David J. Leak

Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production.

We describe the metabolic engineering of two strains of Geobacillus thermoglucosidasius to divert their fermentative carbon flux from a mixed acid pathway, to one in which ethanol becomes the major product. This involved elimination of the lactate dehydrogenase and pyruvate formate lyase pathways by disruption of the ldh and pflB genes, respectively, together with upregulation of expression of pyruvate dehydrogenase. Unlike the situation in Escherichia coli, pyruvate dehydrogenase is active under anaerobic conditions in thermophilic bacilli, but expressed sub-optimally for a role as the primary fermentation pathway. Mutants were initially characterised in batch culture using glucose as carbon substrate and strains with all three modifications shown to form ethanol efficiently and rapidly at temperatures in excess of 60 degrees C in yields in excess of 90% of theoretical. The strain containing the 3 modifications, TM242, was also shown to efficiently ferment cellobiose and a mixed hexose and pentose feed.

Biocatalysts for selective introduction of oxygen

Three types of oxygenase biocatalysts are treated in detail in this review: the non-haem iron alkene mono-oxygenases, the haem and vanadium haloperoxidases, and flavin-based Baeyer–Villiger mono-oxygenases. Other oxygenases are briefly included for comparison. Characteristics of the biocatalysts are presented, and the scope and limitations concerning their applicability for the selective introduction of oxygen are discussed. Key issues include catalytic activity, productivity, cloning and expression, as well as process engineering aspects. Various bottlenecks are identified for the different biocatalysts and measures to increase the number of oxygenase reactions in practical use are discussed.

Development of a versatile shuttle vector for gene expression in Geobacillus spp.

An improved, versatile shuttle vector has been created for the metabolic engineering of Geobacillus spp. As kanamycin is the most thermo-tolerant of commonly used antibiotics, the gene encoding a thermostable kanamycin nucleotidyltransferase, together with the origin of replication from the G. stearothermophilus plasmid pBST1 were cloned into the Escherichia coli cloning vector pUC18. The resulting vector, named pUCG18, replicated in both organisms and could be transformed with an efficiency of 1 x 10(4) transformants per microg of DNA in G. thermoglucosidasius and was stable up to 68 degrees C with antibiotic selection. It was used to demonstrate expression of the pyruvate decarboxylase (pdc) gene from Zymomonas palmae in G. thermoglucosidasius at 45 degrees C. Sequence analysis of the pBST1 derived origin of replication revealed homology with a family of theta replicons that have previously only been found in strains of Bacillus megaterium.

The microbial production of epoxides

Abstract Chiral compounds in which biological activity resides in only one of the enantiomers are, increasingly, being demanded in enantiomerically pure form. The use of biocatalysts for production of epoxides as chiral intermediates in the synthesis of these compounds has realistic commercial prospects, but also presents formidable technical problems.

The Development of Metabolomic Sampling Procedures for Pichia pastoris, and Baseline Metabolome Data

Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris.

Chemoenzymic resolution and deracemisation of (±)-1-methyl-1,2-epoxycyclohexane: the synthesis of (1-S, 2-S)-1-methylcyclohexane-1,2-diol

Abstract Corynebacterium C12 epoxide hydrolase transform (±)-1-methyl-1,2-epoxy-cyclohexane 1 to the (1- S , 2- S )-1-methylcyclohexane-1,2-diol 2 leaving the (1- S , 2- R )-epoxide 3 unchanged. The diol 2 is converted to the the (1- R , 2- S )-epoxide 4 by sulfonation-ring closure. A one pot combination of Corynebacterium C12 epoxide hydrolase and acid catalysed ring opening converts (±)-1-methyl-1,2-epoxycyclohexane 1 to (1- S , 2- S )-1-methylcyclohexane-1,2-diol 2 .

In vivo Studies of Primary Alcohols, Aldehydes and Carboxylic Acids as Electron Donors for the Methane Mono-oxygenase in a Variety of Methanotrophs

Summary: C2 to C4 primary alcohols and their corresponding aldehydes were oxidized by type I, type II and type X obligate methanotrophs. Reducing equivalents from each oxidation step could be utilized, in vivo, to stimulate methane mono-oxygenase activity. As neither oxidation step produces NADH directly, these observations are presented as evidence for reverse electron transport in methanotrophs. In type II methanotrophs, 5 mM-acetate, propionate and butyrate also stimulate methane mono-oxygenase activity apparently by inducing the breakdown of poly-β-hydroxybutyrate. subsequent metabolism of β-hydroxybutyrate giving rise to NADH.

Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156

ABSTRACT Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc2155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzymes specificity for epoxidation.

Genetic tool development underpins recent advances in thermophilic whole‐cell biocatalysts

The environmental value of sustainably producing bioproducts from biomass is now widely appreciated, with a primary target being the economic production of fuels such as bioethanol from lignocellulose. The application of thermophilic prokaryotes is a rapidly developing niche in this field, driven by their known catabolic versatility with lignocellulose‐derived carbohydrates. Fundamental to the success of this work has been the development of reliable genetic and molecular systems. These technical tools are now available to assist in the development of other (hyper)thermophilic strains with diverse phenotypes such as hemicellulolytic and cellulolytic properties, branched chain alcohol production and other ‘valuable bioproduct’ synthetic capabilities. Here we present an insight into the historical limitations, recent developments and current status of a number of genetic systems for thermophiles. We also highlight the value of reliable genetic methods for increasing our knowledge of thermophile physiology. We argue that the development of robust genetic systems is paramount in the evolution of future thermophilic based bioprocesses and make suggestions for future approaches and genetic targets that will facilitate this process.

The genus Geobacillus and their biotechnological potential

The genus Geobacillus comprises a group of Gram-positive thermophilic bacteria, including obligate aerobes, denitrifiers, and facultative anaerobes that can grow over a range of 45-75°C. Originally classified as group five Bacillus spp., strains of Bacillus stearothermophilus came to prominence as contaminants of canned food and soon became the organism of choice for comparative studies of metabolism and enzymology between mesophiles and thermophiles. More recently, their catabolic versatility, particularly in the degradation of hemicellulose and starch, and rapid growth rates have raised their profile as organisms with potential for second-generation (lignocellulosic) biorefineries for biofuel or chemical production. The continued development of genetic tools to facilitate both fundamental investigation and metabolic engineering is now helping to realize this potential, for both metabolite production and optimized catabolism. In addition, this catabolic versatility provides a range of useful thermostable enzymes for industrial application. A number of genome-sequencing projects have been completed or are underway allowing comparative studies. These reveal a significant amount of genome rearrangement within the genus, the presence of large genomic islands encompassing all the hemicellulose utilization genes and a genomic island incorporating a set of long chain alkane monooxygenase genes. With G+C contents of 45-55%, thermostability appears to derive in part from the ability to synthesize protamine and spermine, which can condense DNA and raise its Tm.