Stuart M. Thomas

Biocatalysis: applications and potentials for the chemical industry.

The chemical industry is exploring the use of renewable feed stocks to improve sustainability, prompting the exploration of bioprocesses for the production of chemicals. Attractive features of biological systems include versatility, substrate selectivity, regioselectivity, chemoselectivity, enantioselectivity and catalysis at ambient temperatures and pressures. However, a challenge facing bioprocesses is cost competitiveness with chemical processes because capital assets associated with the existing commercial processes are high. The chemical industry will probably use biotechnology with existing feed stocks and processes to extract higher values from feed stocks, process by-products and waste streams. In this decade, bioprocesses that offer either a process or a product advantage over traditional chemical routes will become more widely used.

mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species.

ABSTRACT mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

Cloning and Characterization of a Gene Cluster for Cyclododecanone Oxidation in Rhodococcus ruber SC1

Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C11 to C15), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C5 to C7).

Genetic analysis of a gene cluster for cyclohexanol oxidation in Acinetobacter sp. Strain SE19 by in vitro transposition.

Biological oxidation of cyclic alcohols normally results in formation of the corresponding dicarboxylic acids, which are further metabolized and enter the central carbon metabolism in the cell. We isolated an Acinetobacter sp. from an industrial wastewater bioreactor that utilized cyclohexanol as a sole carbon source. A cosmid library was constructed from Acinetobacter sp. strain SE19, and oxidation of cyclohexanol to adipic acid was demonstrated in recombinant Escherichia coli carrying a SE19 DNA segment. A region that was essential for cyclohexanol oxidation was localized to a 14-kb fragment on the cosmid DNA. Several putative open reading frames (ORFs) that were expected to encode enzymes catalyzing the conversion of cyclohexanol to adipic acid were identified. Whereas one ORF showed high homology to cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871, most of the ORFs showed only moderate homology to proteins in GenBank. In order to assign functions of the various ORFs, in vitro transposon mutagenesis was performed using the cosmid DNA as a target. A set of transposon mutants with a single insertion in each of the ORFs was screened for cyclohexanol oxidation in E. coli. Several of the transposon mutants accumulated a variety of cyclohexanol oxidation intermediates. The in vitro transposon mutagenesis technique was shown to be a powerful tool for rapidly assigning gene functions to all ORFs in the pathway.

Stationary-Phase Quorum-Sensing Signals Affect Autoinducer-2 and Gene Expression in Escherichia coli

ABSTRACT Quorum sensing via autoinducer-2 (AI-2) has been identified in different strains, including those from Escherichia, Vibrio, Streptococcus, and Bacillus species, and previous studies have suggested the existence of additional quorum-sensing signals working in the stationary phase of Escherichia coli cultures. To investigate the presence and global effect of these possible quorum-sensing signals other than AI-2, DNA microarrays were used to study the effect of stationary-phase signals on the gene expression of early exponential-phase cells of the AI-2-deficient strain E. coli DH5α. For statistically significant differential gene expression (P < 0.05), 14 genes were induced by supernatants from a stationary culture and 6 genes were repressed, suggesting the involvement of indole (induction of tnaA and tnaL) and phosphate (repression of phoA, phoB, and phoU). To study the stability of the signals, the stationary-phase supernatant was autoclaved and was used to study its effect on E. coli gene expression. Three genes were induced by autoclaved stationary-phase supernatant, and 34 genes were repressed. In total, three genes (ompC, ptsA, and btuB) were induced and five genes (nupC, phoB, phoU, argT, and ompF) were repressed by both fresh and autoclaved stationary-phase supernatants. Furthermore, supernatant from E. coli DH5α stationary culture was found to repress E. coli K-12 AI-2 concentrations by 4.8-fold ± 0.4-fold, suggesting that an additional quorum-sensing system in E. coli exists and that gene expression is controlled as a network with different signals working at different growth stages.

Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental Brevibacterium Isolate Using mRNA Differential Display

The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.

Differential Gene Expression To Investigate the Effect of (5Z)-4-Bromo- 5-(Bromomethylene)-3-Butyl-2(5H)-Furanone on Bacillus subtilis

ABSTRACT (5Z)-4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming, and biofilm formation of gram-positive bacteria. Using the gram-positive bacterium Bacillus subtilis as a test organism, we observed cell killing by 20 μg of furanone per ml, while 5 μg of furanone per ml inhibited growth approximately twofold without killing the cells. To discover the mechanism of this inhibition on a genetic level and to investigate furanone as a novel antibiotic, full-genome DNA microarrays were used to analyze the gene expression profiles of B. subtilis grown with and without 5 μg of furanone per ml. This agent induced 92 genes more than fivefold (P < 0.05) and repressed 15 genes more than fivefold (P < 0.05). The induced genes include genes involved in stress responses (such as the class III heat shock genes clpC, clpE, and ctsR and the class I heat shock genes groES, but no class II or IV heat shock genes), fatty acid biosynthesis, lichenan degradation, transport, and metabolism, as well as 59 genes with unknown functions. The microarray results for four genes were confirmed by RNA dot blotting. Mutation of a stress response gene, clpC, caused B. subtilis to be much more sensitive to 5 μg of furanone per ml (there was no growth in 8 h, while the wild-type strain grew to the stationary phase in 8 h) and confirmed the importance of the induction of this gene as identified by the microarray analysis.

Pure bacterial isolates that convert p-xylene to terephthalic acid

Abstract. Bacteria that grow on p-xylene, p-toluic acid, and terephthalic acid (TPA) were isolated from a wastewater bioreactor that is used to treat a waste stream that contains all three of these compounds. Although previously described aerobic bacteria degrade p-xylene by initially oxidizing a single methyl group to form p-toluic acid and then cleaving the aromatic ring, some of the bacteria isolated during this study transformed p-xylene by oxidizing both methyl groups to produce TPA.

Operation of mixed-culture immobilized cell reactors for the metabolism of meta- and para-nitrobenzoate by comamonas sp. JS46 and comamonas sp. JS47

The treatment of meta- and para-nitrobenzoic acid in an industrial wastestream by Comamonas sp. JS46 and Comamonas sp. JS47 is investigated. The most important feature of the wastestream is the constantly changing concentration ratio of the two isomers. The most extreme occurrence is considered here: the complete change in feed carbon source from one isomer to the other. A series of immobilized cell airlift reactor experiments are described to examine the operation and response of the system to these changes in the feed carbon source. Separate reactors containing each species immobilized are compared with a reactor containing both species immobilized within the same bead, and to a reactor containing both species with each species confined to separate beads. On the basis of response time necessary to recover the appropriate activity, the reactor containing both species immobilized within the same bead offers the most effective arrangement. Interactions occurring between the two organisms in the coimmobilized system, mediated by the nitrobenzoate metabolites, are discussed relative to the improved response of this arrangement. Copyright 1998 John Wiley & Sons, Inc.

Continuous culture dynamics for aniline metabolism by Pseudomonas sp. CIT1

Inhibition by toxic substrates enables multiple steady states to arise in biodegradation systems. This phenomenon was investigated for the continuous metabolism of aniline by Pseudomonas sp. CIT1. Differences of various metabolic parameters between the two growth regimes (uninhibited and inhibited) and the transient response to a step-up in dilution rate were determined. Regulatory mechanisms consistent with the experimental evidence are proposed. Aniline is the transcriptional inducer of a metabolic pathway that converts aniline to TCA cycle intermediates. The suite of enzymes is coordinately expressed from a single promoter. We followed the level of the pathway mRNA using a fragment containing the catechol 2,3 dioxygenase gene (andioxB) and monitored the pathway enzyme activity using catechol 2,3 dioxygenase (C23D). The inhibited regime resulted in a 60% lower growth yield, near constant levels of C23D monomer, but a 50% reduction in the specific activity of C23D, increased RNA synthesis rates (total and aniline pathway mRNA), and elevated RNA decay rates. Elucidation of regulatory mechanisms indicates that C23D is noncompetitively inhibited by aniline and subject to feedback inhibition by 2-hydroxymuconic semialdehyde (HMS). During uninhibited growth regime operation, metabolism of HMS is the rate-limiting step; in contrast, conversion of aniline to catechol limits growth in the inhibited regime.