Metabolic engineering cofactor

Biosynthetic production of thymidine is overall a complex process combining the controlled introduction of a novel biotransformation step into a biological system with selective enhancement or knock-out of a series of existing metabolic steps. Metabolic engineering to enhance cofactor recycling at both ribonucleotide reduction and dUMP methylation steps has important parallels in other systems, as whole-cell biotransformations are frequently employed as a means to supply, in situ, high-cost and usually labile cofactors. 

Heterogeneous catalysis One-step reactions Simple product recovery Enzyme engineering multi-step reactions no cofactor problems long catalyst lifetime metabolic engineering ... 

There have been many successful cases of the development of metabolically engineered E. coli strains for the production of P(3HB), which is one of the best characterized PHAs. P(3HB) synthesis is initiated by condensation of two acetyl-CoA molecules into acetoacetyl-CoA, subsequently followed by reduction to 3-hydroxybutyryl-CoA using NADPH as a cofactor, and finally 3-hydroxybutyryl-CoA is incorporated into the growing chain of P(3HB) (Lee 1996). Because the P(3HB) synthesis pathway competes with inherent metabolic pathways needing acetyl-CoA, it is very important to increase the acetyl-CoA pool available for the P(3HB) synthesis reaction, resulting in increased P(3HB) yield and productivity. 

Whole-cell biocatalysts are basically one-pot cascade reactions, with various enzymatic reactions being carried out concurrently within individual cells. Compared to purified enzymes, whole-cell biocatalysts are inexpensive and easily scalable and can be stably stored indefinitely. Certain enzymes are unstable and lose activity when purified from cells. Living cells also contain and regenerate otherwise expensive redox cofactors and, with metabolic engineering, can produce desired chemicals from inexpensive carbon and nitrogen sources [66, 67]. On the other hand, a major downside of using whole cells for biotransformations is the increased cost of product extrachon and purification from fermentahon broths. One also has to consider the... 

In addition, cofactor engineering has been used to deliberately modify the intracellular NADH/NAD+ ratio that plays a predominant role in controlling the Lactococcus lactis fermentation pattern. The introduction of the nox gene, which codes for a NADH oxidase (NOX) that converts molecular oxygen to water at the expense of NADH, to a strain with an inactivated copy of the aldB gene for a-acetolactate decarboxylase led to the efficient metabolism of the na-... 

It has already been pointed out that a great deal of intracellular biochemistry is based on cofactors, with these cofactors, in turn, often being derived from nucleotides. However, while this indirectly implies the proficiency of ancient RNA catalysts, it does not prove that such catalysts could have existed. Although there are, for example, protein dehydrogenases and esterases, there are no modem ribozymes with similar activities. Just as engineering a ribozyme self-replicase will be an experimental demonstration that life could have arose via RNA, so the production of artificial ribozymes will be a demonstration that a metabolically complex RNA world may once have existed. 

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