Bioprocess Technology- Fundamentals and Applications Chapt 4_Metabolic basis(14)

Fig. 4.9 Biosynthesis of the aromatic amino acids (shikimic acid pathway). Feed-backinhibition is indicated by backward arrows from the end products. PEP =phosphoenolpyruvate; E4P = erythrose-4-phosphate; DAHP = 3-deoxy-D-arabino-heptulosonate 7-phosphate; DHQ = 3-dehydroquinate; DHS = 3-dehydroshikimate; EPSP =5-enolpyruvoylshikimate-3-phosphate. aroG = DAHP synthase (phe); aroF = DAHP synthase(tyr); aroH = DAHP synthase (trp); pheA = the bifunctional enzyme chorismate mutaseprephenate dehydratase; tyr A = the bifunctional enzyme chorismate mutase prephenatedehydrogenase; trpE = anthranilate synthase.

As an example, a combination of these methods can yield an organism with the followinggenetic properties that overproduces the aromatic amino acid phenylalanine: tyr, trp,pXX/aroF, pheAFBR(FBR = feed back resistant). This organism is auxotrophic for tyrosineand tryptophan (deletions of the trp E operon and tyr A operon) a property which increasesthe flux of metabolites to phenylalanine. The strain must however be supplied with theseamino acids in order to grow. Furthermore this microorganism harbours a multicopy plasmidwhich contain genes for the tyrosine inhibited DAHP-synthase and a feed-back resistant(both allosterically and transcriptionally) prephenate dehydratase. During growth with addedtyrosine and tryptophan there will be no real overproduction of phenylalanine becausetyrosine inhibits synthesis of the plasmid encoded DAHP-synthase(tyr) and phenylalanineregulates the chromosomal DAHP-synthase(phe). When the added tyrosine (and tryptophan)is exhausted from the medium growth stops but DAHP-synthase(tyr) will be produced as aresult of relief of inhibition. This leads to enhanced production of phenylalanine sincephenylalanine itself cannot inhibit DAHP-synthase(tyr) and the chorismate mutaseprephenate dehydratase is a mutant enzyme, insensitive to regulation by phenylalanine.

4.8 Macromolecular products

Macromolecules like proteins (e.g. hydrolytic enzymes or recombinant proteins) orpolysaccharides are important products in the biotechnical industry. It is not possible to giveany general rules regarding the conditions for their production because of the different natureand metabolic affiliation of the various macromolecular species. This circumstancedistinguishes macromolecular products from metabolites derived from catabolic or anabolicpathways. For example, extracellular hydrolytic enzymes like amylases or proteases arespontaneously produced by microorganisms in response to environmental stimuli likepresence or absence of certain nutrients. Such enzymes are in fact part of the catabolicmachinery as is the intracellular enzyme β-galactosidase. The latter is of course under controlof induction and cAMP. To obtain overproduction of such an enzyme it is necessary togenetically modify the regulatory mechanisms e.g. by creating a constitutive mutant that isindependent of induction. This may also be combined with the use of a multicopy plasmidthat harbours the gene as is common practise in production of recombinant proteins. Theproduction of recombinant proteins is further governed by the chosen promoter ruling outnormal regulatory mechanisms. It is important to consider the effects of a too high copynumber or a too strong promoter on the cellular physiology because it may lead to exhaustionof precursors and energy which in turn may trigger some of the starvation responses (Table4.1). Production of recombinant proteins will further be described in chapter 16.

4.9 Secondary metabolism

The preceding sections of this chapter has dealt with the primary metabolism and productsthereof. A great number of industrially important products such as antibiotics are, however,derived from the secondary metabolism. Secondary metabolism, common in plants andmicroorganisms, is characterised by not being essential for growth. The purpose of it orbenefit from it for the organism is still under debate. Usually, secondary metabolites areproduced during the stationary phase. This depends on their formation being repressed duringgrowth by different mechanisms. These include: 1) Carbon catabolite inhibition andrepression i.e. good carbon end energy sources repress formation of enzymes in the

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