Isoprenoid production

#The present invention relates to the manufacture of isoprenoids using molecular biology techniques. In particular, the present invention provides DNAs, vectors and host cells for the efficient production of various enzymnes in the mevalonate pathway or for converting isopentyl pyrophosphate to farnesyl pyrophosphate synthase.

#Astaxanthin is reportedly distributed in a wide variety of organisms such as animals (e.g., birds, such as flamingo and scarlet ibis; fish, such as rainbow trout and salmon), algae and microorganisms. It is also reported that astaxanthip has a strong antioxidation property against oxygen radicals, which is believed to be pharmaceutically useful for protecting living cells against some diseases such as a cancer. Moreover, from a commercial prospective, there is an increasing demand for astaxanthin as a coloring reagent especially in the fish farming industry, such as salmon farming, because astaxanthin imparts a distinctive orange-red coloration to the fish and contributes to consumer appeal.

#Phaffia rhodoyma is known as a carotenogenic yeast strain which produces astaxanthin specifically. Different from the other carotenogenic yeast, Rhodotorula species, such as Phaffia rhodozyma (P. rhodozyma) can ferment some sugars such as D-glucose. This is a commercially important feature. In a recent taxonomic study, the sexual cycle of P. rhodozyma was revealed and its telemorphic state was designated as Xanthophyllomyces dendrorhous (W. I. Golubev; Yeast: 11, 101-110, 1995). Some strain improvement studies to obtain hyper-producers of astaxanthin from P. rhodozyma have been conducted, but such efforts have been restricted to conventional methods including mutagenesis and protoplast fusion in this decade.

#FIG. 1 depicts a deduced biosynthetic pathway from acetyl-CoA to astaxanthin in P. rhodozyma.

#FIG. 2 shows the expression study by using an artificial mvk gene obtained from an artificial nucleotide addition at the amino terminal end of a pseudo-mvk gene from P. rhodozyma. The cells from 50 ml of broth were subjected to 10% sodium dodecyl sulfide—polyacrylamide gel electrophoresis (SDS-PAGE). Lane 1, E. coli (M15 (pREP4) (pQE30) without IPTG); Lane 2, E. coli (M15 (pREP4) (pQE30) with 1 mM IPTG); Lane 3, Molecular weight marker (105 kDa, 82.0 kDa, 49.0 kDa, 33.3 kD and 28.6 kDa, up to down, BIO-RAD); Lane 4, E. coli (M15 (pREP4) (pMK1209 #3334) without IPTG); Lane 5, E. coli (M15 (pREP4) (pMK1209 #3334) with 1 mM IPTG).

#The present invention provides an isolated DNA sequence which codes for enzymes which are involved in a biological pathway that includes the mevalonate pathway or the reaction pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate. The enzymes of the present invention may be exemplified by those involved in the mevalonate pathway or the reaction pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate in Phaffia rhodozyma. These sequences include, for example, 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate pyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

#The present invention has utility in the production of the compounds involved in the mevalonate pathway and the carotenogenic pathway and various products derived from such compounds. The compounds involved in the mevalonate pathway are acetoacetyl-CoA, 3-hydroxymethyl-3-glutaryl-CoA, mevalonic acid, mevalonate-phosphate, mevalonate-pyrophosphate and isopentenyl-pyrophosphate. Subsequently, isopentenyl-pyrophosphate is converted to geranylgeranyl-pyrophosphate through geranyl-pyrophosphate and farnesyl-pyrophosphate via the “Isoprene Biosynthesis” reactions as indicated in FIG. 1.

#The compounds involved in the carotenogenic pathway are geranylgeranyl-pyrophosphate, phytoene, tycopene, β-carotene and astaxanthin. Among the compounds involved in the above-mentioned biosynthesis, geranyl-pyrophosphate may be utilized for the production of ubiquinone. Farnesyl-pyrophosphate may be utilized for the production of sterols, such as cholesterol and ergosterol. Geranylgeranyl-pyrophosphate is used to produce vitamin K, vitamin E, chlorophyll and the like. Thus, the present invention has particular utility for the biological production of isoprenoids. As used herein, the term “isoprenoids” is intended to mean a series of compounds having an isopentenyl-pyrophosphate as a skeleton unit. Further examples of isoprenoids are vitamin A and vitamin D3.