Optimized compatible set of BioBrick™ vectors for metabolic pathway engineering

The BioBrick™ paradigm for the assembly of enzymatic pathways is being adopted and becoming a standard practice in microbial engineering. We present a strategy to adapt the BioBrick™ paradigm to allow the quick assembly of multi-gene pathways into a number of vectors as well as for the quick mobilization of any cloned gene into vectors with different features for gene expression and protein purification. A primary BioBrick™ (BB-eGFP) was developed where the promoter/RBS, multiple cloning sites, optional protein purification affinity tags and reporter gene were all separated into discrete regions by additional restriction enzymes. This primary BB-eGFP then served as the template for additional BioBrick™ vectors with different origins of replication, antibiotic resistances, inducible promoters (arabinose, IPTG or anhydrotetracycline), N- or C-terminal Histidine tags with thrombin cleavage, a LacZα reporter gene and an additional origin of mobility (oriT). All developed BioBricks™ and BioBrick™ compatible vectors were shown to be functional by measuring reporter gene expression. Lastly, a C₃₀ carotenoid pathway was assembled as a model enzymatic pathway to demonstrate in vivo functionality and compatibility of this engineered vector system.     

SNA – a toolbox for the stoichiometric analysis of metabolic networks

Background  

Despite recent algorithmic and conceptual progress, the stoichiometric network analysis of large metabolic models remains a computationally challenging problem.     

Can sugars be produced from fatty acids? A test case for pathway analysis tools

Motivation: In recent years, several methods have been proposedfor determining metabolic pathways in an automated way basedon network topology. The aim of this work is to analyse thesemethods by tackling a concrete example relevant in biochemistry.It concerns the question whether even-chain fatty acids, beingthe most important constituents of lipids, can be convertedinto sugars at steady state. It was proved five decades agothat this conversion using the Krebs cycle is impossible unlessthe enzymes of the glyoxylate shunt (or alternative bypasses)are present in the system. Using this example, we can comparethe various methods in pathway analysis. Results: Elementary modes analysis (EMA) of a set of enzymescorresponding to the Krebs cycle, glycolysis and gluconeogenesissupports the scientific evidence showing that there is no pathwaycapable of converting acetyl-CoA to glucose at steady state.This conversion is possible after the addition of isocitratelyase and malate synthase (forming the glyoxylate shunt) tothe system. Dealing with the same example, we compare EMA withtwo tools based on graph theory available online, PathFindingand Pathway Hunter Tool. These automated network generatingtools do not succeed in predicting the conversions known fromexperiment. They sometimes generate unbalanced paths and revealproblems identifying side metabolites that are not responsiblefor the carbon net flux. This shows that, for metabolic pathwayanalysis, it is important to consider the topology (includingbimolecular reactions) and stoichiometry of metabolic systems,as is done in EMA. Contact: ldpf{at}minet.uni-jena.de; schuster{at}minet.uni-jena.de Supplementary information: Supplementary data are availableat Bioinformatics online. FOOTNOTES Associate Editor: Alfonso Valencia Received on July 24, 2008; revised on September 18, 2008; accepted on September 18, 2008     

Two (completely) rate-limiting steps in one metabolic pathway? The resolution of a paradox using bacteriorhodopsin liposomes and the control theory

Proton pumping by bacteriorhodopsin and charge-compensating ion movement can both and simultaneously behave as the rate-limiting step in light-driven proton uptake into bacteriorhodopsin liposomes. This apparently excessive control exerted on the net proton influx is possible because of the negative (-1) 'control coefficient' of the net proton influx with respect to the proton leaks. Furthermore, the property of bacteriorhodopsin that it is inhibited by the membrane potential is responsible for the transfer of part of the control on the net proton influx from the first, irreversible, step in the pathway (i.e. bacteriorhodopsin) to the second, reversible, step (i.e., charge-compensating ion movement).     

An evolving paradigm for the secretory pathway?

The paradigm that the secretory pathway consists of a stable endoplasmic reticulum and Golgi apparatus, using discrete transport vesicles to exchange their contents, gained important support from groundbreaking biochemical and genetic studies during the 1980s. However, the subsequent development of new imaging technologies with green fluorescent protein introduced data on dynamic processes not fully accounted for by the paradigm. As a result, we may be seeing an example of how a paradigm is evolving to account for the results of new technologies and their new ways of describing cellular processes.     

CelloSelect – A synthetic cellobiose metabolic pathway for selection of stable transgenic CHO cell lines

Current protocols for generating stable transgenic cell lines mostly rely on antibiotic selection or the use of specialized cell lines lacking an essential part of their metabolic machinery, but these approaches require working with either toxic chemicals or knockout cell lines, which can reduce productivity. Since most mammalian cells cannot utilize cellobiose, a disaccharide consisting of two β-1,4-linked glucose molecules, we designed an antibiotic-free selection system, CelloSelect, which consists of a selection cassette encoding Neurospora crassa cellodextrin transporter CDT1 and β-glucosidase GH1-1. When cultivated in glucose-free culture medium containing cellobiose, CelloSelect-transfected cells proliferate by metabolizing cellobiose as a primary energy source, and are protected from glucose starvation. We show that the combination of CelloSelect with a PiggyBac transposase-based integration strategy provides a platform for the swift and efficient generation of stable transgenic cell lines. Growth rate analysis of metabolically engineered cells in cellobiose medium confirmed the expansion of cells stably expressing high levels of a cargo fluorescent marker protein. We further validated this strategy by applying the CelloSelect system for stable integration of sequences encoding two biopharmaceutical proteins, erythropoietin and the monoclonal antibody rituximab, and confirmed that the proteins are efficiently produced in either cellobiose- or glucose-containing medium in suspension-adapted CHO cells cultured in chemically defined media. We believe coupling heterologous metabolic pathways additively to the endogenous metabolism of mammalian cells has the potential to complement or to replace current cell-line selection systems.     

A novel metabolic pathway for glucose production mediated by α-glucosidase-catalyzed conversion of 1,5-anhydrofructose

α-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form α-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for α-glucosidases. α-Glucosidase-catalyzed hydration produced α-glucose from AF, which plateaued with time. Combined reaction with α-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger α-glucosidase (31AG), which is stable in organic solvent, produced ethyl α-glucoside from AF in 80% ethanol. The findings indicate that α-glucosidases catalyze trans-addition. This is the first report of α-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.     

Parasite polyclonal activators: new targets for vaccination approaches?

Taking into consideration that the immune response following infection promotes the expansion of lymphocyte clones that are essentially non-specific, ensuring both parasite evasion and persistence inside the host, what would be the major consequences of this polyclonal response to the development of immunopathology? We favor the hypothesis that the polyclonal B cell responses triggered by the infection is responsible of the host susceptibility and is a major contributor to the maintenance of a progressive disease. In particular, the activation of B cells by parasite mitogens would contribute to the class determination of T cell responses and to the inhibition of macrophages - target cells for parasite multiplication and also responsible for parasite clearance. We also envisage that the activation of T cells by parasite 'superantigens', and the ensuing energy and deletion of these cells, processes that are frequently observed, would contribute for the immunosuppression as well as to parasite escape and persistence in the host. We had concentrated our efforts on the study of the non-specific aspects of the immune response following Trypanosoma cruzi infection. We aimed at finding new strategies to modulate and control the mechanisms leading to both the immunosuppression and the development of chronic auto-immunity leading to rational vaccine approaches against parasite infection and immunopathology.     

Who's your neighbor? New computational approaches for functional genomics

Several recently developed computational approaches in comparative genomics go beyond sequence comparison. By analyzing phylogenetic profiles of protein families, domain fusions, gene adjacency in genomes, and expression patterns, these methods predict many functional interactions between proteins and help deduce specific functions for numerous proteins. Although some of the resultant predictions may not be highly specific, these developments herald a new era in genomics in which the benefits of comparative analysis of the rapidly growing collection of complete genomes will become increasingly obvious.     

Metabolites: a helping hand for pathway evolution?

The evolution of enzymes and pathways is under debate. Recent studies show that recruitment of single enzymes from different pathways could be the driving force for pathway evolution. Other mechanisms of evolution, such as pathway duplication, enzyme specialization, de novo invention of pathways or retro-evolution of pathways, appear to be less abundant. Twenty percent of enzyme superfamilies are quite variable, not only in changing reaction chemistry or metabolite type but in changing both at the same time. These variable superfamilies account for nearly half of all known reactions. The most frequently occurring metabolites provide a helping hand for such changes because they can be accommodated by many enzyme superfamilies. Thus, a picture is emerging in which new pathways are evolving from central metabolites by preference, thereby keeping the overall topology of the metabolic network.     

Fabry disease--a metabolic disorder with a challenge for endocrinologists?

OBJECTIVE: To revisit Fabry disease, a rare X-linked metabolic glycosphingolipid storage disease caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). METHOD: Summary of the existing knowledge of Fabry disease including the clinical feature of Fabry disease and the recent breakthrough in the treatment of Fabry patients with the development of recombinant human alpha-gal A. CONCLUSION: The diffuse organ manifestations of Fabry disease resemble medical endocrinological diseases, and medical endocrinology might be an appropriate speciality to manage the treatment in collaboration with other specialists and clinical geneticists.     

Prebiotic metabolic networks?

A prebiotic origin of metabolism has been proposed as one of several scenarios for the origin of life. In their recent work, Ralser and colleagues (Keller et al, 2014 ) observe an enzyme‐free, metabolism‐like reaction network under conditions reproducing a possible prebiotic environment.     

What can genome-scale metabolic network reconstructions do for prokaryotic systematics?

It has recently been proposed that in addition to Nomenclature, Classification and Identification, Comprehending Microbial Diversity may be considered as the fourth tenet of microbial systematics [Staley JT (2010) The Bulletin of BISMiS, 1(1): 1–5]. As this fourth goal implies a fundamental understanding of microbial speciation, this perspective article argues that translation of bacterial genome sequences into metabolic features may contribute to the development of modern polyphasic taxonomic approaches. Genome-scale metabolic network reconstructions (GSMRs), which are the result of computationally predicted and experimentally confirmed stoichiometric matrices incorporating all enzyme and metabolite components encoded by a genome sequence, provide a platform that can illustrate bacterial speciation. As the topology and the composition of GSMRs are expected to be the result of adaptive evolution, the features of these networks may provide the prokaryotic taxonomist with novel tools for reaching the fourth tenet of microbial systematics. Through selected examples from the Actinobacteria, which have been inferred from GSMRs and experimentally confirmed after phenotypic characterisation, it will be shown that this level of information can be incorporated into modern polyphasic taxonomic approaches. In conclusion, three specific examples are illustrated to show how GSMRs will revolutionize prokaryotic systematics, as has previously occurred in many other fields of microbiology.     

Does free-energy change of individual reactions alone determine the flux through a metabolic pathway?

Most text books of biochemistry present the concept of free energy change clearly under bioenergetics. However, some text books in trying to explain this in more detail later in the book, with more specific examples, often confuse the students. With the possibility of more accurate measurement of reactants and products by high pressure liquid chromatography or other modern techniques, and by selecting an appropriate tissue, it should be possible to avoid errors of this kind, which contradict the basic concepts taught to the students earlier in the course.