Abstract
BackgroundThere are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria.ResultsIn this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites.ConclusionsThere is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to ‘traditional’ metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.
Highlights
There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds
Expression of the two heterologous pathways Dhurrin is a cyanogenic glucoside derived from tyrosine, and its biosynthetic pathway involves two cytochrome P450s that convert tyrosine into p-hydroxymandelonitrile and a UDP-glycosyltransferase that transfers a glucose from UDP-glucose (Fig. 1a)
The second compound we chose is the forskolin precursor 13R-manoyl oxide, a diterpenoid derived from geranylgeranyl diphosphate by the enzymatic action of two diterpene synthases [13] (Fig. 1a)
Summary
There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria. Vavitsas et al Microb Cell Fact (2017) 16:140 are another diverse class of metabolic products They consist of a carbon skeleton that is formed from one or multiple isoprenoid units, further decorated by modifying enzymes [7]. We showed that Synechocystis can produce dhurrin, a cyanogenic glucoside, in a light-dependent manner, using tyrosine and UDP-glucose as precursor molecules [11]
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