OH catechol 2,3-dioxygenase

catechol 1,2-dioxygenase blockage in pathway low affinity COOH _{:0 0 H} COOH OH dicarboxylic acid compound

not produced

semialdehyde / ketol compound not produced

FIGURE 1.11:

Pathways for the degradation o f benzene (R=H) and toluene (R=CH3) by the mutant strain P. putida 2313 developed by Robinson et a l, (1992). C l 2 0 has a low affinity for 3-methyl catechol and a blockage in the pathway at the level o f C 2 3 0 results in accumulation o f 3-methylcatechol.

Fiona Vanier__________________________________________ PhD Thesis Introduction

have been supplied in the form of a review which outlines the necessary biochemical engineering which must be considered during the decision-making stage of process design. The biocatalytic oxidation of aromatic compounds was used as a model system in this review, with a particular emphasis on the TOL pathway (Marshall & Woodley,

1995).

1.5.2 Designing a Feasibility Study 1.5.2.1 Theoretical Evaluations

A theoretical evaluation is necessary for the identification of key pathway characteristics using the literature available. Features which are potentially beneficial and detrimental to the process should be identified, so that possible strategies can be developed which maximise the beneficial features and minimise the detrimental features. The physical and chemical properties of the compounds synthesised during the biotransformation should be determined, since properties such as the low solubility of a substrate may reduce the amount of substrate in contact with the enzyme unless countered by a successful substrate feeding strategy. Toxic and inhibitory effects caused by any of the compounds should be thoroughly investigated, since the maximum concentrations that can be tolerated by the host must be known prior to the design of a feeding or removal process. The properties of the pathway enzymes should also be evaluated, with a particular emphasis on their mineral content and any cofactor requirements. This will ensure that the medium is adequately supplemented with the precursors necessary for’recombinant protein production.

1.5.2.2 Sequencing of Operations

Prior to the assessment of process engineering options, the phases of the process must be identified in order. A whole cell bioconversion consists of a growth phase, a substrate conversion phase and a product recovery phase. The sequencing of these phases (or operations) is largely dependant on the choice of host micro-organism and metabolic pathway. If the activity of the pathway enzymes is growth associated, than it may be beneficial to carry out the growth and substrate conversion phases simultaneously. Separation of these phases in necessary if the amount of enzyme produced during growth is insufficient to promote the bioconversion or if the product, substrate and / or intermediates inhibit growth.

1.5.2.3 P ro c e s s Im p ro v e m e n ts

After the development of the initial process, alterations can then be made in order to improve process efficiency. A method of measuring the success of any improvements is necessary and must be such that a comparison can be made between two or more processes regardless of their operating scale. An indicator of success within a test system is important not only because it is a defined aim and goal, but also because it ensures that only the most relevant areas of process design are covered. This could be the maintenance of the plasmid such that levels of enzyme activity are maintained over a defined period of time, or the achievement of a specific concentration of the product. Improvements may not necessarily involve a change in the process with respect to biochemical engineering. Further genetic engineering may increase the efficiency of the process to a far greater extent than any change in the process environment. Amplification of the genes encoding the enzymes with the greatest influence on the metabolic flux may result in dramatic improvements. On the other hand, changes in the physical and chemical environment can also influence the pathway flux, and the sensitivity of the metabolic pathway to such changes could be tested. Consequently, both development and improvement of the bioprocess involves an integration of the skills of the genetic and biochemical engineer, and the use of a feasibility study as an aid during process design. The initial feasibility study is usually based on theoretical research, so in order to establish whether the guidelines stated are of any practical use, the practical design of the bioprocess should be undertaken using these guidelines.

1.5.3 P r o je c t A im s

The aims of this research were to evaluate all the process engineering options necessary for a multi-step biotransformation, undertake the decision-making stage of process design by following the guidelines in a feasibility study and to put into practise the design of a bioprocess. The model system used for this practical evaluation was made up of a linear portion of the TOL mera-cleavage pathway expressed in a recombinant E. coli host strain JM107, and is shown in Figure 1,12.

The process scheme proposed is shown in Figure 1.13. A theoretical feasibility study will ensure that as much information as possible is known about the process prior to design. The experiments undertaken are dependant on the information collected in the feasibility study. The practical evaluation can be defined as the assessment of the workability of the process by analysis of results obtained from practical data.

Fiona Vanier PhD Thesis Introduction benzoate Moxygenase xylXYZ benzoate dihydrodiol dehydrogenase xylL COOH _{HOOC OH} OH NADH N A C y NAD"^ NADH catechol 2,3-dioxygenase xylE OH O2 L L _ II iT^^OOH' t O O H ^ O H BENZOATE Gene Map: BENZOATE DIHYDRODIOL CATECHOL 2-HYDROXYMUCONIC SEMIALDEHYDE EcoRl _{X h o l}

FIG U RE 1.12:

The metabolic pathway used as part o f the model system for this research.