The dynamic regulation of nitrogen and phosphorus in the early phase of fermentation improves the erythromycin production by recombinant Saccharopolyspora erythraea strain
© Zhang et al.; licensee Springer. 2014
Received: 31 March 2014
Accepted: 14 August 2014
Published: 12 September 2014
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.
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).
Appropriately feeding phosphate combined with rational nitrogen regulation in the early fermentation phase was an effective way to improve erythromycin production.
Erythromycin is a kind of polyketide antibiotic produced by Saccharopolyspora erythraea through industrial fermentation. Recently, erythromycin has received much attention because its semi-synthetically modified derivatives, such as azithromycin, roxithromycin, and clarithromycin, are widely used in the treatment of infectious diseases ,. The high requirement of erythromycin market stimulates the research on the improvement of the production and productivity of erythromycin. It has been reported that the genetic engineering technology, such as Saccharopolyspora erythraea mutB knockout and artificial attB site, have been applied to improve erythromycin production ,. The variation of the medium composition and fermentation conditions also successfully improved the erythromycin productivity ,. Zou et al. reported that feeding corn steep liquor could regulate oxygen uptake rate (OUR) at a suitable level in the early phase of fermentation, elevate the intracellular levels of lactic acid, pyruvic acid, citric acid, and propionic acid, and ultimately enhance the metabolic flux of erythromycin biosynthesis . It is well known that the quality of corn steep liquor is unstable due to its production process and the difficulty to be stored . This led to the severe fluctuation of erythromycin production on industrial scale. In this study, yeast powder, a stable nitrogen source, was used to replace corn steep liquor, so that the erythromycin production could be maintained at a stable level. Our data showed that the erythromycin production was little affected by the substitution of corn steep liquor with yeast powder. Furthermore, it was found that phosphorus content limited the consumption rate of amino nitrogen.
Compared with organic phosphorus, the concentration of inorganic phosphorus can be controlled quantitatively. In most cases, phosphorus is used as a crucial growth-limiting nutrient in antibiotic fermentations. The concentration of soluble phosphate generally fell in the range of 0.0003 to 0.3 mol/L. When the concentration of soluble phosphate was higher than 0.01 mol/L, the biosynthesis of many antibiotics would be suppressed -. It was reported that the initial phosphate concentration of 0.0061 to 0.0096 mol/L was beneficial for the cell growth in the aminoglycoside antibiotic JI-20A fermentation  and feeding phosphorus could provide optimal conditions for penicillin G biosynthesis . In our previous work, it was found that erythromycin A production was increased by 8.7% through the rational regulations of phosphate salt, soybean meal, and ammonium sulfate . However, few studies, to the best of our knowledge, focused on the effects of initial phosphorus and feeding phosphorus in the early stage of fermentation on the erythromycin fermentation. Hence, the effects of adding and feeding potassium dihydrogen phosphate in the early fermentation phase on the erythromycin fermentation were investigated.
Microorganism and culture conditions
The recombinant erythromycin-producing strains Saccharopolyspora erythraea ZL1004 is preserved in our laboratory .
Preliminary seed medium contains (g/L) the following: starch 30, dextrin 40, soybean meal 20, CaCO3 5, NaCl 3, and (NH4)2SO4 2.
Secondary seed medium contains (g/L) the following: starch 30, dextrin 30, soybean meal 30, corn steep liquor 10, CaCO3 5, and antifoam agent 1.25 mL.
In the fermentation in 50-L fermenter, preliminary seed culture was transferred into a 15-L fermenter containing 8 L of the secondary seed medium and cultivated at 34°C for 41 h. Then, the secondary seed culture was transferred into a 50-L fermenter containing 30 L of fermentation medium and cultivated at 34°C for 190 h. The fermenter was an agitated bioreactor equipped with three turbine impellers and devices capable of monitoring more than 14 online measurable parameters (FUS-50, Shanghai Guoqiang Bioengineering Equipment Co., Ltd., Shanghai, China) ,. DO (dissolved oxygen) was monitored with polarographic DO electrode (Mettler-Toledo, Greifensee, Switzerland) and controlled in 40-60% of air saturation by adjusting agitation and aeration during fermentation.
The substitution of corn steep liquor with yeast powder
The original fermentation medium (g/L) consisted of starch 35, dextrin 5, soybean meal 30, corn steep liquor 18, ammonium sulfate 3.0, NaCl 2.0, CaCO3 7.0, and antifoam agent 1.9 mL. For the experiment group, corn steep liquor was replaced by 10 g/L yeast powder. The nitrogen content of 18 g/L corn steep liquor (0.734 g/L) was equal to that of 10 g/L yeast powder (0.736 g/L) based on their total nitrogen amount detected by the Kjeldahl method .
Adding and feeding potassium dihydrogen phosphate in the early stage of fermentation.
Strategy 1: adding potassium dihydrogen phosphate to the initial medium.
Potassium dihydrogen phosphate was added before the sterilization of medium. The concentrations of potassium dihydrogen phosphate in the medium were set at 0.08 and 0.12 g/L, respectively, which were determined based on the difference of soluble phosphate concentration between yeast powder medium (0.07 g/L) and corn steep liquor medium (0.14 g/L).
Strategy 2: Feeding potassium dihydrogen phosphate in the early stage of fermentation
The levels of soluble phosphate and amino nitrogen were chosen as the indicator for switching on and switching off phosphate feeding. When soluble phosphate concentration was about 0.04 g/L, phosphate feed began. When amino nitrogen concentration was near 0.18 g/L, phosphate feed was terminated. The solution of potassium dihydrogen phosphate (10 g/L) was fed at the rate of 10 g/h in mode 3 and 20 g/h in mode 4, respectively.
The scale-up of phosphate feed on 500-L fermenter scale
Preliminary seed medium and secondary seed medium were the same to the media used for 50 L fermentation. The fermentation medium was the yeast powder medium. Preliminary seed culture was transferred into a 100-L fermenter containing 60 L of the secondary seed medium and cultivated at 34°C for 41 h. Then the secondary seed culture was transferred into a 500-L fermenter containing 300 L of fermentation medium and cultivated at 34°C for 190 h (Shanghai Guoqiang Bioengineering Equipment Co., Ltd., Shanghai, China). The solution of potassium dihydrogen phosphate (10 g/L) was fed at a rate of 100 g/h when soluble phosphate concentration fell to about 0.055 g/L and ceased to feed when amino nitrogen concentration was close to 0.18 g/L.
The fermentation with condensed soy protein as nitrogen source
The condensed soy protein medium (g/L) consisted of starch 35, dextrin 5, soybean meal 30, condensed soy protein 7, ammonium sulfate 3.0, NaCl 2.0, CaCO3 7.0, and antifoam agent 1.9 mL. The phosphate feed strategy was the same to that of mode 4.
Fermentation broth was centrifuged at 4,000 rpm for 10 min and PMV (packed mycelium volume) was the percentage of the precipitation (v/v). The concentration of amino nitrogen was detected by formol titration method . Total sugar concentration was assayed by Fehling method after acid hydrolysis . Soluble phosphate level and total phosphorus content were measured with molybdenum blue method .
The erythromycin titer was measured by the modified colorimetric method . The fermentation supernatant was collected after centrifugation and poured into the equal volume of K2CO3 solution (0.35%). Then, this solution was extracted with the equal volume of butyl acetate. Extracted erythromycin (oil phase) was mixed with the 0.1 mol/L hydrochloric acid. The aqueous phase fraction was separated with great care and further mixed with sulfate acid (18 mol/L) for 3 min. Then, the absorbance of this mixed solution was measured at 498 nm with a spectrophotometer. The standard curve of erythromycin titer was obtained using erythromycin A standard sample (97% purity). The erythromycin titer of fermentation broth was calculated according to the standard curve (y = 241.97x + 15.71, R2 = 0.998).
Results and discussion
The erythromycin fermentation with yeast powder as the easily metabolized nitrogen source
Owing to the instability of corn steep liquor, the substitution of corn steep liquor with yeast powder was investigated. The results showed that the erythromycin production of yeast powder medium was reached to 9,746 U/mL at 192 h, which was similar to that with corn steep liquor medium (9,444 U/mL). This result indicated that yeast powder was suitable to replace corn steep liquor for the erythromycin production.
The effects of the addition of potassium dihydrogen phosphate on erythromycin fermentation
The effects of feeding potassium dihydrogen phosphate on the fermentation process
The erythromycin production under mode 3 (12,077 U/mL) and mode 4 (11,734 U/mL) were increased by 24.0% and 20.4%, respectively, as compared with that of the control. These data showed that feeding inorganic phosphate to improve the consumption rate of amino nitrogen was an effective way to improve erythromycin production. In our further study, feeding strategy was tested on 500-L fermenter scale.
Feeding potassium dihydrogen phosphate on 500-L fermenter scale
The erythromycin fermentation with condensed soy protein as easily metabolized nitrogen source
In this paper, a stable nitrogen source, yeast powder was introduced into erythromycin industrial fermentation medium in the place of the unstable nitrogen source, corn steep liquor. Considering the relatively low phosphorus content of yeast powder, a strategy of adding and feeding potassium dihydrogen phosphate was tested and the results showed that phosphate could enhance the cell growth in the early phase of fermentation and ultimately improved the erythromycin production. This strategy of feeding phosphate was successfully applied to 500-L fermenter. The further fermentation with a low-phosphorus-content nitrogen source (condensed soy protein) supported that phosphate feed combined with rational nitrogen regulation was an effective way to improve erythromycin production.
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), 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).
- 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. 10.1023/B:ABIM.0000046985.66328.7aView ArticleGoogle Scholar
- 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. 10.1016/S0149-2918(03)80095-4View ArticleGoogle Scholar
- 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. 10.1007/s00253-011-3650-3View ArticleGoogle Scholar
- 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. 10.1128/AEM.06034-11View ArticleGoogle Scholar
- 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. 10.1016/j.biortech.2009.01.064View ArticleGoogle Scholar
- 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. 10.1016/j.biortech.2007.08.050View ArticleGoogle Scholar
- 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 m 3 scale. Bioresour Technol 2009, 100: 1406–1412. 10.1016/j.biortech.2008.09.017View ArticleGoogle Scholar
- 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. 10.1021/jf201792uView ArticleGoogle Scholar
- Chu J, Li YR: Modern concepts of industrial fermentation. Chemical Industry Press, Bei Jing; 2006.Google Scholar
- 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.Google Scholar
- 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. 10.1023/A:1005357930000View ArticleGoogle Scholar
- 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.Google Scholar
- 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. 10.1016/j.bej.2005.06.008View ArticleGoogle Scholar
- 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. 10.1016/j.biortech.2013.02.023View ArticleGoogle Scholar
- 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. 10.1128/AEM.02770-07View ArticleGoogle Scholar
- 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. 10.1002/jctb.1440View ArticleGoogle Scholar
- 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. 10.1007/s00769-010-0677-6View ArticleGoogle Scholar
- 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. 10.1007/s00449-013-0883-9View ArticleGoogle Scholar
- 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.Google Scholar
- 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. 10.1007/s10295-008-0407-9View ArticleGoogle Scholar
- 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. 10.1007/BF00406458View ArticleGoogle Scholar
- Akinyanju J, Smith RJ: Accumulation of ppGpp and pppGpp during nitrogen deprivation of the cyanophyte Anabaena cylindrica . FEBS Lett 1979, 107: 173–176. 10.1016/0014-5793(79)80489-5View ArticleGoogle Scholar
- 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.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.