G enetic M anipulation

1.3.1 Mutation & Strain Selection

Prior to the use of recombinant DNA technology, the commercial success of strain development and improved productivity was dependant on mutation and strain selection. Methods commonly used for mutation include treatment with UV radiation and chemical reagents such as NTG. After treatment with an appropriate mutagen, the microbial strain able to express the desired phenotypic characteristics is isolated using strain selection techniques. Illustrations of the use of this technique include the development of cultures for chlortetracycline production (Stanbury & Whittaker, 1993), penicillin production (Cruegar & Cruegar, 1990) and cis,cis muconic acid production (Yoshikawa et a l, 1993).

The molecular mode of action of some mutagens is fairly well known, but what can never be predicted is the effect of a mutation on a specific gene or complex process.

The appearance of mutants is also dependant on factors such as the base sequence of the gene to be mutated, location of "hot spots", the repair system of the cell and the occurrence of suppressor mutations often resulting in reversion to wild type (Jenkins et a l, 1987). The process of mutation, strain selection and the application of an appropriate screening program can be lengthy and expensive, and some compromise must be made between the suitability of a mutant strain and its productivity. In addition, the search for producers of compounds which do not give the producing organism any selective advantage means random isolation procedures need to be adopted. Therefore a more exact and accurate method of producing strains which over accumulate desired products is useful.

1.3.2 Recombinant DNA Technology

Recombinant DNA technology allows the greater expression of targeted genes within a host micro-organism. The use of plasmids as cloning vectors facilitates the presence of multiple copies of a plasmid construct within a biological cell. High copy number plasmids are frequently used to further increase the gene dosage per cell (Martin-Rendon et a l, 1992), and hence increase the magnitude of gene expression within a fermentation or biotransformation. The use of strong promoters such as lac

promoters (Laffend & Shuler, 1994) will also increase gene expression. This technology also allows the possible transferral of targeted genes into foreign microbial hosts. An enzyme can be produced not only in the original micro-organism, but also in another microbe which may have properties making it more suitable for use in a particular biotransformation. This technique has been used to synthesise products such as ethanol (Ohta et a l, 1991), tyrosine and phenylalanine (Ikeda & Katsumata, 1992) and indigo (Flores et a l, 1996). Although interest in the use of recombinant DNA technology has increased, its use for industrial strain improvement has a long way to go before it can compete with well established mutation techniques. Reasons for this may be due to the lack of basic knowledge of the genetics of some industrial micro­ organisms and the relative ease of application of mutation and strain selection procedures, despite the problems associated with them.

The manipulation of a partial or whole metabolic pathway as opposed to the individual genes within the pathway suggests another application of recombinant DNA technology. This application involves the exploitation of a series of sequential biochemical reactions in order to accumulate a desired precursor - and is known as metabolic pathway engineering.

Fiona Vanier__________________________________________ PhD Thesis Introduction

1.3.3 M etabolic Pathway Engineering 1.3.3.1 Definition and Categorisation

Naturally occurring metabolic networks are not optimised for the aims necessary for practical applications. Consequently, the success of a bioprocess involving product over-accumulation is dependant on genetic modification of the metabolic pathway. Metabolic pathway engineering involves the manipulation of a series of sequential biochemical reactions in their entirety, and has been defined as the improvement of cellular activities by manipulation of cellular functions using recombinant DNA technology (Bailey, 1991). The effects of genetically altering a metabolic pathway must be experimentally confirmed since individual pathways do not function in isolation from other pathways in the cell. Experimental confirmation is especially important so as to avoid any "metabolic surprises" which may accompany a proposed change.

The methods of genetic manipulation discussed in this chapter are shown in Figure 1.1. The uses of metabolic engineering are divided into the four categories shown (Cameron & Tong, 1993), each of which will be covered in more detail. One should appreciate that these categories are arbitrary and some examples fit into more than one category. For example, the work of Wood & Ingram (1992) involves the improvement of ethanol production by recombinant K. oxytoca and the extension of the substrate range to incorporate cellulose.

1.3.3.2 Improved Production of Chemicals

A great deal of work has been done on ethanol production, since it is widely used for the production of alcoholic beverages and as an alternative transportation fuel. The main organisms of interest are E, coli, Z. mobilis, Erwina sp., and Klebsiella sp. In most cases, the host micro-organisms are ethanol producers but the yield from the native pathways is relatively low. The reason for such low yields is the synthesis of mixed fermentation products such as acetate, formate, lactate and butanediol - as well as ethanol. The introduction of new genetic material which favours ethanol as the principle fermentation product led to the improved production of ethanol to levels comparable to those found in industrially used micro-organisms. Earlier work involved the expression of the gene(s) encoding alcohol dehydrogenase II and pyruvate decarboxylase from Z. mobilis in host organisms such as E. coli (Ingram et a l, 1987),

GENETIC MANIPULATION