Sol M. Resnick's research while affiliated with Queen's University Belfast and other places

Chemical conjugation of the influenza peptide antigen M2E to different variants of virus-like particles (VLPs) was investigated. Wild-type cowpea chlorotic mottle virus (CCMV) and two novel cysteine mutants of CCMV, all expressed in Pseudomonas fluorescens, were utilized in this study. Two different conjugation schemes, primary amine-directed and cysteine-directed, were tested and compared. Both strategies were successfully used to attach M2E peptides to the surface of these VLPs. Ultimately, the cysteine-directed conjugation strategy using the CCMV cysteine mutant particles displayed key advantages over the primary amine-directed strategy.

Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens. The human g-csf gene was optimized for expression in P. fluorescens and cloned into a set of periplasmic expression vectors. These plasmids were transformed into a variety of P. fluorescens host strains each having a unique phenotype, to evaluate soluble expression in a 96-well growth and protein expression format. To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfēnex Expression Technology™ toolbox were screened in parallel using biolayer interferometry (BLI) to quantify active G-CSF binding to its receptor. A subset of these strains was screened by LC-MS analysis to assess the quality of the expressed G-CSF protein. A single strain with an antibiotic resistance marker insertion in the pfaI gene was identified that produced>99% Met-GCSF. A host with a complete deletion of the autotransporter-coding gene pfaI from the genome was constructed, and expression of soluble, active Met-GSCF in this strain was observed to be 350mg/L at the 1 liter fermentation scale.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Aromatic ring hydroxylating dioxygenases play a key role in the biodegradation of numerous environmental pollutants, both in the natural environment (via natural attenuation) and in the engineered bioremediation systems. Recent structural and mechanistic information, together with enzyme engineering and strain construction strategies should allow the development of engineered microorganisms with new and/or optimized degradation abilities. The continued application of these approaches should also facilitate the development of Rieske non-heme iron dioxygenases with requisite selectivities for specific opportunities in target direct biocatalysis or metabolic engineering.

In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD). The antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR). We have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales.

Since the initial discovery of toluene dioxygenase (TDO) by David Gibson and coworkers (Yeh et al. 1977; Subramanian et al. 1979), aromatic hydrocarbon dioxygenases have been reported to catalyze the initial reaction in the bacterial biodegradation of a diverse array of aromatic and polyaromatic hydrocarbons, aromatic acids, chlorinated aromatic, and heterocyclic aromatic compounds. To date, more than 100 aromatic compound dioxygenases have been described in the literature based on biological activity or nucleotide sequence identity. These enzymes are cofactor-requiring multicomponent heteromultimeric proteins (EC 1.14.12) that catalyze the initial activation through reductive dihydroxylation of their substrates, and are distinct from aromatic ring-cleavage (or ring-fission) dioxygenases (EC 1.13.11), which act on the catechol intermediates in many of the same catabolic pathways. This chapter will focus primarily on the aromatic ringhydroxylating dioxygenases (Rieske non-heme iron dioxygenases; EC 1.14.12), which initiate an attack on aromatic hydrocarbons, heterocycles and related compounds carrying various substituents (Cl, NO2). The aspects described will relate to dioxygenase discovery, classification, enzymology, structure, electron transport, mechanism and applications.

The purple nonsulfur bacterium Rhodobacter capsulatus strain B10 grew phototrophically on the aromatic compound hippurate (N-benzoyl-L-glycine) and related benzoyl amino acids. Absorption spectra, extraction, and GC/MS analysis of culture supernatants showed that hippurate was stoichiometrically converted to benzoate and glycine, with the latter used as a carbon or nitrogen source for growth. This conclusion was supported by detection of the enzyme hippuricase in permeabilized intact cells. Chemotrophic growth on hippurate by Rba. capsulatus, either at full or reduced oxygen tensions, was not observed. The type strain of Rhodobacter sphaeroides as well as four strains of Rhodopseudomonas palustris also grew phototrophically on hippurate, while several other aromatic-degrading species of purple bacteria did not.

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the α subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenylcis-3,4-dihydrodiol [77 and 60% (−)-(3S,4R), respectively] to that formed by wild-type NDO [>98% (+)-(3R,4S)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrenecis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36. A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluenecis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (−)-biphenylcis-(3S,4R)-dihydrodiol and (−)-phenanthrenecis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively.