Continuous carbon source supply is essential for high rifamycin productivity of Amycolatopsis mediterranei in nitrate-stimulated fermentation revealed by a metabolomic study

Abstract

Amycolatopsis mediterranei U32 is an industrial strain capable of producing therapeutically useful rifamycin SV. In early days of fermentation studies, nitrate was found to increase the yield of rifamycin along with globally, affecting both carbon and nitrogen metabolism in favor of antibiotic biosynthesis; thus, the nitrate-stimulating effect (NSE) hypothesis was proposed. Although GlnR is likely the master regulator of the pleotropic effect of NSE, the global metabolism affected by NSE has never been systematically examined. In this study, we use mass spectrometry-based metabolomics to quantitatively monitor the metabolomic responses of A. mediterranei U32 to nitrate supplementation. The concentrations of many metabolites involved in central carbon metabolism, including glucose 6-phosphate, glucose 1-phosphate, UDP-glucose, and acetyl-coenzyme A, decrease significantly after the addition of 80 mM potassium nitrate to the medium. We find that the rifamycin SV production yield could be increased by the addition of glucose during the logarithmic growth phase. Moreover, at multiple time points during glucose supplementation in the mid- and late-exponential phases, the yield of rifamycin SV further increases, reaching 354.3%. Quantitative real-time PCR assays of the key genes corresponding to the synthesis of the rifamycin SV precursor combined with data from metabolomics analysis confirm that carbon source deficiency is compensated for after glucose supplementation and that the expression of genes involved in the pathway of 3-amino-5-hydroxybenzoic acid synthesis by UDP-glucose and glutamine is significantly increased. This preliminary exploration of dynamic metabolomic profiles has the potential to increase our understanding of the NSE.

1. Introduction

Rifamycin is an ansamycin antibiotic that effectively inhibits bacterial RNA polymerase via specific binding to its β subunit [1], encoded by rpoB [2], so it has been demonstrated to be a broad-spectrum antibiotic against pathogenic bacterial infections [3,4] and is particularly effective for tuberculosis chemotherapy [5]. A significant increase in rifamycin SV productivity in the industrial strain Amycolatopsis mediterranei U32 via the addition of nitrate into its fermentation medium was reported a few decades ago, and the increase in the specific yield of rifamycin was found to be directly correlated in a dose-dependent manner with the increase in the specific activity of glutamine synthase (GS), which produces glutamine to provide the nitrogen source for the synthesis of the key starter unit of rifamycin, 3-amino-5-hydroxybenzoic acid (AHBA) [6,7]. Thus, the “nitrate-stimulating effect (NSE)” was coined [8,9] and has been demonstrated in the industrial production of other antibiotics, such as rifamycin B [10], lincomycin [11–13] and azalomycin B [14]. Further physiological and biochemical studies also indicated that NSE is likely pleotropic, affecting both nitrogen and carbon primary metabolism toward the production of antibiotics [15].

Notably, the atypical OmpR/PhoB subfamily response regulator GlnR was characterized as an essential regulatory protein for the transcription of the GS encoding the glnA gene in streptomycetes first [16] and then in many other actinomycetes, including strains of A. mediterranei [17,18] and mycobacteria [19]. Research has shown that GlnR is a global transcription factor that is bound to the promoter regions of many metabolic genes [20], globally regulating their expression [21] not only for nitrogen metabolism [22–24] but also for other metabolic processes, such as carbon [25–27], phosphate [28,29] and even antibiotic biosynthesis [18,30]. A subsequent genomic study of A. mediterranei U32 revealed its carbon and nitrogen metabolic pathways as well as biosynthesis-related gene clusters, particularly for rifamycin, the rif cluster [31]. The following transcriptome study revealed their expression properties, particularly those related to the biosynthesis of secondary metabolites [32], and thus, this study well integrated the knowledge of NSE to facilitate the acquisition of the molecular mechanism of the GlnR-mediated global regulatory function. Nitrate, which is mediated by functional GlnR, induces the expression of the nasACKBDEF operon [33], and this additional nitrogenous substrate likely maintains the healthy growth status of the cells during fermentation [33]. Moreover, nitrate may also function as a signal to activate the transcription factor GlnR and further affect global metabolism, largely af...