Title of Invention	PRODUCTION OF ISOPRENOIDS
Abstract	The present invention relates to a process for the production of isoprenoids, in particular Coenzyme Q-10 by microorganisms. More particularly, the present invention relates to a process for increased production of Coenzyme Q-10 by microorganisms of the genus Rhodobacter, preferably of the species R. sphaeroides which have been transformed with one or more gene(s) of the mevalonate (mev) operon from a different microorganism, preferably of the genus Paracoccus, more preferably of the species P. zeaxanthinifaciens, whereby the mev operon is mutated leading to an increased coenzyme Q-10 production. Sequences carrying such a mutation as well as a microorganism carrying such a mutated mev operon are also included.

Title of Invention

PRODUCTION OF ISOPRENOIDS

Abstract

The present invention relates to a process for the production of isoprenoids, in particular Coenzyme Q-10 by microorganisms. More particularly, the present invention relates to a process for increased production of Coenzyme Q-10 by microorganisms of the genus Rhodobacter, preferably of the species R. sphaeroides which have been transformed with one or more gene(s) of the mevalonate (mev) operon from a different microorganism, preferably of the genus Paracoccus, more preferably of the species P. zeaxanthinifaciens, whereby the mev operon is mutated leading to an increased coenzyme Q-10 production. Sequences carrying such a mutation as well as a microorganism carrying such a mutated mev operon are also included.

Full Text	Production of isoprenoids The present invention relates to a process for the production of isoprenoids, in particular Coenzyme Q-10 by microorganisms. More particularly, the present invention relates to a process for increased production of Coenzyme Q-10 by microorganisms of the genus Rhodobacter, preferably of the species R. sphaeroides which have been transformed with one or more gene(s) of the mevalonate (mev) operon from a different microorganism, preferably of the genus Paracoccus, more preferably of the species P. zeaxanthinifaciejis, whereby the mev operon is mutated leading to an increased coenzyme Q-10 production. Sequences carrying such a mutation as well as a microorganism carrying such a mutated mev operon are also included. Coenzyme Q-10 (2,3-dimethoxy-dimethyl-6-decaprenyl-l,4-benzoquinone), also known as ubiquinone-10, is a Upid-soluble benzoquinone having an isoprenoid side chain comprised often C-5 isoprenoid units. Coenzyme Q-10 (hereafter abbreviated as CoQIO) is found in microorganisms and plants, as well as in animals. It is the most prevalent form of ubiquinone in humans and most mammals. There is established' aiiti growing evidence that CoQl 0 is an important factor in the health status of humans and their protection from diseases. The medical and health beneficial effects of CoQIO have been associated with its two main physiological functions., namely to function as an essential cofactor of the mitochondrial electron transport chain (which is coupled to the synthesis of adenosine triphosphate) and to act as a lipid soluble antioxidant. The health benefits of CoQIO have led to increased commercial importance of this compound. CoQl 0 can be produced by chemical synthesis or by fermentation using microorganisms. These microorganisms may be natural CoQl 0 producers that have been improved for CoQIO production by genetic engineering, or they may not naturally produce CoQIO but have been manipulated by genetic engineering to be able to synthesize it. In bacteria, the quinoid ring of ubiquinones is derived from chorismate, a central intermediate in the biosynthesis of aromatic compounds, while the isoprenoid tail of ubiquinones is derived from the C-5 compound isopentenyl pyrophosphate (PP). The length of the isoprenoid tail added to the quinoid ring depends of the particular prenyltransferase enzyme that exists in the bacterium. For example, in Escherichia coli, octaprenyl pyrophosphate synthase catalyzes the formation of octaprenyl pyrophosphate (C-40) from farnesyl pyrophosphate (FPP, C-15) and five IPP units. Addition of this molecule to the quinoid ring results in formation of ubiquinone-8. In Paracoccus and Rhodobacter species, decaprenyl pyrophosphate (DPP) synthase catalyzes the formation of DPP (C-50) from FPP (C-15) and seven IPP units. Addition of DPP to the quinoid ring then results in formation of ubiquinone-10 (CoQl 0). hi nature, two different pathways are known for the biosynthesis of IPP (Figure 1). The mevalonate pathway, as its name implies, utilizes mevalonate as a central intermediate and has been well studied in eukaryotes. The mevalonate pathway was thought for many years to be the universal pathway of PP synthesis in nature. However, in the last decade a second pathway of PP biosynthesis, called non-mevalonate pathway or the MEP pathway (as it has 2C-methyl-D-erythritol 4-j>hosphate as an intermediate) was discovered. The MEP pathway has so far been shown to exist in many eubacteria and in the plastid compartment of higher plants. Based on the presence in R. sphaeraides of the yaeM gene (now called ispC) coding for 1-deoxy-D-xylulose 5-phosphate reductoisomerase and on inspection of the nearly completed genome sequence of R. sphaeroides, it appears that this bacterium uses exclusively the MEP.pathway ;for biosynthesis, of IPP. Additional evidence supporting the exclusive use of the MEP pathway in R. spJiaeroides is the finding that a closely related species, Rhodobacter capsulatus, uses only the MEP pathway for isoprenoid biosynthesis. WO 02/26933 Al discloses methods for increasing the production of CoQl 0 in Rhodobacter sphaeroides by overexpressing a few native and/or heterologous genes coding for some of the enzymes of the MEP pathway. However, the overexpression of these genes resulted in only very modest improvement in CoQIO production. As mentioned above, some bacteria use only the mevalonate pathway for biosynthesis of IPP. Paracoccus zeaxanthinifaciens is an example of such a bacterium, hi P. zeaxanthinifaciens, the genes coding for the five enzymes of the mevalonate pathway, plus the gene coding for PP isomerase (see Figure 1), are clustered together in a single transcriptional unit on the chromosome, i.e., an operon called hereinafter the mevalonate (mev) operon (Hiimbelin et at., Gene 297, 129-139, 2002). It has now been found that the production of isoprenoids, in particular CoQIO can be significantly increased by the introduction of a mutated mev operon into a microorganism which is naturally deficient of one or more gene(s) of the mev operon, i.e. naturally using the non-mevalonate pathway for the production of isoprenoids, wherein either the complete mev operon or one or more gene(s) of said mev operon comprising one or more rmrtation(s) may be introduced. The one or more mutation may be for instance in one or all of the following genes: mvaA encoding hydroxymethylghitaryl-CoA reductase, idi encoding isopentenyl diphosphate isomerase, hcs encoding hydroxymethylghitaryl-CoA synthase, mvk encoding mevalonate kinase, ^wzfc encoding phosphomevalonate kinase, and mvd encoding diphosphomevalonate decarboxylase. Thus, the present invention provides a polynucleotide sequence comprising one or more gene(s) encoding a protein having hydroxymethylglutaryl-CoA reductase activity, isopentenyl diphosphate isomerase activity, hydroxymethylglutaryl-CoA synthase activity, mevalonate kinase activity, phosphomevalonate kinase activity, and/or diphosphomevalonate decarboxylase activity, wherein said polynucleotide sequence carries one or more mutation(s) leading to an improved production of isoprenoids when present in a microorganism. The improvement in isoprenoid production may be measured for instance by a comparison of the production in a wild-type microorganism not carrying said polynucleotide sequences, i.e. not using the mev pathway, with the microorganism carrying a polynucleotide sequence as of the present invention. In one embodiment, the present invention is directed to a polynucleotide sequence which is obtainable from SEQ ID NO: 1 or 2, i.e. comprising genes of the wild-type mev operon, or a fragment thereof, wherein the fragment has the activity of at least one of the genes of the mev operon, e.g., mvaA encoding hydroxymetbylglutaryl-CoA reductase., idi encoding isopentenyl diphosphate isomerase, hcs encoding hydroxymethylglutaryl-CoA synthase, mvk encoding mevalonate kinase, pmk encoding phosphomevalonate kinase, and mvd encoding diphosphomevalonate decarboxylase. Preferably, the mutated polynucleotide sequence is represented by SEQ ID NO:3 or a fragment thereof, leading to an increase in e.g. CoQIO production when present in a microorganism. In one aspect, the present invention relates to a DNA sequence comprising a mev operon carrying a mutation, said DNA sequence being represented by SEQ ID NO:3. The term "improved isoprenoid production", in particular improved CoQl 0 production, as used herein means for instance an increase of at least about 10% obtained by a microorganism carrying a. polynucleotide comprising one or more mumu The complete mev operon or one or more gene(s) thereof carrying one or more mutation(s) may be for instance either totally synthesized or partially isolated and synthesized. An isolated mev operon or one or more gene(s) thereof may originate from any microorganism using the mevalonate pathway, i.e. wherein one or more gene(s) included within said operon are naturally occurring. Preferably, the microorganism belongs to the genus Paracoccus, more preferably from Paracoccus zeaxanthinifaciens, such as, e.g. Paracoccus sp. Rl 14 or P. zeaxanthinifaciens ATCC 21588. Allelic variations and mutants of the DNA sequence of the operon which are fully functional are also comprised by the above term. The complete mev operon of P. zeaxanthinifaciens ATCC 21588 is shown in SEQ ID NO:1, the complete mev operon of Paracoccus sp. Rl 14 is shown hi SEQ ID N0:2 (see also SEQ ID NO:42 of WO 02/099095). Strain Paracoccus sp. Rl 14 is a derivative of P. zeaxanthinifaciens ATCC 21588 and has been deposited under the terms of the Budapest Treaty with ATCC under the Patent Deposit Designation PTA-3335 on April 24, 2001. With respect to Paracoccus species and strains which may be used for the present invention and the taxonomic reclassification of Flavobacterium sp. as Paracoccus reference is made to WO 02/099095, pages 47ff. Examples of such species which maybe used are P. marcusii, P. carotinifaciens, P. solventivorans,P. zeaxanthinifaciens or Paracoccus sp. R114, The mev operon of the present invention carries one or more mutation(s) which may be located at any position within the operon, leading to an alteration in the activity of one or rno.re,gene(s) within said operon, resulting in an improved production of isoprenoids within a microorganism carrying such polynucleotide. In one embodiment, the mev operon carries at least one mutation, which is preferably located in the has gene of the mev operon, for instance in the hcs gene of the mev operon of Paracoccus zeaxanthinifaciens, e.g. in the hcs gene of the mev operon of Paracoccus sp. Rl 14 or P. zeaxanthinifaciens ATCC 21588. More preferably, the mutated mev operon as used in the present invention is represented by SEQ ID NO:3. The sequences of the wild~ type hcs gene and the respective protein of Paracoccus sp. R114 are shown in SEQ ID NO:4 and 5, respectively. In one embodiment, the present invention is directed to a polynucleotide comprising one or more imitation(s) in the hcs gene, preferably a polynucleotide shown in SEQ ID NO:6 encoding a protein having hydroxymethylglutaryl-CoA synthase shown in SEQ ID NO:7, wherein a glutamine present on position 90 in SEQ ID NO:5 is replaced by a lysine. Thus, it is an object of the present invention to provide a polynucleotide sequence as described before comprising one or more gene(s) of a mev operon, said polynucleotide being selected from the group consisting of: (a) polynucleotides encoding a polypeptide comprising the amino acid sequence according the SEQ ID NO:7, (h) polynucleotides comprising the nucleotide sequence according to SEQ ID NO:6, (c) polynucleotides comprising a nucleotide sequence encoding a fragment or derivative of a polypeptide encoded by a polynucleotide of (a) or (b) wherein hi said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said fragment or derivative has the activity of hydroxymethylglutaryl-CoA synthase (Hcs); (d) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polypeptide as defined in any one of (a) to (c) and which encodes a Hcs protein. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living microorganism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition'anid'stiirbe isolated in that such vector or composition is not part of its natural environment. An isolated polynucleotide or nucleic acid as used herein may be a DNA or KNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5'-end and one on the 3'-end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, a nucleic acid includes some or all of the 5'-non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence. The term "isolated polynucleotide" therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences, it also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state. A gene may include coding sequences, non-coding sequences such as for instance untranslated sequences located at the 3'- and 5'-ends of the coding region of a gene, and regulatory sequences. Moreover, a gene refers to an isolated nucleic acid molecule as defined herein. It is furthermore appreciated by the skilled person that DNA sequence polymorphisms that lead to changes in the amino acid sequences of proteins may exist within a population. The invention also relates to an isolated polynucleotide hybridisable under stringent conditions, preferably under highly stringent conditions, to a polynucleotide as of the present invention, such as for instance a polynucleotide shown in SEQ ID NO:6. As used herein, the term "hybridizing" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, most preferably at least 95% homologous to each other typically remain hybridized to each other. A preferred, non-limiting example of such hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in Ix SSC, 0.1% SDS at 50°C, preferably at 55°C, more preferably at 60°C and even more ' preferably at"65°C. Highly stringent conditions include, for example, 2 h to 4 days incubation at 42°C using a digoxigenin (DIG)-labeled DNA probe (prepared by using a DIG labeling system; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in a solution such as DigEasyHyb solution (Roche Diagnostics GmbH) with or without 100 ju.g/ml salmon sperm DNA, or a solution comprising 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 0.02% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine, and 2% blocking reagent (Roche Diagnostics GmbH), followed by washing the filters twice for 5 to 15 minutes in 2x SSC and 0.1% SDS at room temperature and then washing twice for 15-30 minutes in 0.5x SSC and 0.1% SDS or O.lx SSC and 0.1% SDS at 65-68°C. The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N. Y.). Of course, a polynucleotide which hybridizes only to a poly (A) sequence (such as the 3'-terminal poly (A) tract of roRNAs), or to a complementary stretch of T (or U) residues, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone). The present invention can be used fro the production of isoprenoids, in particular ubiquinones, preferably CoQIO. Preferably, the polynucleotides comprising one or more mutation(s), i.e. either the complete mev operon or only fragments thereof, are introduced into a suitable microorganism which is originally deficient of one or more gene(s) of the mev operon, i.e. naturally using only the MEP pathway for the production of isoprenoids. The mutated mev operon or one or more gene(s) thereof as described above may be for instance introduced into a microorganism of the genus Rhodobacter. In particular, the present invention is directed to a process for the production of isoprenoids, in particular ubiquinones, preferably CoQIO, comprising (1) introducing a polynucleotide as of the present invention into a microorganism which is originally deficient of said polynucleotides, and (2) cultivating the microorganism of step (1) under conditions that allow the production of the isoprenoid. Preferably, the mutated mev operon or fragments thereof of a microorganism belonging to the genus Paracoccus is introduced into a microorganism belonging to the genus Rhodobacter. More preferably, a polynucleotide comprising one or more mutation(s) in the gene encoding hydroxymethylglutaryl-CoA synthase is introduced into said microorganism, and most preferred is the introduction of a polynucleotide sequence represented by SEQ ID NO:3. Any microorganism which is originally deficient of one or more gene(s) of the mev operon, i.e. using the MEP pathway for the production of isoprenoids, may be used for the purpose of the present invention. In particular microorganisms of the genus Rhodobacter may be used for the introduction of a mutated polynucleotide as of the present invention, such as for instance R. sphaeroides, R. adriaticus, R. capsulatus, R. sulfidophilus, or R. veldkampii. A preferred strain is R. sphaeroides, even more preferred is R. sphaeroides ATCC 35053. It is understood that the microorganisms as named herein also include synonyms or basonyms of such species having the same physico-chemical properties, as defined by the International Code of Nomenclature of Prokaryotes. The present invention further provides recombinant microorganisms which having introduced a polynucleotide described above, i.e. comprising one of more gene(s) of a mev operon carrying one or more mutation(s), wherein the respective wild-type microorganism is originally deficient of one or more gene(s) of the mev operon, i.e. using the MEP pathway for production of isoprenoids, leading to an improved production of isoprenoids, particularly ubiquinones, preferably CoQIO, compared to the respective wild-type microorganism. Besides the mutation within the one or more gene(s) of the mev operon as described above, the recombinant microorganism may contain further modifications/alterations as long as they lead to an improvement in the production of isoprenoids within said microorganism. In one embodiment, such further modification is the introduction of a DNA sequence encoding a protein having decaprenyl diphosphate synthase activity into said recombinant microorganism, preferably obtainable from a microorganism of the genus Paracoccus, e.g., P. zeaxanthinifaciens, in particular P. zeaxanthinifaciens ATCC 21588. Most preferred is a polynucleotide sequence as of SEQ ID NO:8 or a polynucleotide encoding a protein as of SEQ ID NO:9. Thus, the present invention is directed to a process fox the production of isoprenoids, preferably CoQl 0 as above wherein a DNA sequence encoding a protein having decaprenyl diphosphate synthase activity is further introduced into a microorganism which is naturally using the MEP pathway and has one or more gene(s) of the mev operon 3,jqqm,prjsmg.pne or more mutation(s) introduced as described above, e.g. a microorganism of the genus Rhodobacter. Any DNA sequence encoding a protein having decaprenyl diphosphate synthase activity may be used for the present invention. An example of such a DNA is the ddsA gene or a partial sequence thereof coding for a protein having decaprenyl diphosphate synihase activity of a microorganism of the genus Paracoccus, for instance of Paracoccus zeaxanthinifaciens, more preferred of P. zeaxanthinifaciens ATCC 21588. Most preferred is the ddsA gene from P. zeaxanthinifaciens ATCC 21588 as represented by SEQ ED NO:6, coding for decaprenyl diphosphate synthase (SEQ ID NO:7). Thus, it is an aspect of this invention to provide a process for the production of CoQl 0 comprising (1) introducing both a mevalonate (mev) operon and a DNA sequence encoding a protein having decaprenyl diphosphate synthase activity of a microorganism belonging to the genus Paracoccus into a microorganism belonging to the genus Rhodobacter, wherein said mev operon carries one or more mutation(s) and (2) cultivating the modified Rhodobacter strain. The term "introducing into" is used in the present specification and claims in connection with the transformation of a microorganism or host organism, e.g. of the genus Rhodobacter, to comprise any method well-known to a person skilled in the art which may be used to efficiently bring genetic material, in particular the mutated mevalonate operon or one or more gene(s) thereof, into the host organism, i.e. in a way that it is expressed by the organism. Introduction may be effected, e.g. by vectors, preferably expression plasmids or by integration into the host's genome in accordance with standard methods. A preferred method is the introduction of genes via plasmids, such as for instance genes which are cloned in the expression plasmid pBBR-K-PcrtE (the construction of this plasmid has been described in detail in Example 6, page 91, lines 12-27 of WO 02/099095) under the control of the PcrtE promoter. A preferred method for introducing the DNA such as for instance the expression plasmid into a microorganisms of the genus Rhodobacter is the conjugational transfer of plasmids, such as for instance conjugational transfer of a plasmid from E. coli SI7-1 to a Rhodobacter strain (Nishimura et al., Nucl. Acids Res. 18, 6169, 1990; Simon etal., Bio/Technology 1983, 784-91). The mutation(s) of the me\> operon or of one or more gene(s) thereof may be generated by for instance PCR site directed mutagenesis using a method with which a person skilled in the art is familiar and which needs no specific explanation. Further mutagenesis methods, which may be also used for the purpose of the present invention and which are known to the skilled person include for instance UV irradiation, transposition or chemical mutagenesis. The screening method for identification of mutants showing increased isoprenoid, e.g. CoQIO, productivity may be for instance selected from direct measurement of isoprenoids, e.g. CoQ 10, by UV light, HPLC, NMR or thin layer chromatographyr Production of isoprenoids, e.g. CoQ 10, may be also measured indirectly via measuring an increase in carotenoid color intensity, which a person skilled in the art is familiar with. The host transformed with the mutated mev operon or one or more gene(s) thereof as of the present invention may be cultivated in accordance with known methods, viz. in a medium containing for instance carbon and nitrogen sources, inorganic salts, etc., which may be assimilated by the host and under temperature, pH and aeration conditions suitable for efficient growing and expression of the desired product, in particular CoQIO. Isolation from the fermentation broth and/or the transformant, i.e. the microorganism in which me mutated mevalonate operon of a microorganism belonging e.g. to the genus Paracoccus has been introduced, and, optionally, purification and further processing of the obtained isoprenoid, in particular CoQIO, including for instance lormuianons 01 sucn produced CoQIO for human or animal usage may be effected in accordance with methods well-known in the art. For use in animal health and •nutrition, however, no specific purification may be necessary. In this case the produced isoprenoids like CoQl 0 together with the biomass and/or other components of the fermentation broth may be further processed to yield a commercially attractive product. The process of the present invention results in higher yields of isoprenoids, such as for instance CoQIO. The increase may be for instance at least about 10% compared to processes using a non-recombinant strain of for instance Rhodobacter or using a recombinant strain such as for instance Rhodobacter carrying the wild-type mev operon of e.g. aParacoccus strain. Figure 1 represents the pathway for CoQIO biosynthesis in R. sphaeroides, which uses the MEP pathway for IPP formation. The boxed region indicates the reaction sequence that comprises the mevalonate pathway (leading to formation of IPP), plus the IPP isomerase step. The mevalonate pathway does not naturally occur in R. sphaeroides. In P. zeaxanthinifaciens, the genes coding for the five enzymes of the mevalonate pathway plus IPP isomerase form an operon, called hereinafter the mevalonate operon. The following Examples illustrate the invention without restricting it in any way. Example 1: Bacteria and culture conditions Rliodobacter sphaeroides strain ATCC 35053 (obtained from the American Type Culture Collection, Manassas VA, USA) was used as the base host for construction of recombinant strains having improved production of CoQIO. All R. sphaeroides strains were grown at 3-0°C in medium RSI 00. The composition and preparation of medium RSI 00 is summarized in Table I; Fifty iag/1 kanamycin was added to the me.dium.for, growth of recombinant strains. E. coli strains were grown at 37°C in LB medium (Becton Dickinson, Sparks, MD, USA). For maintenance of plasmids in recombinant E. coli strains, ampicillin (100 mg/1) and/or kanamycin (25-50 mg/1, depending on the plasmid) were added to the culture medium. Liquid cultures of E. coli and R. sphaeroides were routinely grown aerobically in a rotary shaker at 200 rpm. When solid media were required, agar (1.5% final concentration) was added. Table 1. Composition and preparation of medium RSI 00 (Table Removed) Example 2: Analytical assay for CoQIO 400 fj.1 of whole cultivation broth (see Example 5) were transferred to a disposable o mi polypropylene centrifuge tube. 4ml of stabilized extraction solution (0.5 g/1 BHT in 1:1 (v/v) DMSO/THF) were added and the samples were mixed for 20 min in a laboratory shaker (DCA, Germany) to enhance extraction. Finally, the samples were centrifuged and the supernatants transferred to amber glass vials for analysis by reverse phase HPLC. This method was developed for the simultaneous determination of ubiquinones and their corresponding hydroquinones, with a clear separation of CoQIO from the carotenoids phytoene, spheroidenone, spheroidene and neurosporene. Chromatography was performed using an Agilent 1100 HPLC system (Agilent Technologies, USA) equipped with a temperature-controlled autosampler and a diode array detector. The method parameters were as follows: Column YMC Carotenoid C30 column 3 micron, steel, 150 mm x 3.0 mm I.D. (YMC, Part No. CT99S031503QT) Guard column Security Guard CIS (ODS, Octadecyl) 4 mm length x 3.0 mm I.D. (Phenomenex, Part No. AJO-4287) Typical column pressure 60 bar at start Flow rate 0.5 ml/min Mobile phase Gradient profile Mixture of acetonitrile(A):methanol(B):TBME(C) %B 15 15 100 15 15 %A 60 60 00 . 60 60 25 25 25 25 Time (min) 0 13 20 22. , . 25 Post time Injection volume Column temperature Detection 4 min. 10 fil 15°C Three wavelengths weie used for detection of specific compounds according to Table 2. Table 2. HPLC retention times and wavelengths used (Table Removed) Calculations: Calculations were based on peak areas. Example 3: Cloning of mutated mev operon from P. zeaxanthinifaciens Construction of plasmids pBBR-K-7??ev-op-wt and pBBR-K-mgy-op-Rl 14. The construction of plasmid pBBR-K-?«ev-op-up-4 (plasmid containing the first 4 genes of the mevalonate operon) is described in detail in Example 13 (page 105, line 10 to page 106, line 8) of WO 02/099095. Using P. zeaxanthinifaciens Rl 14 genomic DNA as template, PCR is performed with primers hcs-5326 (SEQ ID NO: 10) and mvd-9000 (SEQ ID NO:11). Primer hcs-5326 corresponds to the sequence of the P. zeaxanthinifaciens mev operon from nucleotide 3321 to 3340 of SEQ ID NO:2 while primer mvd-9000 corresponds to the reverse complement of the sequence from nucleotides 6977 to 69J>6 of SEQ ID NO:2. The obtained PCR product of 3675 bp is cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA), resulting into plasmid TOPO-pCR2.1-/«ev-op-d-3wt. This plasmid thus contains the downstream half of the mevalonate operon including the 3' end of has and the last three genes, mvk, pmk and mvd. Plasmids pBBR-K-7?zev-op-up-4 and TOPO-pCR2.1-7?zev-op-d-3wt are digested with the restriction endonucleases Sad and Ndel and the resulting 3319 bp fragment from TOPO-pCR2.1-??2ev-op-d-3wt is ligated with the 8027 bp fragment from pBBR-K-/?zev-op-up-4. The resulting plasmid, pBBR-K-?«ev-op-Rl 14, contains the complete mevalonate operon from P. zeaxanthinifaciens Rl 14 including its (putative) promoter. Construction of plasmids pBBR-K-mev-op-wt-Pc?l£-crt.£ and pBBR-K-??iev-op-Rl 14-fcrtE-crtE The construction of plasmid pBBR.-K-PcrtE-crtE is described in detail in Example 6 (page 92, lines 10-17) of WO 02/099095. Plasmid pBBR-K-Pcrt£-crt£ was cut with Noel and the 1.33 kb fragment was isolated and inserted into the Ecll36H site of pBBR-K-mev-op-up-4. The orientation of the insert was checked and the plasmid which carried the crtE gene in the same orientation as the mevalonate operon genes was designated pBBR-K-mev-op-up-4-'PcrtE-crtE-2. Plasmid pBBR-K-mev-op-up-4-Pc7tE-c/-tE-2 was cut with Sphl and Spel and the resulting 5566 bp fragment containing the crtE gene was isolated. This fragment was ligated with the 7132 bp Sphl-Spel fragment obtained after a restriction digest of pBBR-K-/nev-op-wt or pBBR-K-mev-op-Rl 14 using the same enzymes, resulting in plasmid pBBR-K-jnev-op-vrt-PcrtE-crtE and pBBR-K-/nev-op-Rl 14-PcrtE-crtE, respectively. Construction of plasmids pBBK-K.-mev~oT>-vft-PcrtE~ddsAwt and pBBR-K-mgv-op-Rl 14-VcrtE-ddsA,* The construction of plasmid pBBR-K-Pcr/Ji is described in detail in Example 6 (page 91, lines 12-27) of WO 02/099095. The ddsA gene from P. zeaxanthinifaciens strain ATCC 21588 (designated ddsAva) was amplified by PCR (GC-rich PCR System, Roche Molecular Biochemicals, Mannheim, Germany) using the primers dds-Nde (SEQ ID NO: 12) and dds-Bam (SEQ ID NO: 13) and cloned into pCR2.1-TOPO (hivitrogen, Carlsbad, CA, USA) to result in plasmid TOPO- "Plasmid TQPO-dJiS^wt.-was,RUt,with. JVdel and BainHL and the obtained fragment of 1005 bp containing the ddsA gene was cloned into plasmid pBBR-K-Pcr/.E cut with Ndel and BairiHl, resulting in plasmid pBBR.-'K-PcrtE-ddsA^t. A recognition site for the endonuclease EcoKL within the ddsA gene was eliminated by mtroducing a silent mutation with the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) using the oligonucleotides dds-R-1 (SEQ ID NO: 14) and dds-R-2 (SEQ ID NO: 1 5). The resulting plasmid pBBR.-K-'PcrtE-ddsAwt-'R. was cut with EcoKL and Maml and the fragment 1278 bp fragment containing the ddsA gene was inserted into TOPO-ddsA^ cut with .EcoRI and EcdKV, resulting in the plasmid pCR2.1-TOPO-d&J4wt-R. This plasmid was cut with CelR and Xbal and the obtained fragment of 121 1 bp containing the ddsA gene was ligated with a 11,6 kb restriction fragment obtained from digestion of pBBR-K-mev-op-vfi-~?crtE-crtE andpBBR-K-mev-op-RlH-Pcr^-crz'E1, respectively, with CelR and Blnl. The resulting plasmids were named pBBR-K-meri'-op-wt-'PcrtE-ddsAvn and pBBR-K-7?zev-op-Rl 14-PcrtE-ddsAvn, respectively. Construction of plasmid Plasmid pBBR-K.-m&>-op-4-&9-PcrtE-ddsAw is obtained by two rounds of PCR site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and primers mut4-89-l-fw (SEQ ID NO: 16) and mut4-89-1-rev (SEQ ID NO: 17). Both primers are complementary to each other and contain the desired mutation A instead of C at position 2949 of SEQ ID NO:3, corresponding to position 268 of SEQ ID NO:4. The first mutagenesis reaction is set up as follows according to the manufacturer's instructions: 5 jil lOx reaction buffer, 10 ng plasmid DNA pBBR-K-jnev-op-wt-PcrtE-J^wt, 125 ng primer mut4-89-l-fw, 125 ngmut4-89-l-rev, 1 /il dNTP mix, 3 /tl QuikSolution and 2.5 U PfuTurbo DNA polymerase are mixed in a final volume of 50 /zl. The cycling is carried out by using the following parameters: 1 cycle: 95°C for 1 min; 18 cycles: 95°C for 50 sec, 60°C forSO sec, 68°C for 30 min; 1 cycle: 68°C for 7 min. After cooling the reaction mix to 37°C, 10 U of the restriction endonuclease Dpnl is added and the reaction is incubated at 37°C for 2 hours. Escherichia coli XLlO-Gold Ulrracompetent cells (Stratagene, La Jolla, CA, USA) are transformed with the Z)pHl-treated DNA according to the manufacturer's protocol. The resulting plasmid pBBR-K-7?iev-op-4-89-l-Pcr££'- Example 4: Introduction of the mutated mev operon into R. sphaeroides ATCC 35053 Transformation of Z coli S17-1 (Simon et al, Bio/Technology 11, 784-791, 1983) with plasmids carrying the mutated mev operon and subsequent transfer of plasmids from E. coli S17-1 to R. sphaeroides ATCC 35053 by conjugation were performed using standard procedures (Nishimura et al, Nucl. Acids Res. 18, 6169, 1990; Simon et al., Bio/Technology 1983, 784-91). A spontaneous rifampicin resistant mutant of R. sphaeroides ATCC 35053 was first isolated by growing strain ATCC 35053 in RSI 00 liquid medium supplemented with 100 mg/1 rifampicin, plating the cells on RSI 00 plates containing 100 mg/1 rifampicin, and isolating a single colony. For the conjugation, one milliliter aliquots of cultures of the recipient cells (rifampicin-resistant R. sphaeroides ATCC 35053) grown in RSI 00 medium containing 100 mg/1 rifampicin and the donor cells (E. coli SI 7-1 carrying the plasmid to be transferred, grown in LB broth containing 50 mg/1 kanamycin) were pelleted by centrifugation. The supernatant was discarded, and the cells were washed twice with fresh RS 1 00 medium to remove antibiotics. Each pellet was then resuspended in 1 ml of fresh RSI 00 medium. Fifty microliters of donor cells and 0.45 ml of recipient cells were mixed, pelleted by centrifugation, resuspended in 0.03 ml of fresh RSI 00 medium and spotted onto an RSI 00 plate. After overnight incubation at 30°C the cells were harvested with an inoculating loop and resuspended in 0.3 ml of RS100 medium. Dilutions of this suspension were spread onto RSI 00 plates containing 100 mg/1 rifampicin and 50 mg/1 kanamycin and incubated at 30°C. Colonies (putative transformed cells of R. sphaeroides ATCC 35053) were picked from the plates, grown in liquid RSI 00 medium containing 50 mg/1 kanamycin and the presence of the plasmid was tested in a PCR reaction with an annealing temperature of 56°C and an elongation time of 1 min, 15 sec using the following two different primer pairs: pBBR-K-up (SEQ ID NO:20)/PcrtE-2442 (SEQ ID NO:21) KanSout (SEQ ID NO:22)/mvaA3256 (SEQ ID NO:23) Positive, clones were, streaked onto. RSI 00 plates containing 50 mg/1 kanamycin to obtain single colonies. One single colony from each clone was again grown in liquid RSI 00 medium containing 50 mg/1 kanamycin, and the presence of the expected plasmid was confirmed by PCR as described above. The resulting recombinant strain was named Example 5: Production of CoQIO in transformed strains of JZ. sphaeroides ATCC 35053 R. sphaeroides strains ATCC 35053, ATCC and ATCC 35Q53/p'BER.-K.-mev-op-4-89-PcrtE-ddsAWt were grown in shake flask cultures in RSI 00 medium. Cultures containing the recombinant R. sphaeroides contained 50 mg/1 kanamycin. Twenty five-milliliter cultures were grown at 30°C in 250-ml baffled Erlenmeyer flasks with shaking at 200 rpm. For testing CoQIO production, frozen glycerolized stock cultures of the/?, sphaeroides strains were used to inoculate 25-ml seed cultures. After growth of the seed cultures for 24-28 hours, suitable volumes of the cultures were used to inoculate the experimental flasks such that the initial optical density at 660 nanometers (OD66o) was 0.16. Two milliliter samples were taken aseptically at 24 hour intervals. Analyses included growth (measured as ODggo), pH, glucose in the culture supernatant and CoQIO and carotenoids (determined by HPLC) as described in Example 2. The results are summarized in Table 3. These results clearly show that the expression of the cloned mutated mevalonate operon from P. zeaxanthinifaciens significantly improved CoQIO production inR. sphaeroides. Table 3. Production of CoQIO in transformed R. sphaeroides ATCC 35053 strains (Table Removed) Specific Formation is expressed as mg/1 CoQIO produced/ODeeo- Claims 1. A polynucleotide sequence comprising one or more gene(s) encoding a protein having hydroxymethylglutaryl-CoA reductase activity, isopentenyl diphosphate isomerase activity, hydroxymethylglutaryl-CoA synthase activity, mevalonate kinase activity, phosphomevalonate kinase activity, and/or diphosphomevalonate decarboxylase activity, wherein said polynucleotide sequence carries one or more mutation(s) leading to an improved production of isoprenoids when present in a microorganism. 2. A polynucleotide sequence according to claim 1 which is obtainable from SEQ ID NO: 1 or 2 or a fragment thereof and which carries one or more mutation(s). 3. A polynucleotide sequence according to claim 1 or 2 comprising one or more mutation(s) in the hcs gene encoding a protein having hydroxymethylglutaryl-CoA synthase activity. 4 A polynucleotide sequence according to any one of claims 1 to 3 which is represented by SEQ ID NO:3 or a fragment thereof. 5. A polynucleotide sequence according to any one of claims 1 to 4 selected from the group consisting of: (a) polynucleotides encoding a polypeptide comprising the amino acid sequence according the SEQ ID NO: 7, (b) polynucleotides comprising the nucleotide sequence according to SEQ ID NO:6, (c) polynucleotides comprising a nucleotide sequence encoding a fragment or derivative of a pQlypeptide,,encoded,hy a polynucleotide of (a) or (b) wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said fragment or derivative has the activity of hydroxymethylglutaryl-CoA synthase; (d) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polypeptide as defined in any one of (a) to (c) and which encodes a Hcs protein. 6. Use of a polynucleotide according to any one of claims 1 to 5 for the production of isoprenoids, preferably Co-enzyme Q10. 7. A process for the production of isoprenoids comprising: (a) introducing a polynucleoti.de sequence according to any one of claims 1 to 5 into a microorganism which is originally deficient of said polynucleotides, and (b) cultivating the microorganism of step (a) under conditions that allow the production ofisoprenoids. 8. The process according to claim 7 wherein the microorganism in which the polynucleotide is introduced is selected from the genus Rhodobacter., preferably Khodobacter sphaeroides. 9. The process according to claims 7 or 8 wherein the introduced polynucleotide is ohtainable from a microorganism selected' from the genus Paracoccits, preferably P. zeaxanthimfaciens. 10. A microorganism, comprising a polynucleotide sequence according to any one. of claims 1 to 5. . 1 1 . The microorganism according to claim 1 0 which, is selected from the genus Khodobacter, preferably Rhodobacter sphaeroides. 12. The microorganism according to claims 10 01 1 1 wherein the polyrmclsotide according to any one of claims 1 to 5 obtainable from a microorganism selected from the genus Paracoccus is introduced, 13. Use of a microorganism according to :any one of claims 10 to 12 for the production of isoprenoids. .

Full Text

Production of isoprenoids
The present invention relates to a process for the production of isoprenoids, in particular Coenzyme Q-10 by microorganisms. More particularly, the present invention relates to a process for increased production of Coenzyme Q-10 by microorganisms of the genus Rhodobacter, preferably of the species R. sphaeroides which have been transformed with one or more gene(s) of the mevalonate (mev) operon from a different microorganism, preferably of the genus Paracoccus, more preferably of the species P. zeaxanthinifaciejis, whereby the mev operon is mutated leading to an increased coenzyme Q-10 production. Sequences carrying such a mutation as well as a microorganism carrying such a mutated mev operon are also included.
Coenzyme Q-10 (2,3-dimethoxy-dimethyl-6-decaprenyl-l,4-benzoquinone), also known as ubiquinone-10, is a Upid-soluble benzoquinone having an isoprenoid side chain comprised often C-5 isoprenoid units. Coenzyme Q-10 (hereafter abbreviated as CoQIO) is found in microorganisms and plants, as well as in animals. It is the most prevalent form of ubiquinone in humans and most mammals. There is established' aiiti growing evidence that CoQl 0 is an important factor in the health status of humans and their protection from diseases. The medical and health beneficial effects of CoQIO have been associated with its two main physiological functions., namely to function as an essential cofactor of the mitochondrial electron transport chain (which is coupled to the synthesis of adenosine triphosphate) and to act as a lipid soluble antioxidant.
The health benefits of CoQIO have led to increased commercial importance of this compound. CoQl 0 can be produced by chemical synthesis or by fermentation using microorganisms. These microorganisms may be natural CoQl 0 producers that have been
improved for CoQIO production by genetic engineering, or they may not naturally produce CoQIO but have been manipulated by genetic engineering to be able to synthesize it.
In bacteria, the quinoid ring of ubiquinones is derived from chorismate, a central intermediate in the biosynthesis of aromatic compounds, while the isoprenoid tail of ubiquinones is derived from the C-5 compound isopentenyl pyrophosphate (PP). The length of the isoprenoid tail added to the quinoid ring depends of the particular prenyltransferase enzyme that exists in the bacterium. For example, in Escherichia coli, octaprenyl pyrophosphate synthase catalyzes the formation of octaprenyl pyrophosphate (C-40) from farnesyl pyrophosphate (FPP, C-15) and five IPP units. Addition of this molecule to the quinoid ring results in formation of ubiquinone-8. In Paracoccus and Rhodobacter species, decaprenyl pyrophosphate (DPP) synthase catalyzes the formation of DPP (C-50) from FPP (C-15) and seven IPP units. Addition of DPP to the quinoid ring then results in formation of ubiquinone-10 (CoQl 0).
hi nature, two different pathways are known for the biosynthesis of IPP (Figure 1). The mevalonate pathway, as its name implies, utilizes mevalonate as a central intermediate and has been well studied in eukaryotes. The mevalonate pathway was thought for many years to be the universal pathway of PP synthesis in nature. However, in the last decade a second pathway of PP biosynthesis, called non-mevalonate pathway or the MEP pathway (as it has 2C-methyl-D-erythritol 4-j>hosphate as an intermediate) was discovered. The MEP pathway has so far been shown to exist in many eubacteria and in the plastid compartment of higher plants.
Based on the presence in R. sphaeraides of the yaeM gene (now called ispC) coding for 1-deoxy-D-xylulose 5-phosphate reductoisomerase and on inspection of the nearly completed genome sequence of R. sphaeroides, it appears that this bacterium uses exclusively the MEP.pathway ;for biosynthesis, of IPP. Additional evidence supporting the exclusive use of the MEP pathway in R. spJiaeroides is the finding that a closely related species, Rhodobacter capsulatus, uses only the MEP pathway for isoprenoid biosynthesis.
WO 02/26933 Al discloses methods for increasing the production of CoQl 0 in Rhodobacter sphaeroides by overexpressing a few native and/or heterologous genes coding for some of the enzymes of the MEP pathway. However, the overexpression of these genes resulted in only very modest improvement in CoQIO production.
As mentioned above, some bacteria use only the mevalonate pathway for biosynthesis of IPP. Paracoccus zeaxanthinifaciens is an example of such a bacterium, hi P. zeaxanthinifaciens, the genes coding for the five enzymes of the mevalonate pathway, plus the gene coding for PP isomerase (see Figure 1), are clustered together in a single
transcriptional unit on the chromosome, i.e., an operon called hereinafter the mevalonate (mev) operon (Hiimbelin et at., Gene 297, 129-139, 2002).
It has now been found that the production of isoprenoids, in particular CoQIO can be significantly increased by the introduction of a mutated mev operon into a microorganism which is naturally deficient of one or more gene(s) of the mev operon, i.e. naturally using the non-mevalonate pathway for the production of isoprenoids, wherein either the complete mev operon or one or more gene(s) of said mev operon comprising one or more rmrtation(s) may be introduced. The one or more mutation may be for instance in one or all of the following genes: mvaA encoding hydroxymethylghitaryl-CoA reductase, idi encoding isopentenyl diphosphate isomerase, hcs encoding hydroxymethylghitaryl-CoA synthase, mvk encoding mevalonate kinase, ^wzfc encoding phosphomevalonate kinase, and mvd encoding diphosphomevalonate decarboxylase.
Thus, the present invention provides a polynucleotide sequence comprising one or more gene(s) encoding a protein having hydroxymethylglutaryl-CoA reductase activity, isopentenyl diphosphate isomerase activity, hydroxymethylglutaryl-CoA synthase activity, mevalonate kinase activity, phosphomevalonate kinase activity, and/or diphosphomevalonate decarboxylase activity, wherein said polynucleotide sequence carries one or more mutation(s) leading to an improved production of isoprenoids when present in a microorganism. The improvement in isoprenoid production may be measured for instance by a comparison of the production in a wild-type microorganism not carrying said polynucleotide sequences, i.e. not using the mev pathway, with the microorganism carrying a polynucleotide sequence as of the present invention.
In one embodiment, the present invention is directed to a polynucleotide sequence which is obtainable from SEQ ID NO: 1 or 2, i.e. comprising genes of the wild-type mev operon, or a fragment thereof, wherein the fragment has the activity of at least one of the genes of the
mev operon, e.g., mvaA encoding hydroxymetbylglutaryl-CoA reductase., idi encoding isopentenyl diphosphate isomerase, hcs encoding hydroxymethylglutaryl-CoA synthase, mvk encoding mevalonate kinase, pmk encoding phosphomevalonate kinase, and mvd encoding diphosphomevalonate decarboxylase. Preferably, the mutated polynucleotide sequence is represented by SEQ ID NO:3 or a fragment thereof, leading to an increase in e.g. CoQIO production when present in a microorganism.
In one aspect, the present invention relates to a DNA sequence comprising a mev operon carrying a mutation, said DNA sequence being represented by SEQ ID NO:3.
The term "improved isoprenoid production", in particular improved CoQl 0 production, as used herein means for instance an increase of at least about 10% obtained by a
microorganism carrying a. polynucleotide comprising one or more mumu The complete mev operon or one or more gene(s) thereof carrying one or more mutation(s) may be for instance either totally synthesized or partially isolated and synthesized. An isolated mev operon or one or more gene(s) thereof may originate from any microorganism using the mevalonate pathway, i.e. wherein one or more gene(s) included within said operon are naturally occurring. Preferably, the microorganism belongs to the genus Paracoccus, more preferably from Paracoccus zeaxanthinifaciens, such as, e.g. Paracoccus sp. Rl 14 or P. zeaxanthinifaciens ATCC 21588. Allelic variations and mutants of the DNA sequence of the operon which are fully functional are also comprised by the above term. The complete mev operon of P. zeaxanthinifaciens ATCC 21588 is shown in SEQ ID NO:1, the complete mev operon of Paracoccus sp. Rl 14 is shown hi SEQ ID N0:2 (see also SEQ ID NO:42 of WO 02/099095).
Strain Paracoccus sp. Rl 14 is a derivative of P. zeaxanthinifaciens ATCC 21588 and has been deposited under the terms of the Budapest Treaty with ATCC under the Patent Deposit Designation PTA-3335 on April 24, 2001. With respect to Paracoccus species and strains which may be used for the present invention and the taxonomic reclassification of Flavobacterium sp. as Paracoccus reference is made to WO 02/099095, pages 47ff. Examples of such species which maybe used are P. marcusii, P. carotinifaciens, P. solventivorans,P. zeaxanthinifaciens or Paracoccus sp. R114,
The mev operon of the present invention carries one or more mutation(s) which may be located at any position within the operon, leading to an alteration in the activity of one or rno.re,gene(s) within said operon, resulting in an improved production of isoprenoids within a microorganism carrying such polynucleotide.
In one embodiment, the mev operon carries at least one mutation, which is preferably located in the has gene of the mev operon, for instance in the hcs gene of the mev operon of Paracoccus zeaxanthinifaciens, e.g. in the hcs gene of the mev operon of Paracoccus sp. Rl 14 or P. zeaxanthinifaciens ATCC 21588. More preferably, the mutated mev operon as used in the present invention is represented by SEQ ID NO:3. The sequences of the wild~ type hcs gene and the respective protein of Paracoccus sp. R114 are shown in SEQ ID NO:4 and 5, respectively.
In one embodiment, the present invention is directed to a polynucleotide comprising one or more imitation(s) in the hcs gene, preferably a polynucleotide shown in SEQ ID NO:6
encoding a protein having hydroxymethylglutaryl-CoA synthase shown in SEQ ID NO:7, wherein a glutamine present on position 90 in SEQ ID NO:5 is replaced by a lysine.
Thus, it is an object of the present invention to provide a polynucleotide sequence as described before comprising one or more gene(s) of a mev operon, said polynucleotide being selected from the group consisting of:
(a) polynucleotides encoding a polypeptide comprising the amino acid sequence according the SEQ ID NO:7,
(h) polynucleotides comprising the nucleotide sequence according to SEQ ID NO:6,
(c) polynucleotides comprising a nucleotide sequence encoding a fragment or derivative
of a polypeptide encoded by a polynucleotide of (a) or (b) wherein hi said derivative one or
more amino acid residues are conservatively substituted compared to said polypeptide, and
said fragment or derivative has the activity of hydroxymethylglutaryl-CoA synthase (Hcs);
(d) polynucleotides the complementary strand of which hybridizes under stringent
conditions to a polypeptide as defined in any one of (a) to (c) and which encodes a Hcs
protein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living microorganism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition'anid'stiirbe isolated in that such vector or composition is not part of its natural environment.
An isolated polynucleotide or nucleic acid as used herein may be a DNA or KNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5'-end and one on the 3'-end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, a nucleic acid includes some or all of the 5'-non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence. The term "isolated polynucleotide" therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced
by PCR or restriction endonuclease treatment) independent of other sequences, it also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.
A gene may include coding sequences, non-coding sequences such as for instance untranslated sequences located at the 3'- and 5'-ends of the coding region of a gene, and regulatory sequences. Moreover, a gene refers to an isolated nucleic acid molecule as defined herein. It is furthermore appreciated by the skilled person that DNA sequence polymorphisms that lead to changes in the amino acid sequences of proteins may exist within a population.
The invention also relates to an isolated polynucleotide hybridisable under stringent conditions, preferably under highly stringent conditions, to a polynucleotide as of the present invention, such as for instance a polynucleotide shown in SEQ ID NO:6.
As used herein, the term "hybridizing" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, most preferably at least 95% homologous to each other typically remain hybridized to each other.
A preferred, non-limiting example of such hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in Ix SSC, 0.1% SDS at 50°C, preferably at 55°C, more preferably at 60°C and even more ' preferably at"65°C.
Highly stringent conditions include, for example, 2 h to 4 days incubation at 42°C using a digoxigenin (DIG)-labeled DNA probe (prepared by using a DIG labeling system; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in a solution such as DigEasyHyb solution (Roche Diagnostics GmbH) with or without 100 ju.g/ml salmon sperm DNA, or a solution comprising 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 0.02% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine, and 2% blocking reagent (Roche Diagnostics GmbH), followed by washing the filters twice for 5 to 15 minutes in 2x SSC and 0.1% SDS at room temperature and then washing twice for 15-30 minutes in 0.5x SSC and 0.1% SDS or O.lx SSC and 0.1% SDS at 65-68°C.
The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N. Y.). Of course, a polynucleotide which hybridizes only to a poly (A) sequence (such as the 3'-terminal poly (A) tract of roRNAs), or to a complementary stretch of T (or U) residues, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
The present invention can be used fro the production of isoprenoids, in particular ubiquinones, preferably CoQIO. Preferably, the polynucleotides comprising one or more mutation(s), i.e. either the complete mev operon or only fragments thereof, are introduced into a suitable microorganism which is originally deficient of one or more gene(s) of the mev operon, i.e. naturally using only the MEP pathway for the production of isoprenoids.
The mutated mev operon or one or more gene(s) thereof as described above may be for instance introduced into a microorganism of the genus Rhodobacter.
In particular, the present invention is directed to a process for the production of isoprenoids, in particular ubiquinones, preferably CoQIO, comprising (1) introducing a polynucleotide as of the present invention into a microorganism which is originally deficient of said polynucleotides, and (2) cultivating the microorganism of step (1) under conditions that allow the production of the isoprenoid. Preferably, the mutated mev operon or fragments thereof of a microorganism belonging to the genus Paracoccus is introduced into a microorganism belonging to the genus Rhodobacter. More preferably, a polynucleotide comprising one or more mutation(s) in the gene encoding hydroxymethylglutaryl-CoA synthase is introduced into said microorganism, and most preferred is the introduction of a polynucleotide sequence represented by SEQ ID NO:3.
Any microorganism which is originally deficient of one or more gene(s) of the mev operon, i.e. using the MEP pathway for the production of isoprenoids, may be used for the purpose of the present invention. In particular microorganisms of the genus Rhodobacter may be used for the introduction of a mutated polynucleotide as of the present invention, such as for instance R. sphaeroides, R. adriaticus, R. capsulatus, R. sulfidophilus, or R. veldkampii. A preferred strain is R. sphaeroides, even more preferred is R. sphaeroides ATCC 35053.
It is understood that the microorganisms as named herein also include synonyms or basonyms of such species having the same physico-chemical properties, as defined by the International Code of Nomenclature of Prokaryotes.
The present invention further provides recombinant microorganisms which having introduced a polynucleotide described above, i.e. comprising one of more gene(s) of a mev operon carrying one or more mutation(s), wherein the respective wild-type microorganism is originally deficient of one or more gene(s) of the mev operon, i.e. using the MEP pathway for production of isoprenoids, leading to an improved production of isoprenoids, particularly ubiquinones, preferably CoQIO, compared to the respective wild-type microorganism.
Besides the mutation within the one or more gene(s) of the mev operon as described above, the recombinant microorganism may contain further modifications/alterations as long as they lead to an improvement in the production of isoprenoids within said microorganism.
In one embodiment, such further modification is the introduction of a DNA sequence encoding a protein having decaprenyl diphosphate synthase activity into said recombinant microorganism, preferably obtainable from a microorganism of the genus Paracoccus, e.g., P. zeaxanthinifaciens, in particular P. zeaxanthinifaciens ATCC 21588. Most preferred is a polynucleotide sequence as of SEQ ID NO:8 or a polynucleotide encoding a protein as of SEQ ID NO:9.
Thus, the present invention is directed to a process fox the production of isoprenoids,
preferably CoQl 0 as above wherein a DNA sequence encoding a protein having
decaprenyl diphosphate synthase activity is further introduced into a microorganism which
is naturally using the MEP pathway and has one or more gene(s) of the mev operon
3,jqqm,prjsmg.pne or more mutation(s) introduced as described above, e.g. a microorganism
of the genus Rhodobacter.
Any DNA sequence encoding a protein having decaprenyl diphosphate synthase activity may be used for the present invention. An example of such a DNA is the ddsA gene or a partial sequence thereof coding for a protein having decaprenyl diphosphate synihase activity of a microorganism of the genus Paracoccus, for instance of Paracoccus zeaxanthinifaciens, more preferred of P. zeaxanthinifaciens ATCC 21588. Most preferred is the ddsA gene from P. zeaxanthinifaciens ATCC 21588 as represented by SEQ ED NO:6, coding for decaprenyl diphosphate synthase (SEQ ID NO:7).
Thus, it is an aspect of this invention to provide a process for the production of CoQl 0 comprising (1) introducing both a mevalonate (mev) operon and a DNA sequence encoding a protein having decaprenyl diphosphate synthase activity of a microorganism belonging to

the genus Paracoccus into a microorganism belonging to the genus Rhodobacter, wherein said mev operon carries one or more mutation(s) and (2) cultivating the modified Rhodobacter strain.
The term "introducing into" is used in the present specification and claims in connection with the transformation of a microorganism or host organism, e.g. of the genus Rhodobacter, to comprise any method well-known to a person skilled in the art which may be used to efficiently bring genetic material, in particular the mutated mevalonate operon or one or more gene(s) thereof, into the host organism, i.e. in a way that it is expressed by the organism. Introduction may be effected, e.g. by vectors, preferably expression plasmids or by integration into the host's genome in accordance with standard methods. A preferred method is the introduction of genes via plasmids, such as for instance genes which are cloned in the expression plasmid pBBR-K-PcrtE (the construction of this plasmid has been described in detail in Example 6, page 91, lines 12-27 of WO 02/099095) under the control of the PcrtE promoter. A preferred method for introducing the DNA such as for instance the expression plasmid into a microorganisms of the genus Rhodobacter is the conjugational transfer of plasmids, such as for instance conjugational transfer of a plasmid from E. coli SI7-1 to a Rhodobacter strain (Nishimura et al., Nucl. Acids Res. 18, 6169, 1990; Simon etal., Bio/Technology 1983, 784-91).
The mutation(s) of the me\> operon or of one or more gene(s) thereof may be generated by for instance PCR site directed mutagenesis using a method with which a person skilled in the art is familiar and which needs no specific explanation. Further mutagenesis methods, which may be also used for the purpose of the present invention and which are known to the skilled person include for instance UV irradiation, transposition or chemical mutagenesis. The screening method for identification of mutants showing increased isoprenoid, e.g. CoQIO, productivity may be for instance selected from direct measurement of isoprenoids, e.g. CoQ 10, by UV light, HPLC, NMR or thin layer chromatographyr Production of isoprenoids, e.g. CoQ 10, may be also measured indirectly via measuring an increase in carotenoid color intensity, which a person skilled in the art is familiar with.
The host transformed with the mutated mev operon or one or more gene(s) thereof as of the present invention may be cultivated in accordance with known methods, viz. in a medium containing for instance carbon and nitrogen sources, inorganic salts, etc., which may be assimilated by the host and under temperature, pH and aeration conditions suitable for efficient growing and expression of the desired product, in particular CoQIO.
Isolation from the fermentation broth and/or the transformant, i.e. the microorganism in which me mutated mevalonate operon of a microorganism belonging e.g. to the genus Paracoccus has been introduced, and, optionally, purification and further processing of the

obtained isoprenoid, in particular CoQIO, including for instance lormuianons 01 sucn produced CoQIO for human or animal usage may be effected in accordance with methods well-known in the art. For use in animal health and •nutrition, however, no specific purification may be necessary. In this case the produced isoprenoids like CoQl 0 together with the biomass and/or other components of the fermentation broth may be further processed to yield a commercially attractive product.
The process of the present invention results in higher yields of isoprenoids, such as for instance CoQIO. The increase may be for instance at least about 10% compared to processes using a non-recombinant strain of for instance Rhodobacter or using a recombinant strain such as for instance Rhodobacter carrying the wild-type mev operon of e.g. aParacoccus strain.
Figure 1 represents the pathway for CoQIO biosynthesis in R. sphaeroides, which uses the MEP pathway for IPP formation. The boxed region indicates the reaction sequence that comprises the mevalonate pathway (leading to formation of IPP), plus the IPP isomerase step. The mevalonate pathway does not naturally occur in R. sphaeroides. In P. zeaxanthinifaciens, the genes coding for the five enzymes of the mevalonate pathway plus IPP isomerase form an operon, called hereinafter the mevalonate operon.
The following Examples illustrate the invention without restricting it in any way. Example 1: Bacteria and culture conditions
Rliodobacter sphaeroides strain ATCC 35053 (obtained from the American Type Culture Collection, Manassas VA, USA) was used as the base host for construction of recombinant strains having improved production of CoQIO. All R. sphaeroides strains were grown at 3-0°C in medium RSI 00. The composition and preparation of medium RSI 00 is summarized in Table I; Fifty iag/1 kanamycin was added to the me.dium.for, growth of recombinant strains. E. coli strains were grown at 37°C in LB medium (Becton Dickinson, Sparks, MD, USA). For maintenance of plasmids in recombinant E. coli strains, ampicillin (100 mg/1) and/or kanamycin (25-50 mg/1, depending on the plasmid) were added to the culture medium. Liquid cultures of E. coli and R. sphaeroides were routinely grown aerobically in a rotary shaker at 200 rpm. When solid media were required, agar (1.5% final concentration) was added.
Table 1. Composition and preparation of medium RSI 00
(Table Removed)

Example 2: Analytical assay for CoQIO
400 fj.1 of whole cultivation broth (see Example 5) were transferred to a disposable o mi polypropylene centrifuge tube. 4ml of stabilized extraction solution (0.5 g/1 BHT in 1:1 (v/v) DMSO/THF) were added and the samples were mixed for 20 min in a laboratory shaker (DCA, Germany) to enhance extraction. Finally, the samples were centrifuged and the supernatants transferred to amber glass vials for analysis by reverse phase HPLC. This method was developed for the simultaneous determination of ubiquinones and their corresponding hydroquinones, with a clear separation of CoQIO from the carotenoids phytoene, spheroidenone, spheroidene and neurosporene. Chromatography was performed using an Agilent 1100 HPLC system (Agilent Technologies, USA) equipped with a temperature-controlled autosampler and a diode array detector. The method parameters were as follows:

Column

YMC Carotenoid C30 column
3 micron, steel, 150 mm x 3.0 mm I.D.
(YMC, Part No. CT99S031503QT)

Guard column

Security Guard CIS (ODS, Octadecyl) 4 mm length x 3.0 mm I.D. (Phenomenex, Part No. AJO-4287)

Typical column pressure 60 bar at start
Flow rate 0.5 ml/min

Mobile phase Gradient profile

Mixture of acetonitrile(A):methanol(B):TBME(C)
%B 15 15
100
15
15
%A 60 60 00 . 60 60
25 25
25 25
Time (min) 0
13 20
22. , . 25

Post time Injection volume Column temperature Detection

4 min. 10 fil 15°C
Three wavelengths weie used for detection of specific compounds according to Table 2.

Table 2. HPLC retention times and wavelengths used

(Table Removed)
Calculations: Calculations were based on peak areas.
Example 3: Cloning of mutated mev operon from P. zeaxanthinifaciens
Construction of plasmids pBBR-K-7??ev-op-wt and pBBR-K-mgy-op-Rl 14.
The construction of plasmid pBBR-K-?«ev-op-up-4 (plasmid containing the first 4 genes of the mevalonate operon) is described in detail in Example 13 (page 105, line 10 to page 106, line 8) of WO 02/099095.
Using P. zeaxanthinifaciens Rl 14 genomic DNA as template, PCR is performed with primers hcs-5326 (SEQ ID NO: 10) and mvd-9000 (SEQ ID NO:11). Primer hcs-5326 corresponds to the sequence of the P. zeaxanthinifaciens mev operon from nucleotide 3321 to 3340 of SEQ ID NO:2 while primer mvd-9000 corresponds to the reverse complement of the sequence from nucleotides 6977 to 69J>6 of SEQ ID NO:2. The obtained PCR product of 3675 bp is cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA), resulting into plasmid TOPO-pCR2.1-/«ev-op-d-3wt. This plasmid thus contains the downstream half of the mevalonate operon including the 3' end of has and the last three genes, mvk, pmk and mvd.
Plasmids pBBR-K-7?zev-op-up-4 and TOPO-pCR2.1-7?zev-op-d-3wt are digested with the restriction endonucleases Sad and Ndel and the resulting 3319 bp fragment from TOPO-pCR2.1-??2ev-op-d-3wt is ligated with the 8027 bp fragment from pBBR-K-/?zev-op-up-4. The resulting plasmid, pBBR-K-?«ev-op-Rl 14, contains the complete mevalonate operon from P. zeaxanthinifaciens Rl 14 including its (putative) promoter.
Construction of plasmids pBBR-K-mev-op-wt-Pc?l£-crt.£ and pBBR-K-??iev-op-Rl 14-fcrtE-crtE
The construction of plasmid pBBR.-K-PcrtE-crtE is described in detail in Example 6 (page 92, lines 10-17) of WO 02/099095. Plasmid pBBR-K-Pcrt£-crt£ was cut with Noel and the 1.33 kb fragment was isolated and inserted into the Ecll36H site of pBBR-K-mev-op-up-4. The orientation of the insert was checked and the plasmid which carried the crtE gene in the same orientation as the mevalonate operon genes was designated pBBR-K-mev-op-up-4-'PcrtE-crtE-2.
Plasmid pBBR-K-mev-op-up-4-Pc7tE-c/-tE-2 was cut with Sphl and Spel and the resulting 5566 bp fragment containing the crtE gene was isolated. This fragment was ligated with the 7132 bp Sphl-Spel fragment obtained after a restriction digest of pBBR-K-/nev-op-wt or pBBR-K-mev-op-Rl 14 using the same enzymes, resulting in plasmid pBBR-K-jnev-op-vrt-PcrtE-crtE and pBBR-K-/nev-op-Rl 14-PcrtE-crtE, respectively.
Construction of plasmids pBBK-K.-mev~oT>-vft-PcrtE~ddsAwt and pBBR-K-mgv-op-Rl 14-VcrtE-ddsA,*
The construction of plasmid pBBR-K-Pcr/Ji is described in detail in Example 6 (page 91, lines 12-27) of WO 02/099095.
The ddsA gene from P. zeaxanthinifaciens strain ATCC 21588 (designated ddsAva) was amplified by PCR (GC-rich PCR System, Roche Molecular Biochemicals, Mannheim, Germany) using the primers dds-Nde (SEQ ID NO: 12) and dds-Bam (SEQ ID NO: 13) and cloned into pCR2.1-TOPO (hivitrogen, Carlsbad, CA, USA) to result in plasmid TOPO-
"Plasmid TQPO-dJiS^wt.-was,RUt,with. JVdel and BainHL and the obtained fragment of 1005 bp containing the ddsA gene was cloned into plasmid pBBR-K-Pcr/.E cut with Ndel and BairiHl, resulting in plasmid pBBR.-'K-PcrtE-ddsA^t. A recognition site for the endonuclease EcoKL within the ddsA gene was eliminated by mtroducing a silent mutation with the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) using the oligonucleotides dds-R-1 (SEQ ID NO: 14) and dds-R-2 (SEQ ID NO: 1 5). The resulting plasmid pBBR.-K-'PcrtE-ddsAwt-'R. was cut with EcoKL and Maml and the fragment 1278 bp fragment containing the ddsA gene was inserted into TOPO-ddsA^ cut with .EcoRI and EcdKV, resulting in the plasmid pCR2.1-TOPO-d&J4wt-R. This plasmid was cut with CelR and Xbal and the obtained fragment of 121 1 bp containing the ddsA gene was ligated with a 11,6 kb restriction fragment obtained from digestion of pBBR-K-mev-op-vfi-~?crtE-crtE andpBBR-K-mev-op-RlH-Pcr^-crz'E1, respectively, with CelR and
Blnl. The resulting plasmids were named pBBR-K-meri'-op-wt-'PcrtE-ddsAvn and pBBR-K-7?zev-op-Rl 14-PcrtE-ddsAvn, respectively.
Construction of plasmid
Plasmid pBBR-K.-m&>-op-4-&9-PcrtE-ddsAw is obtained by two rounds of PCR site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and primers mut4-89-l-fw (SEQ ID NO: 16) and mut4-89-1-rev (SEQ ID NO: 17). Both primers are complementary to each other and contain the desired mutation A instead of C at position 2949 of SEQ ID NO:3, corresponding to position 268 of SEQ ID NO:4. The first mutagenesis reaction is set up as follows according to the manufacturer's instructions: 5 jil lOx reaction buffer, 10 ng plasmid DNA pBBR-K-jnev-op-wt-PcrtE-J^wt, 125 ng primer mut4-89-l-fw, 125 ngmut4-89-l-rev, 1 /il dNTP mix, 3 /tl QuikSolution and 2.5 U PfuTurbo DNA polymerase are mixed in a final volume of 50 /zl. The cycling is carried out by using the following parameters: 1 cycle: 95°C for 1 min; 18 cycles: 95°C for 50 sec, 60°C forSO sec, 68°C for 30 min; 1 cycle: 68°C for 7 min. After cooling the reaction mix to 37°C, 10 U of the restriction endonuclease Dpnl is added and the reaction is incubated at 37°C for 2 hours. Escherichia coli XLlO-Gold Ulrracompetent cells (Stratagene, La Jolla, CA, USA) are transformed with the Z)pHl-treated DNA according to the manufacturer's protocol.
The resulting plasmid pBBR-K-7?iev-op-4-89-l-Pcr££'- Example 4: Introduction of the mutated mev operon into R. sphaeroides ATCC
35053
Transformation of Z coli S17-1 (Simon et al, Bio/Technology 11, 784-791, 1983) with plasmids carrying the mutated mev operon and subsequent transfer of plasmids from E.
coli S17-1 to R. sphaeroides ATCC 35053 by conjugation were performed using standard procedures (Nishimura et al, Nucl. Acids Res. 18, 6169, 1990; Simon et al., Bio/Technology 1983, 784-91).
A spontaneous rifampicin resistant mutant of R. sphaeroides ATCC 35053 was first isolated by growing strain ATCC 35053 in RSI 00 liquid medium supplemented with 100 mg/1 rifampicin, plating the cells on RSI 00 plates containing 100 mg/1 rifampicin, and isolating a single colony. For the conjugation, one milliliter aliquots of cultures of the recipient cells (rifampicin-resistant R. sphaeroides ATCC 35053) grown in RSI 00 medium containing 100 mg/1 rifampicin and the donor cells (E. coli SI 7-1 carrying the plasmid to be transferred, grown in LB broth containing 50 mg/1 kanamycin) were pelleted by centrifugation. The supernatant was discarded, and the cells were washed twice with fresh RS 1 00 medium to remove antibiotics. Each pellet was then resuspended in 1 ml of fresh RSI 00 medium. Fifty microliters of donor cells and 0.45 ml of recipient cells were mixed, pelleted by centrifugation, resuspended in 0.03 ml of fresh RSI 00 medium and spotted onto an RSI 00 plate. After overnight incubation at 30°C the cells were harvested with an inoculating loop and resuspended in 0.3 ml of RS100 medium. Dilutions of this suspension were spread onto RSI 00 plates containing 100 mg/1 rifampicin and 50 mg/1 kanamycin and incubated at 30°C. Colonies (putative transformed cells of R. sphaeroides ATCC 35053) were picked from the plates, grown in liquid RSI 00 medium containing 50 mg/1 kanamycin and the presence of the plasmid was tested in a PCR reaction with an annealing temperature of 56°C and an elongation time of 1 min, 15 sec using the following two different primer pairs:
pBBR-K-up (SEQ ID NO:20)/PcrtE-2442 (SEQ ID NO:21) KanSout (SEQ ID NO:22)/mvaA3256 (SEQ ID NO:23)
Positive, clones were, streaked onto. RSI 00 plates containing 50 mg/1 kanamycin to obtain single colonies. One single colony from each clone was again grown in liquid RSI 00 medium containing 50 mg/1 kanamycin, and the presence of the expected plasmid was confirmed by PCR as described above. The resulting recombinant strain was named
Example 5: Production of CoQIO in transformed strains of JZ. sphaeroides ATCC
35053
R. sphaeroides strains ATCC 35053, ATCC and ATCC 35Q53/p'BER.-K.-mev-op-4-89-PcrtE-ddsAWt were grown in shake flask cultures in RSI 00 medium. Cultures containing the recombinant R. sphaeroides contained 50 mg/1 kanamycin. Twenty five-milliliter cultures were grown at 30°C in 250-ml baffled
Erlenmeyer flasks with shaking at 200 rpm. For testing CoQIO production, frozen glycerolized stock cultures of the/?, sphaeroides strains were used to inoculate 25-ml seed cultures. After growth of the seed cultures for 24-28 hours, suitable volumes of the cultures were used to inoculate the experimental flasks such that the initial optical density at 660 nanometers (OD66o) was 0.16. Two milliliter samples were taken aseptically at 24 hour intervals. Analyses included growth (measured as ODggo), pH, glucose in the culture supernatant and CoQIO and carotenoids (determined by HPLC) as described in Example 2. The results are summarized in Table 3. These results clearly show that the expression of the cloned mutated mevalonate operon from P. zeaxanthinifaciens significantly improved CoQIO production inR. sphaeroides.
Table 3. Production of CoQIO in transformed R. sphaeroides ATCC 35053 strains
(Table Removed)

Specific Formation is expressed as mg/1 CoQIO produced/ODeeo-

Claims
1. A polynucleotide sequence comprising one or more gene(s) encoding a protein
having hydroxymethylglutaryl-CoA reductase activity, isopentenyl diphosphate isomerase
activity, hydroxymethylglutaryl-CoA synthase activity, mevalonate kinase activity,
phosphomevalonate kinase activity, and/or diphosphomevalonate decarboxylase activity,
wherein said polynucleotide sequence carries one or more mutation(s) leading to an
improved production of isoprenoids when present in a microorganism.
2. A polynucleotide sequence according to claim 1 which is obtainable from SEQ ID
NO: 1 or 2 or a fragment thereof and which carries one or more mutation(s).
3. A polynucleotide sequence according to claim 1 or 2 comprising one or more
mutation(s) in the hcs gene encoding a protein having hydroxymethylglutaryl-CoA
synthase activity.
4 A polynucleotide sequence according to any one of claims 1 to 3 which is represented by SEQ ID NO:3 or a fragment thereof.
5. A polynucleotide sequence according to any one of claims 1 to 4 selected from the group consisting of:
(a) polynucleotides encoding a polypeptide comprising the amino acid sequence
according the SEQ ID NO: 7,
(b) polynucleotides comprising the nucleotide sequence according to SEQ ID NO:6,
(c) polynucleotides comprising a nucleotide sequence encoding a fragment or derivative
of a pQlypeptide,,encoded,hy a polynucleotide of (a) or (b) wherein in said derivative one or
more amino acid residues are conservatively substituted compared to said polypeptide, and
said fragment or derivative has the activity of hydroxymethylglutaryl-CoA synthase;
(d) polynucleotides the complementary strand of which hybridizes under stringent
conditions to a polypeptide as defined in any one of (a) to (c) and which encodes a Hcs
protein.

6. Use of a polynucleotide according to any one of claims 1 to 5 for the production of
isoprenoids, preferably Co-enzyme Q10.
7. A process for the production of isoprenoids comprising:

(a) introducing a polynucleoti.de sequence according to any one of claims 1 to 5 into a
microorganism which is originally deficient of said polynucleotides, and
(b) cultivating the microorganism of step (a) under conditions that allow the production
ofisoprenoids.
8. The process according to claim 7 wherein the microorganism in which the
polynucleotide is introduced is selected from the genus Rhodobacter., preferably
Khodobacter sphaeroides.
9. The process according to claims 7 or 8 wherein the introduced polynucleotide is
ohtainable from a microorganism selected' from the genus Paracoccits, preferably P.
zeaxanthimfaciens.
10. A microorganism, comprising a polynucleotide sequence according to any one. of
claims 1 to 5. .
1 1 . The microorganism according to claim 1 0 which, is selected from the genus Khodobacter, preferably Rhodobacter sphaeroides.
12. The microorganism according to claims 10 01 1 1 wherein the polyrmclsotide
according to any one of claims 1 to 5 obtainable from a microorganism selected from the
genus Paracoccus is introduced,
13. Use of a microorganism according to :any one of claims 10 to 12 for the production
of isoprenoids. .

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=j2EXLo/RWIVroW6w5QEKKQ==&loc=+mN2fYxnTC4l0fUd8W4CAA==

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Patent Number

269673

Indian Patent Application Number

715/DELNP/2007

PG Journal Number

45/2015

Publication Date

06-Nov-2015

Grant Date

30-Oct-2015

Date of Filing

25-Jan-2007

Name of Patentee

DSM IP ASSETS B.V

Applicant Address

HET OVERLOON 1, NL-6411 TE HEERLEN, THE NETHERLANDS

Inventors:

#	Inventor's Name	Inventor's Address
1	BERRY, ALAN	5185 DUGUID ROAD, FAYERREVILLE, NEW YORK 13066, USA
2	MANHART, CHRISTIAN	ST. ALBAN RHEINWEG 164, CH-4052 BASEL, SWITZERLAND
3	SIMIC, PETRA	GUNDELDINGERSTRASSE 209, CH-4053 BASEL, SWITZERLAND

PCT International Classification Number

C12N 15/53

PCT International Application Number

PCT/EP2005/008702

PCT International Filing date

2005-08-11

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	04019646.1	2004-08-19	EUROPEAN UNION