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.

Keywords: glucose supplementation; metabolome; nitrate-stimulating effect; rifamycin SV production.

Figure 1

Cell growth, rifamycin SV production and glucose concentration in B and BK (A) Cell growth, (B) rifamycin SV production, (C) glucose concentration in B and BK; Black line is nitrate-free Bennet medium, red line is Bennet medium with 80 mM KNO3. Error bars represent standard deviations of three biological replicates.

Figure 2

NSE results in divergent concentration changes of metabolites involved in carbon and nitrogen metabolism Data shown are the ratio of metabolite concentrations in A.mediterranei U32 grown at 48 h in BK medium versus B medium. Data shown are the means of six independent experiments.

Figure 3

Glucose concentration, cell growth and rifamycin SV production of U32 in BK after different glucose supplementation strategies (A) Adding 1 mL glucose solution once at 48 h. (B) Adding 2 mL glucose solution once at 48 h. (C) Repeatedly adding 1 mL glucose solution at 48, 60, 72, 84, 96 and 108 h. ‘+’ means supplying 1 mL glucose solution, ‘++’ means supplying 2 mL glucose solution. Data are shown as the mean ± SD (n = 3).

Figure 4

The transcription of key genes involved in carbohydrate metabolism and rifamycin SV biosynthesis after multiple-time point glucose supplementation (A) The expressions of genes involved in carbohydrate metabolism. (B) The expressions of genes involved in rifamycin SV biosynthesis. The results showed transcription at 12 h and 24 h with or without glucose supplementation, ‘+’ means adding glucose, the time zero point of sugar addition was used for comparison. The legend shows the time after the first sugar supplementation (48 h), rpoB is used as the reference gene. Error bars represent standard deviations of three biological replicates.

Figure 5

Comparison of the expressions of genes and dynamic metabolome after multiple-time point glucose supplementation Transcription data shown are –ΔΔCt at 60 h (12 h after glucose supplementation); 48 h without glucose supplementation is used as the control group, and rpoB is used as the reference gene. Metabolome data shown are the ratio of both samples above. The solid arrow is a one-step reaction, the dotted arrow is a multi-step reaction, the green arrow is downregulation, and the red arrow is upregulation; Red font with red box means the ratio is greater than 2, pink font with pink box means the ratio is 1.5-2, and black font indicates little change. Data are shown as the mean ± SD (n = 6 independent experiments).