US 20070077616A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0077616 A1 KEASLING et al. (43) Pub. Date: Apr. 5, 2007 (54) BIOSYNTHESIS OF ISOPENTENYL Publication Classification PYROPHOSPHATE (51) Int. Cl. CI2P 33/00 (2006.01) (76) Inventors: Jay KEASLING, Berkeley, CA (US); CI2P 17/00 (2006.01) Vincent Martin, Montreal (CA); CI2P 9/00 (2006.01) Douglas Pitera, Oakland, CA (US); CI2P 7/04 (2006.01) Seon-Won Kim, Jeongdon-myeon CI2P 5/00 (2006.01) (KR); Sydnor T. Withers III, CI2P 5/02 (2006.01) Richmond, CA (US); Yasuo Yoshikuni, CI2N 9/10 (2006.01) Berkeley, CA (US); Jack Newman, San CI2N I/2 (2006.01) Francisco, CA (US); Artem C7H 2L/04 (2006.01) Valentinovich Khlebnikov, Mountain CI2N 15/74 (2006.01) View, CA (US) (52) U.S. Cl. ............................ 435/52; 435/117; 435/131; 435/157; 435/166; 435/167; 435/252.3; 435/193; 435/471; Correspondence Address: 536/23.2; 435/252.33 BOZICEVIC, FIELD & FRANCIS LLP 1900 UNIVERSITY AVENUE (57) ABSTRACT SUTE 200 EAST PALO ALTO, CA 94.303 (US) Methods for synthesizing isopentenyl pyrophosphate are provided. A first method comprises introducing into a host microorganism a plurality of heterologous nucleic acid (21) Appl. No.: 11/610,686 sequences, each coding for a different enzyme in the meva lonate pathway for producing isopentenyl pyrophosphate. A related method comprises introducing into a host microor (22) Filed: Dec. 14, 2006 ganism an intermediate in the mevalonate pathway and at least one heterologous nucleic acid sequence, each sequence coding for an enzyme in the mevalonate pathway necessary Related U.S. Application Data for converting the intermediate into isopentenyl pyrophos phate. The invention also provides nucleic acid sequences, (62) Division of application No. 10/006,909, filed on Dec. enzymes, expression vectors, and transformed host cells for 6, 2001, now Pat. No. 7,172,886. carrying out the methods. Patent Application Publication Apr. 5, 2007 Sheet 1 of 5 US 2007/007761.6 A1 MEVALONATE PATHWAY AND PREFERRED ENZYMES AND SEQUENCES FOR PRODUCING ISOPENTENYL PYROPHOSPHATE STEP PREFERRED PREFERRED ENZYME SEQUENCE O O 2- -- ?' - Acetyl-CoA Acetyl-CoA Acetoacetyl-CoA thiolase SEQ ID NO 1 //C O . Acetoacetyl-CoA HMG-CoA synthase SEQ ID NO2 C O HC JXOl -CoA HMG-CoA Truncated HMG-CoA reductase SEQID NO 3 O HC OH Mevalonate FIG 1A Patent Application Publication Apr. 5, 2007 Sheet 2 of 5 US 2007/007761.6 A1 MEVALONATE PATHWAY AND PREFERRED ENZYMES AND SEQUENCES FOR PRODUCING ISOPENTENYL PYROPHOSPHATE (CONTINUED) STEP PREFERRED PREFERRED ENZYME SEQUENCE O - O OH Mewalionate Mevalonate kinase SEQ ID NO 4 O SpH JXC CH HO e--- O Mevalonate 5-phosphate Phosphomevalonate kinase SEQ ID NO 5 O Sso OH OH HO e---- C| O| Mevalonate 5-pyrophosphate Mevalonate pyrophosphate decarboxylase SEQID NO 6 C H ----- O O Isopentenyl pyrophosphate FIG 1B Patent Application Publication Apr. 5, 2007 Sheet 3 of 5 US 2007/007761.6 A1 12 ETE.coli DH.10B pBBRIMCS-3, E. E.coli DPDXR1 pBBRMBI-2, |||||||||||||||||||||||||||||||||||||||||| E H O mM nM 10 mM. 20 nM 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||nM Mew Mew Mew Mew Mew FIG. 2 pAC -LYC only pBADMEVT, pBBRMBI-2, p.AC LYC FIG. 3 Patent Application Publication Apr. 5, 2007 Sheet 4 of 5 US 2007/007761.6 A1 2. 5 2 5 0. 5 O pTRCADS only pBBRMBI-2 & pTRCADS FIG. 4 Patent Application Publication Apr. 5, 2007 Sheet 5 of 5 US 2007/007761.6 A1 8000 7000 5 O 5 20 25 Retention Time (min) FIG. 5 250000 200000 SOCOC Casbene (Ricinus communis L.) retention time at 16.60 0000 5000 mtz F.G. 6 US 2007/007761.6 A1 Apr. 5, 2007 BIOSYNTHESIS OF SOPENTENYL increase isopentenyl pyrophosphate production. Croteau et PYROPHOSPHATE al. describe in U.S. Pat. No. 6,190,895 the nucleic acid sequences that code for the expression of 1-deoxyxylulose TECHNICAL FIELD 5-phosphate synthase, an enzyme used in one biological 0001. The present invention relates to the biosynthesis of pathway for the synthesis of isopentenyl pyrophosphate. isopentenyl pyrophosphate (IPP) and isoprenoids derived Low yields of isopentenyl pyrophosphate remain, however, therefrom. More particularly, the invention relates to meth since several more enzymes are needed to catalyze other ods for biosynthesizing isopentenyl pyrophosphate, and to steps in this isopentenyl pyrophosphate biosynthetic path way. Further, the reference does not address an alternative nucleic acid sequences, enzymes, expression vectors, and pathway for isopentenyl pyrophosphate biosynthesis, transformed host cells for carrying out the methods. namely the mevalonate pathway. BACKGROUND 0006 Thus, the current invention is directed toward Solving these and other disadvantages in the art by increas 0002) Isoprenoids are compounds derived from the five ing the typically low yields associated with conventional carbon molecule, isopentenyl pyrophosphate. Investigators synthesis of isopentenyl pyrophosphate and isoprenoids. have identified over 29,000 individual isoprenoid com Specifically, the current invention is directed toward iden pounds, with new ones continuously being discovered. Iso tification of new methods for the synthesis of isopentenyl prenoids are often isolated from natural products, such as pyrophosphate, as isopentenyl pyrophosphate represents the plants and microorganisms, which use isopentenyl pyro universal precursor to isoprenoid synthesis. phosphate as a basic building block to form relatively complex structures. Vital to living organisms, isoprenoids SUMMARY OF THE INVENTION serve to maintain cellular fluidity and electron transport, as 0007 Accordingly, it is an object of the present invention well as function as natural pesticides, to name just a few of to overcome the above-mentioned disadvantages of the prior their roles in vivo. Furthermore, the pharmaceutical and art by providing a method for synthesizing isopentenyl chemical communities use isoprenoids as pharmaceuticals, pyrophosphate in a host microorganism, comprising the step nutriceuticals, flavoring agents, and agricultural pest control of introducing into the host microorganism a plurality of agents. Given their importance in biological systems and heterologous nucleic acid sequences, each coding for a usefulness in a broad range of applications, isoprenoids have different enzyme in the mevalonate pathway for producing been the focus of much attention by scientists. isopentenyl pyrophosphate. 0003 Conventional means for obtaining isoprenoids include extraction from biological materials (e.g., plants, 0008. It is another object of the invention to provide such microbes, and animals) and partial or total organic synthesis a method wherein the plurality of heterologous nucleic acid in the laboratory. Such means, however, have generally sequences is contained in at least one extrachromosomal proven to be unsatisfactory. For example, organic synthesis expression vector. is usually complex since several steps are required to obtain 0009. It is still another object of the invention to provide the desired product. Furthermore, these steps often involve Such a method wherein the isopentenyl pyrophosphate is the use of toxic solvents, which require special handling and further synthesized into an isoprenoid. disposal. Extraction ofisoprenoids from biological materials may also require toxic solvents. In addition, extraction and 0010. It is yet another object of the invention to provide purification methods usually provide a low yield of the such a method wherein the isoprenoid is selected from the desired isoprenoid, as biological materials typically contain group consisting of a monoterpene, sesquiterpene, diterpene, only small quantities of these compounds. Unfortunately, the sesterterpene, triterpene, tetraterpene, and a steroid. difficulty involved in obtaining relatively large amounts of 0011. It is a further object of the invention to provide such isoprenoids has limited their practical use. In fact, the lack a method wherein the plurality of heterologous nucleic acid of readily available methods by which to obtain certain sequences further comprises a DNA fragment coding for an isoprenoids has slowed down the progression of drug can enzyme capable of converting isopentenyl pyrophosphate to didates through clinical trials. Furthermore, once an iso dimethylallyl pyrophosphate. prenoid drug candidate has passed the usual regulatory scrutiny, the actual synthesis of the isoprenoid drug may not 0012. It is still a further object of the invention to provide lend itself to a commercial scale. a method wherein the host microorganism is a prokaryote. 0004 As a solution to such problems, researchers have 0013. It is an additional object of the invention to provide looked to biosynthetic production of isoprenoids. Some a method wherein the prokaryote is Escherichia coli. Success has been obtained in the identification and cloning 0014 Is it still another object of the invention to provide of the genes involved in isoprenoid biosynthesis. For a method for synthesizing isopentenyl pyrophosphate in a example, U.S. Pat. No. 6.291,745 to Meyer et al. describes host microorganism, wherein the method comprises intro the production of limonene and other metabolites in plants. ducing into the host microorganism an intermediate in the Although many of the genes involved in isoprenoid biosyn mevalonate pathway and at least one heterologous nucleic thesis may be expressed in functional form in Escherichia acid sequence, each said sequence coding for an enzyme in coli and other microorganisms, yields remain