Genes involved in isoprenoid compound production

Details Genes involved in isoprenoid compound production Genes involved in isoprenoid compound production

2001-08-22

2003-12-09

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing hydrocarbon

C435S167000, C435S252300, C435S183000, C435S325000, C435S254200, C536S023200

Reexamination Certificate

active

06660507

ABSTRACT:

FIELD OF THE INVENTION
This invention is in the field of microbiology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes useful for microbial production of isoprenoid compounds.
BACKGROUND OF THE INVENTION
Isoprenoids are an extremely large and diverse group of natural products that have a common biosynthetic origin, i.e., a single metabolic precursor, isopentenyl diphosphate (IPP). The group of natural products known as isoprenoids includes all substances that are derived biosynthetically from the 5-carbon compound isopentenyl diphosphate. Isoprenoid compounds are also referred to as “terpenes” or “terpenoids”, which is the term used in the designation of the various classes of these examples (Spurgeon and Porter, Biosynthesis of Isoprenoid Compounds, pp 3-46, A Wiley-Interscience Publication (1981)).
Isoprenoids are ubiquitous compounds found in all living organisms. Some of the well-known examples of isoprenoids are steroids (triterpenes), carotenoids (tetraterpenes), and squalene, just to name a few.
For many years, it was accepted that IPP was synthesized through the well-known acetate/mevalonate pathway. However, recent studies have demonstrated that the mevalonate-dependent pathway does not operate in all living organisms. An alternate mevalonate-independent pathway for IPP biosynthesis was initially characterized in bacteria and later also in green algae and higher plants (Horbach et al.,
FEMS Microbiol. Lett.
111:135-140 (1993); Rohmer et al,
Biochem.
295: 517-524 (1993); Schwender et al.,
Biochem.
316: 73-80 (1996); Eisenreich et al.,
Proc. Natl. Acad. Sci. USA
93: 6431-6436 (1996)).
Many steps in both the mevalonate-independent and mevalonate-dependent isoprenoid pathways are known. For example, the initial steps of the alternate pathway involve the condensation of 3-carbon molecules (pyruvate and C1 aldehyde group, D-glyceraldehyde 3-phosphate), to yield the 5-carbon compound D-1-deoxyxylulose-5-phosphate. Lois et al. has reported a gene, dxs, that encodes D-1-deoxyxylulose-5-phosphate synthase (DXS) and that catalyzes the synthesis of D-1-deoxyxylulose-5-phosphate in
E. coli
(
Proc. Natl. Acad. Sci. USA
95: 2105-2110 (1998)).
Next, the isomerization and reduction of D-1-deoxyxylulose-5-phosphate yields 2-C-methyl-D-erythritol-4-phosphate. One of the enzymes involved in the isomerization and reduction process is D-1-deoxyxylulose-5-phosphate reductoisomerase (DXR). Takahashi et al. reported that the dxr gene product catalyzes the formation of 2-C-methyl-D-erythritol-4-phosphate in the alternate pathway in
E. coli
(
Proc. Natl. Acad. Sci. USA
95: 9879-9884 (1998)).
Steps converting 2-C-methyl-D-erythritol-4-phosphate to isopentenyl monophosphate are not well characterized, although some steps are known. 2-C-methyl-D-erythritol-4-phosphate is converted into 4-diphosphocytidyl-2C-methyl-D-erythritol in a CTP dependent reaction by the enzyme encoded by non-annotated gene ygbP. Rohdich et al. reported that the YgbP protein in
E. coli
catalyzes the reaction mentioned above. Recently, ygbP gene was renamed as ispD as a part of the isp gene cluster (SwissProt #Q46893) (
Proc. Natl. Acad. Sci. USA
96:11758-11763 (1999)).
Then the 2 position hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol can be phosphorylated in an ATP dependent reaction by the enzyme encoded by the ychB gene. Luttgen et al. has reported that the YchB protein in
E. coli
phosphorylates 4-diphosphocytidyl-2C-methyl-D-erythritol, resulting in 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate. Recently, the ychB gene was renamed as ispE as a part of the isp gene cluster (SwissProt#P24209) (Luttgen et al.,
Proc. Natl. Acad. Sci. USA
97:1062-1067 (2000)).
Herz et al. reported that the ygbB gene product in
E. coli
converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. 2C-methyl-D-erythritol 2,4-cyclodiphosphate can be further converted into carotenoids through the carotenoid biosynthesis pathway (
Proc. Natl. Acad. Sci. USA
97:2486-2490 (2000)). Recently, the ygbB gene was renamed as ispF as a part of the isp gene cluster (SwissProt #P36663).
The reaction catalyzed by the YgbP enzyme is carried out in a CTP dependent manner. Thus, CTP synthase plays an important role in the isoprenoid pathway. PyrG encoded by the pyrG gene in
E. coli
was determined to encode CTP synthase (Weng et al.,
J. Biol. Chem.,
261:5568-5574 (1986)).
Following several reactions not yet characterized, isopentenyl monophosphate is formed. Isopentenyl monophosphate is converted to isopentenyl diphosphate (IPP) by isopentenyl monophosphate kinase, encoded by the ipk gene, and that is identical to the above mentioned yhcB (ispE) gene (Lange and Croteau,
Proc. Natl. Acad. Sci. USA
96:13714-13719 (1999)).
Cunningham et al. (
J of Bacteriol.
182:5841-5848, (2000)) has reported that the lytB gene in
E. coli
that is thought to encode an enzyme of the deoxyxylulose-5-phosphate pathway that catalyzes a step at or subsequent to the point at which the pathway branches to form IPP and dimethylallyl diphosphate. LytB gene is also found in other microorganisms such as Acinetbacter and Synechocystis, (GenBank Accession Numbers AF027189 and U38915, respectively).
Prenyltransferases constitute a broad group of enzymes catalyzing the consecutive condensation of isopentenyl diphosphate (IPP) resulting in the formation of prenyl diphosphates of various chain lengths. Homologous genes of prenyl transferase have highly conserved regions in their amino acid sequences. Ohto et al. reported three prenyl transferase genes in cyanobacterium
Synechococcus elongatus
(
Plant Mol. Biol.
40:307-321 (1999)). They are geranylgeranyl (C20) diphosphate synthase, farnesyl (C15) diphosphate synthase and another prenyltransferase that can catalyze the synthesis of five prenyl diphosphates of various lengths.
Further down in the isoprenoid biosynthesis pathway, more genes are involved in the synthesis of specific isoprenoids. As an example, the crtN gene was found in
Heliobacillus mobilis
(Xiang et al.,
Proc. Natl. Acad. Sci. USA
95:14851-14856 (1998)) to encode diapophytoene dehydrogenase is a part of the carotenoid biosynthesis pathway.
Although most of the genes involved in the isoprenoid pathways are known, the genes involved in the isoprenoid pathway of methanotrophic bacteria are not described in the existing literature. However, there are many pigmented methylotrophic and methanotrophic bacteria, which suggests that the ability to produce carotenoid pigments is widespread in these bacteria and therefore the genes must be widespread in these bacteria. Applicants have isolated a number of unique open reading frames encoding enzymes of the isoprenoid biosynthesis pathway from a Methylomonas sp.
Applicants have solved the stated problem by isolating genes containing 9 open reading frames (ORFs) encoding enzymes involved in isoprenoid synthesis.
SUMMARY OF THE INVENTION
The present invention provides an isolated nucleic acid molecule encoding a isoprenoid biosynthetic enzyme, selected from the group consisting of: (a) an isolated nucleic acid molecule encoding the amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18 and 24; (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1× SSC, 0.1% SDS, 65° C. and washed with 2× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS; and (c) an isolated nucleic acid molecule that is complementary to (a) or (b).
Additionally the invention provides polypeptides encoded by the present genes and chimera where the genes are under the control of suitable regulatory sequences. Similarly the invention provides transformed organisms, including bacteria, yeast, filamentous fungi, and green plants expressing one or more of the present genes and gene products.
The present invention provides methods of obtaining all or substantial portions of the instant genes through gene amplification

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