Primary metabolism regulation and antibiotic biosynthesis by industrial bacteria, Streptomyces

Ce travail décrit l’analyse de la distribution des flux de carbones au sein de deux souches de Streptomyces coelicolor A3(2) : la souche sauvage nommée M145 et son mutant M1146 incapable de produire les antibiotiques actinorhodine, undecylprodigiosine, et l’antibiotique dépendant du calcium. Metabolite Balance Analysis et Isotopomer Balance Analysis sont mis en œuvre pour proposer un modèle de distribution des flux de carbones de S. coelicolor en phase exponentielle de croissance. Les souches M145 et M1146 sont cultivées dans un milieu minimum limitant en azote et leurs comportements métaboliques sont comparés. Dans la souche non productrice M1146, un taux de croissance plus élévé, un flux plus important dans la voie des pentoses phosphates, un flux plus faible au niveau du cycle de Krebs ainsi qu’une activité respiratoire plus faible sont mis en évidence. Cela traduit le coût énergétique important associé à la production d’actinorhodine par M145. De plus, ce travail propose un rôle important de la nicotinamide nucléotide transhydrogénase pour le maintien de l’homéostasie du NADPH lors de la production d’actinorhodine par M145. Comme il existe de bonnes corrélations entre les données expérimentales et celles issues de la modélisation au niveau des bilans carbones, des bilans de pouvoir réducteur et des échanges gazeux, il sera intéressant d’utiliser cette modélisation avec la technique de Flux Balance Analysis pour prédire les variations de la distribution des flux de carbones dans des mutants de S. coelicolor pour lesquels des gènes auraient été sur-exprimés ou délétés.This work describes an analysis of carbon flux distribution in two strains of Streptomyces coelicolor A3(2), namely the wild type strain M145 and its derivative M1146 that is no longer able to produce the antibiotics actinorhodin, undecylprodigiosin and the calcium dependent antibiotic. Metabolite Balance Analysis and Isotopomer Balance Analysis were used to propose a model for carbon flux distribution in S. coelicolor during the exponential phase of growth. Strains M145 and M1146 were grown under nitrogen limitation in minimal medium and their metabolic behaviour were compared. In the non-producing strain M1146, a higher growth rate, a higher flux via the pentose phosphate pathway, a decreased flux through the TCA cycle and a decreased respiratory activity were evidenced. This highlighted the high energetic cost for actinorhodin production in M145. In this paper, we also propose a key role for the nicotinamide nucleotide transhydrogenase in NADPH homeostasis in M145 during actinorhodin production. As there are good correlations between experimental data and the model in terms of carbon balance, reducing power balance and gas exchanges, this model will be of great interest for Flux Balance Analysis to predict carbon-flux distribution changes in S. coelicolor strains in which gene are deleted or overexpressed

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

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

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

Two lignocellulosic agricultural residues, sunflower stalks and rape straw, were investigated as potential low-cost, non-food substrates for the production of triacylglycerols by the oleaginous, lignocellulolytic bacteria Streptomyces lividans. Chemical analysis of each type of residue revealed similar cell wall compositions in the polysaccharides and lignins of the two feedstocks, with high lignin β-O-4 bond content compared to other angiosperms’ lignin. Growing tests of Streptomyces lividans TK 24 were performed before and after sequential water and ethanol extraction by assessing bacterial fatty acid accumulation. All extracted and non-extracted samples were found to be substrates of the bacteria with fatty acid production ranging between 19% and 44% of the production obtained with arabinose as a reference substrate. The maximum conversion rate was obtained with the less lignified, non-extracted sample. This study suggests that lignocellulosic residues from oleaginous crops could be advantageously valorized by microbial bioconversion processes for the production of lipids of interest

Bioconversion of agricultural lignocellulosic residues into branched-chain fatty acids using

Two lignocellulosic agricultural residues, sunflower stalks and rape straw, were investigated as potential low-cost, non-food substrates for the production of triacylglycerols by the oleaginous, lignocellulolytic bacteria Streptomyces lividans. Chemical analysis of each type of residue revealed similar cell wall compositions in the polysaccharides and lignins of the two feedstocks, with high lignin β-O-4 bond content compared to other angiosperms’ lignin. Growing tests of Streptomyces lividans TK 24 were performed before and after sequential water and ethanol extraction by assessing bacterial fatty acid accumulation. All extracted and non-extracted samples were found to be substrates of the bacteria with fatty acid production ranging between 19% and 44% of the production obtained with arabinose as a reference substrate. The maximum conversion rate was obtained with the less lignified, non-extracted sample. This study suggests that lignocellulosic residues from oleaginous crops could be advantageously valorized by microbial bioconversion processes for the production of lipids of interest

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

<div><p>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 <i>Streptomyces coelicolor</i> 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 phospho<i>enol</i>pyruvate 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 <i>S. coelicolor</i> as a platform for the production of bio-based products of industrial interest.</p></div

Precursors, energy and reducing power needs for 1 g dry biomass synthesis in <i>S. coelicolor</i>.

<p>Positive values correspond to metabolite consumptions and negative values correspond to metabolite productions.</p

Carbon flux distribution in <i>S. coelicolor</i> M145.

<p>Grey arrows represent carbon fluxes within the central metabolism of <i>S. coelicolor</i> M145. Arrow widths are proportional to carbon fluxes. Flux values in the central metabolism are included in boxes with solid lines, flux values to biomass are included in boxes with dotted lines. The upper numbers represent average actual fluxes (μmol (g dry mass)⁻¹ h⁻¹), the lower number represent average normalized fluxes (μmol (μmol glucose)⁻¹). Positive values correspond to consumption and negative values to production.</p

Kinetic parameters and GC-MS analyses.

<p>Kinetic parameters were experimental values obtained as described in 2.19.1. Mass Isotopic Distributions were normalized as described in section 2.19.4. Values were means of two independent experiments and RSD their relative standard deviations. Glu: Glutamic acid; Thr: Threonine; Asp: Aspartic acid; Ala: Alanine; Val: Valine.</p><p>For <i>q_S</i>, <i>γ_ACT</i>, <i>q_O2</i> and <i>γ_CO2</i>, positive values correspond to metabolite consumption and negative values correspond to metabolite production.</p

Biomass composition of <i>S. coelicolor</i> M145 cells (μmol (g dry cells)⁻¹).

a<p>UDP-N-Acetylglucosamine</p>b<p>UDP-N-Acetylmuramic acid</p

Macromolecules content of <i>S. coelicolor</i> M145 cells.

a<p>Biomass composition (% dry cell weight) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084151#pone.0084151-Borodina2" target="_blank">[71]</a>.</p>b<p>Calculated from our composition of building blocks (% dry cell weight)</p>c<p>Relative standard deviation (%) between published and calculated values.</p