Background The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited. Results In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E.limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth. Conclusions The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E.limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.
Figure 1: Ray Tracing Visualization Toolkit. An extensible GUI, together with a ray state recording library and a collection of visualization components, integrate existing client renderers via a flexible plug-in architecture to support visual analysis of ray-based rendering algorithms. Here, rtVTK is used to visually debug an ill-behaved path tracer. The previously undiscovered bug is not obvious in either a low-quality image with relatively few samples per pixel (top left) or a high-quality image with many more samples per pixel (bottom left). However, when viewing the ray state directly with rtVTK (middle, right), the problem is immediately obvious: some shadow rays originating from diffuse surfaces (circled) are not directed toward any light source in the scene. Once identified, the correction was trivial; however, in its previous state, the renderer was incorrect, and likely would have remained so without the ability to visualize the ray tracing process itself. AbstractThe Ray Tracing Visualization Toolkit (rtVTK) is a collection of programming and visualization tools supporting visual analysis of ray-based rendering algorithms. rtVTK leverages layered visualization within the spatial domain of computation, enabling investigators to explore the computational elements of any ray-based renderer. Renderers utilize a library for recording and processing ray state, and a configurable pipeline of loosely coupled components allows run-time control of the resulting visualization. rtVTK enhances tasks in development, education, and analysis by enabling users to interact with a visual representation of ray tracing computations.
Background: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.Results: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product yields during growth.Conclusions: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.
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