Development of in silico strategies to photoautotrophically produce poly-β-hydroxybutyrate (PHB) by cyanobacteria

Abstract

Poly(3-hydroxybutyrate) (PHB), a biopolymer similar to polypropylene, is currently produced by heterotrophic bacteria. However, the elevated cost of raw material used as carbon and energy sources are promoting the development of new sustainable bioprocess based on photoautotrophic routes costs. In unbalanced growth conditions, some cyanobacteria can accumulate PHB using CO2 as the sole carbon source, becoming an option for PHB production. In this work, we study in silico PHB production with the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). We consider two different strategies for in silico strain design: coupled growth-PHB production and a two-stage fermentation strategy. For both strategies we consider a GEnome-scale Model (GEM) of Synechocystis, which was modified to account for PHB production. The coupled approach relies on a bilevel optimization problem that identifies gene knock-outs needed to obtain a mutant that couples PHB production with biomass formation. For this case, two mutants that successfully achieve the required coupling are obtained, and the best one is analyzed in terms of intervention strategies, flux distributions and productivities. For the two-stage fermentation strategy, we consider two bioreactors in series. The first one is optimized for biomass production under balanced growth conditions, and the second one is optimized for PHB production under nitrogen starvation conditions. Both stages are simulated within dfba, a python tool for solving the Dynamic Flux Balance Analysis (DFBA) problem which provides the solution of a bioreactor model subject to the mass balances of the GEM. Numerical results for the two-stage approach provide a PHB concentration of 4.764 g PHB/L while the mutant provides a lower concentration of 0.391 g PHB/L. Nevertheless, both strategies provide high PHB content per cell dry weight (cdw), which makes results attractive for photosynthetic PHB production.