Info

P.Dehyd t CM

Prephenate

| P.Dehydrog

Phenylpyruvate Pretyrosine

Tyrosine i t CM

Prephenate

| P.Dehydrog

FlO. 3.32. Control of the aromatic amino acid family in Corynebac-terium glutamicum.

---a Feedback inhibition control

CM Chorismate mutase

DS DAHP synthase

P.Dehyd Prephenate dehydratase P.Dehydrog Prephenate dehydrogenase SK Skikimate kinase

The transformed strain was capable of producing 28 g dm ~3 phenylalanine and the levels of all three enzymes coded for by the vector were amplified approximately seven fold. If the original tryptophan producing strain was transformed with a plasmid containing only DAHP synthase and chorismate mutase, then tyrosine was overproduced (26 g dm-3). Thus, genetic engineering techniques allowed a tryptophan producer to be redesigned into either a phenylalanine or tyrosine producer.

The development of B. sphaericus strains overproducing biotin was discussed in our consideration of the use of analogue resistant mutants. Although B. sphaericus is an intrinsically good biotin producer one of the limitations to its commercial exploitation is its complex nutritional requirements (Brown et al., 1991). It uses carbon sources such as glucose inefficiently and requires complex organic nitrogen sources. Thus, workers have cloned the biotin genes from B. sphaericus into E. coli. Again, it is important to appreciate that it was the analogue resistant genes that were cloned allowing deregulated production in a more commercially amenable host (Osawa et al., 1989; Brown et al., 1991). The two gene cassettes which were cloned were under the control of inducible promoters so that production could be initiated after the growth phase and therefore maintain plasmid stability. Yields of up to 45 mg dm-3 were achieved.

A different application of recombinant DNA technology is seen in the modification of the ICI pic Pru-teen organism, Methylomonas methylotrophus. The efficiency of the organisms' ammonia utilization was improved by the incorporation of a plasmid containing the glutamate dehydrogenase gene from E. coli (Windon et al, 1980). The wild type M. methylotrophus contained only the glutamine synthetase/glutamate synthase system which, although having a lower Km value than glutamate dehydrogenase, consumes a mole of ATP for every mole of NH3 incorporated. Glutamate dehydrogenase, on the other hand, has a lower affinity for ammonia but does not consume ATP. In the commercial process ammonia was in excess because methanol was the limiting substrate so the expenditure of ATP in the utilization of ammonia was wasteful of energy. The manipulated organism was capable of more efficient NH3 metabolism, which resulted in a 5% yield improvement in carbon conversion. However, the strain was not used in the industrial process due to problems of scale-up.

The application of in vitro recombinant DNA technology to the improvement of secondary metabolite production may not be as advanced as it is for primary metabolites, but it has made a very significant contribution. Techniques have been developed for the genetic manipulation of streptomycetes (Hopwood et al., 1985a) and the filamentous fungi (Elander, 1989) and a number of different strategies have been devised for cloning secondary metabolism genes (Hunter and Baumberg, 1989). In all the streptomycete systems so far studied the genes for the biosynthesis of a secondary metabolite are clustered. Furthermore, these clusters also contain the genes for regulation, resistance, export and extracellular processing. Work of this type has not only increased the basic understanding of the molecular genetics of secondary metabolism, but it has also facilitated strain improvement.

An excellent example of the application of recombinant DNA technology to the improvement of secondary metabolite production is provided by the work of the Lilly Research Laboratories group (Skatrud, 1992). These workers attempted to increase cephalosporin C synthesis by Cephalosporium acremonium by increasing the gene dosage at limiting steps in the pathway. Four critical steps were involved:

(i) Identifying the biochemical rate limiting step in the cephalosporin C industrial fermentation.

L-a-aminoadipic acid + L-Cyst + L-Val ACV synthetase

L-a-aminoadipic acid + L-Cyst + L-Val ACV synthetase

C02H O

Isopenicillin N

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