Mechanistic insights into ammonium-driven metabolic regulation for enhanced nemadectin biosynthesis in Streptomyces cyaneogriseus

Abstract

Nemadectin, a milbemycin-class macrocyclic lactone antibiotic produced by Streptomyces cyaneogriseus, is a potent broad-spectrum insecticide with excellent environmental compatibility. Its derivative moxidectin, featuring a C-23 methoxime modification, demonstrates enhanced insecticidal activity and has become a commercially successful agrochemical. This study reveals ammonium regulation effectively boosts nemadectin biosynthesis in S. cyaneogriseus, with mechanistic insights gained through integrated multi-omics analysis. Transcriptomic profiling showed ammonium sulfate supplementation significantly upregulates the nemadectin biosynthetic gene cluster, including polyketide synthase (PKS) genes, backbone modification genes, and pathway-specific transcription factors, while also enhancing the expression of Avenolide-like signaling molecules and global transcription factor Afskne. Metabolomic dynamics revealed reinforced precursor biosynthesis through coordinated metabolic reprogramming: enhanced acetyl-CoA production, reinforced Embden–Meyerhof–Parnas pathway and amino acid/acyl-CoA metabolism, coupled with reduced tricarboxylic acid cycle activity. Systematic integration of physiological phenotyping, metabolite profiling, and transcriptional regulation data comprehensively elucidated the ammonium-driven overproduction mechanism, providing critical insights for developing advanced fermentation strategies and genetic engineering approaches in industrial antibiotic production.

Keywords

Nemadectin / Streptomyces cyaneogriseus / Ammonium regulation / Multi-omics analysis

References

[1] Amon J, Titgemeyer F, Burkovski A. A genomic view on nitrogen metabolism and nitrogen control in mycobacteria. J Mol Microbiol Biotechnol, 2008, 17(1): 20-29

[2] Chen Y, Wang Z, Chu J, Zhuang Y, Zhang S, Yu X. 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

[3] Doscher ME, Wood IB, Pankavich JA, Ricks CA. Efficacy of nemadectin, a new broad-spectrum endectocide, against natural infections of canine Gastrointestinal helminths. Vet Parasitol, 1989, 34(3): 255-259

[4] Fernández-Moreno MA, Caballero J, Hopwood DA, Malpartida F. The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the BldA tRNA gene of streptomyces. Cell, 1991, 66(4): 769-780

[5] Guo J, Zhao J, Li L, Chen Z, Wen Y, Li J. The pathway-specific regulator aver from streptomyces avermitilis positively regulates avermectin production while it negatively affects oligomycin biosynthesis. Mol Genet Genomics, 2010, 283(2): 123-133

[6] Hong M, Mou H, Liu X, Huang M, Chu J. 13 C-assisted metabolomics analysis reveals the positive correlation between specific erythromycin production rate and intracellular propionyl-CoA pool size in saccharopolyspora Erythraea. Bioprocess Biosyst Eng, 2017, 40(9): 1337-1348

[7] Ikeda H, Ōmura S. Avermectin biosynthesis. Chem Rev, 1997, 97(7): 2591-2610

[8] Kitani S, Miyamoto KT, Takamatsu S, Herawati E, Iguchi H, Nishitomi K, Uchida M, Nagamitsu T, Omura S, Ikeda H, Nihira T. Avenolide, a streptomyces hormone controlling antibiotic production in. roc of the Natl Acad Sci, 2011, 108(39): 16410-16415

[9] Li C, He H, Wang J, Liu H, Wang H, Zhu Y, Wang X, Zhang Y, Xiang W. Characterization of a LAL-type regulator NemR in nemadectin biosynthesis and its application for increasing nemadectin production in streptomyces cyaneogriseus. Sci China Life Sci, 2019, 62(3): 394-405

[10] Liao C-H, Yao L, Xu Y, Liu W-B, Zhou Y, Ye B-C. Nitrogen regulator GlnR controls uptake and utilization of non-phosphotransferase-system carbon sources in actinomycetes. Proc Natl Acad Sci, 2015, 112(51): 15630-15635

[11] Liu P, Wang S, Li C, Zhuang Y, Xia J, Noorman H. Dynamic response of Aspergillus Niger to periodical glucose pulse stimuli in Chemostat cultures. Biotechnol Bioeng, 2021, 118(6): 2265-2282

[12] Lyons ET, Tolliver SC, Drudge JH, Granstrom DE, Collins SS, Stamper S. Critical and controlled tests of activity of moxidectin (CL 301, 423) against natural infections of internal parasites of equids. Vet Parasitol, 1992, 41(3): 255-284

[13] Martín JF, Sola-Landa A, Santos-Beneit F, Fernández-Martínez LT, Prieto C, Rodríguez-García A. Cross-talk of global nutritional regulators in the control of primary and secondary metabolism in streptomyces. Microb Biotechnol, 2011, 4(2): 165-174

[14] Prichard R, Ménez C, Lespine A. Moxidectin and the avermectins: consanguinity but not identity. Int J Parasitology: Drugs Drug Resist, 2012, 2: 134-153

[15] Pullan ST, Chandra G, Bibb MJ, Merrick M. Genome-wide analysis of the role of GlnR in streptomyces Venezuelae provides new insights into global nitrogen regulation in actinomycetes. BMC Genomics, 2011, 12(1): 175

[16] Rajkarnikar A, Kwon HJ, Ryu YW, Suh JW. Catalytic domain of AfsKav modulates both secondary metabolism and morphologic differentiation in streptomyces avermitilis ATCC 31272. Curr Microbiol, 2006, 533204-208

[17] Song X, Zhang Z, Yu J, Zhang Y, Xue J, Guo Y, Liang J, Ren M, Ling Q, Mohsin A, Qian J, Wang Z, Wang Y. Oxygen uptake rate (OUR) guided nitrogen control strategy for improving nemadectin production by streptomyces cyaneogriseus ssp. Noncyanogenus. Process Biochem, 2025, 150: 248-256

[18] Technikova-Dobrova Z, Damiano F, Tredici SM, Vigliotta G, di Summa R, Palese L, Abbrescia A, Labonia N, Gnoni GV, Alifano P. Design of mineral medium for growth of actinomadura sp. ATCC 39727, producer of the glycopeptide A40926: effects of calcium ions and nitrogen sources. Appl Microbiol Biotechnol, 2004, 65(6): 671-677

[19] Tiffert Y, Franz-Wachtel M, Fladerer C, Nordheim A, Reuther J, Wohlleben W, Mast Y. Proteomic analysis of the GlnR-mediated response to nitrogen limitation in streptomyces coelicolor M145. Appl Microbiol Biotechnol, 2011, 89(4): 1149-1159

[20] Uchida M, Takamatsu S, Arima S, Miyamoto KT, Kitani S, Nihira T, Ikeda H, Nagamitsu T. Total synthesis and absolute configuration of avenolide, extracellular factor in streptomyces avermitilis. J Antibiot, 2011, 64(12): 781-787

[21] Wang H, Li C, Zhang B, He H, Jin P, Wang J, Zhang J, Wang X, Xiang W. Complete genome sequence of streptomyces cyaneogriseus ssp. noncyanogenus, the thermotolerant producer of commercial antibiotics nemadectin. J Biotechnol, 2015, 204: 1-2

[22] Yao L-L, Liao C-H, Huang G, Zhou Y, Rigali S, Zhang B, Ye B-C. GlnR-mediated regulation of nitrogen metabolism in the actinomycete saccharopolyspora Erythraea. Appl Microbiol Biotechnol, 2014, 98(18): 7935-7948

[23] Yu J, Wang Z, Wang J, Mohisn A, Liu H, Zhang Y, Zhuang Y, Guo M. Physiological metabolic topology analysis of Halomonas elongata DSM 2581T in response to sodium chloride stress. Biotechnol Bioeng, 2022, 119(12): 3509-3525

[24] Zhang A, Yang S-T. Engineering Propionibacterium Acidipropionici for enhanced propionic acid tolerance and fermentation. Biotechnol Bioeng, 2009, 104(4): 766-773

[25] Zhang A, Yang S-T. Propionic acid production from glycerol by metabolically engineered Propionibacterium Acidipropionici. Process Biochem, 2009, 44(12): 1346-1351

[26] Zhang Q, Hang H, Tian X, Zeng W, Yu Z, Wang X, Tang Y, Zhuang Y, Chu J. Combined available nitrogen resources enhanced erythromycin production and preliminary exploration of metabolic flux analysis under nitrogen perturbations. Bioprocess Biosyst Eng, 2019, 42(11): 1747-1756

[27] Zhu J, Chen Z, Li J, Wen Y. AvaR1, a butenolide-type autoregulator receptor in streptomyces avermitilis, directly represses avenolide and avermectin biosynthesis and multiple physiological responses. Front Microbiol, 2017, 8: 2577

[28] Zou X, Hang H-f, Chu J, Zhuang Y-p, Zhang S-l. Enhancement of erythromycin A production with feeding available nitrogen sources in erythromycin biosynthesis phase. Bioresour Technol, 2009, 100(13): 3358-3365

Funding

Hebei Provincial Key Research Projects(2025ZD1203400)

RIGHTS & PERMISSIONS

The Author(s)