Escherichia coli isoprenoid pathways

Recently, a potential cytosolic component of the MEP precursor pathway, xylulose kinase, has been cloned and tested for function in an Escherichia coli complementation system. " The kinase activates exogenous xylulose in the cytoplasm. DXP is the precursor for DXS, which resides in the plastid, suggesting the activated substrate must be transported into the plastid. Another xylulose kinase homologue in Arabidopsis that contains a plastid targeting sequence was not active in the E. coli system, suggesting that it may have some other function in the plastid. Perhaps plant and bacterial tissue cultures may be fed xylulose to condition accumulation of isoprenoid metabolites. 

Sprenger, G.A. et al.. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol, Proc. Natl. Acad Sci. USA 94, 12857, 1997. Lange, B.M. et al., A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway, Proc. Natl. Acad Sci. USA 95, 2100, 1998. Lois, L.M. et al., Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1- deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis, Proc. Natl. Acad. Sci. USA 95, 2105, 1998. 

Wang, C.W., Oh, M.K., and Liao, J.C., Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli, Biotechnol. Bioeng. 62, 235, 1999. 

Rodriguez-Concepcion, M., Campos, N., Lois, L.M. et al. (2000) Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli. FEBS Letters, 473, 328-332. 

Pitera, D.J., Paddon, C.J., Newman, J.D. and Keasling, J.D. (2007) Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metabolic Engineering, 9, 193-207. 

DUVOLD, T., CALI, P., BRAVO, J.-M., ROHMER, M., Incorporation of 2-C-methyl-D-erythritol, a putative isoprenoid precursor in the mevalonate-independent pathway, into ubiquinone and menaquinone of Escherichia coli, Tetrahedron Lett., 1997, 38, 6181-6184. 

Figure 9.2. The inherent metabolic flexibility of the isoprenoid pathway leading to the synthesis of some carotenoid pigments. Genes coding for two enzymes capable of acting on carotenoid structures were introduced into Escherichia coli which had already been transformed to give it the capacity to make p,p-carotene. Both of the two introduced new enzymes (one shown with red arrows and the other with blue arrows) acted on multiple substrates because of their lack of specificity. The resulting matrix of transformations means that nine different products can be made by just two tailoring enzymes. (Adapted from Umeno et al. ° who used data from Misawa et al. °)...

From the many enzymes that are known to make and break C-C bonds, we first chose the two transferases, transketolase (TKT) and transaldolase (TAL), both from the Gram-negative bacterium Escherichia coli. While project B21 evolved, we learned that this microorganism holds other and so far unknown enzymes which are of interest for asymmetric syntheses. One transketolase-like enzyme, 1-deoxy-D-xylulose 5-phosphate synthase (DXS), turned out to be the first enzyme of a novel biosynthetic pathway leading to isoprenoids in bacteria, algae, and plants. The other, fructose 6-phosphate aldolase (ESA) - while similar to transaldolase - allows the direct use of the inexpensive dihydroxyacetone in aldol condensations. 

In bacteria, the thiazole moiety (42) of thiamine is derived from 1-deoxy-D-xylulose 5-phosphate (43) that can also serve as a precursor for pyridoxal in many eubacteria (Fig. 5) and for isoprenoids via the nonmevalonate pathway (cf. isoprenoid cofactors). The sulfur atom is derived from the persulfide that also serves as precursor for iron/sulfur clusters and for biotin (6) and thiooctanoate (7) (Fig. 1). C2 and N3 of the thiazole moiety of thiamine have been reported to stem from tyrosine in Escherichia coli and from glycine in Bacillus subtilis, respectively. Yeasts use ADP-ribulose (44) derived from NAD as precursor (24). 

The MVA pathway was accepted as the unique biosynthetic pathway for the formation of aU isoprenoids in aU living organisms. Discrepancies with this general assertion appeared, however, as early as the 1950s (1, 2). For instance, -labeled MVA was not incorporated into chloroplast isoprenoids (e.g., carotenoids 25 and phytol 24 from chlorophylls Fig. 6), whereas it was well incorporated into phytosterols 27 synthesized in the cytoplasm. Unexpected labeling patterns were found in the prenyl chain of ubiquinone 22 in Escherichia coli at incorporation of C-labeled acetate. Finally, the labeling pattern in an isoprene unit from the sesquiterpenic pentalenene 21 series from a Streptomyces species at incorporation of uniformly... 

Of the two existing isoprenoid biosynthetic pathways (Fig. 3), DXP is used by most prokaryotes for production of IPP and dimethylallyl diphosphate (DMAPP) [65,66]. With the available knowledge of the genes involved in the DXP pathway, several groups have studied the impact of changed expression levels of these genes on the production of reporter terpenoids. Farmer and liao reconstructed the isoprene biosynthetic pathway in Escherichia coli (E. colt) to produce lycopene, which was used as an indication... 

Anthony JR, Anthony LC, Nowroozi F, Kwon G, Newman JD, Keasling JD (2009) Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metab Eng 11 13-19... 

Ajikumar PK, Xiao WH, Tyo KE, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stepha-nopoulos G (2010) Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330 70-74... 

Leonard E, Koffas MA (2007) Engineering of artificial plant cytochrome P450 enzymes for synthesis of isoflavones by Escherichia coli. Appl Environ Microbiol 73 7246-7251 Kizer L, Pitera DJ, Pfleger BE, Keasling JD (2008) Application of functional genomics to pathway optimization for increased isoprenoid production. Appl Environ Microbiol 74 3229-3241... 

As microbial hosts, Escherichia coli and Saccharomyces cerevisiae have often been employed in pathway engineering of functional isoprenoids [95, 98]. 

Anthony JR, Anthony LC, Nowroozi F, Kwon G, Newman JD, Keasling JD (2009) Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metab Eng 11 13-19 Aubel D, Morris R, Lennon B, Rimann M, Kaufmann H, Folcher M, Bailey JE, Thompson CJ, Fussenegger M (2001) Design of a novel mammalian screening system for the detection of bioavailable, non-cytotoxic streptogramin antibiotics. J Antibiot 54 44-55 Baltz RH (2006) Molecular engineering approaches to peptide, polyketide and other antibiotics. Nat Biotechnol 24 1533-1540... 

The evidence that (- )-shikimic acid plays a central role in aromatic biosynthesis was obtained by Davis with a variety of nutritionally deficient mutants of Escherichia coli. In one group of mutants with a multiple requirement for L-tyrosine, L-phenylalanine, L-tryptophan and p-aminobenzoic acid and a partial requirement for p-hydroxybenzoic acid, (—)-shikimic acid substituted for all the aromatic compounds. The quintuple requirement for aromatic compounds which these mutants displayed arises from the fact that, besides furnishing a metabolic route to the three aromatic a-amino acids, the shikimate pathway also provides in micro-organisms a means of synthesis of other essential metabolites, and in particular, the various isoprenoid quinones involved in electron transport and the folic acid group of co-enzymes. The biosynthesis of both of these groups of compounds is discussed below. In addition the biosynthesis of a range of structurally diverse metabolites, which are derived from intermediates and occasionally end-products of the pathway, is outlined. These metabolites are restricted to certain types of organism and their function, if any, is in the majority of cases obscure. 

Lee, S.H. et al. (2007) Increased beta-carotene production in recombinant Escherichia coli harboring an engineered isoprenoid precursor pathway with mevalonate addition. BiotechnoL Progr, 23, 599-605. 

Ajikumar PK et al (2010) Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330 70-74... 

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