IMPROVEMENT IN AUREOFUSCIN PRODUCTION BY Streptomyces aureofuscus WITH THE ADDITION OF ACETATE AND PROPIONATE SODIUM MELHORIA NA PRODUÇÃO AUREOFUSCIN POR Streptomyces aureofaciens

Aureofuscin is an important tetraene macrolides antibiotic produced in submerged culture by Streptomyces aureofuscus isolated from the soil in China. In the present work, the effects of the addition of precursor on cell growth and the kinetics of aureofuscin production were investigated during submerged cultivation of Str. aureofuscus. The sodium acetate and sodium propionate are more suitable precursor than alcohol, sodium butyrate, n-propanol, nbutanol. The addition of acetate and propionic sodium at a ratio of 5:1 at a total concentration of 1.5mg mL after 24 h showed stimulatory effects on aureofuscin production, reaching 3.708 mg mL (approximately 267% increases in aureofuscin production, compared with the control culture). Moreover , A further enhancement in aureofuscin production was achieved by cultivation in a 5-L stirred-tank bioreactor under controlled pH conditions under the above optimum condition. The optimal fermentation conditions were that 4L/min ventilatory capacity, 220r/min rotational speed, dissolves oxygen not be lower than 20%, fermentation period 84h and pH should control nearby 5.5, and the maximum yield of aureofuscin of 3.99 mg mL was achieved after 84 h When the sodium acetate and sodium propionate were added as a mixture of acetate and propionate at a ratio of 5:1 and a final concentration of 1.5 mg mL after 24 h cultivation.


INTRODUCTION
Aureofuscin is an important antifungal antibiotic produced by Streptomyces aureofuscus isolated from the soil in China.Its structure is similar to natamycin (natamycin = pimaricin).Both are tetraene macrolides antibiotic.Figure1 shows the Chemic structure of Aureofuscin and Natamycin.However, aureofuscin has more antibacterial activity than natamycin against Saccharomyces cerevisiae, Aspergillus niger, Penicillium chrysogenum, and Fusarium.Aureofuscin has the chemical formula C 25 H 37 NO 10 , a molecular mass of 511 (IPRS 1975).Aureofuscin belongs to the polyene antibiotic group and the biosynthesis of the aureofuscin basic chain follows the biosynthesis of fatty acids, which is composed of the decarboxylative condensation of simple carboxylic acids (APARICIO et al 2004).Therefore, the addition of low-molecular weight precursors such as carboxylic acids and amino acids was considered to be an inexpensive, scalable and easy approach to increase the production of different antibiotics, such as in the case of rapamycin (CHENG YR et al 1995), tylosin (NGUYEN et al 1995), pneumocandin (Petersen et al 2001), rampolanin (BRUNATI et al 2005), glycopeptide antibioticA40926 (JOVETIC et al 2008), bitespiramycin (LI ZL et al 2009), and natamycin (ELSAYED et al 2013).To our knowledge, natamycin has been extensively used as a food preservative.However, aureofuscin has not been well researched or industrially developed because of its low yields from fermentation since 1975.Therefore, if the yield of aureofuscin could be enhanced, it could be a promising antifungal agent to be used industrially as a food preservative.
In this study, we attempt to increase the production of aureofuscin by adding precursors.We investigated the effect of concentration and adding time of sodium acetate, sodium propionate, sodium butyrate, alcohol, n-propanol on aureofuscin production.After full optimization of the precursor addition strategy, the optimal fermentation condition (stir speed, the kinetics of cell growth and aureofuscin production) was investigated to increase the production of aureofuscin.

Microorganisms and Cultivation conditions
Str.aureofuscus was obtained from the strain preservation center in Shenyang Agricultural University (strain numbers SYAU0709).

Sample preparation and dry cell weight determination
Sample was 10 mL fermentation broth in the case of bioreactor cultures, it was in the form of flask.All samples were taken at 72h during cultivation and were centrifuged at 4000 rpm for 20 min.The supernatant was frozen at −20•C for antibiotic determination, and the remaining centrifuged cells were washed twice using distilled water, followed by centrifugation.The centrifuge tubes were then dried to a constant weight at 80•C to determine the dry cell weight.(JIE WEI et al 2008) For the detection of aureofuscin, the maximum absorption wavelength was measured from 200nm to 400 nm with an ultraviolet spectrophotometer.To analyze aureofuscin, a 1 ml portion of the culture broth was extracted by adding 9 ml of methanol, shaking vigorously, centrifuging at high speed (9200g,10000 r/min, 10 min), and saving the supernatant.

Aureofuscin bioassay and HPLC analysis
High Performance Liquid Chromatography (HPLC) analysis was performed using a Waters C18 column as the stationary phase and methanol-waterglacial acetic acid (48:32:1, v/v) as the mobile phase.The flow rate was 1.0 ml/min, UV wavelengh at 302.5 nm.A known, pure sample of aureofuscin was used as the internal standard.

Effect of the different addition on aureofuscin production
In this experiment, six parallel cultivations were conducted to investigate the effect of the different addition on aureofuscin production.The sodium acetate, sodium propionate, sodium butyrate, alcohol, n-propanol, n-butanol were added separately at the beginning of the cultivation time at concentrations 0.5mg mL −1 .Figure2 indicates that aureofuscin production increased upon supplementation of the fermentation medium with sodium acetate or sodium propionate.

Figure 2. Effect of different addition on aureofuscin production
The maximal aureofuscin production of 2.133 mg mL −1 was achieved in sodium propionate supplemented culture.The addition of acetate to the fermentation medium also showed a stimulatory effect on aureofuscin production.The maximal aureofuscin production of 2.065 mg mL −1 was obtained.On the other hand, the stimulatory effect of butyrate and alcohol on volumetric aureofuscin production was much lower compared with acetate and propionate.The specific aureofuscin production was lower for n-propanol and n-butanol than the control.Based on these results, the addition of sodium acetate or sodium propionate to the fermentation medium at a low concentration of 0.5mg mL −1 was chosen as the best precursors for aureofuscin production in the subsequent experiments.

Effect of the addition concentrations on aureofuscin production
To better understand the stimulatory effect of different carboxylic acids on aureofuscin production, the antibiotic-specific production yields are reported in Figure 3.As shown, the yield of aureofuscin production increased gradually by increasing either sodium acetate or sodium propionate concentrations in the culture from 0.5 to 3 mg mL −1 and decreased thereafter.The increase in aureofuscin production in acetate-and propionatesupplemented cultures was not related to the change in the biomass but to the increased cell productivity.The maximal values of aureofuscin prouction of 3.125 mg mL −1 and 3.247 mg mL −1 in cultures supplemented with 1.5 mg mL −1 sodium acetate and sodium propionate, respectively, were higher than those reported in previously published work using the same strain in batch culture under optimal cultivation conditions in medium without carboxylic acid addition(2.94mg mL −1 and 2.897 mg mL −1 )  Experiments were conducted to investigate the effect of concomitant addition of acetate and propionate at different ratios (sodium acetate: sodium propionate ratios ranging from 1:9 to 9:1).The concentration of total acetate and propionate supplemented for all ratios used was 1.5 mg mL −1 .As shown in Figure4, cell growth decreased gradually when the acetate ratio in the mixture was increased from 1:9 to 9:1.On the other hand, the effectiveness of acetate and propionate supplementation on aureofuscin production was more pronounced at all applied ratios when a mixture was used compared with single acetate and propionate experiments.The maximal aureo fuscin production of 3.575 mg mL −1 was achieved in culture supplemented with sodium acetate: sodium propionate at a ratio of 5:1.This value was higher than the control culture by approximately 157% and was also higher than in cultures individually supplemented with acetate or propionate by approximately 14% and 9.9%, respectively.
The observed stimulatory effects of the addition of a carboxylic acid mixture of acetic and propionic acids at a ratio of 7:1 were also reported in the previous study of Elsayed Ahmed et al.,in which acetate-propionate increased natamycin production in a submerged culture of Str.natalensis by approximately 150% (3.98 g L −1 )when added to the production medium at a concentration of 2 g L −1 .(Both acetate-propionate addition were added simultaneously as a mixture to a final concentration of 1.5 mg mL −1 at the beginning of the cultivation time before inoculation.)

Effect of the addition time of acetate and propionate on aureofuscin production
In addition to the effect of the ratio of acetate: propionate ratios on aureofuscin production, the time of addition also played a significant role in this enhancement effect.The maximal yield of aureofuscin production was achieved when sodium acetate and sodium propionate (ratio 5:1) were added at the beginning or during the first 24 h of cultivation.If sodium acetate and sodium propionate were added after 72 h (during the late exponential growth phase-early stationary phase), the positive effect of the addition of acetate and propionate on aureofuscin biosynthesis was almost negligible (Figure 5).The maximal aureofuscin production of 3.708 mg mL −1 was achieved when the acetatepropionate mixture was added after 12h.However, cell growth was slightly influenced by the addition of sodium acetate-propionate.It was clearly observed that the earlier the addition time, the higher the negative effect on biomass production.Once cells entered the stationary phase (after 72 h), no effect of acetate-propionate mixture on biomass was observed.The results also showed that to achieve the stimulatory effect of acetate-propionate mixture, they should be added to the cultivation medium during the first 24h of cultivation.This maybe because the aureofuscin biosynthesis in this strain started during the exponential growth phase parallel to cell growth.This was attributed to the fact that propionic acid needs to be converted into propionyl-CoA to act as a precursor for polyene ring synthesis.The enzyme system controlling this step is more active during the exponential growth phase (JING K et al 2011;BIBB MJ 2005;GIL;CAMPELO DIEZ 2003;MARTIN JF;MCDANIEL LE 1976).

Aureofuscin production during batch cultivation in a 5-Lstirred-tank bioreactor with and without sodium acetate-propionate addition
Based on the above studies, cultivations were transferred to bioreactor level to investigate the possibility of industrialization of this process.Medium composition and cultivation conditions were the same as in shake flasks.The pH of the culture was controlled at 7.2 at the beginning by the addition of NaOH to the culture medium.Figure 6 shows cell growth, aureofuscin production, pH value, dissolved oxygen (DO) and reducing sugar (%) in culture medium in a 5-L bioreactor.During cultivation without sodium acetate-propionate addition, the maximal cell dry weight (CDW) was 32 mg mL −1 .This value was higher than the biomass obtained in sodium acetate-propionate supplemented culture by approximately 45%.(Closed and open symbols correspond to results obtained in cultivations without and with sodium acetate-propionate addition, respectively.The sodium acetate and sodium propionate were added as a mixture of acetate and propionate at a ratio of 5:1 and a final concentration of 1.5 mg mL −1 after 24 h cultivation.).
The maximal value of aureofuscin in acetate-propionate supplemented culture was 3.99 mg mL −1 after 84 h (approximately 183% higher than the control culture).The increase in aureofuscin production was directly related to higher cell productivity in the sodium acetatepropionate supplemented culture.However, the changes in the pH in both cultures were similar.The pH value decreased gradually from 7.2 to approximately 5.2 during the first 48h of cultivation time and increased again gradually to neutral pH after 120 h.During the growth phase, the pH drop as a result of excretion of TCA organic acids such as pyruvic ,α-ketoglutaric and lactic acids in low concentration.The excess carbon in medium could be used to produce organic acids if not oxidized aerobically to CO 2 for energy production (El-Enshasy et al 2003,2008;Ayar-Kayali H and Tarhuan L 2006).In the subsequent incubation period, the pH increased gradually as a result of the decrease of cell growth and the increase in aureofuscin production.
In both cultures, the reducing sugar concentration in the bioreactor decreased at a faster rate and the reducing sugar was completely consumed after 84 h.This was because bioreactor cultivation provides better oxygen availability in the cultivation vessel due to the continuous aeration and better agitation; this increased oxygen availability is positively reflected on the physiological activity of aerobic microorganisms.The higher cell metabolic activities led to higher cell growth and antibiotic production rates.In general, the changes in dissolved oxygen DO in both cultures were similar, decreasing successively during the exponential growth phase (the first 72h of cultivation), increasing gradually thereafter and reaching approximately 85% saturation by the end of the cultivation.
From the obtained results, we infer that the addition of a mixture of acetate and propionate sodium at a ratio of 5:1 in a total concentration of 1.5 mg mL −1 at the beginning 24h of the cultivation is an easily applicable and inexpensive strategy to increase aureofuscin production in submerged culture by Str.aureofuscus.This approach could also be scalable for large-scale production of aureofuscin as it also showed higher aureofuscin yields in smallscale bioreactor culture.Production of other polyene antibiotics, which share the same pathway of polyene ring synthesis via the condensation of shortchain carboxylic acids, could also be improved through the addition of a short-chain carboxylic acid mixture as shown in this research.To the best of our knowledge, this is the first report using an acetatepropionate mixture as a precursor to enhance aureofuscin production in Str.aureofuscus at both the shake-flask and bioreactor levels.

Significance and Impact
The production of aureofuscin is very low in the wild-type strain.We attempt to increase the production of aureofuscin with adding precursors.To our knowledge, this approach has not been attempted in S. aureofuscus before.

Figure 3 .
Figure 3.Effect of the addition concentrations of sodium acetate and sodium propionate on aureofuscin production

Figure 4 .
Figure 4. Effect of different sodium acetate : sodium propionate ratios on cell growth and aureofuscin production by Str.aureofuscus in shake-flask cultivation.(Both acetate-propionate addition were added simultaneously as a mixture to a final concentration of 1.5 mg mL −1 at the beginning of the cultivation time before inoculation.)

Figure 5 .
Figure 5.Effect of addition time on cell growth and aureofuscin production by Str.aureofuscus in shake-flask cultivation.(The sodium acetate and sodium propionate were added as a mixture of acetate and propionate at a ratio of 5:1 and a final concentration of 1.5 mg mL −1 .)

Figure 6 .
Figure 6.Kinetics of cell growth and aureofuscin production by Str.aureofuscus in a 5-L bioreactor (working volume 3 L, agitation 220 rpm, aeration 1 v v −1 min −1 , temp.29•C) with and without sodium acetatepropionate.(Closed and open symbols correspond to results obtained in cultivations without and with sodium acetate-propionate addition, respectively.The sodium acetate and sodium propionate were added as a mixture of acetate and propionate at a ratio of 5:1 and a final concentration of 1.5 mg mL −1 after 24 h cultivation.).