth Phase ◽  
Batch Cultures ◽  
Growth Defects ◽  
Chemostat Cultures ◽  
Glucose Tolerant

ABSTRACT The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc−) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc− S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C2 compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc− strain without these growth defects. An acetate-independent Pdc− mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C2 independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc− S. cerevisiae mutant (TAM) that could grow in batch cultures (μmax = 0.20 h−1) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter−1 was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass−1 h−1 during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose−1.

scholarly journals Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export

2004 ◽  
Vol 70 (5) ◽  
pp. 2898-2905 ◽  
Author(s):  
Antonius J. A. van Maris ◽  
Aaron A. Winkler ◽  
Danilo Porro ◽  
Johannes P. van Dijken ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Alcoholic Fermentation ◽  
Lactic Acid Production ◽  
Lactate Production ◽  
Pyruvate Decarboxylase ◽  
Metabolic Energy ◽  
Anaerobic Growth ◽  
Chemostat Cultures ◽  
Engineered Strain

ABSTRACT Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.

scholarly journals Complete and efficient conversion of plant cell wall hemicellulose into high-value bioproducts by engineered yeast

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liang Sun ◽  
Jae Won Lee ◽  
Sangdo Yook ◽  
Stephan Lane ◽  
Ziqiao Sun ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cellulosic Biomass ◽  
Hemicellulose Hydrolysate ◽  
Acetyl Coa ◽  
Engineered Yeast ◽  
Fermentation Inhibitor ◽  
Acid Lactone

AbstractPlant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae. The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an enginered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass.

scholarly journals Subtelomeric Repeat Amplification Is Associated With Growth at Elevated Temperature in yku70 Mutants of Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1039-1051
Author(s):  
Barbara Fellerhoff ◽  
Friederike Eckardt-Schupp ◽  
Anna A Friedl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elevated Temperature ◽  
Alternative Pathway ◽  
Dna Amplification ◽  
Strand Break ◽  
Significant Loss ◽  
Growth Defect ◽  
Slight Reduction ◽  
Telomeric Sequences ◽  
Repair Defect

Abstract Inactivation of the Saccharomyces cerevisiae gene YKU70 (HDF1), which encodes one subunit of the Ku heterodimer, confers a DNA double-strand break repair defect, shortening of and structural alterations in the telomeres, and a severe growth defect at 37°. To elucidate the basis of the temperature sensitivity, we analyzed subclones derived from rare yku70 mutant cells that formed a colony when plated at elevated temperature. In all these temperature-resistant subclones, but not in cell populations shifted to 37°, we observed substantial amplification and redistribution of subtelomeric Y′ element DNA. Amplification of Y′ elements and adjacent telomeric sequences has been described as an alternative pathway for chromosome end stabilization that is used by postsenescence survivors of mutants deficient for the telomerase pathway. Our data suggest that the combination of Ku deficiency and elevated temperature induces a potentially lethal alteration of telomere structure or function. Both in yku70 mutants and in wild type, incubation at 37° results in a slight reduction of the mean length of terminal restriction fragments, but not in a significant loss of telomeric (C1-3A/TG1-3)n sequences. We propose that the absence of Ku, which is known to bind to telomeres, affects the telomeric chromatin so that its chromosome end-defining function is lost at 37°.

Formation of acetaldehyde from threonine by lactic acid bacteria

1976 ◽  
Vol 43 (1) ◽  
pp. 75-83 ◽  
Author(s):  
G. J. Lees ◽  
G. R. Jago
Keyword(s):  
Free Amino ◽  
Thiol Group ◽  
Nutritional Requirement ◽  
Threonine Dehydratase ◽  
Threonine Aldolase ◽  
Streptococcus Cremoris ◽  
Dependent Inhibition ◽  
Ph Dependent ◽  
Dehydratase Activity ◽  
Derivatives Of

SummaryGroup N streptococci were found to cleave threonine to form acetaldehyde and glycine. Threonine aldolase, the enzyme catalysing this reaction, was found in all strains exceptStreptococcus cremorisZ8, an organism which had been shown previously to have a nutritional requirement for glycine. The enzyme was strongly inhibited by glycine and cysteine. The inhibition showed characteristics of allosteric inhibition and was pH-dependent. Inhibition by glycine, but not by cysteine, was highly specific. Analogues and derivatives of cysteine which contained a thiol group and a free amino group inhibited the activity of threonine aldolase. The presence of a carboxyl group was not necessary for inhibition. The cleavage of threonine by wholecell suspensions was stimulated by either an energy source to aid transport, or by rendering the cells permeable to substrate with oleate. Threonine did not appear to be degraded by enzymes other than threonine aldolase, as threonine dehydratase activity was low and NAD- and NADP-dependent threonine dehydrogenases were absent.

scholarly journals Comparative studies on O-acetylhomoserine sulfhydrylase: physiological role and characterization of the Aspergillus nidulans enzyme.

1993 ◽  
Vol 40 (3) ◽  
pp. 421-428 ◽  
Author(s):  
J Brzywczy ◽  
S Yamagata ◽  
A Paszewski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Aspergillus Nidulans ◽  
Comparative Studies ◽  
Alternative Pathway ◽  
Physiological Role ◽  
Oligomeric Protein ◽  
Cysteine Synthesis ◽  
Sulfhydryl Compounds

O-acetylhomoserine sulfhydrylase (OAH SHLase) from Aspergillus nidulans is an oligomeric protein with a broad substrate specificity with regard to sulfhydryl compounds. As its Saccharomyces cerevisiae counterpart the enzyme also reacts with O-acetylserine and is inhibited by carbonyl reagents but not by antiserum raised against the yeast enzyme. In contrast to Saccharomyces cerevisiae the enzyme is not essential for Aspergillus nidulans as indicated by the completely prototrophic phenotype of OAH SHLase-negative mutants. Its major physiological role in Aspergillus nidulans seems to be recycling of the thiomethyl group of methylthio-adenosine but it is also a constituent of the alternative pathway of cysteine synthesis.

scholarly journals Molecular Basis for Anaerobic Growth of Saccharomyces cerevisiae on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis

2004 ◽  
Vol 70 (4) ◽  
pp. 2307-2317 ◽  
Author(s):  
Marco Sonderegger ◽  
Marie Jeppsson ◽  
Bärbel Hahn-Hägerdal ◽  
Uwe Sauer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Global Gene Expression ◽  
Anaerobic Growth ◽  
Flux Analysis ◽  
Atp Production ◽  
Redox Imbalance ◽  
Chemostat Cultures

ABSTRACT Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a Saccharomyces cerevisiae strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production.

Sign in / Sign up

Export Citation Format

Share Document