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Cancers, hepatitis B, leukaemia Cancers, amyotrophic lateral sclerosis, genital warts Cancers, AIDS-related complex, osteopetrosis Hepatitis vaccine sidered (Ward et al., 1990; Dunn-Coleman el al., 1991). Bovine chymosin is an aspartyl protease extracted from the abomasum of unweaned calves and is used as a milk-clotting agent in the manufacture of cheese. Its natural source means that the enzyme is available only in limited quantities which made the possibility of producing it as an heterologous protein an attractive proposition. This process utilizes the filamentous fungus Aspergillus niger var. awamori which was claimed to have the following advantages as a potential host organism:
It is capable of secreting large amounts of protein.
The organism is regarded as safe.
Secretion from a eukaryotic cell is achieved via the endoplasmic reticulum and the Golgi apparatus. Entry into the secretory pathway is determined by the presence of a short, hydrophobic 'signal' sequence on the /Vterminal end of secreted proteins. The signal sequence directs the protein to sites on the endoplasmic reticulum (ER) from where transport into the lumen of the ER occurs. The signal sequence is cleaved by a luminal signal peptidase and the protein passes through the ER and Golgi body, before being packaged into secretory vesicles and exported beyond the cell membrane (Curran and Bugeja, 1993).
Several expression vectors were constructed incorporating the organism's glucoamylase gene and prochy-mosin cDNA. The inclusion of the prochymosin cDNA between the glucoamylase promoter and terminator resulted in a strain which accumulated only 15 mg dm"3 chymosin in the medium despite high intracellular mRNA and chymosin levels. This implied that the enzyme was not being secreted. To facilitate secretion, the prochymosin cDNA was incorporated between the glucoamylase coding region and the glucoamylase terminator. In this case, the organism should synthesize a 'fusion protein' consisting of chymosin attached to the carboxyl terminus of glucoamylase. The fusion protein was secreted and cleaved autocatalytically liberating the chymosin which was produced at 150 mg dm"3.
It is fascinating to appreciate that further improvement of this strain has been achieved by mutant induction and selection. Mutagenesis followed by random selection resulted in the isolation of a superior producer which was subsequently shown to produce very low amounts of the native aspartyl protease. Armed with this information the Genencor group cloned the aspartyl protease and then deleted the gene from the strain. Thus, the insight gained from the random screen allowed the directed in vitro approach to be used. The improved strain produced 250 mg dm"3 chymosin. This strain was then used in further mutation and selection programmes. An automated microtitre plate screen was developed which has already been described in an earlier section of this chapter and the results are shown in Table 3.6.
The strains developed using the automated screen were then mutated and subjected to a directed screen. Spores which survived nitrosoguanidine exposure were plated onto glycerol media containing 1% deoxyglu-cose. Deoxyglucose is a toxic compound which Allen et al. (1989) used to isolate resistant mutants of Neu-rospora crassa capable of producing higher levels of extracellular enzymes normally repressed by glucose. The best resistant strains (termed dgr) produced in excess of 1200 mg dm"3. Parasexual crosses between improved strains showed that two different loci were involved in resistance to deoxyglucose.
The chymosin story is an excellent example of the integration of recombinant DNA technology, mutant induction and automated random selection, directed selection and parasexual genetics in the development of a commercial strain.
(ii) The use of recombinant DNA technology for the improvement of native microbial products
Recombinant DNA technology has been used widely for the improvement of native microbial products. Frequently, this has involved 'self cloning' work where a chromosomal gene is inserted into a plasmid and the plasmid incorporated into the original strain and maintained at a high copy number. Thus, this is not an example of recombination because the engineered strain is altered only in the number of copies of the gene and does not contain genes which were present originally in a different organism. However, the techniques employed in the construction of these strains are the same as those used in the construction of chimeric strains, so it is logical to consider this aspect here.
The first application of gene amplification to industrial strains was for the improvement of enzyme production. Indeed, some regulatory mutants isolated by conventional means owed their productivity to their containing multiple copies of the relevant gene as well as the regulatory lesion. For example, the E. coli ¡3-galactosidase constitutive mutants isolated by Horiuchi et al. (1963) in chemostat culture also contained up to four copies of the lacZ gene. According to Demain (1990), during the 1960s and early 1970s the number of gene copies were increased by using plasmids or trans ducing phage in the same species. The production of yS-galactosidase, penicillinase, chloramphenicol transacetylase and aspartate transcarbamylase were all increased by transferring plasmids containing the structural gene into recipient cultures, especially when the plasmid replicated faster than the host chromosome. The advent of recombinant DNA technology increased the applicability of this approach by allowing the construction of vectors containing the desired gene and enabling the transfer of DNA to other species. Table 3.9 includes examples of gene amplification giving rise to improved enzyme yields.
The development of the process for the production of a lipase for use in washing powders by Novo Industri is an excellent example of the application of recombinant DNA technology to enzyme fermentations, although the technical details of the exercise are unavailable (Upshall, 1992). A lipase was isolated from a fungus which was unsuitable for commercial development. A cDNA clone coding for the lipase was prepared and transformed into an industrial Aspergillus strain used for enzyme production. The recombinant Aspergillus produced the lipase at high levels. Only 8 months elapsed between cloning the gene and the first commercial fermentation. Thus, recombinant DNA technology allowed the exploitation of a valuable microbial enzyme by facilitating its production in a well-established commercially acceptable strain.
The first successful application of genetic engineering techniques to the production of amino acids was obtained in threonine production with E. coli. Debabov (1982) investigated the production of threonine by a threonine analogue resistant mutant of E. coli K12. The entire threonine operon was introduced into a plasmid which was then incorporated into the organism by transformation. The plasmid copy number in the cell was approximately twenty and the activity of the threonine operon enzymes (measured as homoserine dehy drogenase activity) was increased 40-50 times. The manipulated organism produced 30 g dm-3 threonine, compared with 2-3 g dm 3 by the non-manipulated strain. Miwa et al. (1983) utilized similar techniques in constructing an E. coli strain capable of synthesizing 65 g dm-3 threonine. It is important to appreciate that the genes which were amplified in these production strains were already resistant to feedback repression so that the multi-copies present in the modified organism were expressed and not subject to control. Thus, the recombinant DNA techniques built on the achievements made with directed mutant isolation.
The application of genetic engineering to the industrially important corynebacteria was hampered for some years by the lack of suitable vectors. However, vectors have been constructed from corynebacterial plasmids and transformation and selective systems developed. The first patents for suitable vectors were registered by the two Japanese companies Ajinomoto (1983) and Kyowa Hakko Kogyo (1983) and now a range of vectors is available with kanamycin, chloramphenicol and hy-gromycin as common selectable resistance markers (Martin, 1989). Transformation has been achieved using protoplasts and, more recently, electroporation has been used successfully to introduce the required DNA (Dunican and Shivnan, 1989). These systems have enabled not only the use of recombinant DNA technology for strain improvement but have also facilitated the detailed investigation of the molecular biology of these important amino acid and nucleotide producers; for example the molecular organization of the pathway to the aspartate family of amino acids in C. glutamicum has been elucidated.
It is not surprising that the improvement of threonine productio n was the first reported use of recombinant DNA technology with amino acid producing corynebacteria (Shiio and Nakamori, 1989). It may be recalled from the discussion of the development of the
Table 3.9. Examples of the enhancement of enzyme production by gene amplification (After Demain, 1990)
«-Amylase a-Amylase a-Amylase
Eco RI restriction endonuclease DNA polymerase
Bacillus amyloliquefaciens Bacillus stearothermophilus Bacillus stearothermophilus E. coli
Bacillus subtilis Bacillus stearothermophilus Bacillus brevis
10 Sibakov et al. (1983)
100 Tsukagoshi et al.
loo Kelley et al. (1987)
early threonine producers that C. glutamicum was not particularly amenable for threonine over-production using auxotrophs and analogue resistant mutants. Over-production was achieved by incorporating a DNA fragment coding for homoserine dehydrogenase from a Breuibacterium lactofermentum threonine producer into a plasmid and introducing the modified plasmid back into the Breuibacterium. A similar approach was used for homoserine kinase and a strain was developed with remarkably increased homoserine kinase and dehydrogenase activities which produced 33 g dm"3 threonine (Morinaga et al., 1987).
The application of recombinant DNA technology to the development of processes for the production of phenylalanine is an excellent illustration of the interrelationship between mutant development and genetic engineering. Phenylalanine is a precursor of the sweetener, aspartame, and is thus an exceptionally important fermentation product. Backman et al. (1990) described the rationale used in the construction of an E. coli strain capable of synthesizing commercial levels of phenylalanine. E. coli was chosen as the producer because of its rapid growth, the availability of recombinant DNA techniques and the extensive genetic database.
The control of the biosynthesis of the aromatic family of amino acids in E. coli is shown in Fig. 3.30. The first step in the pathway is catalysed by three isoenzymes of dihydroxyacetone phosphate (DAHP) synthase, each being susceptible to one of the three end products of the aromatic pathway, phenylalanine, tyrosine or tryptophan. Control is achieved by both repression of enzyme synthesis and inhibition of enzyme activity. Within the common pathway to chorismic acid the production of shikimate kinase is also susceptible to repression. The conversion of chorismic acid to prephenic acid is catalysed by two isoenzymes of chorismate mutase, each being susceptible to feedback inhibition and repression by one of either tyrosine or phenylalanine. Each isoenzyme also carries an additional activity associated with either the phenylalanine or tyrosine branch. The tyrosine sensitive isoenzyme carries prephenate dehydrogenase activity (the next enzyme in the route to tyrosine) whilst the phenylalanine sensitive enzyme carries prephenate dehydratase (the next enzyme in the route to phenylalanine).
The regulation of gene expression was modified by:
(i) Both tyrosine sensitive and phenylalanine sensitive DAHP synthase and shikimic kinase are regulated by the repressor protein coded by the tyrR gene. The tyrR gene had been cloned and
D-Erythrose 4-phosphate + Phosphoenolpyruvate
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