Escherichia Coli As A Carotenoid Production System

Escherichia coli was the first host for functional expression of carotenogenic genes in order to analyze the function of the corresponding enzymes (10,11). Later, this bacterium was very useful in the heterologous overexpression of individual carotenogenic enzymes as functional proteins for their purification and biochemical characterization (12).

Escherichia coli is a very convenient host for heterologous carotenoid production. Because of its fast and easy cultivation in substantial quantity, it can be transformed simultaneously with several plasmids as long as they belong to different incompatibility groups, i.e., possessing different origins of replication. For their stable maintenance in the cells, it is essential that each plasmid carries a different antibiotic resistance. Only when this selection pressure is maintained spontaneous plasmid loss prevented. Several useful plasmids for cotransformation are available. Convenient vectors are pUC-related plasmids with the pMB1 origin of replication and ampicillin resistance, pACYC184 with a p15A origin of replication and chloramphenicol resistance, pRK404 with an RK2 origin of replication and tetracycline resistance, and pBBR1MCS2 with a SC101 origin of replication and kanamy-cin resistance. These plasmids can be used for expression of individual genes or groups of genes which mediate the formation of certain basic carotenoid structures. For example, one plasmid may carry the genes necessary to obtain certain carotenoid intermediates and others the genes for systematic modifications of the structure (13,14). It should be pointed out that E. coli is noncarotenogenic which increases the flexibility to build up a desired carotenoid synthesis pathway. However, E. coli has to cope with the drain of prenyl pyrophosphates when carotenogenesis is established. Other plasmids may be used to overexpress certain genes to enhance the E. coli metabolic capacity for the supply of carotenoid precursors. Details on the choice of plasmids and growth conditions for carotenoid producing E. coli transformants are given in a recent publication (15).

In the majority of bacteria, including E. coli, formation of prenyl pyrophosphates, which are the precursors of carotenoids, proceed via a reaction sequence which is different from the mevalonate pathway typically found in fungi and animals (16). It is referred to either as the 1-deoxyxylulose-5-phosphate pathway, because this is the first intermediate, or as the 2-C-methyl-D-erythritol-4-phosphate pathway, because this is the first product which is converted to prenyl pyrophosphate exclusively. To date, most of the reaction steps have been elucidated. Initially, a C2 unit from pyruvate is condensed to glyceraldehyde by a thia-min-dependent 1-deoxyxylulose-5-phosphate synthase (Figure 7.2). Then, the product is converted to 2-C-methyl-D-erythrol-4-phosphate by 1-deoxyxylulose-5-phosphate reduc-toisomerase. The following reactions involve the formation of 4-diphosphocytidyl-2-C-methylerythrol in a CTP-dependent reaction and phosphorylation at position 2 by adenosine triphosphate. Subsequently, cytidine-5-phosphate is released and via 2-C-methyl-D-erythritol-2,4-cyclodiphosphate 1-HO-2-methyl-2-butenyl-4-diphosphate is formed.

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