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Controlling the feed rate of glucose and propanol for the enhancement of erythromycin production and exploration of propanol metabolism fate by quantitative metabolic flux analysis

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Abstract

In this paper, several different fermentation experiments were designed to address whether modulating glucose and propanol feeds could benefit the production level of erythromycin during pilot plant (30 L) fermentation. Results showed that glucose feed rate (determined by a set high or low culture pH) had no effect on erythromycin production, indicating that glucose was not the limiting factor for erythromycin biosynthesis under these conditions. It was found that decreasing glucose feed could stimulate the consumption of propanol, and the high erythromycin production (12.49 ± 0.50 mg ml−1) was achieved by controlling the feed rates of glucose and propanol. The quantitative metabolic flux analysis disclosed that high propanol consumption increased the pool size of propionyl-CoA (~2.147 mmol g−1 day−1) and methylmalonyl-CoA (~1.708 mmolg−1 day−1). It was also found that 45–77 % of the propanol went into the TCA cycle which strengthened the conclusion that blocking the propionate pathway to TCA cycle could lead to a significant increase in erythromycin production in carbohydrate-based media (Reeves et al. Ind Microbiol Biotechnol 7:600–609, 2006). In addition, the results also suggested that a relative low intracellular ATP level resulting from low glucose feed did not limit the erythromycin biosynthesis, and a relatively high NADPH should be beneficial for erythromycin biosynthesis.

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Acknowledgments

This work was financially supported by a grant from the Major State Basic Research Development Program of China (973 Program, No. 2012CB721006), National Natural Science Foundation of China (No. 21276081), the National Scientific and Technological Major Special Project (Significant Creation of New drugs, No. 2011ZX09203-001-03), and Research Fund for the Doctoral Program of Higher Education of China (No. 20110074110015).

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, P.O. box 329, 200237, Shanghai, People’s Republic of China

    Yong Chen, Mingzhi Huang, Zejian Wang, Ju Chu, Yingping Zhuang & Siliang Zhang

  2. Food Science and Engineering College, Qingdao Agriculture University, 266109, Qingdao, Shandong, People’s Republic of China

    Yong Chen

Authors
  1. Yong Chen
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  2. Mingzhi Huang
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  3. Zejian Wang
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  4. Ju Chu
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  5. Yingping Zhuang
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Corresponding authors

Correspondence to Mingzhi Huang or Ju Chu.

Appendix: Biochemical reactions in the metabolic model

Appendix: Biochemical reactions in the metabolic model

  1. (a)

    Uptake reactions

    1. 1.

      Glucose + ATP → glucose-6-phosphate

    2. 2.

      Palmitic acid + ATP + 8CoASH → 8acetyl-CoA + 7FADH + 7NADH + AMP + 2Pi

    3. 3.

      Stearic acid + ATP + 9CoASH → 9acetyl-CoA + 8FADH + 8NADH + AMP + Pi

    4. 4.

      Arachidic acid + ATP + 10CoASH → 10acetyl-CoA + 9FADH + 9NADH + AMP + Pi

    5. 5.

      Oleic acid + ATP + 9CoASH → 9acetyl-CoA + 7FADH + 8NADH + AMP + Pi

    6. 6.

      Linoleic acid + ATP + 9CoASH + NADPH → 9acetyl-CoA + 6FADH + 8NADH + AMP + 2Pi

    7. 7.

      Linolenic acid +ATP + 9CoASH + NADPH → 9acetyl-CoA + FADH + 8NADH + AMP + Pi

    8. 8.

      C117.17H218.37O15.86(soybean oil) → C3H8O3(glycerol) + C114.17H210.37O12.86(fatty acid)

    9. 9.

      Fatty acid + 6.4ATP + 50.1CoA + 3.9NADPH → 50.1acetyl-CoA + 40.9FADH + 50.7NADH + 6.4AMP

    10. 10.

      Glycerol → glycerate-3-phosphate + NADH

    11. 11.

      Propanol → Propionyl-CoA + 2NADH

  2. (b)

    Embden–Meyerhoff–Parnas pathway

    1. 1.

      Glucose-6-phosphate → Fructose-6-phosphate

    2. 2.

      Fructose-6-phosphate + ATP → 2glycerate-3-phosphate + ADP + Pi

    3. 3.

      Glycerate-3-phosphate → Pyruvate + ATP + NADH

  3. (c)

    TCA cycle

    1. 1.

      Pyruvate + CoASH → acetyl-CoA + ATP + NADH + CO2

    2. 2.

      Acetyl-CoA + oxaloacetic acid → citrate

    3. 3.

      Citrate → α-ketoglutaric acid + NADH + CO2

    4. 4.

      α-Ketoglutaric acid + CoASH → succinyl-CoA + NADH + CO2

    5. 5.

      Succinyl-CoA + ADP → succinate + ATP + NADH

    6. 6.

      Succinyl-CoA → oxaloacetic acid

  4. (d)

    Pentose–phosphate cycle

    1. 1.

      Glucose-6-phosphate → Ribulose-5-phosphate + 2NADPH + CO2

    2. 2.

      3Ribulose-5-phosphate → 2Fructose-6-phosphate + glycerate-3-phosphate

  5. (e)

    Mitochondrial reactions

    1. 1.

      Pyruvate + ATP + CO2 → oxaloacetic acid

    2. 2.

      Pyruvate → organic acids

    3. 3.

      NADH + 0.5O2 → 2.5ATP

    4. 4.

      FADH + 0.5O2 → 1.5ATP

  6. (f)

    Erythromycin pathway

    1. 1.

      Succinyl-CoA → methylmalonyl-CoA

    2. 2.

      Propionyl-CoA + ATP + CO2 → methylmalonyl-CoA

    3. 3.

      Propionyl-CoA + oxaloacetic acid → methylmalonyl-CoA + Pyruvate

    4. 4.

      Propionyl-CoA + 6methylmalonyl-CoA + 2glucose + 9NADPH → erythromycin + 2NADH + 6CO2

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Chen, Y., Huang, M., Wang, Z. et al. Controlling the feed rate of glucose and propanol for the enhancement of erythromycin production and exploration of propanol metabolism fate by quantitative metabolic flux analysis. Bioprocess Biosyst Eng 36, 1445–1453 (2013). https://doi.org/10.1007/s00449-013-0883-9

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  • DOI: https://doi.org/10.1007/s00449-013-0883-9

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