Abstract
Kluyveromyces marxianus ATCC 26,548 was cultivated in aerobic chemostats with [1-13C] and [U-13C] glucose as carbon source under three different growth conditions (0.10, 0.25, and 0.5 h−1) to evaluate metabolic fluxes. Carbon balances closed always within 97–102%. Growth was carbon limited, and the cell yield on glucose was the same. The extracellular side-product formation was very low, totaling 0.0008 C-mol C-mol−1 substrate at 0.5 h−1. The intracellular flux ratios did not show significant variation for metabolic flux analysis from labelling and biomass composition and metabolic flux ratio analysis from labelling. The observed strictly oxidative metabolism and the stability of the metabolism in terms of fluxes even at high growth rates, without triggering out the synthesis of by-products, is an extremely desired condition that underlines the potential of K. marxianus for biotechnological biomass-related applications and the comprehension of the metabolic pools and pathways is an important step to engineering this organism.
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Data availability
The data that support the findings of this study are available on Tables S4 and S5.
Abbreviations
- ACA:
-
Acetaldehyde
- AcCoA (orACCOA):
-
Acetyl-CoA
- AKG:
-
α-Ketoglutarate
- Ala:
-
Alanine
- Asp:
-
Aspartate
- C1 (orCO):
-
Carbon
- CER:
-
Carbon exchange rate
- cyt:
-
Cytosolic
- D:
-
Dilution rate
- DW:
-
Dry weight
- E4P:
-
Erythrose-4-phosphate
- ED:
-
Entner-Doudoroff
- EMP:
-
Embden–Meyerhoff–Parnas
- f :
-
Flux (least-squares solution)
- F6P:
-
Fructose-6-phosphate
- FlR:
-
Additional flux ratio
- FUM:
-
Fumarate
- G3P:
-
Glyceraldehyde-3-phosphate
- G6P:
-
Glucose-6-phosphate
- G6Pdh:
-
Glucose-6-phosphate dehydrogenase
- GC–MS:
-
Gas chromatography-mass spectrometry
- Glu:
-
Glutamate
- GLY (or Gly):
-
Glycine
- GLYC:
-
Glycerin
- His:
-
Histidine
- Ile:
-
Isoleucine
- l:
-
Labeled fraction
- Leu:
-
Leucine
- Lys:
-
Lysine
- m0 :
-
Lowest mass
- mn :
-
Highest mass
- m n :
-
Corrected intensity of the isotopomeric mass fraction with n labeled C-atoms
- mit:
-
Mitochondrial
- MDV:
-
Mass isotopomer distribution vector
- MDVunlabeled,n :
-
Mass distribution of an unlabeled fragment of length n
- MFA:
-
Metabolic flux analysis
- MFR:
-
Metabolic flux ratio
- NADPH:
-
Nicotinamide adenine dinucleotide phosphate (reduced form)
- NMR:
-
Nuclear magnetic resonance spectroscopy
- OAA:
-
Oxaloacetate
- OD600 :
-
Optical density at 600 nm
- OGA:
-
Oxoglutarate
- OUR:
-
Oxygen uptake rate
- OUT :
-
For biomass formation
- p:
-
Purity of the labeled substrate
- P5P:
-
Pentose-5-phosphate
- PEN15:
-
Pentose with carbons 1, 2, 3, 4 and 5
- PEN25:
-
Pentose with carbons 2, 3, 4, 5 and 6
- PEP:
-
Phosphoenolpyruvate
- PGI:
-
Phosphoglucoisomerase
- PGM:
-
Phosphoglucomutase
- Phe:
-
Phenylalanine
- PPP:
-
Pentose–phosphate (PP) pathway
- PYR:
-
Pyruvate
- qCO2 :
-
Specific rate of CO2 production
- qO2 :
-
Specific rate of O2 consumption
- RQ:
-
Quotient between the rates of CO2 formation and O2 consumption
- S:
-
Substrate concentration in the bioreactor
- SF :
-
Substrate concentration in the feeding medium
- S7P:
-
Sedoheptulose 7-phosphate
- SER (or Ser):
-
Serine
- SFL:
-
Summed fractional labeling
- TCA:
-
Tricarboxylic acids
- THR (or Thr):
-
Threonine
- Tyr:
-
Tyrosine
- U:
-
Uniformelly labelled
- v:
-
Metabolic flux
- Val:
-
Valine
- v.v.m:
-
Air volume culture volume−1 min−1
- X:
-
Biomass concentration in the bioreactor
- YX/S :
-
Biomass yield on glucose
- µ S :
-
Specific rate of substrate consumption
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Acknowledgements
The author thanks Thomas Grotkjær for kindly providing the software utilized for SFL-based metabolic flux analysis, Andreas Gombert, Christoph Wittmann and Elmar Heinzle for the support and lab facilities, and to Michael Fritz for his skilled technical assistance.
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Fonseca, G.G. Metabolic engineering of Kluyveromyces marxianus for biomass-based applications. 3 Biotech 12, 259 (2022). https://doi.org/10.1007/s13205-022-03324-x
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DOI: https://doi.org/10.1007/s13205-022-03324-x