Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria.

George M Taylor,Andrew Hitchcock,John T Heap

doi:10.1093/nar/gkab791

George M Taylor, Andrew Hitchcock + Show 1 more

Open Access

https://doi.org/10.1093/nar/gkab791

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Abstract

Cyanobacteria are simple, efficient, genetically-tractable photosynthetic microorganisms which in principle represent ideal biocatalysts for CO2 capture and conversion. However, in practice, genetic instability and low productivity are key, linked problems in engineered cyanobacteria. We took a massively parallel approach, generating and characterising libraries of synthetic promoters and RBSs for the cyanobacterium Synechocystis sp. PCC 6803, and assembling a sparse combinatorial library of millions of metabolic pathway-encoding construct variants. Genetic instability was observed for some variants, which is expected when variants cause metabolic burden. Surprisingly however, in a single combinatorial round without iterative optimisation, 80% of variants chosen at random and cultured photoautotrophically over many generations accumulated the target terpenoid lycopene from atmospheric CO2, apparently overcoming genetic instability. This large-scale parallel metabolic engineering of cyanobacteria provides a new platform for development of genetically stable cyanobacterial biocatalysts for sustainable light-driven production of valuable products directly from CO2, avoiding fossil carbon or competition with food production.

Highlights

Future sustainable economies consistent with net zero greenhouse gas emissions and limiting climate change will require radical changes in global carbon flows, including the development and large-scale deployment of technologies which capture and recycle carbon
At the start of this study there was a lack of promoter and ribosome-binding sites (RBSs) parts suitable for controlling and tuning gene expression in cyanobacteria ( some examples have been published [54,55])
Genetic stability is essential for industrial microorganisms, so the reported genetic instability of genetically modified cyanobacteria [38] is a key challenge for cyanobacterial synthetic biology [8,32,73,74,75,76]

Summary

Introduction

Future sustainable economies consistent with net zero greenhouse gas emissions and limiting climate change will require radical changes in global carbon flows, including the development and large-scale deployment of technologies which capture and recycle carbon. Cyanobacteria are the simplest and most genetically-tractable organisms capable of oxygenic photosynthesis, using CO2 and sunlight as their sole carbon and energy sources, respectively. Their photosynthetic yield [1] and growth rate [2] are similar to fast-growing microalgae, and greater than terrestrial plants [3,4].

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