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Acta Microbiologica Sinica 2017, 57(2): 281-292 http://journals.im.ac.cn/actamicrocn DOI: 10.13343/j.cnki.wsxb.20160290 Research Article * 300457 M1 M2 GC-MS GC-MS 232 70 PCA PLS 21 5.36 g/l 5.

1 Acta Microbiologica Sinica 2017, 57(2): DOI: /j.cnki.wsxb Research Article * M1 M2 GC-MS GC-MS PCA PLS g/l 5.92 g/l 10.4% [1] (Avermectin AVM) (Streptomyces avermitilis) A1a/A1b B1a/B1b A2a/A2b B2a/B2b B1a B1a (2013CB734004) ( ) * Tel gaoqiang@tust.edu.cn

2 282 Peng Cao et al. Acta Microbiologica Sinica, 2017, 57(2) [2-3] [4] [5] [6] (Streptomyces avermitilis) N- -N-( ) (MSTF) (GC-MS) (Agilent Technologies Palo Alto CA USA) ZWYR-D2403 ( ) ( ) M1 (g/l) (NH 4 ) 2 SO CoCl Na 2 MoO MnSO CH 3 CH 2 COONa PEG B CaCO ( ph ) ph C 25 min M2 M1 Plackett-Burman (g/l) (NH 4 ) 2 SO CoCl Na 2 MoO MnSO CH 3 CH 2 COONa PEG B CaCO ( ph ) ph C 25 min [7] d ( ) M1 M2 20 ml 8000 r/min 5 min 20 ml 4 C 0.6% (W/V) NaCl 6000 r/min 4 C 5 min 4 5 min 80 C [8] actamicro@im.ac.cn

3 , 2017, 57(2) min 50 mg 1.5 ml [9] ml (V/V) 1 min 2 min 4 [10] 1.5 GC-MS GC-MS [11] 1.6 GC-MS MSD NIST08 SIMICA v11.5 (PCA) (PLS) [12-13] M1 M2 2 GC-MS 232 NIST08 70 (18 ) (11 ) (8 ) (15 ) 1 1 (TCA) (EMP) M1 M2 1 1 PCA PCA R 2 (X) R 2 (Y) PCA (>0.8) [14] 2 PCA M1 M2

4 284 Peng Cao et al. Acta Microbiologica Sinica, 2017, 57(2) Class Amino acids Organic acids Fatty acids Saccharides Others Table 1. Identified metabolites 1. GC-MS Intracellular metabolites identified by GC-MS Alanine Glutamine Proline Aspartic acid Glycine Sarcosine Cysteine Isoleucine Serine Aspartic acid Leucine Valine Tyrosine Tryptophan Lysine Glutamic acid Methionine Threonine Acetic acid Lactic acid Propionate Gluconic acid Malic acid Pyruvic acid Citric acid Fumaric acid Succinic acid Oxalic acid α-ketoglutaric acid Oxaloacetic acid 9,12-Octadecadienoic acid Heptadecanoic acid Octadecanoic acid 9-Octadecenoic acid Hexadecanoic acid Palmitic acid Pentanoic acid Oleic acid Galactose Glucose Fructose Mannitol Trehalose Lactose Talose Lyxose Glucopyranose Erythrose-4-phosphate 7-Sedoheptulose-phosphate 3-Phosphoglycerate Cellobiose Glucose-6-phosphate Xylulose-5-phosphate Inositol Methanediimine Sorbitol Adenine Furanone Urea 5-Methyluridine Ribitol Lyxose β-hydroxypyruvic acid 2-Oxoadipic acid Guanine 1-Hexanol Imidazoleacetic acid Phosphate Estrone Succinylacetone Arabinofuranose 1. PCA Figure 1. PCA score plots of avermectin biosynthesis production in different culture media. 2. PCA Figure 2. PCA loading plots of avermectin biosynthesis production in different culture media.

, 2017, 57(2) 285 PLS 3 40 8 4 PLS VIP 26 6-4- 5- α- 3. PLS Figure 3. PLS score plots analysis of avermectin biosynthesis process.

Variable importance of the projection plots for intracellular metabolites along component 1 (VIP1) plot generated by PLS.

1: glucose; 2: glucose-6- phosphate; 3: lactose; 4: pyruvic acid; 5: erythrose-4- phosphate; 6: xylulose-5-phosphate; 7: α-ketoglutaric acid; 8:

5 , 2017, 57(2) 285 PLS PLS VIP α- 3. PLS Figure 3. PLS score plots analysis of avermectin biosynthesis process VIP VIP PLS M1 M2 (EMP) (TCA) d M M VIP Figure 4. Variable importance of the projection plots for intracellular metabolites along component 1 (VIP1) plot generated by PLS. X-axis represents each metabolite. 1: glucose; 2: glucose-6- phosphate; 3: lactose; 4: pyruvic acid; 5: erythrose-4- phosphate; 6: xylulose-5-phosphate; 7: α-ketoglutaric acid; 8: citric acid; 9: oxaloacetic acid; 10: propionic acid; 11: tyrosine; 12: leucine; 13: phenylalanine; 14: threonine; 15: lysine; 16: proline; 17: valine; 18: isoleucine; 19: hexadecanoic acid; 20: heptadecanoic acid; 21: oleic acid; 22: unknown 1; 23: unknown 2; 24: unknown 3; 25: unknown 4; 26: unknown 5.

6 286 Peng Cao et al. Acta Microbiologica Sinica, 2017, 57(2) 5. M1 M2 Figure 5. Variations of metabolites associated with glycolysis pathway in different culture media. PYR: pyruvic acid; 3PG: 3-phosphoglyceric acid; LAC: lactic acid; PPA: propionic acid; G6P: glucose-6-phosphate; X5P: xylulose-5-phosphate; E4P: erythrose-4-phosphate; OAA: oxaloacetic acid; CIT: citric acid; AKG: α-oxoglutarate; SUC: succinic acid. Each value is the mean of five replicates±sd. P<0.05. CoA TCA 4 d M2 CoA CoA [15] M2 M1 M1 4 d HMP d M2 α- M1 M2 TCA M2 M1 2 d M2 L- M d M2 L- M actamicro@im.ac.cn

7 , 2017, 57(2) M1 M2 Figure 6. Variations of amino acids identified in S. avermitilis in different culture media. Tyr: tyrosine; Trp: tryptophane; Leu: leucine; Pro: proline; Val: valine; Thr: threonine; Lys: lysine; Ile: isoleucine. Each value is the mean of five replicates±sd. P< A(MB-CoA) 2- A(MM-CoA) A(Malonyl-CoA) L- L- MB-CoA MM-CoA Malonyl-CoA CoA [16-17] CoA MM-CoA M1 M [18] 7 M2 M1 3 4 d

8 288 Peng Cao et al. Acta Microbiologica Sinica, 2017, 57(2) 3 Novák [18] M (EMP, PPP, TCA) M g/l EMP ( h) 8 36 h 0.5 g/l 7. M1 M2 Figure 7. Variations of fatty acids identified in S. avermitilis. HEX: hexadecanoic acid; HEP: heptadecanoic acid; OA: oleic acid. Each value is the mean of five replicates±sd. P< Figure 8. The effect of adding precursors of central carbon metabolism on avermectin production. Each value is the mean of three replicates±sd. P<0.05. A: lactate; B: pyruvate; C: sodium succinate. actamicro@im.ac.cn

9 , 2017, 57(2) g/l 5.53 g/l 1.0 g/l [19] 36 h 1.0 g/l 5.36 g/l 5.83 g/l M g/l ( h) TCA 12 h 1.0 g/l 5.36 g/l 5.55 g/l 48 h M g/l 9 72 h 96 h 96 h 1.5 g/l 5.75 g/l 72 h 1.5 g/l 5.68 g/l M g/l ( h) 10 0 h 1.5 g/l 2.0 g/l 9. Figure 9. The effect of adding precursors of amino acid metabolism on avermectin production. Each value is the mean of three replicates±sd. P<0.05. A: valine; B: isoleucine; C: threonine.

10 290 Peng Cao et al. Acta Microbiologica Sinica, 2017, 57(2) M2 M1 PCA PLS α- 10. Figure 10. The effect of adding soybean oil on avermectin production. Each value is the mean of three replicates ±SD. P< h 1.0 g/l 5.81 g/l M2 M3 M2 TCA EMP TCA EMP [20] β -CoA M3 12 h 1.0 g/l 36 h 0.5 g/l 1.0 g/l 48 h 1.0 g/l 72 h 1.5 g/l 96 h 1.5 g/l 11 M g/l 5.36 g/l 10.4% 3 M2 (EMP) (TCA) M1 (HMP) M1 11. (M2 M3) Figure 11. The dynamic changes of avermectin production in M2 and M3. SSA: succinic acid disodium salt, LAC: lactic acid, PYR: pyruvic acid, SOB: soybean oil, ILE: isoleucine, VAL: valine. Each value is the mean of three replicates±sd. P<0.05. actamicro@im.ac.cn

11 , 2017, 57(2) 291 M2 M3 M2 10.4% 5.92 g/l [1] Burg RW, Miller BM, Baker EE, Birnbaum J, Currie SA, Hartman R, Kong YL, Monaghan RL, Olson G, Putter I, Tunac JB, Wallick H, Stapley EO, Oiwa R, Ōmura S. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrobial Agents and Chemotherapy, 1979, 15(3): [2] Yoon YJ, Kim ES, Hwang YS, Choi CY. Avermectin: biochemical and molecular basis of its biosynthesis and regulation. Applied Microbiology and Biotechnology, 2004, 63(6): [3] Ikeda H, Nonomiya T, Usami M, Ohta T, Ōmura S. Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(17): [4] Chen JS, Liu M, Liu XT, Miao J, Fu CZ, Gao HY, Müller R, Zhang Q, Zhang LX. Interrogation of Streptomyces avermitilis for efficient production of avermectins. Synthetic and Systems Biotechnology, 2016, 1(1): [5] Ryu YG, Butler, MJ, Chater KF, Lee KJ. Engineering of primary carbohydrate metabolism for increased production of actinorhodin in Streptomyces coelicolor. Applied and Environmental Microbiology, 2006, 72(11): [6] Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta, 2008, 76(5): [7] Gao H, Liu M, Liu JT, Dai HQ, Zhou XL, Liu XY, Zhuo Y, Zhang WQ, Zhang LX. Medium optimization for the production of avermectin B1a by Streptomyces avermitilis 14-12A using response surface methodology. Bioresource Technology, 2009, 100(17): [8] Ding MZ, Zhou X, Yuan YJ. Metabolome profiling reveals adaptive evolution of Saccharomyces cerevisiae during repeated vacuum fermentations. Metabolomics, 2010, 6(1): [9] Bo T, Liu M, Zhong C, Zhang Q, Su QZ, Tan ZL, Han PP, Jia SR. Metabolomic analysis of antimicrobial mechanisms of ε-poly-l-lysine on Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry, 2014, 62(19): [10] Guo G, Tian PP, Tang D, Wang XX, Yang HJ, Cao P, Gao Q. Metabolomics analysis between wild-type and industrial strains of Streptomyces avermitilis based on gas chromatography-mass spectrometry strategy//zhang TC, Nakajima M. Advances in applied biotechnology. Berlin Heidelberg: Springer, 2015: [11] Guo G, Tang D, Tian PP, Shi XF, Cao P, Gao Q. Optimized sample preparation for metabolome studies on Streptomyces avermitilis by GC-MS. Biotechnology Bulletin, 2015, 31(5): (in Chinese),,,,,. GC-MS., 2015, 31(5): [12] Fiehn O. Metabolomics-the link between genotypes and phenotypes. Plant Molecular Biology, 2002, 48(1/2): [13] Aa JY. Analysis of metabolomic data: principal component analysis. Chinese Journal of Clinical Pharmacology and Therapeutics, 2010, 15(5): (in Chinese).., 2010, 15(5): [14] Wold S, Geladi P, Esbensen K, Öhman J. Multi-way principal components-and PLS-analysis. Journal of Chemometrics, 1987, 1(1): [15] Dı kbas N. Determination of antibiotic susceptibility and fatty acid methyl ester profiles of Bacillus cereus strains

12 292 Peng Cao et al. Acta Microbiologica Sinica, 2017, 57(2) isolated from different food sources in Turkey. African Journal of Biotechnology, 2010, 9(11): [16] Dotzlaf JE, Metzger LS, Foglesong MA. Incorporation of amino acid-derived carbon into tylactone by Streptomyces fradiae GS14. Antimicrobial Agents and Chemotherapy, 1984, 25(2): [17] Chen TS, Inamine ES. Studies on the biosynthesis of avermectins. Archives of Biochemistry and Biophysics, 1989, 270(2): [18] Novák J, Hájek P, Rezanka T, Vanĕk Z. Nitrogen regulation of fatty acids and avermectins biosynthesis in Streptomyces avermitilis. FEMS Microbiology Letters, 1992, 72(1): [19] Madden T, Ward JM, Ison AP. Organic acid excretion by Streptomyces lividans TK24 during growth on defined carbon and nitrogen sources. Microbiology, 1996, 142(11): [20] Shen ZB, Chen GH, Chen CH. Study on effect of soybean oil on fermentation of erythromycin and its mechanism. Chinese Journal of Antibiotics, 2006, 31(11): (in Chinese),,.., 2006, 31(11): Enhanced avermectin production by rational feeding strategies based on comparative metabolomics Peng Cao, Dong Hu, Jun Zhang, Bianqiang Zhang, Qiang Gao * Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin , China Abstract: [Objective] In order to reveal key metabolites and metabolic pathways of avermectin, comparative metabolomics approach was used to analyze the difference of key intracellular metabolites of Streptomyces avermitilis in different fermentation media. Then the rational feeding approach was used to enhance avermectin production. [Methods] Mycelial samples in M1 and M2 media were analyzed by GC-MS based comparative intracellular metabolomics. Based on the deficiency of relative metabolic pathways, single precursor supplement and combined precursors supplement were exerted, and the optimized medium M3 was obtained to enhance avermectin production. [Results] In total 232 intracellular metabolites were identified and 70 of them were accurately matched, and 21 metabolites influencing avermectin biosynthesis were finally verified by PCA and PLS analyses. Among them, lactic acid, pyruvic acid, succinic acid, threonine, isoleucine, valine and lipids determined the avermectin titer more obviously. When supplying with these precursors together to M2 at different time points, the titer increased by 10.4% from 5.36 g/l to 5.92 g/l. [Conclusion] The production of avermectin increased apparently with the help of comparative metabolomics analysis and rational feeding optimization. It also provides a new approach to enhance the yield of bioproducts. Keywords: Streptomyces avermitilis, avermectin, metabolomics, rational feeding Supported by the National Basic Research Program (973 Program) of China (2013CB734004) and by the National Natural Science Foundation of China ( , ) * Corresponding author. Tel: ; gaoqiang@tust.edu.cn Received: 18 July 2016; Revised: 7 September 2016; Published online: 18 September 2016 actamicro@im.ac.cn

L 8 0 g L ~0 700 g L % 6 00% 1 50% 1 3 NaCl 0 50% NH 4 2 SO % KH 2 PO % CaCO % 0 03% ~ 0 04% 2 24 h 0 02

L 8 0 g L ~0 700 g L % 6 00% 1 50% 1 3 NaCl 0 50% NH 4 2 SO % KH 2 PO % CaCO % 0 03% ~ 0 04% 2 24 h 0 02 38 5 Vol 38 No 5 2014 9 Journal of Jiangxi Normal UniversityNatural Science Sep 2014 1000-5862201405-0501-05 1 1* 2 2 1 3300222 330052 Streptomyces lincolnensis 13 8 6% 12 4% Q 93 A 0 1 2 Streptomyces