Fructose metabolism in Chromohalobacter salexigens: interplay between the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways

Background The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner–Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner–Doudoroff and Embden–Meyerhof–Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner–Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden–Meyerhof–Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described. Conclusions Overall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism.University of Murcia and University of Seville was supported by projects: BIO2015-63949-R, BIO2014-54411-C2-1-REuropa MINECO/FEDER RTI2018-094393-B-C21Fundación Séneca (Grant no. 19236/PI/14

Genetic reanalysis of patients with a difference of sex development carrying the NR5A1/SF-1 variant p.Gly146Ala has discovered other likely disease-causing variations.

NR5A1/SF-1 (Steroidogenic factor-1) variants may cause mild to severe differences of sex development (DSD) or may be found in healthy carriers. The NR5A1/SF-1 c.437G>C/p.Gly146Ala variant is common in individuals with a DSD and has been suggested to act as a susceptibility factor for adrenal disease or cryptorchidism. Since the allele frequency is high in the general population, and the functional testing of the p.Gly146Ala variant revealed inconclusive results, the disease-causing effect of this variant has been questioned. However, a role as a disease modifier is still possible given that oligogenic inheritance has been described in patients with NR5A1/SF-1 variants. Therefore, we performed next generation sequencing (NGS) in 13 DSD individuals harboring the NR5A1/SF-1 p.Gly146Ala variant to search for other DSD-causing variants and clarify the function of this variant for the phenotype of the carriers. Panel and whole-exome sequencing was performed, and data were analyzed with a filtering algorithm for detecting variants in NR5A1- and DSD-related genes. The phenotype of the studied individuals ranged from scrotal hypospadias and ambiguous genitalia in 46,XY DSD to opposite sex in both 46,XY and 46,XX. In nine subjects we identified either a clearly pathogenic DSD gene variant (e.g. in AR) or one to four potentially deleterious variants that likely explain the observed phenotype alone (e.g. in FGFR3, CHD7). Our study shows that most individuals carrying the NR5A1/SF-1 p.Gly146Ala variant, harbor at least one other deleterious gene variant which can explain the DSD phenotype. This finding confirms that the NR5A1/SF-1 p.Gly146Ala variant may not contribute to the pathogenesis of DSD and qualifies as a benign polymorphism. Thus, individuals, in whom the NR5A1/SF-1 p.Gly146Ala gene variant has been identified as the underlying genetic cause for their DSD in the past, should be re-evaluated with a NGS method to reveal the real genetic diagnosis

Nuevos aspectos de la regulación del metabolismo de acetato en Escherichia coli= New insights into the regulation of acetate metabolism in Escherichia coli

Objetivos: (i) caracterizar los mutantes delecionados en las rutas de consumo/producción de acetato y determinar las consecuencias de su deleción a distintos niveles celulares, (ii) estudiar la regulación in vivo del metabolismo de acetato por acetilación de proteínas en E. coli , (iii) describir el contexto genómico de los genes de las enzimas responsables de la acetilación de proteínas en E. coli (cobB y patZ) y estudiar su regulación transcripcional, (iv) discernir el papel de la acetilación de proteínas en las diferencias metabólicas observadas entre las cepas de E. coli K12 y BL21, (v) identificar los patrones de acetilación de proteínas en distintas condiciones y fondos genéticos en E. coli y (vi) caracterizar la funcionalidad de la acetilación de proteínas en el metabolismo del acetato y la regulación transcripcional. Metodología: durante esta Tesis se utilizaron la cepa de Escherichia coli BW25113 y sus mutantes delecionados en algunos genes de interés. Además se utilizaron técnicas para determinar expresión génica (RT-PCR, DNA-microarrays y vectores sonda de promotor), actividades enzimática en extractos crudos y enzimas purificadas, western blot, cuantificación de metabolitos por HPLC y espectrometría de masas de alta resolución para proteómica. Resultados. La deleción de la mayor vía de producción de acetato alteró el metabolismo central a distintos niveles, desde la transcripción a los niveles energéticos celulares, mostrando la conexión de este metabolismo y el central, y también mostrando la importancia de un funcionamiento equilibrado de este metabolismo. La sirtuina CobB y la acetiltransferasa PatZ alteraron la actividad in vivo de la enzima acetil-CoA sintetasa. El gen cobB se expresa constitutivamente mientras que la expresión génica de patZ se activa transcripcionalmente por el factor de transcripción CRP, al igual que el gen de la enzima acetil-CoA sintetasa, teniendo dos sitios de unión en su región aguas arriba de su secuencia. La ausencia de las proteínas CobB y PatZ en la cepa BL21 alteró mas severamente el metabolismo del acetato que en la cepa K12. Esto indicó que probablemente la regulación diferencial de estas enzimas puede ser la clave de porque ambas cepas tienen un metabolismo del acetato distinto. La actividad desacetilasa de CobB es global y contribuye a la desacetilación de numerosos substratos , generando un gran efecto sobre la fisiología. La acetilación de la isocitrato liasa contribuye al ajuste fino del ciclo del glioxilato y la acetilación de la lisina 154 de RcsB previene la unión de este factor de transcripción al ADN. Este último efecto de la acetilación activa la biosíntesis de flagelos y proteínas relacionadas con la motilidad, e incrementa la susceptibilidad a estrés. La deleción de la proteín-acetiltransferasa patZ aumenta la acetilación en cultivos con acetato. Esto sugiere que tal vez el papel de esta proteína sea regular los niveles de agentes acetilantes en la célula. Este hecho esto explicaría la activación transcripcional simultanea de los genes de patZ y su sustrato acs. Conclusiones. Los resultados presentados en esta Tesis ofrecen nuevos aspectos de las funciones del metabolismo del acetato y la acetilación de proteinas en E. coli. El metabolismo de acetato está directamente unido al metabolismo central, regulando los niveles de metabolitos clave como el acetil-CoA y el acetil-fosfato, que son clave para la acetilación enzimática y química de los residuos de lisina de las proteínas. Esta regulación post-traduccional es importante para el control del metabolismo pero también en otras funciones celulares como pueden ser la movilidad y la supervivencia a estrés. La proteín-acetiltransferasa PatZ no es responsable, directamente, de la mayor parte de las acetilaciones en E. coli, pero juega un papel importante en la regulación de la acetilación química en E. coli. Summary Objectives: (i) to characterise knockout mutants of the acetate producing/consuming pathways and to determine the consequences of the deletion at different metabolic levels; (ii) to study the regulation in vivo of the acetate metabolism by protein lysine acetylation in E. coli; (iii) to describe the genomic context of the genes that encode for the enzymes involved in protein lysine acetylation in E. coli (cobB and patZ) and to study their transcriptional regulation; (iv) to decipher the role of lysine acetylation in the different acetate metabolism observed between the BL21 and K12 E.coli strains; (v) to identify protein acetylation patterns in different growing conditions and genomic backgrounds; (iv) and to characterise the function of protein lysine acetylation in acetate metabolism and transcriptional regulation. Methodology: the strain Escherichia coli BW25113 and its knockout mutants deleted in the pathways of interest were used during this PhD thesis. Several techniques were used in order to achieve the objectives mentioned above, such as RT-PCR, promoter probe plasmids, DNA microarray, enzyme activities in cell crude extracts and purified enzymes, metabolite measurement by HPLC and High resolution MS proteomics. Results and discussion. The deletion of the main acetate-producing pathway altered central metabolism at different levels, from the transcripts to the energetic levels, showing the link between this metabolism and central carbon metabolism, and also the importance of a proper-balance of acetate metabolism. The sirtuin CobB and the acetyltransferase PatZ altered the activity of acetyl-CoA synthetase in vivo. the cobB gene is expressed constitutively while the patZ gene expression is activated transcriptionally by the transcription factor CRP, similarly to what has been described for the acetyl-CoA synthetase gene, having two putative binding sites in its upstream region. The absence of the CobB and PatZ protein in the BL21 strain altered more severely the acetate metabolism than in the K12. This indicates that probably the differential metabolic regulation of these enzymes could be the key for this different acetate production. Further, the deacetylase activity of CobB is global, contributes to the deacetylation of a big number of substrates and has a major impact on bacterial physiology. Acetylation of isocitrate lyase contributes to the fine-tuning regulation of the glyoxylate shunt and the acetylation of lysine 154 of RcsB prevents DNA binding. This last effect activates flagella biosynthesis and motility proteins, and increases susceptibility to acid stress. Besides, deletion of the acetyltransferase patZ increased acetylation especially in acetate cultures. These results suggest that the role of PatZ could be the regulation of the levels of acetylating agents in the cell. In fact this last finding would explain the simultaneous transcriptional activation of the protein acetyltransferase (patZ) and acetyl CoA synthetase (acs). Conclusions. The results presented in this PhD thesis offered new insights into the roles of acetate metabolism and lysine acetylation in E. coli. Acetate metabolism is linked to central metabolism regulating the levels of key metabolites, such as acetyl-CoA and acetyl-phosphate, both having been shown involved in protein lysine acetylation. This post-translational modification mechanism is important in the control of metabolism, but also in other important physiological roles such as motility and stress survival. Also, the protein acetyltransferase, PatZ, is not a major protein-acetyltransferase in E. coli, but rather might play a role in the regulation of chemical acetylation in E. coli

Acetate metabolism regulation in Escherichia coli: carbon overflow, pathogenicity, and beyond

International audienceAcetate is ubiquitously found in natural environments. Its availability in the gut is high as a result of the fermentation of nutrients, and although it is rapidly absorbed by intestinal mucosa, it can also be used as carbon source by some members of gut microbiota. The metabolism of acetate in Escherichia coli has attracted the attention of the scientific community due to its role in central metabolism and its link to multiple physiological features. In this microorganism, acetate is involved directly or indirectly on the regulation of functional processes, such as motility, formation of biofilms, and responses to stress. Furthermore, it is a relevant nutrient in gut, where it serves additional roles, which regulate or, at least, modulate pathophysiological responses of E. coli and other bacteria. Acetate is one of the major by-products of anaerobic (fermenting) metabolism, and it is also produced under fully aerobic conditions. This acetate overflow is recognized as one of the major drawbacks limiting E. coli's productivity in biotechnological processes. This review sums up current knowledge on acetate metabolism in E. coli, explaining the major milestones that have led to deciphering its complex regulation in the K-12 strain. Major differences in the metabolism of acetate in other strains will be underlined, with a focus on strains of biotechnological and biomedical interest

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

International audience4-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

An insight into the role of phosphotransacetylase (pta) and the acetate/acetyl-CoA node in Escherichia coli

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

An insight into the role of phosphotransacetylase (<it>pta</it>) and the acetate/acetyl-CoA node in <it>Escherichia coli</it>

Abstract Background Acetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance. The main problems related to the use of E. coli as cellular factory are i) the deficient utilization of carbon source due to the excretion of acetate during aerobic growth, ii) the inhibition of cellular growth and protein production by acetate and iii) the need for cofactor recycling (namely redox coenzymes and free CoASH) to sustain balanced growth and cellular homeostasis. Results This work analyzes the effect of mutations in the acetate excretion/assimilation pathways, acetyl-CoA synthethase (acs) and phosphotransacetylase (pta), in E. coli BW25113 grown on glucose or acetate minimal media. Biomass and metabolite production, redox (NADH/NAD+) and energy (ATP) state, enzyme activities and gene expression profiles related to the central metabolism were analyzed. The knock-out of pta led to a more altered phenotype than that of acs. Deletion of pta reduced the ability to grow on acetate as carbon source and strongly affected the expression of several genes related to central metabolic pathways. Conclusion Results showed that pta limits biomass yield in aerobic glucose cultures, due to acetate production (overflow metabolism) and its inefficient use during glucose starvation. Deletion of pta severely impaired growth on acetate minimal medium and under anaerobiosis due to decreased acetyl-coenzyme A synthethase, glyoxylate shunt and gluconeogenic activities, leading to lower growth rate. When acetate is used as carbon source, the joint expression of pta and acs is crucial for growth and substrate assimilation, while pta deletion severely impaired anaerobic growth. Finally, at an adaptive level, pta deficiency makes the strain more sensitive to environmental changes and de-regulates the central metabolism.</p

Functional analysis of isoprenoid precursors biosynthesis by quantitative metabolomics and isotopologue profiling

International audienceIsoprenoids are amongst the most abundant and diverse biological molecules and are involved in a broad range of biological functions. Functional understanding of their biosynthesis is thus key in many fundamental and applicative fields, including systems biology, medicine and biotechnology. However, available methods do not yet allow accurate quantification and tracing of stable isotopes incorporation for all the isoprenoids precursors

Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae

Spatial clustering of enzymes has proven an elegant approach to optimize metabolite transfer between enzymes in synthetic metabolic pathways. Among the multiple methods used to promote colocalisation, enzyme fusion is probably the simplest. Inspired by natural systems, we have explored the metabolic consequences of spatial reorganizations of the catalytic domains of Xanthophyllomyces dendrorhous carotenoid enzymes produced in Saccharomyces cerevisiae. Synthetic genes encoding bidomain enzymes composed of CrtI and CrtB domains from the natural CrtYB fusion were connected in the two possible orientations, using natural and synthetic linkers. A tridomain enzyme (CrtB, CrtI, CrtY) harboring the full β-carotene producing pathway was also constructed. Our results demonstrate that domain order and linker properties considerably impact both the expression and/or stability of the constructed proteins and the functionality of the catalytic domains, all concurring to either diminish or boost specific enzymatic steps of the metabolic pathway. Remarkably, the yield of β-carotene production doubled with the tridomain fusion while precursor accumulation decreased, leading to an improvement of the pathway efficiency, when compared to the natural system. Our data strengthen the idea that fusion of enzymatic domains is an appropriate technique not only to achieve spatial confinement and enhance the metabolic flux but also to produce molecules not easily attainable with natural enzymatic configurations, even with membrane bound enzymes. Keywords: Metabolic engineering, Synthetic biology, Metabolic flux, Enzyme spatial proximity, Carotenoids, Multidomain enzyme

Inferring assembly-curving trends of bacterial micro-compartment shell hexamers from crystal structure arrangements

Bacterial microcompartments (BMC) are complex macromolecular assemblies that participate to varied chemical processes in about one fourth of bacterial species. BMC-encapsulated enzymatic activities are segregated from other cell contents by means of semipermeable shells, justifying why BMC are viewed as prototype nano-reactors for biotechnological applications. Herein, we undertook a comparative study of trends of self-assembly of BMC hexamers (BMC-H), the most abundant shell constituents. Published and new microscopy data show that some BMC-H, like -carboxysomal CcmK, tend to assemble flat whereas other BMC-H often build curved-implying objects. Inspection of available crystal structures presenting BMC-H in tiled arrangements permitted to identify two major assembly modes with a striking connection with experimental trends. All-atom molecular dynamics (MD) supported that BMC-H bending is triggered robustly only from the disposition adopted by BMC-H that form curved objects experimentally, conducting to almost identical arrangements to those found in structures of recomposed BMC shells. Simulations on ensembles of planar-behaving hexamers, which were previously reconfigured to comply with such disposition, confirmed that bending is defined by assembly details, rather than by BMC-H identity. Finally, although no common atomic determinants could be identified as responsible of BMC-H spontaneous curvature, an inter-hexamer ionic pair was pinpointed as contributor to hold a subset of BMC-H in low bending dispositions. These results are expected to improve our understanding of the variable mechanisms of biogenesis characterized for BMC, and of possible strategies to regulate BMC size and shape