Functional characterisation of alkane-degrading monooxygenases in Rhodococcus jostii strain 8

Short-chain alkanes are gaseous hydrocarbons that contribute to photochemical pollution and ozone production in the atmosphere. A number of studies have shown that Rhodococcus species possess the ability to metabolite a wide range of hydrocarbons since they contain multiple hydrocarbon-degrading enzymes such as soluble diiron monooxygenases (SDIMOs) and alkB-type alkane monooxygenases. This study aimed to investigate the role of multiple alkane-degrading enzymes in the metabolism of gaseous alkanes in Rhodococcus jostii strain 8. R. jostii strain 8 was isolated from a propane enrichment culture using petroleum-contaminated soil as an inoculum. R. jostii strain 8 could grow on ethane, propane, butane, octane, naphthalene and some potential intermediates in alkane metabolism. Oxidation studies showed that R. jostii strain 8 is likely to oxidise propane via both terminal and sub-terminal oxidation of propane and that these activities are induced in propane-grown cells. Alcohol dehydrogenase assays were carried out in order to determine cofactor and substrate ranges of these enzymes. Results showed that alcohol dehydrogenases involved in the metabolism of gaseous alkanes are NDMA-dependent. The size of the genome sequence of R. jostii strain 8 is 8.5 Mbp with a G+C content of 67%. The closest relative of R. jostii strain 8, based on 16S rRNA sequence, is Rhodococcus jostii RHA1 with 99% identity. However, growth profiles and a number of catabolic genes in the genome of R. jostii strain 8 clearly indicated that this bacterium is different from R. jostii RHA1. R. jostii strain 8 contains two alkane-degrading enzyme systems – a propane monooxygenase and an alkB-type alkane monooxygenase. Polypeptide analysis on cell-free extracts from cells grown on gaseous alkanes using SDS-PAGE indicated that propane monooxygenase is inducible during growth on propane. Expression studies using RT-qPCR of prmA and alkB showed that prmA was highly expressed during growth on propane. The exact involvement of alkB-type alkane monooxygenase in the degradation of alkanes was still unclear. A gene transfer system for R. jostii strain 8 was established. Marker-exchanged mutagenesis of prmA and alkB was attempted. Construction of mutated-prmA and mutated-alkB plasmids was achieved. Electroporation conditions were successfully optimised in order to transfer linear DNA into R. jostii strain 8. However, mutants lacking active prmA or alkB are still needed to further study their phenotypes and to provide more evidence supporting the role of these two enzymes

Molecular characterization of three linked genes, fixB, βshbd and adh1 from Clostridium acetobutylicum P262

Bibliography: pages 140-158.Clostridium acetobutylicum P262 is an endospore-forming Gram-positive anaerobic bacterium, and it has been used in the industrial production of acetone and butanol for many years. The aim of this study was to characterize the upstream region of the βhbd gene which is linked to the adh1 gene, and to investigate the expression of these linked genes by transcriptional analysis. The upstream region of the βhbd gene was isolated from a gene bank of C. acetobutylicum P262, constructed using the pWE15 cosmid vector. Characterization of this upstream region was done initially at the nucleotide sequence level. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1002-bp which encoded a protein of 334 amino acid residues with a calculated Mᵣ of 35,679. This protein showed significant amino acid homology to the fixB protein of Rhizobium meliloti and Azorhizobium caulinodans and the electron transport flavoproteins from humans and rats. Studies on the expression of the three linked genes fixB, βhbd and adh1 were carried out at the transcriptional level. Northern and primer extension analyses indicated that all of the three genes were independently transcribed throughout the various stages of the acetone-butanol-ethanol (ABE) fermentation in C. acetobutylicum P262. Each of the genes produced mRNA transcripts of approximately 1.4 kb. The βhbd and adh1 genes were shown to have at least two major and one minor transcriptional start sites in C. acetobutylicum P262. Transcription was initiated at the same promoter region of the fixB gene in both C. acetobutylicum P262 and Escherichia coli. the βhbd gene was shown to have a stronger promoter region than those of the fixB and adh1 genes based on the lacZ-fusion studies in E. coli. The βhbd and adh1 genes encode the 3-hydroxybutyryl-CoA dehydrogenase (BHBD) and the NADPH-dependent alcohol dehydrogenase (ADH), respectively. The BHBD enzyme is part of the central fermentation pathway and is required for acid and solvent production, whereas the ADH is part of a branched solvent pathway and is only required for solvent production. Analysis of mRNA transcription and the identification of transcription initiation sites, indicated that each of these two genes was independently and constitutively transcribed throughout the acidogenic, sol ventogenic and sporulation stages in C. acetobutylicum P262 and in exponential E. coli cells. These results suggest that the adh1 gene is not part of a branched solvent pathway which is only induced and transcribed during the solventogenic phase

Exploiting the Anaerobic Expression of Pyruvate Dehydrogenase for the Production of Biofuels

The Pyruvate dehydrogense complex (PDH) is a primarily aerobic enzyme which catalyses pyruvate to acetyl-CoA and carbon dioxide. Its counterpart in anaerobic metabolism is pyruvate formate lyase (Pfl) which converts pyruvate to acetyl-CoA and formate. A novel fermentation pathway involving PDH rather than Pfl (or equivalent), which retains the reducing equivalents from pyruvate oxidation, could provide a novel route for ethanol production, as well as changes in redox balance opening up opportunities for the production of higher alcohols such as butanol. Utilising PDH for the production of biofuels has been investigated in three microorganisms: Geobacillus thermodenitrificans, Bacillus subtilis, and E. coli. Geobacillus thermodenitrificans does express Pfl, thus PDH is always active in the G. thermodenitrificans regardless of whether the bacterium is growing in aerobic or anaerobic conditions. To utilise this PDH in the production of ethanol a bi-functional alcohol dehydrogenase (AdhE) was introduced to G. thermodenitrificans K1041. Further optimisation of ethanol production was achieved by knocking-out lactate dehydrogenase (Ldh), which would otherwise compete with ethanol for flux from acetyl-CoA, and activity of the PDH promoter verses potential alternative promoters to increase the expression of the native PDH was investigated. Like G. thermodenitrificans, Bacillus subtilis also does not have a PFL pathway, but does have a native Adh so can undergo fermentation, albeit poorly. To increase ethanol production competing fermentation pathways were knocked-out, however this resulted in strains which were unable to grow anaerobically. The activity of the native PDH promoter was investigated, and PDH subsequently upregulated. The production of 1-butanol from B. subtilis was also achieved using expression of Clostridial genes encoding a butanol synthetic pathway from a plasmid and from chromosomal integrations. PDH in Gram-negative bacteria such as E. coli are not active during anerobic growth due to fermentation resulting in elevated levels of intracellular NADH; which in turn triggers negative feedback inhibition of PDH. A consequence of this is E. coli strains which are engineered to produce increased titres of ethanol by knocking-out pfl are unable to grow anaerobically. To alleviate this problem a PDH from gram-positive bacteria was expressed in E. coli. The effect of these PDH was also used to assess their potential benefits on 1-butanol in E. coli, by introducing Clostridial genes encoding a butanol synthetic pathway via plasmids

The transition between growth and sporulation in Physarum polycephalum CL

The overall aim of this project was to examine the relationship between the cell cycle and sporulation in Physarum polycephalum: in particular, to determine when sporulation specific genes were transcribed. The work fell into three sections. The first made use of the anti-fungal agent nocodazole to determine if a plasmodium competent to sporulate after 72 h starvation was in the G1 or the G2 phase of the cell cycle. Clear evidence was obtained that differentiation was initiated from the G2 phase of the cell cycle. The effect of blocking mitosis with nocodazole on the subsequent DNA replication was investigated in both growing and starving plasmodia. It had previously been reported, and was shown in this work, that P. polycephalum has no G1 phase in the cell cycle during growth. The fact that growing and starving plasmodia responded similarly to nocodazole with regard to the onset of DNA synthesis indicated that there was no prolonged G1 period during the starvation period in P. polycephalum. It is postulated that nocodazole may interfere with a temperature-sensitive pathway that controls both the increase in thymidine kinase activity and metaphase onset. The second part of the investigation was to approach the problem of pinpointing when in the G2 phase of the cell cycle, there was sporulation specific transcription. It was assumed that this question might be answered by differential screening of a genomic library of P. polycephalum, using as probes radiolabelled copy DNA prepared from poly(A)+ RNA from growing and starving plasmodia. The first requirement was a genomic library of P. polycephalum CL DNA. Of the two phage vectors, Charon 4AP and lambda1059 which were compared, the latter proved to be superior as it was shown that a genomic library prepared in Charon 4AP would be diluted by the presence of a considerable number of non-recombinant phage. To generate libraries of P. polycephalum DNA it was necessary to digest it with suitable restriction endonucleases. P. polycephalum DNA was partially digested with either Sau3A or BamHI and the 15-25 kb fragments were isolated by electroelution. These fragments were then used to generate two genomic libraries. In each case only one type of recombinant phage was created which was derived from lambda1059 and contained a fragment of Physarum DNA. The DNA used to prepare these gene banks was found to be contaminated by a second type of DNA. This contaminating DNA was tentatively identified as mitochondrial in origin. This difficulty was eliminated when Physarum DNA was isolated by the method of Hardman & Jack (1978). DNA was partially digested with Sau3A and the 15-25 kb fragments isolated. A genomic library was prepared in lambda1059 and restriction analysis of a random sample of phage showed that all were derived from lambda1059 and all had restriction patterns different from the parental phage. Hybridization of [32P] nick-translated Physarum DNA to filter replicas of phage identified the inserts as Physarum DNA. The third part of the work involved the isolation of RNA from P. polycephalum. A requirement for screening the library was the preparation of undegraded poly(A)+ RNA from which copy DNA probes could be made. Initially an attempt was made to isolate RNA that was being actively translated on polysomes at the time of isolation. However, all attempts to prepare polysomes in sufficient quantity were unsuccessful. Cytoplasmic RNA was isolated from growing plasmodia but was highly contaminated by a polysaccharide material. This contaminant was removed by cetyltrimethylammonium bromide precipitation. Examination of the RNA, after electrophoresis under denaturing conditions showed that the RNA was very susceptible to degradation even when prepared in the presence of two inhibitors of RNase activity, RNasin and vanadyl ribonucleoside complex. Less degraded RNA was isolated in a buffer containing 4M guanidine thiocyanate, an inhibitor of RNase activity. This total RNA preparation was less degraded than the cytoplasmic RNA. When poly(A)+ RNA was isolated by oligo (dT) cellulose chromatography it directed the synthesis of very short copy DNA. The purest and most undegraded RNA was isolated by a modified version of the method described by Cox & Smulian (1983). After the initial isolation procedure the poly(A)+ RNA was further purified by a cetyltrimethylammonium bromide precipitation and a phenol/chloroform extraction. The poly(A)+ RNA was used as a template for the synthesis of cDNA in vitro which was found to be 200-900 nucleotides in length. This cDNA hybridized to filter replicas of recombinant phage. The overall conclusion from this work was that the molecular genetical techniques applied in this study have a good potential for investigating the detailed sequence of events in sporulation of Physarum polycephalum

Engineering lipases and solvents for trans/-esterification of used vegetable oils

Diminishing petroleum reserves and increasing environmental awareness has led to an urgent need to develop alternative fuels, such as biodiesel. However, the conventional method to produce biodiesel uses environmentally harmful chemical catalysts. A relatively new development in the production of biodiesel is through enzymatic trans/- esterification with a lipase catalyst. Despite several advantages, there are a few technical and economical obstacles that limit this process: (1) immiscibility of the hydrophilic methanol and hydrophobic triglyceride which results in the formation of an interface leading to mass transfer resistance, (2) insufficient availability of large quantities of inexpensive lipase suitable for catalysis, and (3) stripping of essential water from the active site by the strong polarity of methanol causing a reduction in enzyme activity after multiple reuses. Each obstacle is addressed by: · Examining the effect of 15 organic solvents on activity of lipase from three sources, namely Candida antarctica, Pseudomonas cepacia, and Thermomyces lanuginosus, in the trans/-esterification of used vegetable oil with the goal of recommending the best solvent through solvent engineering. · Cloning and over-expressing recombinant lipase from T lanuginosus in tobacco for the enzymatic production of biodiesel in order to develop an abundant inexpensive biocatalyst. · Investigating the effects of reaction parameters on the trans/-esterification of used vegetable oil and their effects on enzymatic activity over consecutive reactions with a view to lowering costs. The major findings are: (i) there appears to be a correlation between the solvent\u27s hydrophobicity (log P) and biodiesel yield, (ii) the choice of lipase can have a considerable effect on the reaction kinetics and biodiesel yield, (iii) a thermophilic fungus lipase gene can be constitutively expressed in tobacco without adversely affecting plant growth or development, (iv) plants systems offer a promising platform for producing recombinant enzymes for biodiesel production, (v) the type of methanol addition does not appear to greatly affect the biodiesel yield when fresh enzyme is used, but does significantly affect the enzyme\u27s activity during subsequent reuse, and (vi) optimization of reaction parameters such as methanol addition, reaction temperature, and solvent can minimize enzymatic deactivation and increase enzyme reusability without significantly affecting biodiesel yield

MOLECULAR RESOLUTION OF MARINE NEMATODES FOR IMPROVED ASSESSMENT OF BIODIVERSITY

Free-living nematodes are abundant in all marine habitats, highly diverse and can be important ecological indicators for monitoring anthropogenic impacts on the environment. Despite such attributes, nematode diagnostics has traditionally relied on detailed comparison of morphological characters which is often difficult and laborious, and as a result there is an increasing 'black hole' in faunal inventories where the biodiversity of groups such as nematodes is typically underestimated. Molecular methods offer a potentially efficient alternative approach to studying the biodiversity of marine nematode communities, and the main focus of this thesis was to apply molecular ecological tools for improved understanding of nematode diversity in marine and estuarine environments. Denaturing gradient gel electrophoresis (DGGE) has been evaluated as a novel tool for the identification of marine nematodes and for rapid assessment of their diversity based on amplification of the nuclear 18S rRNA gene. This approach successfully identified nematode taxa based on banding pattern and was also able to detect the most abundant taxa in samples from marine and estuarine environments. A DNA barcoding approach based on the 18S rRNA gene was applied for the first time in marine nematology, in an attempt to speed up the identification process. The success rate of this approach, across a range of nematode groups, was found to be close to 97%. A combined morphometrics and molecular approach was also undertaken to investigate cosmopolitanism and cryptic speciation by analysing populations of a cosmopolitan marine nematode, Terschellingia longicaudata, from different geographical regions. Results suggest that Terschellingia longicaudata is indeed truly cosmopolitan, with a wide geographic distribution. Two haplotypes that were divergent from most T. longicaudata were also identified in this study, indicating possible novel cryptic lineages or previously undescribed species of the genus. The final focus of this thesis was to develop methods for the molecular investigation of nematodes stored in formalin and other organic compounds. The effectiveness of formalin as a short term preservative was first evaluated, since this would allow morphological and molecular work to be conducted on the same specimen. Amplifiable DNA could be routinely obtained from specimens stored in formalin for periods of up to nine days. In addition the effectiveness of other organic solvents for the preservation of both molecular and morphological integrity of marine nematodes was investigated. The final part of this study developed and optimized a novel DNA extraction technique that could be employed to recover DNA from archived formalin fixed marine nematode specimens so as to carry out subsequent molecular analysis such as PCR amplification and sequencing.Plymouth Marine Laborator

Towards spatial computing and chemical information storage in soft materials using DNA programming

Living organisms possess the ability to form and recover complex patterns in prescribed locations at length scales of hundreds of microns. During the past 15 years, experimentalists within the fields of DNA nanotechnology and synthetic biology have developed a variety of systems capable of self-assembly and reorganization at the nanoscale using synthetic oligonucleotide building blocks to mimic the functions of biological tissues and to provide new routes of manipulating materials with molecular programs. Programming ‘smart and responsive’ nano- and micromaterials using DNA circuits has the potential to impact numerous applications including molecular diagnostics, biodefense, drug delivery systems, and low-energy information storage. In this thesis, I present and develop computational and experimental systems that leverage oligonucleotide strand displacement reaction networks, digital maskless photolithographic technology, and microfluidic delivery methods to design DNA-functionalized micro-materials that process and store chemical information spatiotemporally. These systems couple reactions, transport, and feedback control to achieve specific temporal concentration profiles at specific points in hydrogel substrates. First, I developed a reaction-diffusion waveguide designed to coordinate spatiotemporal sensing and regulation of synthetic DNA- based materials using autocatalysis. I discuss the design requirements for this architecture and the results of in silico and experimental analyses of the components of this system. Based on the operational requirements of this system, I then designed a DNA-compatible hydrogel microfabrication method that accommodates UV photo-directed release of oligonucleotides from defined regions of a hydrogel, which can be used to initiate downstream reaction-diffusion processes in materials. Building on this platform, I constructed a reaction-diffusion system that enables shape programming of biomolecular attractor patterns in photopatterned poly(ethylene-glycol) diacrylate microgels. These patterns were able to heal their structure in response to spatial perturbation. Finally, I develop and discuss a model of a reaction-diffusion associative memory, consisting of a distributed network of nodes that store and repair spatial chemical patterns