A Combined Sensitivity and Metabolic Flux Analysis Unravel the Importance of Amino Acid Feeding Strategies in Clavulanic Acid Biosynthesis

Sánchez, Claudia; Rúa, Natalia Eugenia Gómez; Quintero, Juan Carlos; Ochoa, Silvia; Ríos, Rigoberto

doi:10.1007/978-3-319-01568-2_25

Cited by 5 publications

(9 citation statements)

References 6 publications

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“…On the other hand, the effect of the oxygen flux variation on the main intracellular fluxes is shown in Figure B. It is observed that the clavulanic acid flux ( v 84 ) reaches a very high value compared with the values reported experimentally . Furthermore, the values reached by the fluxes of the precursors v 36 , v 37 , v 39 are unrealistically high, and this may be the case for other values predicted by the model.…”

Section: Resultsmentioning

confidence: 79%

“…Clavulanic acid (CA) is a potent β‐lactamase inhibitor used to counteract the microbial resistance created by some pathogenic microorganisms capable of synthesizing β‐lactamase enzymes . Streptomyces clavuligerus (S. clavuligerus) can produce a large number of compounds, among which cephamycins and clavulanic acid are the most actively studied . However, despite the importance of those products for the pharmaceutical industry, there are still concerns about the economic feasibility in the clavulanic acid production process.…”

Section: Introductionmentioning

confidence: 99%

“…1 Streptomyces clavuligerus (S. clavuligerus) can produce a large number of compounds, among which cephamycins and clavulanic acid are the most actively studied. 2 However, despite the importance of those products for the pharmaceutical industry, there are still concerns about the economic feasibility in the clavulanic acid production process. In an effort to overcome these problems, genetic modifications have been performed to improve process productivity.…”

Section: Introductionmentioning

confidence: 99%

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Flux balance analysis in the production of clavulanic acid byStreptomyces clavuligerus

Sánchez

Quintero

Ochoa

2015

Biotechnol Progress

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In this work, in silico flux balance analysis is used for predicting the metabolic behavior of Streptomyces clavuligerus during clavulanic acid production. To choose the best objective function for use in the analysis, three different optimization problems are evaluated inside the flux balance analysis formulation: (i) maximization of the specific growth rate, (ii) maximization of the ATP yield, and (iii) maximization of clavulanic acid production. Maximization of ATP yield showed the best predictions for the cellular behavior. Therefore, flux balance analysis using ATP as objective function was used for analyzing different scenarios of nutrient limitations toward establishing the effect of limiting the carbon, nitrogen, phosphorous, and oxygen sources on the growth and clavulanic acid production rates. Obtained results showed that ammonia and phosphate limitations are the ones most strongly affecting clavulanic acid biosynthesis. Furthermore, it was possible to identify the ornithine flux from the urea cycle and the α-ketoglutarate flux from the TCA cycle as the most determinant internal fluxes for promoting clavulanic acid production.

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Section: Resultsmentioning

confidence: 79%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Flux balance analysis in the production of clavulanic acid byStreptomyces clavuligerus

Sánchez

Quintero

Ochoa

2015

Biotechnol Progress

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“…To solve the overdetermined system the least‐squares method was used . The matrix A is nonsingular and well posed, with a condition number of 31.24, which indicates a low propagation error. Furthermore, sensitivity analysis of the stoichiometric matrix indicates that only the fluxes of reactions 3, 4, 5, 6, 9 and 14 in Table are more sensitive to measurement errors in the specific rates of leucine, lysine and methionine.…”

Section: Gene Disruptionmentioning

confidence: 99%

An LDHa single allele CHO cell mutant exhibits altered metabolic state and enhanced culture performance

Wilkens

Vishwanathan

Baltes

et al. 2019

J of Chemical Tech & Biotech

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BACKGROUND Reducing lactate production in animal cell cultures has been reported to improve cell culture performance and productivity of recombinant protein. A novel genome editing tool, CRISPR/Cas, has been used widely to induce double‐strand breaks in the genome and introduce targeted mutations efficiently. RESULTS In the present work, we used a publicly available human‐codon optimized CRISPR/Cas system to introduce mutations in one of the LDHa gene copies to obtain an LDHa single‐allele knockout Chinese hamster ovary (CHO) cell clone to analyze its effect over cell metabolism. Fed‐batch cultures were conducted in order to evaluate the culture performance of mutant cells. Results show that cell growth was reduced and metabolism was modified by the LDHa single‐allele knockout, whereas specific protein rate and volumetric production were greatly enhanced. Additionally, the first in‐depth analysis of the metabolic effects of LDHa single allele knockout is presented. CONCLUSION By using CRISPR/Cas we were able to knock down LDHa, showing improved culture productivity. Metabolic flux analysis revealed higher fluxes in the TCA cycle in the mutant cell than in the parental cell, indicating that LDHa mutant cells have an enhanced energy metabolism, which can be attributed mainly to a higher catabolism of amino acids. © 2018 Society of Chemical Industry

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“…At the same time, it gives the possibility to control and determine the best stimulating substrates to intensify the production of certain compounds (Sánchez et al, 2014), potentially allowing the increase of productivity, or to capture changes in the flow of metabolites in genetically modified microorganisms (with an increased production of amino acids) (Sánchez et al, 2014).…”

mentioning

confidence: 99%

Metabolomics and fluxomics in biotechnology: current trends

Wojtowicz¹,

Młynarz²

2016

bta

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Metabolomics and fluxomics are two very rapidly developing fields of science. They provide a wide range of information on different biological systems and are the last link in the biology "omics" studies chain: genomics-transcriptomics-proteomics-metabolomics. Metabolomics belongs to systems biology and it refers to changes occurring in low molecular weight compounds (<1500 Da) and reveals significant information about the actual state of examined organisms in relation to a reference group. Metabolic flux analysis provides very important information about the flux metabolites in a pathway of a living organism based on 13 C enriched isotopically substrates. Both these sciences use very advanced mathematical approaches in order to gain the highest possible output originating from living systems. All features of both methods allow their use in with adopting of appropriate methodology, in a particular field of biotechnology, including medicine, pharmaceutical science and industry, food processing, toxicology, plant cultivation, and animal breeding.

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A Combined Sensitivity and Metabolic Flux Analysis Unravel the Importance of Amino Acid Feeding Strategies in Clavulanic Acid Biosynthesis

Cited by 5 publications

References 6 publications

Flux balance analysis in the production of clavulanic acid byStreptomyces clavuligerus

Flux balance analysis in the production of clavulanic acid byStreptomyces clavuligerus

An LDHa single allele CHO cell mutant exhibits altered metabolic state and enhanced culture performance

Metabolomics and fluxomics in biotechnology: current trends

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