Nearly all bacteria require iron for growth as it is utilised in many major biological processes. Despite its indispensability, iron causes problems of toxicity, poor solubility and poor bioavailability. Highly efficient iron acquisition and storage systems are used to overcome these problems. Gram-negative bacteria take up iron and haem complexes via specific outer-membrane (OM) receptors. Translocation across the OM is dependent on the cytosolic membrane potential and the energy transducing TonB-ExbB-ExbD system. Iron-complexes thus delivered to the periplasm are in tum translocated across the cytoplasmic membrane (CM) to the cytosol via binding-protein dependent ATP-binding cassette (ABC) transporters. DNA array analysis of global iron-dependent gene regulation in Escherichia coli K-12 has revealed several novel Fe-repressed genes likely to specify new components of iron uptake. Such genes include the yncD and yncE genes. yncD and yncE are divergently orientated in the E. coli genome being separated by 241 bp of non-coding DNA. yncD encodes a possible TonBdependent outer-membrane ferric-siderophore receptor and, like yncE, is conserved in other E. coli strains and Salmonella. YncD appears to possess all the structural features of typical TonBdependent OM proteins, including an N-terminal signal sequence directing export across the CM and a C-terminal TonB box. However, the yncD gene is, at best, only weakly Fur and iron regulated. In contrast, the yncE gene is strongly Fur and iron regulated (up to 18 fold) suggesting that the intergenic Fur-box like sequence acts upon the yncE promoter. yncE encodes a predicted periplasmic protein of unknown function. Close homologues of Y ncE are multi-domain proteins from archaebacteria and are annotated as cell surface antigens/proteins, although their precise functions are unclear. In the research described in this thesis, Y ncE has been over-produced and purified, and antibodies have been successfully raised. Western blotting has confirmed that YncE levels are iron and Fur regulated, and that levels are maximal in the early/mid logarithmic phase of growth. Analysis of subcellular fractions show that Y ncE is mostly located in the periplasm, as anticipated. The N-terminus of isolated YncE is processed consistent with its periplasmic location. Difference spectroscopy indicated that YncE-His6 bind haems in an approximate ratio of 1:1, and BiAcore analysis confirmed haem binding with a high affinity (KD value of -6 nM). However, spectroscopic analysis of native (tag free) Y ncE did not show any clear interaction with haem, thus indicating that YncE-His6 binds haem via its His tag. Studies with strains containing yncD-or yncE-lacZ transcriptional fusions confirmed that expression of yncE is growth-phase dependent, with maximal expression in the exponential phase. yncE transcription was observed to be induced by Bip as expected. On the other hand, although yncD transcription had a similar growth-phase dependency, the overall degree of expression was far less (-20 fold). Furthermore, Bip was not found to induce expression, indicating thatyncD, in contrast to yncE, is not Fe2 + -Fur repressed. Crystals of YncE-His6 were obtained. The crystals are monoclinic, space group P2., a=69.7A, b=108.8IA, c=85.37A and 13=104.96°. Complete data sets for the native (2.0 A) and two isomorphous derivatives (Au and Ag, 2.6A and 3.oA, respectively) allowed the structure to be solved. Model building and refinement gave a seven bladed B-propeller structure for Y ncE. Superimposition of the methanol dehydrogenase (MDH; a PQQ containing B-propeller protein) and YncE structures showed that similar residues are located in the two proteins function in MDH as pyrroloquinoline quinone (PQQ) ligands. Y ncE appears to support binding of a PQQ-like cofactor in that it has a similar hydrophobic edge (Phe62) and several positively charged residues all residing on the top of the B-propeller domain (ArgI14, Lys199, His285, Asn287, Lys303). In addition, it was possible to test whether YncD is required for transport of PQQ into the periplasm for glucose dehydrogenase (GCD) activity which is required for glucose-dependent growth in the absence of the glucose transport systems. The phenotypic characterisation of a yncD null mutation in a glucose/mannose transport negative background (ptsG and manZ) , clearly showed that Y ncD is not required for PQQ enhanced growth of ptsG and ptsG manZ strains on glucose. This clearly shows that YncD is not involved in the acquisition of extracellular PQQ (at least not for GCD utilisation) as was suggested. Also, no PQQ binding to YncE could be detected by BiAcore analysis. This is consistent with no role for YncD in PQQ delivery.

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A method to improve production of Fab' fragments using gas blending and pH-stat feeding strategy in a fermentation process with Escherichia coli has been developed. Regime analysis together with design of experiments (DoE) has been used to evaluate the effect of gas blending and feeding strategy on a Fab' fermentation process. The economic implications of the fermentation strategy have also been considered. Batch-feeding fermentations carried out at 20 L and 450 L scale indicated that cascade control was not sufficient to maintain a constant DOT level throughout the fermentation. DOT levels dropped to zero during induction phase at both scales. Regime analysis was performed based on experimental KLa determination. KLa values of -400 h"1 were observed at 20 L and 450 L scale. Comparison of time of oxygen consumption (tQc) and time of oxygen transfer (t0i) suggested that 02 limitation is present in this fermentation process and that this worsens as the scale increases. A gas blending system at 20 L scale was proposed to address this problem. A factorial 22 experimental design was executed to evaluate independently the effects and interactions of two main engineering factors (related to oxygen transfer in the broth) on Fab' titre: DOT level and agitation rate. Gas blending was successful in maintaining constant levels of DOT at 20 L scale. Fab' production was increased by 77 % at of agitation rate of 500 rpm independent of the DOT level, compared to operation at the same scale but without gas blending. High levels of product localisation in the periplasm of 84 - 93% were also obtained. Furthermore, based on t0c and t0T, it could be suggested that no oxygen limitation is likely to occur in the fermentations performed with gas blending, regardless of the agitation rate. Batch-fed fermentations either with or without gas blending carried out at 20 L scale indicated the presence of glycerol oscillations. A pH-stat feeding strategy in a gas blending system was implemented to address this problem. Results showed that a 2-fold increase in the production of Fab' at 20 L scale, compared to a fermentation operated in a pulsed fed- batch mode could be achieved. A consistently high level (>90%) of product localisation in the periplasm was also achieved and no negative impact on product recovery was observed. Finally, a preliminary economic analysis was performed to estimate the production costs in £/mg of product. The effects of gas blending and feeding strategy at 20 L scale on the final product cost were evaluated by comparing: batch-fed non gas-blending, batch-fed gas blending, and pH-stat gas blending fermentations. A fermentation production cost of £1.45/mg of Fab' was estimated for a pH-stat gas blending system. This resulted in cost savings of up to 75% when compared to the production cost of £5.80/mg of Fab' in the batch-fed system without gas blending. The results obtained in this work provide an impetus for further studies to evaluate the potential of gas blending and pH-stat feeding strategy for the industrial production of Fab' antibody fragments. The use of statistical design of experiments together with regime analysis was found to be a very useful tool to gain a better understanding of this system.

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