posted on 2021-11-22, 23:30authored byBeatrice Battaglino, Wei Du, Cristina Pagliano, Joeri A. Jongbloets, Angela Re, Guido Saracco, Filipe Branco dos Santos
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to oxidize water, cyanobacteria can directly
convert atmospheric CO2 into valuable carbon-based compounds
and meanwhile release O2 to the atmosphere. As such, cyanobacteria
are promising candidates to be developed as microbial cell factories
for the production of chemicals. Nevertheless, similar to other microbial
cell factories, engineered cyanobacteria may suffer from production
instability. The alignment of product formation with microbial fitness
is a valid strategy to tackle this issue. We have described previously
the “FRUITS” algorithm for the identification of metabolites
suitable to be coupled to growth (i.e., side products in anabolic
reactions) in the model cyanobacterium Synechocystis. sp PCC6803. However, the list of candidate metabolites identified
using this algorithm can be somewhat limiting, due to the inherent
structure of metabolic networks. Here, we aim at broadening the spectrum
of candidate compounds beyond the ones predicted by FRUITS, through
the conversion of a growth-coupled metabolite to downstream metabolites
via thermodynamically favored conversions. We showcase the feasibility
of this approach for malate production using fumarate as the growth-coupled
substrate in Synechocystis mutants. A final titer
of ∼1.2 mM was achieved for malate during photoautotrophic
batch cultivations. Under prolonged continuous cultivation, the most
efficient malate-producing strain can maintain its productivity for
at least 45 generations, sharply contrasting with other producing Synechocystis strains engineered with classical approaches.
Our study also opens a new possibility for extending the stable production
concept to derivatives of growth-coupled metabolites, increasing the
list of suitable target compounds.