Carbon-Flux Distribution within Streptomyces coelicolor Metabolism: A Comparison between the Actinorhodin-Producing Strain M145 and Its Non-Producing Derivative M1146

Abstract

Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and 13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.

Enzyme-constrained models and omics analysis of Streptomyces coelicolor reveal metabolic changes that enhance heterologous production

“…Meanwhile, nucleoside triphosphate levels strongly regulate ribosomal RNA synthesis, and we therefore hypothesize that a higher ATP/ADP ratio in M1152 compared to M145 after phosphate depletion may be causing the differences in expression of ribosomal proteins. The observed upregulation of the ATP-synthase gene cluster supports this hypothesis (Figure S9A), and a high level of ATP has been reported in the M1146 strain parental to M1152 (Coze et al, 2013b) which also lacks the 4 major BGCs but does not feature the rpoB mutation. Regardless, neither of these hypotheses can explain why a difference in ribosomal protein expression is only seen in production phase, and the correlation with the metabolic switch indicates that it may as well be related to the lack of the four BGCs or the produced compounds.…”

“…Meanwhile, simulations with EcSco-GEM reveal that more than half of the glutamate-derived nitrogen is excreted as ammonia into the medium ( Figure S6B). A reduced flux through glycolysis has previously been reported for strain M1146 lacking the four BGCs, and it is suggested that this is caused by allosteric inhibition of phosphofructokinase by a higher ATP/ADP ratio in the M1146 strain (Coze et al, 2013a;Esnault et al, 2017).…”

“…The origin of the reduced growth rate of M1152 remains uncertain, while our results indicate that the growth deficiency can be caused by reduced glucose uptake and the resulting reorganization of central carbon metabolism or by the rpoB mutation, possibly in interaction with the BGC deletions. It is intriguing that the M1146 mutant strain in contrast has an increased growth rate compared to M145 (Coze et al, 2013a), as the only difference between these two strains is the rpoB single point mutation, which has been shown to enhance antibiotic production without growth impairment in S. lividans (Hu et al, 2002). However, epistatic interactions involving rpoB has caused unexpected results in S. coelicolor previously (Lopatniuk et al, 2014), and different cultivation conditions when independently comparing strains may contribute to obtaining contradictory results.…”

Disruption of Macrodomain Protein SCO6735 Increases Antibiotic Production in Streptomyces coelicolor

“…Intracellular and extracellular actinorhodin contents were quantified following the protocol (56) with a single modification using 1 m NaOH instead of 1 m KOH. Intracellular actinorhodin content was quantified from 1-ml culture pellet, whereas supernatant was used to quantify the extracellular γ-actinorhodin.…”

Bioconversion of agricultural lignocellulosic residues into branched-chain fatty acids usingStreptomyces lividans

“…Lignocellulosic substrates (1.2 g) were sterilized with 19 ml of water in 150-ml growth flasks, and 1 ml of minimal medium without glucose. (Coze et al, 2013) was added to obtain a final volume of 20 ml. Inoculations were done with 3 × 10 8 spores per flask.…”