scholarly journals Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

2001 ◽  
Vol 67 (1) ◽  
pp. 33-41 ◽  
Author(s):  
Ana Ramos ◽  
Ingeborg C. Boels ◽  
Willem M. de Vos ◽  
Helena Santos
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Pentose Phosphate ◽  
Biosynthetic Pathways ◽  
Glycolytic Intermediates ◽  
Phosphoglyceric Acid ◽  
Exopolysaccharide Biosynthesis ◽  
Fructose 1,6 Bisphosphate ◽  
Sugar Derivatives

ABSTRACT The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation.

scholarly journals Genomic Considerations for the Modification of Saccharomyces cerevisiae for Biofuel and Metabolite Biosynthesis

2020 ◽  
Vol 8 (3) ◽  
pp. 321 ◽  
Author(s):  
James T. Arnone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Native Species ◽  
Genetic Manipulation ◽  
Scale Up ◽  
Biosynthetic Pathways ◽  
Yeast Saccharomyces Cerevisiae ◽  
Position Effects ◽  
Industrial Use ◽  
A Site ◽  
Global Population

The growing global population and developing world has put a strain on non-renewable natural resources, such as fuels. The shift to renewable sources will, thus, help meet demands, often through the modification of existing biosynthetic pathways or the introduction of novel pathways into non-native species. There are several useful biosynthetic pathways endogenous to organisms that are not conducive for the scale-up necessary for industrial use. The use of genetic and synthetic biological approaches to engineer these pathways in non-native organisms can help ameliorate these challenges. The budding yeast Saccharomyces cerevisiae offers several advantages for genetic engineering for this purpose due to its widespread use as a model system studied by many researchers. The focus of this review is to present a primer on understanding genomic considerations prior to genetic modification and manipulation of S. cerevisiae. The choice of a site for genetic manipulation can have broad implications on transcription throughout a region and this review will present the current understanding of position effects on transcription.

scholarly journals Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

2004 ◽  
Vol 70 (12) ◽  
pp. 7277-7287 ◽  
Author(s):  
Christoph Wittmann ◽  
Patrick Kiefer ◽  
Oskar Zelder
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

Evidence for a Transcarboxylase Reaction in Maize Chloroplast Extracts

1981 ◽  
Vol 36 (7-8) ◽  
pp. 688-691 ◽  
Author(s):  
David Pan ◽  
Kim H. Tan
Keyword(s):  
Carbonyl Compound ◽  
Crude Extract ◽  
Pentose Phosphate ◽  
Assay System ◽  
Cooh Group ◽  
Sugar Phosphate ◽  
Considerable Evidence ◽  
Maize Chloroplast ◽  
Fructose 1,6 Bisphosphate

Abstract The authors have suggested (Can. J. Bot. 49, 631 (1971) that the — COOH group of the C4-P carbonyl compound of the thermostable P-enolpyruvate acid carboxylase reaction may transcarboxylase with a pentose phosphate as ac­ ceptor. We now have considerable evidence supporting this consideration. In an assay system (0.8 ml) containing soni­ cated chloroplast extract in 0.1 m Tris-HCl, pH 6.3; and PEP, 0.1 |imol; Mg2+, 0.5 nmol; NADH, 0.25 nmol; sugar phosphate, 0.5 nmol. The amount of 14C 0 2 fixation is con­ siderably enhanced by either ribose-5-phosphate, fructose-1,6-bisphosphate or ribulose-l,5-bisphosphate in the pres­ ence of PEP. The products of the reaction include malate as a product of /?-carboxylation, and glycerate or 3-phosphate glycerate, their proportion being determined by the ac­ ceptor sugar phosphate. The results provide evidence for a "transcarboxylase" presented in the crude extract of Maize chloroplasts.

scholarly journals Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Content Type ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Triosephosphate metabolism by mature boar spermatozoa

1997 ◽  
Vol 9 (6) ◽  
pp. 577 ◽  
Author(s):  
A. R. Jones
Keyword(s):  
Glycolytic Pathway ◽  
Dihydroxyacetone Phosphate ◽  
Glycolytic Intermediates ◽  
Boar Sperm ◽  
Equilibrium Concentrations ◽  
Fructose 1,6 Bisphosphate ◽  
Boar Spermatozoa

Boar sperm rapidly interconverted dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, produced fructose-1,6-bisphosphate, approximately equilibrium concentrations of fructose 6-phosphate and glucose 6-phosphate but not glycerol or glycerol 3-phosphate. In the presence of 3-chloro-1-hydroxypropanone, an inhibitor of stage 2 of the glycolytic pathway, the triosephosphates were metabolized faster, produced less fructose-1,6-bisphosphate, fructose 6-phosphate and glucose 6-phosphate, but not glycerol or glycerol 3-phosphate. This suggests that these cells may have the capacity to convert glycolytic intermediates into a storage metabolite to conserve carbon atoms for the eventual synthesis of lactate.

Molecular approaches to the manipulation of carbon allocation in plants

1993 ◽  
Vol 71 (6) ◽  
pp. 765-778 ◽  
Author(s):  
S. D. Blakeley ◽  
D. T. Dennis
Keyword(s):  
Amino Acids ◽  
Fatty Acids ◽  
Metabolic Pathways ◽  
Carbon Allocation ◽  
Genetic Manipulation ◽  
Biosynthetic Pathways ◽  
Molecular Approaches ◽  
Carbon Substrates ◽  
Storage Organs ◽  
Storage Products

In plants, sucrose is the end product of photosynthesis and is converted to a wide variety of storage compounds in tissues such as seeds and tubers. The allocation of carbon from sucrose to the various metabolic pathways leading to these products will determine the quantity of each synthesized in the respective storage organs. If the level of the enzymes involved in the allocation of carbon could be changed by genetic manipulation, it is probable that the relative yields of the various storage products can also be altered. The initial breakdown of sucrose occurs in the cytosol of the cell. Many biosynthetic pathways, however, including those involved in the synthesis of storage products such as fatty acids, starch, and amino acids, occur in the plastid. The distribution of carbon substrates for these processes will be determined, to a large extent, by the flux of carbon through the glycolytic pathways found in both the cytosolic and plastid compartments. This article will discuss the importance and consequences of compartmentation, review the extent of our understanding of glycolysis and other enzymes and pathways regulating carbon allocation, and will speculate on the potential for the genetic manipulation of these pathways. Key words: genetic manipulation, carbon allocation, metabolism, glycolysis.

Properties of Phosphoenolpyruvate Carboxykinase Operative in C4 Pathway Photosynthesis

1977 ◽  
Vol 4 (2) ◽  
pp. 207 ◽  
Author(s):  
MD Hatch ◽  
S Mau
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Reverse Direction ◽  
Nucleoside Triphosphate ◽  
Dihydroxyacetone Phosphate ◽  
C4 Pathway ◽  
Phosphoglyceric Acid ◽  
Michaelis Constants ◽  
Chloris Gayana ◽  
Fructose 1,6 Bisphosphate ◽  
Chloris Gayana Kunth

A procedure is described for partially purifying phosphoenolpyruvate carboxykinase [ATP : oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] from leaves of Chloris gayana Kunth. In three steps the enzyme was purified about 60-fold with 22% recovery of activity. This procedure removes enzymes, particularly malate dehydrogenase, that preclude the use of a simple spectrophotometric assay for phosphoenolpyruvate carboxykinase. The activity of the enzyme in the direction of oxaloacetate decarboxylation was about 10 times that in the reverse direction. At the optimal pH of 8.0, ATP was the preferred nucleoside triphosphate but CTP, UTP, GTP and ITP were also active. A requirement for Mn2+ could not be replaced by Mg2+. The Michaelis constants for oxaloacetate and ATP were 0.035 mM and 0.024 nM, respectively. The photosynthetic intermediates fructose 1,6-bisphosphate, 3-phosphoglyceric acid and dihydroxyacetone phosphate significantly inhibited the enzyme at concentrations in the region of 1-5 mM. Unlike the phosphoenolpyruvate carboxykinase from other sources, the capacity of the leaf enzyme to catalyse the decarboxylation of oxaloacetate to pyruvate was negligible. The properties of the enzyme are discussed in relation to its proposed role in C4 pathway photosynthesis.

scholarly journals Unleashing natural competence inLactococcus lactisby induction of the competence regulator ComX

2017 ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits ◽  
Derivatives Of

AbstractIn biotechnological work horses likeStreptococcus thermophilusandBacillus subtilisnatural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starterLactococcus lactisnatural competence has not been established to date. However,in silicoanalysis of complete genome sequences of 43L. lactisstrains revealed complete late-competence gene-sets in 2L. lactissubspeciescremorisstrains (KW2 and KW10) and 8L. lactissubspecieslactisstrains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulatorcomXinL. lactissubsp.lactisKF147 resulted in the induction of expression of the canonical competence regulon, and elicited a state of natural competence in this strain. By contrast,comXexpression inL. lactisNZ9000, predicted to encode an incomplete competence gene-set, failed to induce natural competence. Moreover, mutagenesis of thecomEA-ECoperon in strain KF147, abolished thecomXdriven natural competence, underpinning the involvement of the competence machinery. Finally, introduction of nisin-induciblecomXexpression intonisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence also in these strains, expanding this phenotype to otherL. lactisstrains of both subspecies.Significance statementSpecific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the speciesLactococcus lactisare very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end-product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria, and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

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