Impairing photorespiration increases photosynthetic conversion of CO2 to isoprene in engineered cyanobacteria

Photorespiration consumes fixed carbon and energy generated from photosynthesis to recycle glycolate and dissipate excess energy. The aim of this study was to investigate whether we can use the energy that is otherwise consumed by photorespiration to improve the production of chemicals which requires energy input. To this end, we designed and introduced an isoprene synthetic pathway, which requires ATP and NADPH input, into the cyanobacterium Synechocystis sp. 6803. We then deleted the glcD1 and glcD2 genes which encode glycolate dehydrogenase to impair photorespiration in isoprene-producing strain of Synechocystis. Production of isoprene in glcD1/glcD2 disrupted strain doubled, and stoichiometric analysis indicated that the energy saved from the impaired photorespiration was redirected to increase production of isoprene. Thus, we demonstrate we can use the energy consumed by photorespiration of cyanobacteria to increase the energy-dependent production of chemicals from CO2.

Stoichiometry and Kinetics of Sequential Dimethacrylate Enzymolysis

The increasing use of methacrylate-based materials in tissue engineering and dental restorations demands detailed evaluation of enzymolysis of these materials due to toxicity, durability, and biocompatibility concerns. The objective of this study is to develop tools for assessing and ranking the enzymolysis kinetics of dimethacrylate (DMA) compounds. Triethyleneglycol DMA and diurethane DMA are employed as model DMAs for kinetic studies of 2-step enzymolysis by 2 esterases, pseudocholine esterase and cholesterol esterase. In addition, the intermediate hydrolysis products, mono-methacrylates (mono-MAs), are prepared via esterases. The kinetics of DMA enzymolysis are evaluated per the concentrations of DMA. The enzymolysis products are quantified by high-performance liquid chromatography. Additionally, stoichiometric analysis and a Berkeley Madonna model are employed to compare the efficacy of esterases in DMA enzymolysis. The chemical structure of mono-MAs is verified by proton and heteronuclear single quantum coherence (2D 1H-13C) nuclear magnetic resonance spectroscopy and mass spectrometry. In evaluating the ratio of sequential and simultaneous degradations of DMA and mono-MA, the stoichiometric analysis draws the same conclusions without using [mono-MA] as the experimental observation using [mono-MA]. The majority of the 4 esterase-DMA combinations undergo the sequential enzyme-catalyzed hydrolysis, from DMA to mono-MA to diol. However, cholesterol esterase is more effective than pseudocholine esterase in maintaining sequential degradation until >90% of DMA is decomposed. Both enzymolysis steps are first-order reactions. The mono-MAs are more hydrolysis resistant than DMAs. Moreover, esterase efficacy and selectivity on DMA enzymolysis are presented. The stoichiometric analysis provides valuable tools in assessing DMA enzymolysis when mono-MA is difficult to be obtained. The resistance of mono-MAs to enzymolysis suggests a need for thorough toxicity evaluations of these intermediate compounds. It also advocates the alternative approaches in designing and developing durable and biocompatible materials.