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

Stable production of commodity chemicals in cyanobacterial cell factories

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Mathematical modeling for the design of evolution experiments to study the genetic instability of metabolically engineered photosynthetic microorganisms

Engineering the metabolism of photosynthetic microorganisms with the aim of converting CO2 and water, by exploiting solar energy, into end-products of commercial value is a rising interest in the biotechnology field. The producing host that carries a genetic modification not associated with competitive fitness advantage usually experiences a production burden (i.e., a metabolic burden related to product synthesis), leading to genetic instability and abortive production phenotype. The genetic instability of these engineered strains is a major hindrance to the spreading of large-scale photosynthetic cell factory processes. This genetic instability can be studied by means of evolution experiments, which are often time-consuming. In these experiments, the cell population is subjected to a long-term culturing during which the possible variation of the number of producers and of cells that lose the production traits, here defined as retro-mutants, is recorded. Here, a mathematical model that describes the dynamics of retro-mutants into a population of metabolically engineered photosynthetic microorganisms has been developed. The model has been used to simulate evolution experiments, conducted both in continuous (chemostat and turbidostat) and semi-continuous (serial batch transfer) culturing modes. These simulations allowed identifying the set of operative parameters for each cultivation mode that optimizes an evolution experiment in terms of experimental time needed to detect the arising of retro-mutants. Moreover, it has been found that in a scale of number of microbial generations only two parameters, precisely the production burden and the mutation rate, are determinant for the appearance of retro-mutants. These parameters are intrinsic features of any metabolically engineered strain and do not depend on the adopted cultivation system or on the microbial growth kinetics characteristics. This result further extends the applicability of the model also to non-photosynthetic metabolically engineered microorganisms

Binding properties of photosynthetic herbicides with the QB site of the D1 protein in plant Photosystem II: a combined functional and molecular docking study

Photosystem II (PSII) is a multi-subunit enzymatic complex embedded in the thylakoid membranes responsible for the primary photosynthetic reactions vital for plants. Many herbicides used for weed control inhibit PSII by interfering with the photosynthetic electron transport at the level of the D1 protein, through competition with the native plastoquinone for the QB site. Molecular details of the interaction of these herbicides in the D1 QB site remain to be elucidated in plants. Here, we investigated the inhibitory effect on plant PSII of the PSII-inhibiting herbicides diuron, metobromuron, bentazon, terbuthylazine and metribuzin. We combined analysis of OJIP chlorophyll fluorescence kinetics and PSII activity assays performed on thylakoid membranes isolated from pea plants with molecular docking using the high-resolution PSII structure recently solved from the same plant. Both approaches showed for terbuthylazine, metribuzin and diuron the highest affinity for the D1 QB site, with the latter two molecules forming hydrogen bonds with His215. Conversely, they revealed for bentazon the lowest PSII inhibitory effect accompanied by a general lack of specificity for the QB site and for metobromuron an intermediate behavior. These results represent valuable information for future design of more selective herbicides with enhanced QB binding affinities to be effective in reduced amounts