Biosynthesis directed

The nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the chemical carriers of a cell s genetic information. Coded in a cell s DNA is the information that determines the nature of the cell, controls the cell s growth and division, and directs biosynthesis of the enzymes and other proteins required for cellular functions. 

The primary cellular function of mRNA is to direct biosynthesis of the thousands of diverse peptides and proteins required by an organism—perhaps 100,000 in a human. The mechanics of protein biosynthesis take place on ribosomes, small granular particles in the cytoplasm of a cell that consist of about 60% ribosomal RNA and 40% protein. 

A number of mitomycin analogues have been prepared by precursor-directed biosynthesis [104]. A range of amines were fed to S. caespitosus, and novel derivatives of mitomycin C (type I analogues) and mitomycin B (type II analogues) were identified and in some cases (42-46 and 52-56 Scheme 11.4) isolated and characterized. Antibiotic and antitumor activities were comparable to those of mitomycin C, with the type I analogues more active than the type II analogues. 

We may describe the production of diverse penicillins using this strategy as directed biosynthesis using precursor feeding. We have listed some examples of penicillins in Table 6.2. 

Although a range of penicillins could be produced by directed biosynthesis using precursor feeding, this approach is limited by the toxicity of the precursors, the ability of the penicillin producing cells to take up the precursor and by the capability of the acyltransferase to transfer the acyl groups onto the 6-aminopenicillanic add moiety. 

In this chapter, by using the examples of -lactams we have briefly examined how microbial cultures may be used to produce sufficient antibiotics to meet market demands. We have also explained how enzymes (or cells) may be used to biotransform, and thereby diversify, antibiotics. By outlining the history of penicillin production, we explained how analysis and manipulation of culture regimes may be used to enhance the yields of antibiotics (and other secondary products). These studies led to die concept of directed biosynthesis by precursor feeding. 

An alternative strategy for producing new derivatives by directed biosynthesis is to produce mutants in which particular pathways may be blocked or a new pathway created. Again, we will use a specific example to illustrate this approach. 

We complete this section by reminding you that we have now identified three strategies of using directed biosynthesis to produce diversified products. These are ... 

Jacobsen, J.R., Keatinge-Clay, A.T., Cane, D.E. and Khosla, C. (1998) Precursor-directed biosynthesis of 12-ethyl erythromycin. Bioorganic and Medicinal Chemistry, 6, 1171. 

Ward, S.L., Desai, R.P., Hu, Z. et al. (2007) Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues is improved by removal of the initial catalytic sites of the polyketide synthase. Journal of Industrial Microbiology and Biotechnology, 34, 9-15. 

Thiericke, R. and Rohr, J. (1993) Biological variation of microbial metabolites by precursor-directed biosynthesis. Natural Product Reports, 10 (3), 265-289. 

Fig. 4. Structures of plant-derived compounds related to directed biosynthesis/biotechnology aspects.

McCoy E, O Connor SE, Directed biosynthesis of alkaloid analogs in the medicinal plant Catharanthus roseus, J Am Chem Soc 128 14276-14277, 2006. 

Feng S, Li C, Xu X, Wang X, Screening strains for directed biosynthesis of [[)] -D-mono-glucuronide-glycytthizin and kinetics of enzyme production,/Afo/ Catal B EnzymolYi. Si—GJ, 2006. 

RNA directs biosynthesis of various peptides and proteins essential for any living organisms. Protein biosynthesis seems to be catalysed by mRNA rather than protein-based enzymes and occur on the ribosome. On the ribosome, the mRNA acts as a template to pass on the genetic information that it has transcribed from the DNA. The specific ribonucleotide sequence in mRNA forms an instruction or codon that determines the order in which different amino acid residues are to be joined. Each instruction or codon along the mRNA chain comprises a sequence of three ribonucleotides that is specific for a given amino acid. For example, the codon U-U-C on mRNA directs incorporation of the amino acid phenylalanine into the growing protein. 

The biosynthesis of gallic acid (3.47) has been under investigation for more than 50 years. Different biosynthetic routes have been proposed, as depicted in Figure 3-6 (/) direct biosynthesis from an intermediate of the shikimate pathway, (2) biosynthesis via phenylalanine (3.27), cinnamic acid (3.29), />coumaric acid (3.30), caffeic acid (3.32), and 3,4, 5-trihydroxycinnamic acid (3.44), or (3) biosynthesis via caffeic acid (3.32) and protocatechuic acid (3.45). The possibility that different pathways co-existed in different species or even within one species was also considered. 

Table 1.4. IMMUNOSUPPRESSIVE ACTIVITY OF CYCLOSPORINS nat = natural dbs = by directed biosynthesis ps = by partial synthesis syn = by total synthesis + + +, strong immunosuppressive activity + +, moderate activity +, weak activity (+) or -, no significant activity.

Keywords Chalcone synthase superfamily enzyme, Engineered biosynthesis, Precursor-directed biosynthesis, Structure-based engineering, Type III polyketide synthase... 

Bu Lock et al. had shown initially [24] that supplementation of S. avermitilis fermentations with a range of fatty acids resulted in their uptake and incorporation to generate novel avermectins modified at the C25 side chain of the molecule. This approach, described as precursor-directed biosynthesis, has been employed to produce many new antibiotics [25], but the co-expression of the parent molecule interferes with the detection and isolation of the novel analogs. To circumvent these difficulties, the elegant technique of mutational biosynthesis [26] or mutasynthesis [27,28] was developed. In this approach, a mutant of an organism, deficient in the production of an essential precursor for the secondary metabolite of choice, is isolated, and precursor-directed biosynthesis is then employed to generate only the novel analogs. 

Figure 9 Precursor-directed biosynthesis. A block in the 6-dEB pathway was created by a Cys— Ala mutation in the active site of the KS of module 1. Feeding diketides with different a and j substitutions resulted in the 6-dEB analogs in which the starter unit or first extender unit was modified. The stereochemistry of an unsaturated triketide dictated whether it was incorporated into module 2 or 3, leading to either a 14-membered or 16-membered macrolactone. See Sec. VI.C for details.

JR Jacobsen, CR Hutchinson, DE Cane, C Khosla. Precursor directed biosynthesis of novel erythromycin analogs by an engineered polyketide synthase. Science 277 367-369, 1997. 

A third approach is precursor-directed biosynthesis in which altered starter unit functionality is introduced through the incorporation of a synthetic non-natural diketide intermediate into a polyketide product in vivo. This strategy was first... 

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