Linda Thöny-Meyer

Production of glycoprotein vaccines in Escherichia coli

BackgroundConjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.ResultsTwo different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold.ConclusionsThe presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

Enzyme-catalyzed protein crosslinking

The process of protein crosslinking comprises the chemical, enzymatic, or chemoenzymatic formation of new covalent bonds between polypeptides. This allows (1) the site-directed coupling of proteins with distinct properties and (2) the de novo assembly of polymeric protein networks. Transferases, hydrolases, and oxidoreductases can be employed as catalysts for the synthesis of crosslinked proteins, thereby complementing chemical crosslinking strategies. Here, we review enzymatic approaches that are used for protein crosslinking at the industrial level or have shown promising potential in investigations on the lab-scale. We illustrate the underlying mechanisms of crosslink formation and point out the roles of the enzymes in their natural environments. Additionally, we discuss advantages and drawbacks of the enzyme-based crosslinking strategies and their potential for different applications.

Assembly and Function of the Cytochrome cbb Oxidase Subunits in Bradyrhizobium japonicum

The Bradyrhizobium japonicum cbb-type cytochrome oxidase, which supports microaerobic respiration, is a multisubunit enzyme encoded by the genes of the fixNOQP operon. We investigated the contribution of the individual subunits to function and assembly of the membrane-bound complex. In-frame deletion mutants of fixN, fixO, and fixQ, and an insertion mutant of fixP were constructed. All mutants, except the fixQ mutant, showed clearly altered absorption difference spectra of their membranes and decreased oxidase activities, and they were unable to fix nitrogen symbiotically. The presence of the individual subunits was assayed by Western blot analysis, using subunit-specific antibodies, and by heme staining of the c-type cytochromes FixO and FixP. These analyses led to the following conclusions: (i) FixN and FixO are necessary for assembly of the multimeric oxidase, (ii) FixN and FixO assemble independently of FixP, and (iii) FixQ is not required for complex formation and, therefore, does not seem to be an essential subunit. The possible oxidase biogenesis pathway involves the formation of a primary core complex consisting of FixN and FixO, which allows the subsequent association with FixP to form the complete enzyme.

Cytochrome c biogenesis in bacteria : a possible pathway begins to emerge

Cytochrome c biogenesis describes the posttranslational pathway for the conversion of pre‐apocytochrome c into the mature holocytochrome c. It involves an unknown number of consecutive biochemical steps, including translocation of the precursor polypeptide and haem into the periplasm and the covalent linkage between these two molecules. Genetic and molecular analysis of several bacterial mutants suggest that at least eight genes contribute to this process. In this review we summarize the present knowledge of the cytochrome c maturation pathway in bacteria and propose a model in which certain genes and their products are attributed to specific functions.

Characterization of the Escherichia coli CcmH protein reveals new insights into the redox pathway required for cytochrome c maturation

Abstract The CcmH protein of Escherichia coli is encoded by the last gene of the ccm gene cluster required for cytochrome c maturation. A mutant in which the entire ccmH gene was deleted failed to synthesize both indigenous and foreign c-type cytochromes. However, deletion of the C-terminal hydrophilic domain homologous to CycH of other gram-negative bacteria affected neither the biogenesis of indigenous c-type cytochromes nor that of the Bradyrhizobium japonicum cytochrome c550. This confirmed that only the N-terminal domain containing a conserved CXXC motif is required in E. coli. PhoA fusion analysis showed that this domain is periplasmic. Site-directed mutagenesis of the cysteines of the CXXC motif revealed that both cysteines are required for cytochrome c maturation during aerobic growth, whereas only the second cysteine is required for cytochrome c maturation during anaerobic growth. The deficiency of the point mutants was complemented when 2-mercapto-ethanesulfonic acid was added to growing cells; other thiol compounds did not stimulate cytochrome c formation in these strains. We propose a model for the reaction sequence in which CcmH keeps the heme binding site of apocytochrome c in a reduced form for subsequent heme ligation.

Haem-polypeptide interactions during cytochrome c maturation.

Cytochrome c maturation involves the translocation of a polypeptide, the apocytochrome, and its cofactor, haem, through a membrane, before the two molecules are ligated covalently. This review article focuses on the current knowledge on the journey of haem during this process, which is known best in the Gram-negative bacterium Escherichia coli. As haem always occurs bound to protein, its passage across the cytoplasmic membrane and incorporation into the apocytochrome appears to be mediated by a set of proteinaceous maturation factors, the Ccm (cytochrome c maturation) proteins. At least three of them, CcmC, CcmE and CcmF, are thought to interact directly with haem. CcmE binds haem covalently, thus representing an intermediate of the haem trafficking pathway. CcmC is required for binding of haem to CcmE, and CcmF for releasing it from CcmE and transferring it onto the apocytochrome. The mechanism by which haem crosses the cytoplasmic membrane is currently unknown.

Laccase versus Laccase-Like Multi-Copper Oxidase: A Comparative Study of Similar Enzymes with Diverse Substrate Spectra

Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Owing to their broad substrate range, copper-containing laccases are versatile biocatalysts, capable of oxidizing numerous natural and non-natural industry-relevant compounds, with water as the sole by-product. In the present study, 10 of the 11 multi-copper oxidases, hitherto considered to be laccases, from fungi, plant and bacterial origin were compared. A substrate screen of 91 natural and non-natural compounds was recorded and revealed a fairly broad but distinctive substrate spectrum amongst the enzymes. Even though the enzymes share conserved active site residues we found that the substrate ranges of the individual enzymes varied considerably. The EC classification is based on the type of chemical reaction performed and the actual name of the enzyme often refers to the physiological substrate. However, for the enzymes studied in this work such classification is not feasible, even more so as their prime substrates or natural functions are mainly unknown. The classification of multi-copper oxidases assigned as laccases remains a challenge. For the sake of simplicity we propose to introduce the term “laccase-like multi-copper oxidase” (LMCO) in addition to the term laccase that we use exclusively for the enzyme originally identified from the sap of the lacquer tree Rhus vernicifera.

Bacterial tyrosinases: old enzymes with new relevance to biotechnology

Tyrosinases are copper-containing dioxygen activating enzymes found in many species of bacteria and are usually associated with melanin production. These proteins have a strong preference for phenolic and diphenolic substrates and are somewhat limited in their reaction scope, always producing an activated quinone as product. Despite this fact they have potential in several biotechnological applications, including the production of novel mixed melanins, protein cross-linking, phenolic biosensors, production of l-DOPA, phenol and dye removal and biocatalysis. Although most studies have used Streptomyces sp. enzymes, there are several other examples of these proteins that are also of potential interest. For instance a solvent tolerant enzyme has been described, as well as an enzyme with both tyrosinase and laccase activities, enzymes with altered substrate preferences, an enzyme produced as an inactive zymogen as well as examples which do not require auxiliary proteins for copper insertion (unlike the Streptomyces sp. enzymes which do require such a protein). This article will summarise the reports on the biotechnological applications of bacterial tyrosinases as well as the current information available on the different types of this enzyme.

Structure of CcmG/DsbE at 1.14 A resolution: high-fidelity reducing activity in an indiscriminately oxidizing environment

CcmG is unlike other periplasmic thioredoxin (TRX)-like proteins in that it has a specific reducing activity in an oxidizing environment and a high fidelity of interaction. These two unusual properties are required for its role in c-type cytochrome maturation. The crystal structure of CcmG reveals a modified TRX fold with an unusually acidic active site and a groove formed from two inserts in the fold. Deletion of one of the groove-forming inserts disrupts c-type cytochrome formation. Two unique structural features of CcmG-an acidic active site and an adjacent groove-appear to be necessary to convert an indiscriminately binding scaffold, the TRX fold, into a highly specific redox protein.

Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives.

The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA production.