Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0757-y) contains supplementary material, which is available to authorized users.

Terpenoid Metabolic Engineering in Photosynthetic Microorganisms

“…Englund and co-workers produced 13R-manoyl oxide and reached titres of 0.75 mg L −1 [ 101 ]. Manoyl oxide yield improved to 2 mg L −1 by codon-optimizing the terpene synthases and expressing them in a self-replicating vector under a strong promoter [ 82 ].…”

“…An increased biomass productivity caused by a sink effect has been observed in other metabolic engineering studies (e.g., [ 103 ]). Cyanobacteria seem to tolerate the redirection of carbon towards heterologous terpenoid production and increase the flux towards the MEP pathway to compensate for the extra resources consumed [ 82 ]. The MEP pathway provides precursors for carotenoids and chlorophyll, which potentially render any significant imbalance due to heterologous terpenoid production unfavorable for growth.…”

“…The MEP pathway provides precursors for carotenoids and chlorophyll, which potentially render any significant imbalance due to heterologous terpenoid production unfavorable for growth. Nevertheless, negative effects such as growth retardation and reduction in oxygen evolution have been observed routinely [ 78 , 82 , 104 ]. An interesting observation is that knocking out competing pathways does not seem to improve terpenoid production, which is observed when glycogen synthase and squalene hopene cyclase were knocked out in an attempt to enhance limonene and manoyl oxide production, respectively [ 75 , 101 ].…”

“…Cyanobacteria seems to have efficient regulation mechanisms that buffer the effects of metabolic perturbations to maintain homeostasis in primary metabolism. This can be both advantageous (as important native branching pathways are not affected by the heterologous ones) but may also hinder metabolic engineering [ 82 ]. Another noteworthy observation is that different cyanobacterial strains behave very differently.…”

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

“…Cyanobacteria are autotrophic bacteria that can be used for the biosynthesis of a wide variety of natural and heterologous high value metabolic products 1,2,3,4,5,6 . Several hurdles still need to be overcome to expand their commercial viability, most notably, the relatively poor yields compared to heterotrophic bio-platforms (e.g., Escherichia coli and yeast) 7 .…”