scholarly journals Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

2002 ◽  
Vol 68 (11) ◽  
pp. 5422-5428 ◽  
Author(s):  
Mattheos A. G. Koffas ◽  
Gyoo Yeol Jung ◽  
Juan C. Aon ◽  
Gregory Stephanopoulos
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Carboxylase ◽  
Carbon Sources ◽  
Acid Synthesis ◽  
Aspartate Kinase ◽  
Growth Enhancement ◽  
Lysine Production ◽  
Carboxylase Activity ◽  
Amino Acid Synthesis ◽  
Permeabilized Cells

ABSTRACT Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.

scholarly journals Amplified Expression of Fructose 1,6-Bisphosphatase in Corynebacterium glutamicum Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources

2005 ◽  
Vol 71 (12) ◽  
pp. 8587-8596 ◽  
Author(s):  
Judith Becker ◽  
Corinna Klopprogge ◽  
Oskar Zelder ◽  
Elmar Heinzle ◽  
Christoph Wittmann
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Metabolic Effects ◽  
Flux Analysis ◽  
Lysine Production ◽  
Fructose 1,6 Bisphosphatase

ABSTRACT The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfb r . The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.

scholarly journals Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides

2017 ◽  
Author(s):  
Samuel T Coradetti ◽  
Dominic Pinel ◽  
Gina Geiselman ◽  
Masakazu Ito ◽  
Stephen Mondo ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Lipid Metabolism ◽  
Functional Genomics ◽  
Lipid Accumulation ◽  
Protein Modification ◽  
Carbon Sources ◽  
Acid Synthesis ◽  
Rhodosporidium Toruloides ◽  
Chemical Production ◽  
Amino Acid Synthesis

AbstractThe basidomycete yeast Rhodosporidium toruloides (a.k.a. Rhodotorula toruloides) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions into the R. toruloides genome. We identified 1337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We found that both mitochondrial and peroxisomal enzymes were required for growth on fatty acids, with different peroxisomal enzymes required on different fatty acids. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, as well as genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species, identify key biological processes to be further explored and optimized for production of lipid-based bioproducts, and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.

scholarly journals Linking Central Metabolism with Increased Pathway Flux: l-Valine Accumulation by Corynebacterium glutamicum

2002 ◽  
Vol 68 (5) ◽  
pp. 2246-2250 ◽  
Author(s):  
Eva Radmacher ◽  
Adela Vaitsikova ◽  
Udo Burger ◽  
Karin Krumbach ◽  
Hermann Sahm ◽  
...  
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Acid Synthesis ◽  
Acetohydroxy Acid Synthase ◽  
Amino Acid Synthesis ◽  
Branched Chain Amino Acid ◽  
In The Wild ◽  
Valine Biosynthesis ◽  
Branched Chain ◽  
Pathway Flux

ABSTRACT Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete l-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since l-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize l-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for d-pantothenate. When the resulting strain, C. glutamicum 13032ΔilvAΔpanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM l-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the l-valine biosynthesis pathway.

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