Fermentative syntheses incorporation

Metabolic engineering requires an integrated approach of analysis and synthesis. Studies involve expression analysis, metabolite levels, pathway analysis, fermentation experiments, and protein characterization. Synthesis incorporates recombinant DNA techniques such as cloning, transformation, constrnction of vectors and artificial chromosomes. 

Fermentative Synthesis of Structurally Complex Molecules by Incorporation of Labeled Precursors... 

Amino acid separations represent another specific application of the technology. Amino acids are important synthesis precursors - in particular for pharmaceuticals -such as, for example, D-phenylglycine or D-parahydroxyphenylglycine in the preparation of semisynthetic penicillins. They are also used for other chiral fine chemicals and for incorporation into modified biologically active peptides. Since the unnatural amino acids cannot be obtained by fermentation or from natural sources, they must be prepared by conventional synthesis followed by racemate resolution, by asymmetric synthesis, or by biotransformation of chiral or prochiral precursors. Thus, amino acids represent an important class of compounds that can benefit from more efficient separations technology. 

These defects have spurred attempts to prepare analogs. The techniques used have been (1) natural fermentation (in which the penicillin-producing fungus is allowed to grow on a variety of complex natural nutrients from which it selects acids for incorporation into the side chain), (2) biosynthetic production (in which the fermentation medium is deliberately supplemented with unnatural precursors from which the fungus selects components for the synthesis of "unnatural" penicillins), (3) semisynthetic production (in which 6-aminopenicillanic acid (2) is obtained by a process involving fermentation, and suitably activated acids are subsequently reacted chemically with 6-APA to form penicillins with new side chains) and (4) total synthesis (potentially the most powerful method for making deep-seated structural modifications but which is at present unable to compete economically with the other methods). 

However, under normal white fermentation conditions, grape juice is very poor in fatty acids and fermentation is usually carried out under hypoxia conditions (Bertrand and Miele 1984). Under these conditions, yeasts cannot synthesize unsaturated fatty acids. Consequently, Saccharomyces cerevisiae need to use another strategy to fluidize their membranes and the only possibility is incorporating medium chain fatty acids (MCFA) within the phospholipids of the membrane (Roz6s 1992). The effect of a short chain is similar to that of the double bond of a long chain (Quinn and Chapman 1980) and, therefore, the increased synthesis of MCFA could also modulate membrane fluidity. 

Acetic acid is produced at concentrations rarely exceeding 0.7 g/L by yeasts during fermentation. The acid is metabolized by flor yeasts during the biological aging process, its concentration being reduced through consumption via acetyl-CoA for incorporation into the Krebs cycle or fatty acid synthesis. 

Although cephalosporin C is divisable into a-aminoadipic acid, cysteine, and valine, the actual mechanism whereby Cephalosporium sp. incorporates the three amino acids into cephalosporin C has not been established, Arnstein and Morris isolated 8 (a-aminoadipyl) cysteinyl valine from mycelia of Penicillium chrysogenum and suggested that the tripeptide is a precursor in all penicillin biosynthesis.. This same tripeptide also appears to be found in the intracellular pool of Cephalosporium sp.- The final postulated step in the biosynthesis of penicillin is an acyl transfer reaction, or the production of 6-aminopeni-cillanic acid if precursor is not added. Cephalosporium sp. apparently do not produce sidechain amidases or acyl transferases, and no 7-ACA has been reported found in the fermentation. Thus, to obtain clinically useful antibiotics, chemical manipulation of cephalosporin C is necessary. Synthesis of many 7-acyl derivatives was possible once a practical cleavage reaction made available large amounts of 7-ACA from cephalosporin C. of these derivatives, sodium cephalothin was the first... 

As mentioned, the insecticides inhibit chitin synthesis indirectly and they are not useful as fungicides. Polyoxins, however, are structural analogues to uridine diphospate-2-acetamido-2-deoxy-D-glucose, which is the substrate for chitin synthetase, and inhibit the incorporation of 2-aceta-mido-2-deoxy-D-glucose into chitin. It is produced by fermentation of Strep-tori n/ces cacaoi var. asoensis. It is used as a fungicide against powdery mildews in apples and pears, and for many other purposes. Its mammalian toxicity is very low, and it has a no-observed-effect level (NOEL) in rats of 44,000 mg/kg in diet in 2-year studies. The compounds inhibit chitin synthetase from insects, but are not toxic to insects in vivo. 

Although thiazole derivatives are important as pharmaceuticals and are found also in natural products, the fully reduced form has its own importance, and as noted in Chapter 3 it is incorporated in the famous antibiotic penicillin (9.32). Penicillin was first produced by large-scale fermentation procedures, but it can also be made synthetically. The first process was that of J. Sheehan (see Chapter 3, section 3.2.5). A major hurdle to be overcome was the construction of the beta-lactam moiety fused to the thiazolidine ring. The Sheehan synthesis of ben-zylpenicillin is shown in Scheme 9.23, but many other derivatives can be made by modifications of the procedure. The first step involves the... 

Streptomyces strain capable of incorporating the cyclohexyl substituent at C25 from a source that is added to the fermentation medium. Selamectin [34, 35] is a new experimental antiparasitic drug introduced by Pfizer. It is a semisynthetic avermectin-monosaccharide derivative, and it is produced from doramectin by chemical synthesis. 

At higher ethanol concentrations the intracellular alcohol interferes with membrane organization, increasing its fluidity and permeability to ions and small metabolites and inhibiting transport of nutrients. Especially Ca and Mg ions are able to increase the plasma membrane stability. It has been demonstrated that incorporation of unsaturated fatty acids and/or sterol(s) as well as proteolipids into cellular membrane of yeasts helps to alleviate ethanol tolerance. For the synthesis of the unsaturated fatty acids the presence of traces of oxygen under fermentation conditions is required. Further to Ca and Mg ions, other trace elements such as Co, Cu, Mn and Zn " and vitamins, e.g. pantothenate, thiamine, riboflavin, nicotinic acid, pyridoxine, biotin, folic acid and inositol, are essential for the growth and ethanol production by yeasts. 

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