Fig. 3.33. The biosynthetic route to cephalosporin C in Cephalo-
sporium acremonium (Skatrud, 1992).
cef Genes coding for cephalosporin synthesis DAC Deacetylcephalosporin C DACS DAC synthase (commonly known as hydroxylase)
DAOC Deacetoxycephalosporin C
DAOCS DAOC synthase (commonly known as expándase) 1PNS Isopenicillin-N-synthase
(ii) Cloning the gene coding for the enzyme catalysing the rate limiting step.
(iii) Constructing a vector containing the cloned gene and introducing it into the production strain.
(iv) Screening the transformants for increased productivity.
The biosynthetic route to cephalosporin C is shown in Fig. 3.33. Analysis of fermentation broths showed an accumulation of penicillin N which indicated that there was a bottleneck at the next reaction, i.e. the ring expansion step where the 5-membered penicillin N ring is expanded to the 6-membered ring of deacetoxycephalosporin C. This step is catalysed by deacetoxycephalosporin C synthase (DAOCS, commonly called expandase) coded for by the gene cef EF (already previously cloned by Samson et al., 1987). cef EF also codes for the next enzyme in the route, deacetylcephalosporin C synthase (DACS, commonly called hydroxylase). Thus, if the expandase step were rate limiting, introduction of extra copies of cef EF should relieve the limitation and eliminate the accumulation of penicillin N.
The production strain of C. acremonium was transformed with a plasmid containing an exact copy of cef EF and the transformants screened for increased productivity. Approximately one in four transformants were superior producers and one produced almost 50% more antibiotic in a laboratory scale fermentation. Analysis of the transformant showed that a single copy of the transforming DNA had integrated into chromosome III, whereas native cef EF resides in chromosome II. In pilot scale fed-batch fermentations the transformant showed a 15% increase in yield which is still a very significant increase for an industrial strain. As predicted, the transformant did not accumulate penicillin N and the bottleneck appeared to have been relieved. The superior strain was among the first eight transformants examined, whereas 10,000 survivors of a mutagen exposure rendered no improved types. This work illustrates the enormous potential of recombinant DNA technology for secondary metabolite yield improvement, but it must be appreciated that such a programme involves a very considerable financial investment and the product must be sufficiently valuable to enable a good return to be realized on that investment.
IPNE Isopenicillin-N-empimeraseThe brackets indicate that DAOCS and DACS enzyme activities are carried on one polypeptide coded for by the ee/EF gene.
The major difficulty to be overcome in the use of genetically manipulated cultures is their potential instability in large-scale culture, especially continuous processes. The genetically manipulated strain of Meth-ylomonas methylotrophus appears to be stable in continuous culture, but this is not surprising since the modification renders the cell more efficient and, thus, selection force in the chemostat would tend to work to its advantage. However, strains which have been manipulated to produce products which give the cell no selective advantage would be selected against in long-term culture, especially in chemostat systems. Thus, the development of techniques to stabilize manipulated cultures and the incorporation of selectable marker genes consistent with the use of cheap fermentation media are critical to the successful fulfilment of much of the promise of genetic-manipulation techniques. The improved cephalosporin C producer described earlier was transformed with an integrating vector which means that the transformant is stable even in the absence of selective pressure.
The improvement of industrial strains by modifying properties other than the yield of product
The previous sections of this chapter have considered ways of increasing the yields of metabolites produced by industrial micro-organisms. However, the design and economics of a commercial process are influenced by properties of the organism other than its productivity. For example, although a strain may produce a very high level of a metabolite it would be unsuitable for a commercial process if its productivity were extremely unstable, or if the organism's oxygen demand were such that it could not be satisfied in the industrial fermenter available for the process. Therefore, characteristics of the producing organism which affect the process may be critical to its commercial success. Thus, it may be desirable to modify such characteristics of the producing organism which may be achieved by selecting natural and induced variants and recombinants. Naturally, it is crucial that the modified strain retains its desirable productivity so that the screening involved in these procedures should include assay of the yield as well as the characteristics being selected. Some examples of the characteristics which may be important in this context are: strain stability, resistance to phage infection, response to dissolved oxygen, tolerance of medium components, the produc tion of foam, the production of undesirable by-products and the morphological form of the organism.
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