Supplemental Material
A tailoring P450 monooxygenase gene
for FR-008/candicidin biosynthesis
Shi Chen2†, Xiangzhao Mao1†, Yaling Shen1, Yongjun Zhou2, Jialiang Li2,
Lianrong Wang2, Xinyi Tao1, Liang Yang1, Yuxiao Wang1,
Xiufen Zhou2, Zixin Deng2*, Dongzhi Wei1*
1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China
2. Laboratory of Microbial Metabolism and School of Life Science & Biotechnology, Shanghai Jiaotong University, Shanghai 200030, PR China
† Co-first author: These authors contributed equally to this work.
Current addresses: S. C., Department of Cell Biology, Harvard Medical School;
X. M., Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences
*The authors for correspondence: Prof. Dongzhi Wei and Zixin Deng,
Tel: +86-21-64252981 and +86-21-62933404
Fax: +86-21-64250068 and +86-21-62932418
E-mail: and
Optimization of productivity to enable structural and functional analysis of the derivative compound(s) in CS103.
HPLC analysis revealed new peaks possibly representing FR-008/candicidin derivatives in CS103; however production levels were only 1% of the level observed with the wild-type producer, Streptomyces sp. FR-008. We thus attempted to optimize the fermentation process parameters of CS103 to improve its antibiotic yield and facilitate the structural and activity study of the new polyene derivatives in CS103. The optimized medium composition (9) had a positive effect on FR-008/candicidin and a series of its novel derivatives, increasing the yield of the new polyene derivatives in CS103 by 849% (from 3.33 μg/mL to 31.59 μg/mL) when cultured in the optimal medium (9) as compared to the level in the conventional medium.
Then the levels of the fermentation process parameters were optimized by using the Response Surface Methodology (RSM). The CCD design and the corresponding experimental data were shown in Table S1. Data was analyzed by linear multiple regression using the ‘Design Expert’ software (version 7.0, Stat-Ease, Inc., Minneapolis, USA) and the following equation was obtained, describing the relationship between the production (Y) and the test variables in coded factors:
(1)
To validate the regression coefficient, analysis of variance (ANOVA) for CS103 production was performed (Table S2). The best fit of the model was checked by the determination coefficient (R2). In this case, the value of the determination coefficient (R2=0.9663) indicates that only 3.37% of the total variations are not explained by the model. In Table S2 we list the significance of each coefficient which was determined by F-value and P-value. Values of “Prob > F” less than 0.0500 indicate that model terms are significant. In this case A, B, C, D, CD, A2, B2, C2, D2 are significant model terms.
According to the optimized mathematical model, the optimal values of the test variables in coded unit are as follows: X1 =0.230, X2 =-0.020, X3 =0.230, X4 =-0.150 with the corresponding maximum yield of the antibiotic Y =37.13 μg/mL (a possible variation is between 34.49 μg/mL and 39.77 μg/mL) in the confidence range of 95%. Putting the respective values of Xi in Eq. (1), the levels of the fermentation process parameters were at 28.45 h, 9.95%, pH 6.72 and 47 mL, respectively. In order to verify the prediction, an experiment was performed with the optimized concentration. The maximum concentration of antibiotic CS103 obtained experimentally was found to be 38.72 μg/mL.
The optimized fermentation process parameters were obtained as follows: The fermentor was inoculated with 9.95% (v/v) pre-culture, the inoculums age was 28.45 h, operated at pH 6.70. Furthermore, we found the broth volume significantly affected the CS103 production. It was thus obvious that the production of the antibiotic was significantly affected by the dissolved oxygen.
The antibiotic production was performed in a 30 L fermentor (Bioengineering AG, Switzerland), with a working volume of 20 L. The fermentor was inoculated with 9.95% (v/v) pre-culture, and operated at 28 oC and pH 6.7 (with 5N NaOH and HCl). The aeration rate was 200 L/h, with speeds of 300 rpm used for agitation. Maximal fermentation yield (60 μg/mL) was obtained when the fermentation proceeded for about 144 h. From 20 L culture broth, 8 mg of CS103-I and 3 mg of CS103-II were purified by prep-HPLC.
MATERIALS AND METHODS
Bacterial strains and plasmids
The bacterial strains and plasmids used in this study are described in Table S3.
Genetic Procedures.
pJTU28 is a pIJ2925 derivative with a 2.3 kb PstI-KpnI fragment carrying fscP which is derived from a re-ligation of PstI-digested pJTU26. After BssHII digestion, a 396 bp sequence inside fscP gene was removed, followed by an insertion of a 1.4 kb BssHII fragment from pJTU33 carrying the apramycin resistance gene [acc(3)IV] to gain pJTU37. The 3.7 kb BglII fragment including the inserted apramycin resistance gene was recovered from pJTU37 for ligation into the BamHI site of pHZ1358 to generate pJTU50 for targeted replacement of fscP gene. pJTU50 was transferred into Streptomyces sp. FR-008 by conjugation from E. coli ET12567 carrying RP4 derivative pUZ8002 (3). Thiostrepton-sensitive and apramycin-resistance (ThioS-AprR) colonies were counter-selected from the initial ThioR exconjugants after two rounds of nonselective growth, to obtain CS103.
Complementation of CS103 with fscP-fscFE and fscTE
The 1,558 bp fragment was amplified by PCR with the primer fscP-S (5'-CATATGACGACCAGCCCCGGCCCG-3') and fscFE-A (5'- GTCGTCAGCGGTGGTCACGGGGGAG-3'). The PCR product was cloned into the SmaI site of pIJ2925 to generate pJTU587, and then excised as a NdeI-EcoRI fragment and cloned into corresponding sites of pIB139, resulting in pJTU597. One 1.2 kb ApaI-ApaI fragment carrying thiostrepton-resistance gene (Tiangang Liu, personal communication) was inserted into ApaI sites of pJTU597, resulting in the final construct pJTU2219 for the CS103 complementation test.
Growth conditions and fermentation process
Flasks (250 mL) filled with 50 mL of medium composed of glucose 10 g/L, malt extract 3 g/L, yeast extract 3 g/L and peptone 5 g/L, were inoculated in a rotary shaker at 200 rpm and 28 oC for one or two days. Then, 250 mL flasks with 50 mL production medium were inoculated with the seed culture, and this culture was grown under the same conditions as the seed culture. The production medium was: glucose 20 g/L, glycerol 13.89 mL/L, soluble starch 10 g/L, yeast extract 3 g/L, peptone 1.01 g/L, industrial complex aminophenol 5 g/L, ferrous sulfate 0.5 g/L, copper sulfate 0.0428 g/L and sodium chloride 10 g/L(9).
The antibiotic production was performed in a 30 L fermentor (Bioengineering AG, Switzerland), with a working volume of 20 L production medium. The operation parameters were determined according to the results of the optimization experiments.
Optimization of fermentation parameters
A central composite design (CCD) was used to optimize the concentrations of four effective fermentation process parameters in the culture using 250 ml flasks. Each variable was designed at five levels, and the lowest and the highest levels were: inoculums age, 24 h and 32 h; inoculum size, 6% and 14%; the cultivation process pH 4.5 and 8.5; the broth volume in the 250 ml flask, 10 ml and 90 ml, respectively.
We selected a 23 full factorial central composite design (CCD) proposed by Box et al. in our study (1). Table S1 shows the CCD design for the values in coded and the observed values in response. The ‘Design Expert’ software (version 7.0, Stat-Ease, Inc., Minneapolis, USA) was utilized to analyze the significance of experimental results.
Isolation and analysis of the antibiotics
The procedure for the isolation of the novel polyene macrolide complex from the mycelial cake and its purification is based on the method described previously with some modifications (2, 11).
The crude polyene complex was then purified by preparative HPLC on a SB-C8 column ZORBAX (19 mm×150 mm, 5 μm, Agilent Technology Co.). Approximately 8 mg of new heptaene CS103-I and 3 mg of CS103-II were obtained. These materials were used for structural characterization. Electrospray mass spectrometry (ESMS)3 was performed with a Quattro Micro tandem quadrupole mass spectrometer (Waters Corporation, Micromass Ltd., Manchester UK) in positive ion modes.
Concentration of the antibiotics were assayed by high-performance liquid chromatography in the mycelial cake, following the method described by Mao et al. (8). The sample of CS103-I was dissolved in DMSO-d6, and all NMR spectra were recorded at 25°C on a Bruker Avance DRX 500 spectrometer.
Measurement of Antifungal and haemolytic activity
The antibiotic activity was determined using Saccharomyces cerevisiae Y029 as an indicator (3). The method for measurement of haemolytic activity is similar to that described for Amphotericin B (5). Freshly isolated rabbit erythrocytes were used to determine the hemolytic activity. Decarboxylated FR-008/candicidin and FR-008/candicidin were dispersed in DMSO at different concentrations (0.29 to 74 µg/ml and 0.17 to 22 µg/ml, respectively) and incubated for 5 min at 37 oC. Defibrinated rabbit blood in phosphate-buffered saline was then added to a final hematocrit of 2% and incubated at the same temperature for 30 min. Hemolysis was assessed visually or by sedimenting unlysed erythrocytes and determining the absorbance of the supernatant fluid at 560 nm. The value for 100% hemolysis was obtained by hypotonic hemolysis in water.
Table S1. The CCD design and responses for the production of the antibiotic CS103
(μg/mL)
1 / -1.000 / 1.000 / 1.000 / -1.000 / 23.762
2 / 0.000 / 0.000 / 0.000 / -2.000 / 23.583
3 / 0.000 / 0.000 / 0.000 / 0.000 / 35.217
4 / 1.000 / 1.000 / 1.000 / 1.000 / 12.306
5 / 1.000 / -1.000 / -1.000 / -1.000 / 9.989
6 / 1.000 / 1.000 / -1.000 / -1.000 / 7.001
7 / -2.000 / 0.000 / 0.000 / 0.000 / 20.907
8 / -1.000 / -1.000 / -1.000 / 1.000 / 5.675
9 / 0.000 / 0.000 / 0.000 / 0.000 / 35.247
10 / -1.000 / -1.000 / 1.000 / -1.000 / 27.981
11 / 0.000 / 0.000 / 0.000 / 2.000 / 5.671
12 / 1.000 / 1.000 / 1.000 / -1.000 / 26.608
13 / 0.000 / -2.000 / 0.000 / 0.000 / 18.502
14 / 0.000 / 0.000 / 0.000 / 0.000 / 35.021
15 / 0.000 / 0.000 / -2.000 / 0.000 / 3.109
16 / -1.000 / 1.000 / -1.000 / 1.000 / 2.479
17 / 0.000 / 0.000 / 2.000 / 0.000 / 16.909
18 / -1.000 / -1.000 / 1.000 / 1.000 / 7.909
19 / 1.000 / -1.000 / 1.000 / 1.000 / 14.929
20 / 0.000 / 0.000 / 0.000 / 0.000 / 35.328
21 / 1.000 / -1.000 / -1.000 / 1.000 / 6.478
22 / 0.000 / 2.000 / 0.000 / 0.000 / 13.006
23 / -1.000 / 1.000 / -1.000 / -1.000 / 8.778
24 / 1.000 / 1.000 / -1.000 / 1.000 / 4.563
25 / 1.000 / -1.000 / 1.000 / -1.000 / 33.908
26 / -1.000 / 1.000 / 1.000 / 1.000 / 6.352
27 / 0.000 / 0.000 / 0.000 / 0.000 / 35.106
28 / 2.000 / 0.000 / 0.000 / 0.000 / 32.009
29 / 0.000 / 0.000 / 0.000 / 0.000 / 34.987
30 / -1.000 / -1.000 / -1.000 / -1.000 / 8.097
Table S2. The least-squares fit and coefficient estimate
Estimate / Standard
Error / 95% CI
Low / 95% CI
High / F
Value / p-value
Prob > F
Intercept / 35.15 / 1.26 / 32.47 / 37.83
A / 1.96 / 0.63 / 0.62 / 3.30 / 9.70 / 0.0071
B / -1.42 / 0.63 / -2.76 / -0.082 / 5.12 / 0.0389
C / 5.35 / 0.63 / 4.01 / 6.68 / 72.44 / < 0.0001
D / -5.05 / 0.63 / -6.39 / -3.71 / 64.71 / < 0.0001
AB / -0.41 / 0.77 / -2.05 / 1.23 / 0.28 / 0.6032
AC / 1.17 / 0.77 / -0.47 / 2.81 / 2.32 / 0.1486
AD / 0.44 / 0.77 / -1.20 / 2.08 / 0.32 / 0.5794
BC / -0.52 / 0.77 / -2.16 / 1.12 / 0.45 / 0.5113
BD / 0.28 / 0.77 / -1.36 / 1.92 / 0.14 / 0.7177
CD / -3.51 / 0.77 / -5.15 / -1.87 / 20.77 / 0.0004
A2 / -2.80 / 0.59 / -4.06 / -1.55 / 22.78 / 0.0002
B2 / -5.48 / 0.59 / -6.73 / -4.23 / 87.01 / < 0.0001
C2 / -6.92 / 0.59 / -8.17 / -5.66 / 138.59 / < 0.0001
D2 / -5.76 / 0.59 / -7.01 / -4.51 / 96.18 / < 0.0001
The P-Value less than 0.05 are significant.
Table S3. Bacterial strains, phages, and plasmids used in this study
Streptomyces sp. FR-008 / Wild type, FR-008/candicidin producer / Liang, 1987 (6)
Streptomyces sp.CS103 / ΔfscP mutant, AmRThioS, CS103 producer / This work
E. coli ET12567
(pUZ8002) / recF dam – dcm - cml str tet km / MacNeil et al.,1992(7)
Saccharomyces cerevisiae Y029 / To measure the activity of FR-008/candicidin and their derivatives / Lab stock
pIJ2925 / pUC19 derivative, bla，2.7 kb / Janssen et al., 1993(4)
pJTU26 / a pIJ2925 derivative carrying a 6.6 kb KpnⅠfragmant from pHZ145 / Chen et al., 2003 (3)
pJTU28 / a pIJ2925 derivative with a 2.3 kb PstI-KpnI fragment carrying fscP which is derived from a re-ligation of PstI-digested pJTU26 / This work
pHZ1070 / apramycin resistance gene aac(3)Ⅳ containing plasmid, 4.1 kb / Chen et al., 2003 (3)
pJTU33 / a pBluescriptⅡ SK(+) derivative containing a BamHⅠfragment containing an apramycin resistance gene from pHZ1070 / This work
pJTU37 / a pJTU28 derivative with a 396 bp BssHⅡfragment deletion inside the fscP replaced with a 1.4 kb BssHⅡfragment containing an apramycin resistance gene from pJTU33 / This work
pHZ1358 / sti+, oriT, ori(pIJ101), bla, tsr, 10.65 kb / Sun et al., 2003 (10)
pJTU50 / For targeted replacement of fscP gene, the 3.7 kb BglII fragment including inserted apramycin resistance gene was recovered from pJTU37 for ligation into the BamHI site of pHZ1358 / This work
pUZ8002 / MacNeil et al.,1992(7)
pJTU587 / 1,558 bp PCR product (KOD plus); Prime: fscP-Test-S & fscFE-A; SmaI inserted / This work
pJTU597 / 1558 bp NdeI-EcoRI from pJTU587; NdeI-EcoRI inserted / This work
pJTU2219 / 1.2K ApaI-ApaI fragment from one SK(+) derivative (Thio gene inserted in EcoRV site);
ApaI (prepared by CIAP) inserted / This work
Table S4. 1H- and 13C-NMR Data for Compound CS103-I in DMSO-d6 at 25°C