Channeling anabolic side-products towards the production of non-essential metabolites: stable malate production in Synechocystis sp. PCC6803

[Image: see text] Powered by (sun)light to oxidize water, cyanobacteria can directly convert atmospheric CO(2) into valuable carbon-based compounds and meanwhile release O(2) 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

Exploiting Day- and Night-Time Metabolism of Synechocystis sp. PCC 6803 for Fitness-Coupled Fumarate Production around the Clock

Cyanobacterial cell factories are widely researched for the sustainable production of compounds directly from CO2. Their application, however, has been limited for two reasons. First, traditional approaches have been shown to lead to unstable cell factories that lose their production capability when scaled to industrial levels. Second, the alternative approaches developed so far are mostly limited to growing conditions, which are not always the case in industry, where nongrowth periods tend to occur (e.g., darkness). We tackled both by generalizing the concept of growth-coupled production to fitness coupling. The feasibility of this new approach is demonstrated for the production of fumarate by constructing the first stable dual-strategy cell factory. We exploited circadian metabolism using both systems and synthetic biology tools, resulting in the obligatorily coupling of fumarate to either biomass or energy production. Resorting to laboratory evolution experiments, we show that this engineering approach is more stable than conventional methods

Alignment of microbial fitness with engineered product formation: obligatory coupling between acetate production and photoautotrophic growth

Background: Microbial bioengineering has the potential to become a key contributor to the future development of human society by providing sustainable, novel, and cost-effective production pipelines. However, the sustained productivity of genetically engineered strains is often a challenge, as spontaneous non-producing mutants tend to grow faster and take over the population. Novel strategies to prevent this issue of strain instability are urgently needed. Results: In this study, we propose a novel strategy applicable to all microbial production systems for which a genome-scale metabolic model is available that aligns the production of native metabolites to the formation of biomass. Based on well-established constraint-based analysis techniques such as OptKnock and FVA, we developed an in silico pipeline—FRUITS—that specifically ‘Finds Reactions Usable in Tapping Side-products’. It analyses a metabolic network to identify compounds produced in anabolism that are suitable to be coupled to growth by deletion of their re-utilization pathway(s), and computes their respective biomass and product formation rates. When applied to Synechocystis sp. PCC6803, a model cyanobacterium explored for sustainable bioproduction, a total of nine target metabolites were identified. We tested our approach for one of these compounds, acetate, which is used in a wide range of industrial applications. The model-guided engineered strain shows an obligatory coupling between acetate production and photoautotrophic growth as predicted. Furthermore, the stability of acetate productivity in this strain was confirmed by performing prolonged turbidostat cultivations. Conclusions: This work demonstrates a novel approach to stabilize the production of target compounds in cyanobacteria that culminated in the first report of a photoautotrophic growth-coupled cell factory. The method developed is generic and can easily be extended to any other modeled microbial production system

Nonhierarchical Flux Regulation Exposes the Fitness Burden Associated with Lactate Production in <i>Synechocystis</i> sp. PCC6803

Cyanobacteria are mostly engineered to be sustainable cell-factories by genetic manipulations alone. Here, by modulating the concentration of allosteric effectors, we focus on increasing product formation without further burdening the cells with increased expression of enzymes. Resorting to a novel 96-well microplate cultivation system for cyanobacteria, and using lactate-producing strains of <i>Synechocystis</i> PCC6803 expressing different l-lactate dehydrogenases (LDH), we titrated the effect of 2,5-anhydro-mannitol supplementation. The latter acts in cells as a nonmetabolizable analogue of fructose 1,6-bisphosphate, a known allosteric regulator of one of the tested LDHs. In this strain (SAA023), we achieved over 2-fold increase of lactate productivity. Furthermore, we observed that as carbon is increasingly deviated during growth toward product formation, there is an increased fixation rate in the population of spontaneous mutants harboring an impaired production pathway. This is a challenge in the development of green cell factories, which may be countered by the incorporation in biotechnological processes of strategies such as the one pioneered here