Engineering synergetic CO2-fixing pathways for malate production.

Increasing the microbial CO2-fixing efficiency often requires supplying sufficient ATP and redirecting carbon flux for the production of metabolites. However, addressing these two issues concurrently remains a challenge. Here, we present a combinational strategy based on a synergetic CO2-fixing pathway that combines an ATP-generating carboxylation reaction in the central metabolic pathway with the ATP-consuming RuBisCO shunt in the carbon fixation pathway. This strategy provides enough ATP to improve the efficiency of CO2 fixation and simultaneously rewires the CO2-fixing pathway to the central metabolic pathway for the biosynthesis of chemicals. We demonstrate the application of this strategy by increasing the CO2-fixing rate and malate production in the autotroph Synechococcus elongatus by 110% and to 260 µM respectively, as well as increasing these two factors in the heterotrophic CO2-fixing Escherichia coli by 870% and to 387 mM respectively.

Immobilization of fumarase from thermophilic eukaryotic red alga Cyanidioschyzon merolae on ceramic carrier.

Fumarase is an enzyme catalyzing reversible reaction between fumarate and L-malate in the citric acid cycle. Fumarase is used in the industrial production of L-malate, and its immobilization is required for reuse of the fumarases to reduce the cost. Accordingly, understanding the properties of immobilized fumarase is crucial, and several groups report on the storage stability and kinetic parameters of immobilized fumarase. Here we have immobilized fumarase from the thermophilic red alga Cyanidioschyzon merolae (CmFUM) on ceramic beads and investigated its biochemical and physical properties. CmFUM demonstrated sufficient stability and reusability for industry use after immobilization. Notably, the thermostability was dramatically enhanced through immobilization. The Km value and kcat of immobilized CmFUM for fumarate were 1.7 mM and 22.7 s-1 respectively. The Km value for fumarate was lower than that of other reported immobilized fumarases, indicating a high substrate affinity of immobilized CmFUM. Furthermore, the enhanced stability resulting from immobilization partially compensated for the decrease in activity. The high affinity towards fumarate and good thermostability of immobilized CmFUM revealed in this study are advantageous traits for improving enzyme-mediated isomer-specific L-malate production.

Study of silanized nanostructures with immobilized fumarase for production of L-malate.

The conversion of fumaric acid into L-malate by fumarase immobilized on silanized nanostructures was analyzed experimentally. The enzyme was bound to the silanized nanostructures. We carried out scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR) analysis, zeta size analysis and surface area calculation for the characterization of the nanostructures. The effect of initial enzyme concentration and pH on immobilization procedure were investigated and the change of Michaelis-Menten constants (Km and Vmax) with immobilization was examined. The change in the storage stability of the enzyme by immobilization was also investigated. The stability of the immobilized enzyme was very good. We observed that the fumarase was bound to silanized nanostructures [p(HEMA)-3-MTES] in much greater amounts. We have compared the activities of free fumarase and immobilized fumarase and we have observed a significant increase in the activity of the fumarase after immobilization for L-malate production. Moreover, we came to the conclusion that this activity can be better preserved for 30 days compared to free fumarase.

Channeling Anabolic Side Products toward the Production of Nonessential Metabolites: Stable Malate Production in Synechocystis sp. PCC6803.

Powered by (sun)light 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.