The dynamic regulation of nitrogen and phosphorus in the early phase of fermentation improves the erythromycin production by recombinant Saccharopolyspora erythraea strain

Abstract

Background

Erythromycin production often has concern with the consumption rate of amino nitrogen and phosphate, especially in the early fermentation phase. The dynamic regulation of nitrogen and phosphorus was put forward based on the comprehensive analysis of the contents of phosphorus and nitrogen in different nitrogen sources as well as the relations between nitrogen consumption and phosphorus consumption.

Results

Firstly, the unstable nitrogen source, corn steep liquor, was substituted with the stable nitrogen source, yeast powder, with little effects on erythromycin production. Secondly, feeding phosphate in the early fermentation stage accelerated the consumption of amino nitrogen and ultimately increased erythromycin production by approximately 24% as compared with the control (without feeding potassium dihydrogen phosphate). Thirdly, feeding phosphate strategy successfully applied to 500 L fermenter with the final erythromycin concentration of 11839 U/mL, which was 17.3% higher than that of the control. Finally, the application of condensed soy protein (a cheap nitrogen source with low phosphorus content) combined with phosphate feed strategy led to a 13.0% increase of the erythromycin production as compared with the control (condensed soy protein, without feeding potassium dihydrogen phosphate).

Conclusions

Appropriately feeding phosphate combined with rational nitrogen regulation in the early fermentation phase was an effective way to improve erythromycin production.

Keywords

Erythromycin / Potassium dihydrogen phosphate / Feeding strategy / Yeast powder / Condensed soy protein

References

[1.] Mironov VA, Sergienko OV, Nastasyak IN, Danilenko VN. Biogenesis and regulation of biosynthesis of erythromycins in Saccharopolysplra erythraea. Appl Biochem Microbiol, 2004, 40: 613-624.

[2.] Castaldo RS, Celli BR, Gomez F, LaVallee N, Souhrada J, Hanrahan JP. A comparison of 5-day courses of dirithromycin and azithromycin in the treatment of acute exacerbations of chronic obstructive pulmonary disease. Clin Ther, 2003, 25: 542-557.

[3.] Weber JM, Cernota WH, Gonzalez MC, Leach BI, Reeves AR, Wesley RK. An erythromycin process improvement using the diethyl methylmalonate-responsive (Dmr) phenotype of the Saccharopolyspora erythraea mutB strain. Appl Microbiol Biotechnol, 2012, 93: 1575-1583.

[4.] Wu JQ, Zhang QL, Deng W, Qian JC, Zhang SL, Liu W. Toward improvement of erythromycin A production in an industrial Saccharopolyspora erythraea strain via facilitation of genetic manipulation with an artificial attB site for specific recombination. Appl Environ Microbiol, 2011, 77: 7508-7516.

[5.] Zou X, Hang HF, Chu J, Zhuang YP, Zhang SL. Enhancement of erythromycin A production with feeding available nitrogen sources in erythromycin biosynthesis phase. Bioresour Technol, 2009, 100: 3358-3365.

[6.] El-Enshasy HA, Mohamed NA, Farid MA, El-Diwany AI. Improvement of erythromycin production by Saccharopolyspora erythraea in molasses based medium through cultivation medium optimization. Bioresour Technol, 2008, 99: 4263-4268.

[7.] Zou X, Hang HF, Chu J, Zhuang YP, Zhang SL. Oxygen uptake rate optimization with nitrogen regulation for erythromycin production and scale-up from 50 L to 372 m³ scale. Bioresour Technol, 2009, 100: 1406-1412.

[8.] Gao Y, Yuan YJ. Comprehensive quality evaluation of corn steep liquor in 2-keto-L-gulonic acid fermentation. J Agric Food Chem, 2011, 59: 9845-9853.

[9.] Chu J, Li YR. Modern concepts of industrial fermentation, 2006, Bei Jing: Chemical Industry Press.

[10.] Zhang SL, Chu J, Zhuang YP. A multi-scale study of industrial fermentation processes and their optimization. Adv Biochem Eng Biotechnol, 2004, 87: 97-150.

[11.] Reeve LM, Baumberg S. Physiological controls of erythromycin production by Saccharopolyspora erythraea are exerted at least in part at the level of transcription. Biotechnol Lett, 1998, 20: 585-589.

[12.] Chen JF, Shao JW, Zhang YX, Chen H, Guo YH. Control of phosphate concentration on aminoglycoside antibiotic JI-20A fermentation. Microbiology, 2007, 34: 852-855.

[13.] Li XB, Zhao GR, Yuan YJ. A strategy of phosphorus feeding for repeated fed-batch fermentation of penicillin G. Biochem Eng, 2005, 27: 53-58.

[14.] Chen Y, Wang ZJ, Chu J, Zhuang YP, Zhang SL, Yu XG. Significant decrease of broth viscosity and glucose consumption in erythromycin fermentation by dynamic regulation of ammonium sulfate and phosphate. Bioresour Technol, 2013, 134: 173-179.

[15.] Chen Y, Deng W, Wu JQ, Qian JC, Chu J, Zhuang YP, Zhang SL, Liu W. Genetic modulation of the overexpression of tailoring genes eryK and eryG leading to the improvement of erythromycin A purity and production in Saccharopolyspora erythraea fermentation. Appl Environ Microbiol, 2008, 74: 1820-1828.

[16.] Zhang SL, Ye BC, Chu J, Zhuang YP, Guo MJ. From multi-scale methodology to systems biology: to integrate strain improvement and fermentation optimization. Chem Tech Biotechnol, 2006, 81: 734-745.

[17.] Beljkas B, Matic J, Milovanovic I, Jovanov P, Misan A, Saric L. Rapid method for determination of protein content in cereals and oilseeds: validation, measurement uncertainty and comparison with the Kjeldahl method. Accred Qual Assur, 2010, 15: 555-561.

[18.] Chen Y, Huang MZ, Wang ZJ, Chu J, Zhuang YP, Zhang SL. 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, 2013, 36: 1445-1453.

[19.] Yuan AQ, Tao PF, Zhao FY. Detection of phosphate in the solution of zincphosphate with molybdenum blue method. Anal Test Technol Instrum, 2004, 10: 251-253.

[20.] Zou X, Hang HF, Chen CF, Chu J, Zhuang YP, Zhang SL. Application of oxygen uptake rate and response surface methodology for erythromycin production by Saccharopolyspora erythraea. Ind Microbiol Biotechnol, 2008, 35: 1637-1642.

[21.] Friga GM, Borbely G, Farkas GL. Accumulation of guanosine tetraphosphate (ppGpp) under nitrogen starvation in Anacystis nidulans, a cyanobacterium. Arch Microbiol, 1981, 129: 341-343.

[22.] Akinyanju J, Smith RJ. Accumulation of ppGpp and pppGpp during nitrogen deprivation of the cyanophyte Anabaena cylindrica. FEBS Lett, 1979, 107: 173-176.

[23.] Ochi K. Metabolic initiation of differentiation and secondary metabolism by Streptomyces griseus: significance of the stringent response (ppGpp) and GTP content in relation to A factor. J Bacteriol, 1987, 169: 3608-3616.