13C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in Saccharopolyspora erythraea

Hong, Ming; Mou, Han; Liu, Xiaoyun; Huang, Mingzhi; Chu, Ju

doi:10.1007/s00449-017-1792-0

Cited by 20 publications

(11 citation statements)

References 27 publications

(33 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Synthesis of adequate precursor levels for the production of target compounds has been considered one of the crucial strategies to improve the productivity of cell factories in metabolic engineering. The importance of the pool sizes was underlined in several studies for the production of desired compounds [ Hong et al., 2017 ]. For example, acetyl-CoA is a precursor of a variety of biotechnology products, including FAs, 1-butanol, polyhydroxyalkanoates, polyketides, and isoprenoids, etc .…”

Section: Introductionmentioning

confidence: 99%

Engineering precursor pools for increasing production of odd-chain fatty acids in Yarrowia lipolytica

Park

Bordes

Létisse

et al. 2021

Metabolic Engineering Communications

View full text Add to dashboard Cite

Microbial production of lipids is one of the promising alternatives to fossil resources with increasing environmental and energy concern. Odd-chain fatty acids (OCFA), a type of unusual lipids, are recently gaining a lot of interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical, and chemical industries. In this study, we aimed to enhance the pool of precursors with three-carbon chain (propionyl-CoA) and five-carbon chain (β-ketovaleryl-CoA) for the production of OCFAs in Yarrowia lipolytica . We evaluated different propionate-activating enzymes and the overexpression of propionyl-CoA transferase gene from Ralstonia eutropha increased the accumulation of OCFAs by 3.8 times over control strain, indicating propionate activation is the limiting step of OCFAs synthesis. It was shown that acetate supplement was necessary to restore growth and to produce a higher OCFA contents in total lipids, suggesting the balance of the precursors between acetyl-CoA and propionyl-CoA is crucial for OCFA accumulation. To improve β-ketovaleryl-CoA pools for further increase of OCFA production, we co-expressed the bktB encoding β-ketothiolase in the producing strain, and the OCFA production was increased by 33% compared to control. Combining strain engineering and the optimization of the C/N ratio promoted the OCFA production up to 1.87 g/L representing 62% of total lipids, the highest recombinant OCFAs titer reported in yeast, up to date. This study provides a strong basis for the microbial production of OCFAs and its derivatives having high potentials in a wide range of applications.

show abstract

Section: Introductionmentioning

confidence: 99%

Engineering precursor pools for increasing production of odd-chain fatty acids in Yarrowia lipolytica

Park

Bordes

Létisse

et al. 2021

Metabolic Engineering Communications

View full text Add to dashboard Cite

show abstract

“…This was prepared by cultivating Pichia pastoris G/DSEL in 1 L turbine-stirred bioreactor (working volume 0.6 L) and was fed with fully U-13 C (20 g/L, 99%, Cambridge Isotope Laboratories, Inc.) labeled glucose as the sole carbon source. Cultivation took 20 h (Hong et al 2017;Lu et al 2015). Sampling, quenching, and extraction procedures were carried out as described by Carnicer et al (2012), although quenching solution was precooled and maintained at − 80 °C.…”

Section: Preparation Of Uniformly 13 C-labeled Cell Extractsmentioning

confidence: 99%

“…Electrospray ionization parameters were as follows: spray voltage 3000 V, sheath gas pressure 15 arbitrary units, aux gas pressure 10 arbitrary units, ion sweep gas pressure two arbitrary units, capillary temperature 270 °C, and vaporizer temperature 200 °C. Daughter ions, tube lens voltage, and collision energy were optimized individually for each of these compounds (Hong et al 2017).…”

Section: Metabolites Determinationmentioning

confidence: 99%

“…In addition, the partial conversion of pyruvate, nucleotides, and phosphate sugars was observed during extraction with boiling ethanol (Winder et al 2008). In bacteria, the fast filtration method was used and verified to be effective for minimizing the leakage of intracellular metabolites both in Gram-negative and Gram-positive bacteria (Hong et al 2017;Bolten et al 2007). This method can, therefore, be used as an alternative to cold methanol quenching.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Optimized sampling protocol for mass spectrometry-based metabolomics in Streptomyces

Liu

Wang

Sun

et al. 2019

Bioresour. Bioprocess.

Self Cite

View full text Add to dashboard Cite

In quantitative metabolomics studies, the most crucial step was arresting snapshots of all interesting metabolites. However, the procedure customized for Streptomyces was so rare that most studies consulted the procedure from other bacteria even yeast, leading to inaccurate and unreliable metabolomics analysis. In this study, a base solution (acetone: ethanol = 1:1, mol/mol) was added to a quenching solution to keep the integrity of the cell membrane. Based on the molar transition energy (ET) of the organic solvents, five solutions were used to carry out the quenching procedures. These were acetone, isoamylol, propanol, methanol, and 60% (v/v) methanol. To the best of our knowledge, this is the first report which has utilized a quenching solution with ET values. Three procedures were also adopted for extraction. These were boiling, freezing–thawing, and grinding ethanol. Following the analysis of the mass balance, amino acids, organic acids, phosphate sugars, and sugar alcohols were measured using gas chromatography with an isotope dilution mass spectrometry. It was found that using isoamylol with a base solution (5:1, v/v) as a quenching solution and that freezing–thawing in liquid nitrogen within 50% (v/v) methanol as an extracting procedure were the best pairing for the quantitative metabolomics of Streptomyces ZYJ-6, and resulted in average recoveries of close to 100%. The concentration of intracellular metabolites obtained from this new quenching solution was between two and ten times higher than that from 60% (v/v) methanol, which until now has been the most commonly used solution. Our findings are the first systematic quantitative metabolomics tools for Streptomyces ZYJ-6 and, therefore, will be important references for research in fields such as 13C based metabolic flux analysis, multi-omic research and genome-scale metabolic model establishment, as well as for other Streptomyces.

show abstract

“…They are important targets for subsequent strain design because the yield of product synthase may increase with a slowdown of cell growth (Pan and Qiang 2012). In order to verify the effect of the target gene, some targets which are included in the 89 partially essential genes have been validated by knockout experiments in our lab and on the other published papers, including SACE_5639 (Chen et al 2016;Weber et al 2012), SACE_0728 (Mironov et al 2004), SACE_0731 (Minas et al 1998), and SACE_6669 (Hong et al 2017). The knockout results can be found in Table 4.…”

Section: Essential Genes Target Prediction In Silico For Strain Designmentioning

confidence: 99%

In silico reconstruction and experimental validation of Saccharopolyspora erythraea genome-scale metabolic model iZZ1342 that accounts for 1685 ORFs

Zhuang

Huang

Chu

2018

Bioresour. Bioprocess.

Self Cite

View full text Add to dashboard Cite

Background: Saccharopolyspora erythraea (S. erythraea) is a Gram-positive erythromycin-producing filamentous bacterium. The lack of comprehensive S. erythraea genome-scale metabolic models (GEMs) hinders the efficiency of metabolic engineering as well as fermentation process optimization. Results: In this study, the GEMs model of S. erythraea iZZ1342 was reconstructed according to the latest genome annotations, omics databases, and literatures. Compared with the previous S. erythraea model-GSMR, the new model iZZ1342 presented great improvements both on scope and coverage in the number of reactions, metabolites, and annotated genes. In detail, the number of unique reactions in iZZ1342 was increased from 1482 to 1684, the number of metabolites was increased from 1546 to 1614, and the number of unique genes was increased from 1272 to 1342. We also added 1441 gene-protein-reaction associations in iZZ1342 which lacks in the previous model to overcome the limitation in the application of strain designing. Compared with the transcriptomics data obtained from the published literature, 86.3% ORFs and 92.9% reactions in iZZ1342 can be verified. The results of the sensitivity analysis showed the similar trend in the E. coli GEMs. The prediction of growth on available 27 kinds of carbon sources and 33 kinds of nitrogen sources showed the accuracy rate was 77.8 and 87.9%, respectively. Compared with the physiological data obtained from chemostat cultivation, the simulation results showed good consistency. The correlation coefficient between the 13 C-labeled experiment data and the flux simulation result was 0.97. All the above results showed that the iZZ1342 model has good performance. Furthermore, four genes are in the range of successful knockout by comparing these targets with the results which have been earlier published. Conclusion: The new model iZZ1342 improved significantly in model size and prediction performance, which will lay a good foundation to study the systematic metabolic engineering of S. erythraea system in vivo.

show abstract

13C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in Saccharopolyspora erythraea

Cited by 20 publications

References 27 publications

Engineering precursor pools for increasing production of odd-chain fatty acids in Yarrowia lipolytica

Engineering precursor pools for increasing production of odd-chain fatty acids in Yarrowia lipolytica

Optimized sampling protocol for mass spectrometry-based metabolomics in Streptomyces

In silico reconstruction and experimental validation of Saccharopolyspora erythraea genome-scale metabolic model iZZ1342 that accounts for 1685 ORFs

Contact Info

Product

Resources

About