Peps

Carotenoids

Glyceral- + pyruvate dehyde-3-P

1-deoxy-D-xylulose-5-P

IPP + DMAPP

Carotenoids

> GGPP

Figure 7.2 Metabolic engineering of the Escherichia coli deoxyxylulose-5-phosphate pathway and prenyl pyrophosphate formation for optimum precursor supply. Overexpression of the indicated enzymes either relieve a limitation of the metabolic flow or provide a more favorable balance for two-substrate reactions. PEP-S, phosphoenolpyruvate synthase; Dxs, deoxyxylulose-5-phosphate synthase; Dxr, deoxyxylulose-5-phosphate reducto isomerase; Idi, isopentenyl pyrophosphate isom-erase; Gggpps, geranylgeranyl pyrophosphate synthase.

This product may be the branching point for independent routes to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). However, the details of the final steps to IPP and DMAPP are not fully understood yet. See Reference 17 for the newest insight into later reactions of this novel pathway.

Precursor supply for carotenogenesis can be increased in E. coli by overexpression of limiting enzymes of the deoxyxylulose-5-phosphate pathway and subsequent reactions. Supply of prenyl pyrophosphates and subsequent carotenogenesis was stimulated by transformation with the genes encoding 1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and IPP isomerase under a strong promoter (Figure 7.2). Because geranylgeranyl pyrophosphate is the direct substrate for the formation of the first carotenoid in the pathway and its level is comparably low in E. coli, high expression levels of geranylgeranyl pyrophosphate synthase are very important for caro-tenogenesis (18). Another bottleneck for carotenoid biosynthesis in E. coli was relieved in the pathway by overexpressing the gene which encodes phosphoenolpyruvate synthase, a pyruvate consuming enzyme, indicating that the pools of glyceraldehyde 3-phosphate and pyruvate, which both are substrates of 1-deoxy-D-xylulose 5-phosphate synthase, have to be more balanced in the direction of glyceraldehyde 3-phosphate (19). By overexpression of different combinations of the limiting enzymes mentioned above, concentrations of various carotenoids to a final yield of 1.5 mg/g dry weight could be reached (20).

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