Suppressed acid formation by cofeeding of glucose and citrate in Bacillus cultures: emergence of pyruvate kinase as a potential metabolic engineering site

1995 ◽  
Vol 11 (4) ◽  
pp. 380-385 ◽  
Author(s):  
Akshay Goel ◽  
Jinwon Lee ◽  
Michael M. Domach ◽  
Mohammad M. Ataai
Keyword(s):  
Metabolic Engineering ◽  
Pyruvate Kinase ◽  
Acid Formation

13C NMR Evidence for Pyruvate Kinase Flux Attenuation Underlying Suppressed Acid Formation in Bacillus subtilis

2000 ◽  
Vol 16 (2) ◽  
pp. 169-175 ◽  
Author(s):  
C. Phalakornkule ◽  
B. Fry ◽  
T. Zhu ◽  
R. Kopesel ◽  
M.M. Ataai ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pyruvate Kinase ◽  
13C Nmr ◽  
Acid Formation

scholarly journals Inhibition of Pyruvate Kinase From Thermoanaerobacterium saccharolyticum by IMP Is Independent of the Extra-C Domain

2021 ◽  
Vol 12 ◽  
Author(s):  
Christopher A. Fenton ◽  
Qingling Tang ◽  
Daniel G. Olson ◽  
Marybeth I. Maloney ◽  
Jeffrey L. Bose ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Ethanol Production ◽  
Pyruvate Kinase ◽  
Surprising Result ◽  
Sugar Phosphate ◽  
Allosteric Inhibitors ◽  
Truncated Protein ◽  
First Report ◽  
Thermoanaerobacterium Saccharolyticum

The pyruvate kinase (PYK) isozyme from Thermoanaerobacterium saccharolyticum (TsPYK) has previously been used in metabolic engineering for improved ethanol production. This isozyme belongs to a subclass of PYK isozymes that include an extra C-domain. Like other isozymes that include this extra C-domain, we found that TsPYK is activated by AMP and ribose-5-phosphate (R5P). Our use of sugar-phosphate analogs generated a surprising result in that IMP and GMP are allosteric inhibitors (rather than activators) of TsPYK. We believe this to be the first report of any PYK isozyme being inhibited by IMP and GMP. A truncated protein that lacks the extra C-domain is also inhibited by IMP. A screen of several other bacterial PYK enzymes (include several that have the extra-C domain) indicates that the inhibition by IMP is specific to only a subset of those isozymes.

scholarly journals Metabolic Engineering of Acinetobacter baylyi ADP1 for Improved Growth on Gluconate and Glucose

2014 ◽  
Vol 80 (22) ◽  
pp. 7021-7027 ◽  
Author(s):  
Matti Kannisto ◽  
Tommi Aho ◽  
Matti Karp ◽  
Ville Santala
Keyword(s):  
Growth Rate ◽  
Metabolic Engineering ◽  
Carbon Source ◽  
Pyruvate Kinase ◽  
Wax Ester ◽  
Similar Amount ◽  
Acinetobacter Baylyi ◽  
Kinase Gene ◽  
Content Type ◽  
Storage Lipids

ABSTRACTA high growth rate in bacterial cultures is usually achieved by optimizing growth conditions, but metabolism of the bacterium limits the maximal growth rate attainable on the carbon source used. This limitation can be circumvented by engineering the metabolism of the bacterium.Acinetobacter baylyihas become a model organism for studies of bacterial metabolism and metabolic engineering due to its wide substrate spectrum and easy-to-engineer genome. It produces naturally storage lipids, such as wax esters, and has a unique gluconate catabolism as it lacks a gene for pyruvate kinase. We engineered the central metabolism ofA. baylyiADP1 more favorable for gluconate catabolism by expressing the pyruvate kinase gene (pykF) ofEscherichia coli. This modification increased growth rate when cultivated on gluconate or glucose as a sole carbon source in a batch cultivation. The engineered cells reached stationary phase on these carbon sources approximately twice as fast as control cells carrying an empty plasmid and produced similar amount of biomass. Furthermore, when grown on either gluconate or glucose,pykFexpression did not lead to significant accumulation of overflow metabolites and consumption of the substrate remained unaltered. Increased growth rate on glucose was not accompanied with decreased wax ester production, and thepykF-expressing cells accumulated significantly more of these storage lipids with respect to cultivation time.

scholarly journals Characterization of Growth and Acid Formation in a Bacillus subtilis Pyruvate Kinase Mutant

2000 ◽  
Vol 66 (9) ◽  
pp. 4045-4049 ◽  
Author(s):  
B. Fry ◽  
T. Zhu ◽  
M. M. Domach ◽  
R. R. Koepsel ◽  
C. Phalakornkule ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Pyruvate Kinase ◽  
Acid Production ◽  
Metabolic Flux ◽  
Substantial Reduction ◽  
Acid Formation ◽  
High Concentration ◽  
Activity Measurements ◽  
High Concentrations

ABSTRACT Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pykmutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of thepyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO2 production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pykmutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Mg++ - and Ca++-Induced Platelet Aggregation and ADP

1970 ◽  
Vol 24 (03/04) ◽  
pp. 432-437 ◽  
Author(s):  
S Cronberg ◽  
J. P Caen
Keyword(s):  
Platelet Aggregation ◽  
Pyruvate Kinase ◽  
Pyruvic Acid ◽  
Active Principle ◽  
Platelet Suspension ◽  
Edta Plasma ◽  
Phosphoenol Pyruvic Acid

SummaryReports on platelet aggregation after addition of calcium or magnesium to EDTA- PRP or platelet suspensions were confirmed. An aggregating principle was found in the EDTA-plasma and the supernatant of the platelet suspensions. Aggregation by magnesium in a platelet suspension was inhibited by adenosine and phosphoenol- pyruvic acid and pyruvate kinase, which suggested that the active principle was identical with ADP. Degradation of ADP in EDTA plasma was blocked.It thus appears that aggregation induced by calcium or magnesium in EDTA-PRP and platelet suspension was due to accumulation of spontaneously liberated ADP, which was not degraded.

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