Actinobacteria challenge the paradigm: A unique protein architecture for a well-known, central metabolic complex

α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high–molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.

Activating silent glycolysis bypasses in Escherichia coli

All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal, the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal immediately operated in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary for implementing a functional methylglyoxal pathway. Furthermore, we engineered the serine shunt which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection we performed an adaptive laboratory evolution study using an enolase deletion strain. The evolved mutants were shown to use the serine shunt. Our study reveals the flexible redesignation of metabolic pathways to create new metabolite links and rewire central metabolism.

Thermal Stress and Bleaching in the Cnidarian-Dinoflagellate Symbiosis: The Application of Metabolomics

<p>Reef-building corals form critical ecosystems, which provide a diverse range of goods and services. Their success is based on a complex symbiosis between cnidarian host, dinoflagellate algae (genus Symbiodinium) and associated microorganisms (together termed the holobiont). Under functional conditions nutrients are efficiently recycled within the holobiont; however, under conditions of thermal stress, this dynamic relationship can dysfunction, resulting in the loss of symbionts (bleaching). Mass coral bleaching associated with elevated temperatures is a major threat to the long-term persistence of coral reefs. Further study is therefore necessary in order to elucidate the cellular and metabolic networks associated with function in the symbiosis and to determine change elicited by exposure to thermal stress. Metabolomics is the study of small compounds (metabolites) in a cell, tissue or whole organism. The metabolome comprises thousands of components, which will respond rapidly to change, reflecting a combination of genotype, phenotype and the environment. As a result, the study of these metabolic networks serves as a sensitive tool for the detection and elucidation of cellular responses to abiotic stress in complex systems. This thesis presents outputs of gas chromatography-mass spectrometry-based metabolite profiling techniques, which have been applied to the study of thermal stress and bleaching in the cnidarian-dinoflagellate symbiosis. In Chapter 2 these techniques were developed and applied to the model symbiotic cnidarian Aiptasia sp., and its homologous symbiont (Symbiodinium ITS 2 type B1), to characterise both ambient and thermally-induced metabolite profiles (amino and non-amino organic acids) in both partners. Thermal stress, symbiont photodamage and associated bleaching, resulted in characteristic modifications to the free metabolite pools of both partners. These changes differed between partners and were associated with modifications to central metabolism, biosynthesis, catabolism of stores and homeostatic responses to thermal and oxidative stress. In Chapter 3 metabolite profiling techniques (focussing this time on carbohydrate pools) were once again applied to the study of thermally-induced changes to the free pools of the coral Acropora aspera and its symbionts (dominant Symbiodinium ITS 2 type C3) at differing stages of symbiont photodamage and thermal stress. Additionally, targeted analysis was employed to quantify these changes in terms of absolute amounts. Once again exposure to elevated temperatures resulted in symbiont photodamage, bleaching and characteristic modifications to the free metabolite pools of symbiont and host, which differed between partners and with the duration of thermal stress. These changes were associated with increased turnover of a number of networks including: energy-generating pathways, antioxidant networks, ROS-associated damage and damage signalling, and were also indicative of potential alterations to the composition of the associated microbial holobiont. Finally in Chapter 4, metabolite profiling techniques optimized in Chapter 2 and 3 were coupled to 13C labelling in both Aiptasia sp. and A. aspera, in order to further investigate the questions raised in these preceding studies. Once again changes were observed to central metabolism, biosynthesis and alternative energy-generation modes in symbiont and host, in both symbioses. Interestingly however, in all cases there was continued fixation of carbon, production- and translocation of mobile products by the remaining symbionts in hospite. This suggests that even during the later stages of bleaching, symbionts are, at least in part, metabolically functional in terms of photosynthate provision. This study therefore serves as an important first step in developing the application of metabolomics-based techniques to the study of thermal stress in the cnidarian-dinoflagellate symbiosis. The power of these techniques lies in the capacity to simultaneously assess rapid and often post-translational change in a highly repeatable and quantitative manner. With the use of these methods, this study has shown how metabolic, homeostatic and acclimatory networks interact to elicit change in each partner of the symbiosis during thermal stress and how these responses vary between symbiotic partners. Further understanding of these networks, individual sensitivities- and enhanced resistance to thermal stress are essential if we are to better understand the capacity of coral reefs to acclimate and persist in the face of climate change.</p>

Thermal Stress and Bleaching in the Cnidarian-Dinoflagellate Symbiosis: The Application of Metabolomics

<p>Reef-building corals form critical ecosystems, which provide a diverse range of goods and services. Their success is based on a complex symbiosis between cnidarian host, dinoflagellate algae (genus Symbiodinium) and associated microorganisms (together termed the holobiont). Under functional conditions nutrients are efficiently recycled within the holobiont; however, under conditions of thermal stress, this dynamic relationship can dysfunction, resulting in the loss of symbionts (bleaching). Mass coral bleaching associated with elevated temperatures is a major threat to the long-term persistence of coral reefs. Further study is therefore necessary in order to elucidate the cellular and metabolic networks associated with function in the symbiosis and to determine change elicited by exposure to thermal stress. Metabolomics is the study of small compounds (metabolites) in a cell, tissue or whole organism. The metabolome comprises thousands of components, which will respond rapidly to change, reflecting a combination of genotype, phenotype and the environment. As a result, the study of these metabolic networks serves as a sensitive tool for the detection and elucidation of cellular responses to abiotic stress in complex systems. This thesis presents outputs of gas chromatography-mass spectrometry-based metabolite profiling techniques, which have been applied to the study of thermal stress and bleaching in the cnidarian-dinoflagellate symbiosis. In Chapter 2 these techniques were developed and applied to the model symbiotic cnidarian Aiptasia sp., and its homologous symbiont (Symbiodinium ITS 2 type B1), to characterise both ambient and thermally-induced metabolite profiles (amino and non-amino organic acids) in both partners. Thermal stress, symbiont photodamage and associated bleaching, resulted in characteristic modifications to the free metabolite pools of both partners. These changes differed between partners and were associated with modifications to central metabolism, biosynthesis, catabolism of stores and homeostatic responses to thermal and oxidative stress. In Chapter 3 metabolite profiling techniques (focussing this time on carbohydrate pools) were once again applied to the study of thermally-induced changes to the free pools of the coral Acropora aspera and its symbionts (dominant Symbiodinium ITS 2 type C3) at differing stages of symbiont photodamage and thermal stress. Additionally, targeted analysis was employed to quantify these changes in terms of absolute amounts. Once again exposure to elevated temperatures resulted in symbiont photodamage, bleaching and characteristic modifications to the free metabolite pools of symbiont and host, which differed between partners and with the duration of thermal stress. These changes were associated with increased turnover of a number of networks including: energy-generating pathways, antioxidant networks, ROS-associated damage and damage signalling, and were also indicative of potential alterations to the composition of the associated microbial holobiont. Finally in Chapter 4, metabolite profiling techniques optimized in Chapter 2 and 3 were coupled to 13C labelling in both Aiptasia sp. and A. aspera, in order to further investigate the questions raised in these preceding studies. Once again changes were observed to central metabolism, biosynthesis and alternative energy-generation modes in symbiont and host, in both symbioses. Interestingly however, in all cases there was continued fixation of carbon, production- and translocation of mobile products by the remaining symbionts in hospite. This suggests that even during the later stages of bleaching, symbionts are, at least in part, metabolically functional in terms of photosynthate provision. This study therefore serves as an important first step in developing the application of metabolomics-based techniques to the study of thermal stress in the cnidarian-dinoflagellate symbiosis. The power of these techniques lies in the capacity to simultaneously assess rapid and often post-translational change in a highly repeatable and quantitative manner. With the use of these methods, this study has shown how metabolic, homeostatic and acclimatory networks interact to elicit change in each partner of the symbiosis during thermal stress and how these responses vary between symbiotic partners. Further understanding of these networks, individual sensitivities- and enhanced resistance to thermal stress are essential if we are to better understand the capacity of coral reefs to acclimate and persist in the face of climate change.</p>

Model Balancing: A Search for In-Vivo Kinetic Constants and Consistent Metabolic States

Enzyme kinetic constants in vivo are largely unknown, which limits the construction of large metabolic models. Given measured metabolic fluxes, metabolite concentrations, and enzyme concentrations, these constants may be inferred by model fitting, but the estimation problems are hard to solve if models are large. Here we show how consistent kinetic constants, metabolite concentrations, and enzyme concentrations can be determined from data if metabolic fluxes are known. The estimation method, called model balancing, can handle models with a wide range of rate laws and accounts for thermodynamic constraints between fluxes, kinetic constants, and metabolite concentrations. It can be used to estimate in-vivo kinetic constants, to complete and adjust available data, and to construct plausible metabolic states with predefined flux distributions. By omitting one term from the log posterior—a term for penalising low enzyme concentrations—we obtain a convex optimality problem with a unique local optimum. As a demonstrative case, we balance a model of E. coli central metabolism with artificial or experimental data and obtain a physically and biologically plausible parameterisation of reaction kinetics in E. coli central metabolism. The example shows what information about kinetic constants can be obtained from omics data and reveals practical limits to estimating in-vivo kinetic constants. While noise-free omics data allow for a reasonable reconstruction of in-vivo kcat and KM values, prediction from noisy omics data are worse. Hence, adjusting kinetic constants and omics data to obtain consistent metabolic models is the main application of model balancing.

Advances in Wine Fermentation

Fermentation is a well-known natural process that has been used by humanity for thousands of years, with the fundamental purpose of making alcoholic beverages such as wine, and also other non-alcoholic products. From a strictly biochemical point of view, fermentation is a process of central metabolism in which an organism converts a carbohydrate, such as starch or sugar, into an alcohol or an acid. The fermentation process turns grape juice (must) into wine. This is a complex chemical reaction whereby the yeast interacts with the sugars (glucose and fructose) in the must to create ethanol and carbon dioxide. Fermentation processes to produce wines are traditionally carried out with Saccharomyces cerevisiae strains, the most common and commercially available yeast, and some lactic acid bacteria. They are well-known for their fermentative behavior and technological characteristics, which allow obtaining products of uniform and standard quality. However, fermentation is influenced by other factors as well. The initial sugar content of the must and the fermentation temperature are also crucial to preserve volatile aromatics in the wine and retain fruity characters. Finally, once fermentation is completed, and most of the yeast dies, wine evolution continues until the production of the final product.