Streptomyces clavuligerus shows a strong association between TCA cycle intermediate accumulation and clavulanic acid biosynthesis

Abstract

Clavulanic acid (CA) is produced by Streptomyces clavuligerus (S. clavuligerus) as a secondary metabolite. Knowledge about the carbon flux distribution along the various routes that supply CA precursors would certainly provide insights about metabolic performance. In order to evaluate metabolic patterns and the possible accumulation of tricarboxylic acid (TCA) cycle intermediates during CA biosynthesis, batch and subsequent continuous cultures with steadily declining feed rates were performed with glycerol as the main substrate. The data were used to in silico explore the metabolic capabilities and the accumulation of metabolic intermediates in S. clavuligerus. While clavulanic acid accumulated at glycerol excess, it steadily decreased at declining dilution rates; CA synthesis stopped when glycerol became the limiting substrate. A strong association of succinate, oxaloacetate, malate, and acetate accumulation with CA production in S. clavuligerus was observed, and flux balance analysis (FBA) was used to describe the carbon flux distribution in the network. This combined experimental and numerical approach also identified bottlenecks during the synthesis of CA in a batch and subsequent continuous cultivation and demonstrated the importance of this type of methodologies for a more advanced understanding of metabolism; this potentially derives valuable insights for future successful metabolic engineering studies in S. clavuligerus.

Characterization of the Metabolic Response of Streptomyces clavuligerus to Shear Stress in Stirred Tanks and Single-Use 2D Rocking Motion Bioreactors for Clavulanic Acid Production

“…In our case, the decline in RQ was accompanied by a slight decrease in the specific growth rate (Figure 1), and the accumulation of pyruvate and succinate, respectively ( Figure 6). Previously, Ramirez-Malule et al [16] also reported the accumulation of succinate during CA production in 0.3 L of continuous cultivations under different feeding conditions; however, pyruvate was not observed to be accumulated under such conditions. According to Viollier et al [52] the secretion of glycolytic and TCA intermediates in Streptomyces evidences the metabolic imbalance in the central metabolism.…”

“…In the case of S. clavuligerus, little is known about the relationship between shear stress, growth, morphology, and metabolite secretion. The dispersed mycelial morphology has been observed more often in S. clavuligerus cultivations performed in bioreactors and, in contrast to other phylogenetically related species, pellet formation seems to be related with a decrease in the CA production rate [16,17].…”

Genome‐Scale Metabolic Reconstruction of Actinomycetes for Antibiotics Production

“…[38] This simulation allowed explaining a strong association between TCA cycle and CA production, and suggested potential engineering targets in metabolism, such as glutamate dehydrogenase and aspartate aminotransferase, for the enhanced CA overproduction. [38] Despite the efforts made so far, modeling highly complex secondary metabolism should be further systematically challenged. Relevant examples include parsimonious FBA [39] and probabilistic regulatory modeling.…”

“…Simultaneous manipulation of the genes predicted above resulted in the best engineered strain showing 2.3‐fold increase in the tacrolimus production, compared to the wild‐type strain . Finally, a two‐stage optimization was conducted using an updated GEM of S. clavuligerus wherein biomass generation and clavulanic acid (CA) production were first maximized, and the total sum of intracellular fluxes was subsequently minimized along with experimentally obtained constraints for the key metabolites . This simulation allowed explaining a strong association between TCA cycle and CA production, and suggested potential engineering targets in metabolism, such as glutamate dehydrogenase and aspartate aminotransferase, for the enhanced CA overproduction …”

“…(2018) [15] 1522/2304/1927/27 [14] RAVEN Toolbox 2; MetaCyc; KEGG; iMK1208; and manual curation [14] 11: Actinorhodin; calcium-dependent antibiotic; undecylprodigiosin; ectoine; germicidin; flaviolin; geosmin; glbaflavenone; 2methyllisoborneol; hopanoid; and desferrioxamine FSEOF for all the secondary metabolites production; [14] FBA for the triacylglycerol accumulation; [55] tSOT for the actinorhodin production; [41] FBA for the actinorhodin production; [12] MOMA with gene expression data for the metabolic analysis of a phosphofructokinase knockout mutant; [54] and FVA for feed formulation for the actinorhodin production using a zwf knockout strain [53] 4.3-Fold increase in the triacylglycerol content via fadAB knockouts; [55] 2and 1.8-fold increases in the actinorhodin yield via overexpressions of ribulose 5phosphate 3epimerase and NADP-dependent malic enzyme, respectively; [41] and 3-fold increase in the actinorhodin yield via zwf knockout, which was further improved by 18% via glutamate feed [53] 1259/1912/1470/27 [15] [17] 2: Clavulanic acid; and cephamycin C RobOKoD for the clavulanic acid production; [17] FBA for the analysis of relationship between TCA cycle and clavulanic acid production; [38] and FBA for the analysis of growth and clavulanic acid production capabilities [16,17,56] NA a) [23] 1: Tacrolimus (FK506)…”