The aim of the present study was to develop a cultivation system for systems scale studies of metabolic switching in Streptomyces coelicolor
A3(2). This task required the simultaneous observation of several constraints provided by three considerations. (i) The scientific question itself, i.e. batch cultivations with a defined transition phase in response to the depletion of one defined nutrient and a good production phenotype. (ii) The different downstream - ‘omics - analyses and subsequent modelling approaches, i.e. a high degree of reproducibility, with biomass concentrations of >4
g/L CDW available long before nutrient depletion and an absence of analysis-interfering media components. (iii) The growth aspects of the research object S. coelicolor
A3(2) strain M145 in submerged culture, i.e. intra-mycelial nutrient and oxygen supply and the influence of shear forces. The described approach finally led to generic conditions and two cultivation media: SSBM-P for studying metabolic switching in response to phosphate depletion, and SSBM-E which can be used for studying metabolic switching events initiated from the depletion of L-glutamate in the medium. The high reproducibility in biological replicas as demonstrated for the optimized cultivation system and the two different triggering conditions is crucial for systems scale studies of metabolic switching in S. coelicolor
A3(2). Our results, using strain M145, imply that DO levels may influence the cultivation reproducibility. The best reproducibility of antibiotic productivities was obtained by providing a minimum of 50% DO by automatic adjustment of stirrer speed.
As demonstrated using SSBM-P and SSBM-E, respectively, including several biological replicas for high time resolution sampling for transcriptome, proteome and metabolome analysis, the refined cultivation system applying 50% DO also fulfilled the requirement of good reproducibility in full-scale experiments. Different aspects of metabolic switching have already been studied in detail and published by us based on the cultivations and respective fermentation data included in the present study [19
]. These studies confirm the overall high reproducibility between biological replicas cultivated using the optimized system presented here and the quality of derived time-course samples for analyses on the molecular levels of global gene expression (transcriptome analysis), translation (proteome analysis) and intracellular metabolite pools (metabolite profiling), including strain M145 as well as different mutant strains. At this time, several further studies are being summarized that built up on the fundamental results of this study. Results from such studies will further unravel the complex interplay of molecular events during metabolic switching in strain M145 and its derivatives. An integrated molecular study of the effect of phosphate depletion using medium SSBM-P will for example allow for a global analysis of effects from the documented complex crosstalk of central regulators like PhoP, GlnR and AfsR [42
] and its involvement in antibiotic production. SSBM-E may be applied to study combined molecular responses of nitrogen and carbon depletion, possibly triggering a more general stress response. However, the limiting component in this medium, L-glutamate, can obviously serve as both a carbon and nitrogen source as indicated by growth on L-glutamate alone both in (i) shake flask experiments (see Additional file 1: Table S1
) and (ii) 1
L batch fermentation (ferm. #22, Figures
and C), as well as (iii) by 13
C distribution given in Figure
B where carbon from L-glutamate was detected in all analysed metabolites of the central carbon metabolism. On the other hand, ammonium, accumulating in the medium during growth on L-glutamate (fermentations #19a-c in Additional file 1: Figure S1
) and a possible alternative nitrogen source to L-glutamate, did not maintain growth after L-glutamate depletion (Figure
), and cultivation on D-glucose and ammonium did not promote growth in shake flasks ( Additional file 1: Table S1
) and batch fermentation (ferm. #21; Figures
and C). However, in the latter, growth could be triggered when L-glutamate was added after 80
h of cultivation (see fermentation #21 in Additional file 1: Figure S1
). Therefore, if it was desired to study separate depletion of nitrogen and carbon at a systems scale, further media development would be needed. However, this would require extensive additional efforts and possibly an alternative approach of media development than the one applied in the present study.
The most striking result of the present study, however, is the complex interplay of the two sources of carbon and energy, D-glucose and L-glutamate, which need to be present to ensure a sufficiently high growth rate to prevent an early start of antibiotic production prior to either phosphate or L-glutamate depletion. When strain M145 was grown on medium SSBM-P with both carbon sources in excess, after depletion of phosphate at a cell mass of >4
g/L CDW, production of first prodiginines and subsequently actinorhodins was triggered. When D-glucose was excluded and L-glutamate remained the sole carbon source in the medium, culture growth was slightly slower, likely in response to the additional metabolic burden of the need for complete gluconeogenesis to provide precursors for cell wall synthesis and pentoses for nucleic acid biosynthesis. However, the reduction in growth rate was obviously sufficient to trigger production of blue pigmented secondary metabolites at a premature stage, simultaneously with biomass accumulation and prior to the main triggering event of phosphate depletion. Interestingly, only TBP and not RED production was triggered prematurely. This may be explained by the RED production being sensitive to phosphate repression, thus still being dependent on phosphate reduction in addition to a reduced growth rate, as also observed for a number of other antibiotics [31
When the ratio of L-glutamate and phosphate in SSBM-P was altered in a way that when L-glutamate was depleted phosphate stayed in excess (as provided by SSBM-E), culture growth stopped immediately, reducing the carbon dioxide evolution rate abruptly to metabolic maintenance alone. L-glutamate appears to be both the preferred source of nitrogen and carbon, preferred in that sense that alternatives exist for both at the time of L-glutamate depletion: D-glucose was provided in excess at any time during the fermentation, and ammonium has been shown to accumulate during growth phase to significant amounts (fermentations #19a-c in Additional file 1: Figure S1
). The collapse in carbon dioxide production after L-glutamate depletion implies that L-glutamate catabolism provides the main source of energy for biomass accumulation. If solely D-glucose was provided from the beginning as the sole carbon source in combination with inorganic nitrogen provided as ammonium salts, no significant culture growth could be detected in the fermentors.
Growth experiments in the presence of L-glutamate and 13
-D-glucose, and subsequent metabolite analysis revealed carbon from L-glutamate in significant amounts in metabolites derived from intermediates of glycolysis. In turn, some D-glucose derived carbon was found in metabolites closely linked to the TCA cycle, but at considerably lower concentrations than from L-glutamate.
These key results from labelling and cultivation experiments allow for speculations about the function of the central carbon metabolism in strain M145. When both D-glucose and L-glutamate are provided in excess, carbon flow from D-glucose through the Embden-Meyerhof-Parnas pathway and the hexose monophosphate shunt/pentose phosphate pathway (PPP), and L-glutamate through the TCA cycle both result in the production of pyruvate. L-glutamate is catabolised via α-ketoglutarate following deamination and secretion of ammonium ions into the medium, the α-ketoglutarate is decarboxylated to malate, via the TCA cycle, which in turn is decarboxylated to pyruvate via malic enzyme (malate dehydrogenase, decarboxylating; Omara, W. A. M. and Hodgson, D. A., manuscript in preparation). Catabolism of L-glutamate to pyruvate generates ten ATP molecules (presuming complete oxidation of reduced nucleotides). Catabolism of D-glucose via the Emden-Meyerhof-Parnas pathway to two molecules of pyruvate yields seven ATP molecules. Pyruvate must be subsequently decarboxylated via pyruvate dehydrogenase to acetyl-CoA, the precursor of fatty acid production and for both actinorhodin and prodiginine production.
When phosphate was depleted from SSBM-P, the continued presence of both carbon sources ensured the production of actinorhodins from acetyl CoA and also prodiginines with their more complex precursor supply pattern based on acetyl-CoA, malonyl-CoA, proline, serine, glycine and S-adenosyl methionine.
In the fermentation on SSBM-E, L-glutamate depletion after 35 hours caused the collapse in the carbon dioxide evolution rate as a consequence of growth cessation, clearly indicating that the entrance of carbon from D-glucose into the TCA-cycle in the absence of L-glutamate is largely impaired. Although L-glutamate was also used as the source of nitrogen, nitrogen in the form of increasing amounts of ammonium remained in the medium, resulting from L-glutamate catabolism. Therefore, the cessation of biomass accumulation upon L-glutamate exhaustion in SSBM-E medium, was probably due to a number of factors: concomitant exhaustion of a preferred carbon and energy source; exhaustion of a preferred nitrogen source; and a stringent response due to the nutrient down from use of amino acid to that of ammonium as nitrogen source. S. coelicolor
A3(2) has been shown to have a classic stringent response [30
]. After L-glutamate depletion, production of secondary metabolites was still triggered, however, absolute levels were comparably low to those seen during phosphate depletion. The production was also dominated by actinorhodins, which indicated that carbon flow to acetyl-CoA must have been maintained, while the production of RED was still subject to phosphate repression, as discussed above.
Therefore, we need to explain why carbon dioxide production abruptly decreases following L-glutamate exhaustion and why D-glucose as sole carbon and energy source failed to support growth. A common explanation could be a metabolic bottle neck where pyruvate accumulation results from inefficient conversion to acetyl-CoA by pyruvate dehydrogenase (PDH). Pyruvate accumulation would inhibit cell growth via the weak acid effect causing a drop in intracellular pH [44
]. Surowitz and Pfister (1985) previously demonstrated that toxic accumulation of pyruvate during growth of Streptomyces alboniger
on D-glucose was due to an imbalance between the efficiencies of glycolysis and the TCA cycle [45
]. The two pathways could be balanced by the addition of adenine.
The genes characterised as encoding potential pyruvate dehydrogenase components are: SCO2183 aceE1
pyruvate dehydrogenase (PDH) E1 component (EC 18.104.22.168); SCO2181 aceF1
dihydrolipoyllysine-residue acetyltransferase PDH E2 component (EC 22.214.171.124); SCO2180 lpd1
dihydrolipoamide dehydrogenase PDH E3 component (EC 126.96.36.199); SCO7124 aceE3
PDH E1 component (EC 188.8.131.52); SCO7123 aceF3
PDH E2 component (EC 184.108.40.206); SCO2371 aceE2
PDH E1 component (EC 220.127.116.11); SCO1268 aceF4
PDH E2 component (EC 18.104.22.168); SCO1269 aceEA
PDH E1 β subunit (EC 22.214.171.124); SCO1270 aceEB
PDH E1 α subunit (EC 126.96.36.199); SCO0884 lpd2
dihydrolipoamide dehydrogenases E3 component (EC 188.8.131.52); SCO4919 lpd3
dihydrolipoamide dehydrogenase E3 component (EC 184.108.40.206). The lpd2
products could potentially interact with the glycine dehydrogenase (decarboxylating) complex and brancheD-chain α-ketoacid dehydrogenase complex, however, S. coelicolor
does not encode a lipoamide-dependent 2-oxoglutarate dehydrogenase complex. We studied the expression of the strain's pyruvate dehydrogenase (PDH) gene clusters during growth on a variety of carbon sources ( Additional file 1: Figure S2
). All of the PDH clusters were found to be expressed at a constitutive level. Expression of aceE1
was strongly induced on alanine which is equivalent to growing on pyruvate, when deaminated. These four genes were only mildly induced on glutamate but not induced on glucose or glucose plus glutamate. The fact that growth on pyruvate necessarily requires PDH gene expression but growth on glucose does not induce expression of the pyruvate-inducible PDH supports the proposal of a metabolic bottleneck at the stage of PDH.
Growth on L-glutamate as sole carbon source was almost as good as on both carbon sources, even though both are catabolised to pyruvate. The difference between growth on L-glutamate and growth on D-glucose is that growth on the former yields ten molecules of ATP per molecule of pyruvate produced, whereas growth on the sugar only yields 3.5 molecules of ATP per molecule of pyruvate produced. In addition, when catabolising L-glutamate alone some pyruvate will be used in gluconeogenesis, reducing the pyruvate burden for the cell. This proposed pyruvate bottleneck is consistent with the results from the labelling experiment. We clearly observe that not all carbon in PPP is derived from D-glucose which shows a very flexible usage of the two carbon sources when taken up by the cell. Interestingly, the significant proportion of carbon from L-glutamate in the PPP increases further from 22% to 40% from the active growth phase to the secondary metabolite production phase, respectively.
By growing on both D-glucose and L-glutamate in SSBM-P, as in SSBM-E prior to glutamate depletion, metabolism was characterized by a complex interplay of carbon flow from both carbon sources. L-glutamate for ATP generation and acetyl-CoA for antibiotic production were used efficiently even in the presence of high amounts of D-glucose. By that means, the bacterium’s energy and reducing power needs were provided mainly by L-glutamate catabolism with a supporting role of D-glucose. This ensures rapid growth, a rapid accumulation of biomass, the prevention of a premature triggering of and - after metabolic transition - an efficient production of secondary metabolites.