Multiplicity of carotene patterns derives from competition between phytoene desaturase diversification and biological environments.

Phytoene desaturases catalyse from two to six desaturation reactions on phytoene, generating a large diversity of molecules that can then be cyclised and produce, depending on the organism, many different carotenoids. We constructed a phylogenetic tree of a subset of phytoene desaturases from the CrtI family for which functional data was available. We expressed in a bacterial system eight codon optimized CrtI enzymes from different clades. Analysis of the phytoene desaturation reactions on crude extracts showed that three CrtI enzymes can catalyse up to six desaturations, forming tetradehydrolycopene. Kinetic data generated using a subset of five purified enzymes demonstrate the existence of characteristic patterns of desaturated molecules associated with various CrtI clades. The kinetic data was also analysed using a classical Michaelis-Menten kinetic model, showing that variations in the reaction rates and binding constants could explain the various carotene patterns observed. Competition between lycopene cyclase and the phytoene desaturases modified the distribution between carotene intermediates when expressed in yeast in the context of the full ß-carotene production pathway. Our results demonstrate that the desaturation patterns of carotene molecules in various biological environments cannot be fully inferred from phytoene desaturases classification but is governed both by evolutionary-linked variations in the desaturation rates and competition between desaturation and cyclisation steps.

Development of a Biosensor for Detection of Benzoic Acid Derivatives in Saccharomyces cerevisiae.

4-hydroxybenzoic acid (pHBA) is an important industrial precursor of muconic acid and liquid crystal polymers whose production is based on the petrochemical industry. In order to decrease our dependency on fossil fuels and improve sustainability, microbial engineering is a particularly appealing approach for replacing traditional chemical techniques. The optimization of microbial strains, however, is still highly constrained by the screening stage. Biosensors have helped to alleviate this problem by decreasing the screening time as well as enabling higher throughput. In this paper, we constructed a synthetic biosensor, named sBAD, consisting of a fusion of the pHBA-binding domain of HbaR from R. palustris, the LexA DNA binding domain at the N-terminus and the transactivation domain B112 at the C-terminus. The response of sBAD was tested in the presence of different benzoic acid derivatives, with cell fluorescence output measured by flow cytometry. The biosensor was found to be activated by the external addition of pHBA in the culture medium, in addition to other carboxylic acids including p-aminobenzoic acid (pABA), salicylic acid, anthranilic acid, aspirin, and benzoic acid. Furthermore, we were able to show that this biosensor could detect the in vivo production of pHBA in a genetically modified yeast strain. A good linearity was observed between the biosensor fluorescence and pHBA concentration. Thus, this biosensor would be well-suited as a high throughput screening tool to produce, via metabolic engineering, benzoic acid derivatives.