Cm

Prephenate

Pretyrosine

¿Phenylalanine Tyrosjne u

Tryptophan -,

Fig. 3.30. Control of the aromatic amino acid family in Escherichia coli

---• Feedback inhibition control

-• Feedback repression control

CM Chorismate mutase

DS DAHP synthase

P.Dehyd Prephenate dehydratase

P.Dehydrog Prephenate dehydrogenase

SK Shikimate kinase was subjected to in vitro mutagenesis and the wild type gene replaced. This one mutation then resulted in the derepression of both tyrosine and phenylalanine DAHP synthase as well as shikimic kinase.

(ii) The chorismic mutase/prephenate dehydratase protein is under both repression and attenuation control by phenylalanine. These controls were eliminated by replacing the normal promoter with one not containing the regulatory sequences, thus giving a ten times increase in gene expression.

The regulation of enzyme inhibition was modified by:

(i) Chorismic mutase/prephenate dehydratase is subject to feedback inhibition by phenylalanine. It was shown that a certain tryptophan residue was particularly important in the manifestation of feedback inhibition. In vitro mutagenesis was used to delete the tryptophan codon and the modified gene introduced along with the substituted promoter referred to earlier. The enzyme produced by the modified gene was no longer susceptible to phenylalanine, (ii) The rationale was to limitOA the availability of tyrosine such that the tyrosine-sensitive DAHP synthase isoenzyme would not be inhibited. Although the other two isoenzymes would still be susceptible to phenylalanine and tryptophan inhibition, sufficient DAHP would be synthesized by the third isoenzyme (in the absence of tyrosine) to facilitate overproduction of phenylalanine. Traditionally, this objective would have been achieved by using a tyrosine auxotroph fed with limiting tyrosine. However, these workers developed an excision vector system. An excision vector is a genetic element that can carry a cloned gene and can both integrate into, and be excised from, the bacterial chromosome. The vector is based on the bacteriophage lambda and the technology is explained in Fig. 3.31. The excision of the vector can be induced by a temperature shock. Thus, when the vector is excised the progeny of the cell will lose the inserted DNA. The tyrA gene was deleted from the production strain, inserted into the excision vector and transformed back into the organism where the vector became integrated into the chromosome. The fermentation could then be conducted using a cheap medium (the organism was not auxotrophic at inoculation) and allowing growth to an acceptable density. A heat shock may then be used to initiate vector excision, tyrosine auxtrophy and, hence, phenylalanine synthesis.

These efforts should have generated a high-producing strain. However, the tyrosine -sensitive DAHP synthase was susceptible to inhibition by high concentrations of phenylalanine. It will be recalled that the flow of DAHP was intended to come from the deregulated tyrosine sensitive isoenzyme. Thus, the final step in the development of the strain was to render this isoenzyme resistant to phenylalanine inhibition. This was achieved by the selection of mutants resistant to phenylalanine analogues. Thus, the strain was improved using a combination of gene cloning, in vitro mutagenesis and analogue resistance, indicating the importance of the contribution of a range of techniques to strain development. The final strain was capable of producing 50 g dm"3 phenylalanine at a yield of 0.23 ggM glucose and 2 g g ' biomass. The organism produced very low att int

N oLpL cl tyrA

att oRpR rp tyrA

Fig. 3.31. Excision vector technology. An excision vector is repre. sented as a line between two boxes. Above the line and boxes are indicated those genes from bacteriophage lambda that are carried on the vector. oRpR is required for the expression of a repressor determined by cl. This repressor binds to oRpR and oLpL to prevent the expression of other lambda genes. The other genes collectively act to form a recombination activity that allows the vector to integrate into or excise from the bacterial chromosome at the sites (indicated by the boxes) named att. Other genes, such as tyrA (indicated below the excision vector), can be cloned into the excision vector prior to its introduction into a target cell and can thereby be present or absent in the cell in coordination with the vector. Upon entering a cell, the genes for recombination (/V, xis, and int) are expressed because there is not yet any repressor' As repressor accumulates, it shuts off the expression of those genes. In a fraction of the recipient cells, the recombination enzymes cause the vector to integrate into the cell chromosome before those enzymes decay away. That cell and all of its progeny inherit the vector and any gene it might carry (such as tyrA). If the repressor is inactivated, such as by high temperature, new recombination enzymes are formed that excise the vector from the chromosome. In such a cell and all of its progeny, the vector and the gene(s) it might carry are lost (Backman et al., 1990).

amounts of the other products and intermediates of the pathway which the workers claimed was due to the veiy precise manipulation of the strain which avoided the concomitant adverse characteristics associated with many highly mutated organisms.

Ikeda and Katsumata (1992) redesigned a tryptophan producing C. glutamicum strain such that it overproduced either phenylalanine or tyrosine. The regulation of the aromatic pathway in C. glutamicum is shown in Fig. 3.32. Phenylalanine producers which were resistant to phenylalanine analogues were used as sources of genes coding for enzymes resistant to control. Thus, a plasmid was constructed containing genes coding for the deregulated forms of DAHP synthase, chorismate mutase and prephenate dehydratase. The vector was introduced into a tryptophan overproducer having the following features:

(i) Chorismate mutase deficient and, therefore, auxotrophic for both tyrosine and phenylalanine.

(ii) Wild type DAHP synthase.

(iii) Anthranilate synthase partially desensitized to inhibition by tryptophan.

D-Erythrose 4-phosphate + Phosphoenolpyruvate . | DS

Dihydroxyacetone phosphate {DAHP) t

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