Pathway engineering

The work of Frost et al. has focused on the aromatic pathway with respect to the biosynthesis of quinoid organics such as benzoquinone and hydroquinone, as well as adipic acid production [194-197]. The same principles have been used whereby a cloned gene is overexpressed, which allows the recombinant organism to funnel carbon down a specific, selected metabolic pathway. Although currently tlrese compounds are synthesized from petroleum-based feed stocks primarily for economic reasons, environmental concerns and the shortage of these starting 

Another example where metabolic pathway engineering has made a dramatic impact is in the biodegradable polymer field. The polymer of this family most widely studied is poly-P-hydroxybutyrate (PHB) (46). Another member of the PHA family commercialized by Imperial Chemical Industries (ICI), which later became Zeneca under the trade name Biopol, is a copolymer consisting of p-hydroxybutyric acid and P-hydroxyvaleric acid. This biodegradable polymer was first used in plastic shampoo bottles by the Wella Corporation [198]. In the early part of 1996, the Biopol product line, was purchased from Zeneca by the Monsanto Company. 

PHB has been shown to accumulate to levels approaching 90% of the bacterial dry cell weight when these pathway enzymes have been overexpressed [205]. With these efficiencies, it is only a matter of time before the production of these biopolymers can compete economically with the synthetically prepared material [206]. 

The biosynthesis of polyketides is analogous to the formation of long-chain f acids catalyzed by the enzyme fatty acid synthase (FAS). These FASs are Itienzvme complexes that contain numerous enzyme activities. The complexes dense coenzyme A thioesters (usually acetyl, propionyl, or malonyl) followed 

pneumoniae individually in K. pneumoniae using the tac promoter expression plasmid. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth because NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased byproducts and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-PD using two cofactors (NADH and NADPH). 

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Some chemicals such as iadigo, tryptophan, and phenylalanine are overproduced ia bacteria by pathway engineering (36—38). In this method, the enzymes iavolved ia the entire pathway are overproduced. In addition, the host bacterium is also altered such that the carbon flow is directed toward the engiaeered pathway (38). E. colih.2LS been modified to overproduce iadigo and tryptophan and phenylalanine. CoTjnebacteriumglutamicum has been engiaeered to overproduce tryptophan from 28 to 43 g/L. Similarly, attempts are underway to overproduce several vitamins by pathway engineering (34,38). 

Rohlin, L., Oh, M.K., and Liao, J.C., Microbial pathway engineering for industrial processes evolution, combinatorial biosynthesis and rational design, Curr. Opin. Microbiol. 4, 330, 2001. 

Kennedy, J., Murli, S. and Kealey, J.T. (2003) 6-Deoxyerythronolide B analogue production in Escherichia coli through metabolic pathway engineering. Biochemistry, 42, 14342. 

Mutka, S.C., Bondi, S.M., Carney, J.R. et al. (2006) Metabolic pathway engineering for complex polyketide biosynthesis in Saccharomyces cerevisiae. FEMS Yeast Research, 6, 4047. 

In summary, a novel cyclohexyl avermectin analog B1 with enhanced antiparasitic activity was discovered and produced with high selectivity and excellent fermentation titer through deciphering biosynthesis, pathway engineering, and directed evolution. The new product, doramectin, is sold commercially as Dectomax. 

Schijlen E, Ric de Vos CH, Jonker H, van den Broeck H, Molthoff J, van Tunen A, Martens S and Bovy A. 2006. Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit. Plant Biotechnol J 4(4) 433-444. 

Diacids. The microbial generation of mahc, fumaric, and succinic acid essentially imphes Krebs cycle pathway engineering of biocatalytic organisms to overproduce oxaloacetate as the primary four-carbon diacid that subsequently undergoes reduction and dehydration processes (Scheme 2.9). The use of these four-carbon diacids as intermediate chemicals and the state of their desirable microbial production is briefly outlined. 

Outlook Pathway Engineering of Gene Clusters and Biosynthetic... 

Another, more advanced methodology uses a combination of chemoenzymatic synthesis (substrate modifications) together with in vivo pathway engineering, as demonstrated in the recent excellent review by Thibodeaux and Liu [102] on macrolide glycodiversification. 

Example 1 E. coli pathway engineering reduction of acetate production for enhanced recombinant protein production... 

K. Sanford, P. Soucaille, G. Whited and G. Chotani, Genomics to fluxomics and physiomics - pathway engineering,... 

Successful production of chemicals through pathway engineering requires integration of fermentation technologies and metabolic engineering with the goals of... 

K. Sanford, The commerdal production of chemicals using pathway engineering, Biochim. Biophys. Acta 2000, 1543, 434 455. 

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