US 20130224809A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0224809 A1 Bohlmann et al. (43) Pub. Date: Aug. 29, 2013 (54) DITERPENE SYNTHASES AND METHOD Publication Classification FOR PRODUCING DITERPENOIDS (51) Int. Cl. (71) Applicants: Joerg Bohlmann, Vancouver (CA); CI2N 9/88 (2006.01) Philipp Zerbe, North Vancouver (CA) (52) U.S. Cl. CPC ........................................ CI2N 9/88 (2013.01) (72) Inventors: Joerg Bohlmann, Vancouver (CA); USPC ....... 435/127: 536/23.2:435/232; 435/320.1; Philipp Zerbe, North Vancouver (CA) 435/252.3; 435/254.11: 435/419,435/348; 435/325; 435/252.33: 435/254.2:435/155 (21) Appl. No.: 13/694,350 (57) ABSTRACT Provided herein are diterpene synthases (diTPS) and methods (22) Filed: Nov. 21, 2012 for producing diterpenoids. Also provided herein are nucleic acid sequences encoding diTPS, diTPS amino acid Related U.S. Application Data sequences, diTPS proteins, vectors, cells, transgenic organ (60) Provisional application No. 61/562,280, filed on Nov. isms, uses, compositions, methods, processes, and kits 21, 2011. thereof. Patent Application Publication Aug. 29, 2013 Sheet 1 of 12 US 2013/0224809 A1 VIGRIQ?IH Patent Application Publication Aug. 29, 2013 Sheet 2 Of 12 US 2013/0224809 A1 Patent Application Publication Aug. 29, 2013 Sheet 3 of 12 US 2013/0224809 A1 Figure 2 neoabietadiere palustradiene 12 12.2 24 12.6 12.8 13 3.2 13.4 13.6 13.8 14 Time (min) B Abdi TPS4 product (8) 40 80 - 200 240 280 miz Patent Application Publication Aug. 29, 2013 Sheet 4 of 12 US 2013/0224809 A1 Figure 3 Abdi PS4 abietadiene Time min Patent Application Publication Aug. 29, 2013 Sheet 5 of 12 US 2013/0224809 A1 Figure 4 IcuTS Taxus Abdi TPS (ABLAS) AbdiTPS3 AbdiTPS2 (Abiso) AbdiTPS4 (AbCAS) GbLS Gingko 0.1 substitutions site (outgroup) Patent Application Publication Aug. 29, 2013 Sheet 6 of 12 US 2013/0224809 A1 Figure 5 2 ve o cry s ACAS2A classics PaaS-61A + ACAS2AA O “cauclassi ACAS.6A +ACASD2A4A *cGoldass) 8 10 time (min 12 14 s s s s s 4. 80 2 16 200 240 28 mz Patent Application Publication Aug. 29, 2013 Sheet 7 of 12 US 2013/0224809 A1 Patent Application Publication Aug. 29, 2013 Sheet 8 of 12 US 2013/0224809 A1 8. Patent Application Publication Aug. 29, 2013 Sheet 9 of 12 US 2013/0224809 A1 Patent Application Publication Aug. 29, 2013 Sheet 10 of 12 US 2013/0224809 A1 i. : ...& s Patent Application Publication Aug. 29, 2013 Sheet 11 of 12 US 2013/0224809 A1 e Patent Application Publication Aug. 29, 2013 Sheet 12 of 12 US 2013/0224809 A1 US 2013/0224809 A1 Aug. 29, 2013 DITERPENE SYNTHASES AND METHOD can be in vivo or in vitro. Also provided herein are methods FOR PRODUCING DITERPENOIDS for expression of an enzyme with diTPS activity in a host organism. RELATED APPLICATIONS 0008. Also provided herein are diTPS nucleic acid sequences, diTPS amino acid sequences, proteins, vectors, 0001 Benefit of priority is claimed to U.S. Provisional cells, transgenic organisms, uses, compositions, methods, Application Ser. No. 61/562,280, filed Nov. 21, 2011, entitled processes, and kits thereof. For example, a diterpene synthase “Diterpene Synthases and Method for Producing Diterpe (diTPS) polypeptide having the sequence set forth in SEQID noids.” The subject matter of the above-noted application is NO:1; SEQID NO:3, SEQIDNO:5, SEQIDNO:7, an active incorporated by reference in its entirety. fragment thereof, or sequence Substantially identical thereto 0002 This application is related to International PCT are provided. Furthermore, provided herein is a nucleic acid Application No. PCT/CA2012/050837, filed the same day encoding the polypeptide sequence set forth in SEQID NO:1; herewith, entitled "Diterpene Synthases and Method for Pro SEQID NO:3, SEQID NO: 5 or SEQID NO: 7. The nucleic ducing Diterpenoids,” which claims priority to U.S. Provi acid can have the sequence set forth in SEQIDNO: 2: SEQID sional Application Ser. No. 61/562,280. NO: 4, SEQID NO: 6, SEQID NO: 8 or a sequence substan 0003. The subject matter of each of the above-noted appli tially identical thereto. Also provided herein is a nucleic acid cations is incorporated by reference in its entirety. encoding the polypeptide sequence as set forth in SEQ ID NO: 7, an active fragment thereof, or sequence substantially INCORPORATION BY REFERENCE OF identical thereto, wherein the nucleotide at position 621 is SEQUENCE LISTING PROVIDED ON COMPACT replaced by A, the nucleotide at position 402 is replaced by A. DISCS the nucleotide at position 404 is replaced by A or a combina tion thereof. Also provided herein is a diterpene synthase with 0004 An electronic version on compact disc (CD-R) of an active site as disclosed in FIG. 6. the Sequence Listing is filed herewith in duplicate (labeled 0009 Provided herein is a method for producing diterpe Copy #1 Replacement and Copy #2 Replacement), the con noids that includes providing a host containing a nucleotide tents of which are incorporated by reference in their entirety. sequence encoding a diterpene synthase (diTPS), the nucle The computer-readable file on each of the aforementioned otide sequence operatively linked with a regulatory region compact discs, created on Feb. 27, 2013, is identical, 200 that is active in the host, and growing the host thereby kilobytes in size, and titled 234SEQ.002.txt. expressing the nucleotide sequence and producing diterpe noids. FIELD OF THE INVENTION 0010 Also provided herein is a method for producing one or more diterpenoids by introducing into a host capable of 0005 Provided herein are diterpene synthases (diTPS) producing (E.E.E)-geranylgeranyl diphosphate (GGPP) one and methods for producing diterpenoids. Also provided or more nucleotide sequence encoding a diterpene synthase herein are nucleic acid sequences encoding diTPS, diTPS (diTPS), the nucleotide sequence operatively linked with a amino acid sequences, diTPS proteins, vectors, cells, trans regulatory region that is active in the host, and growing the genic organisms, uses, compositions, methods, processes, host thereby expressing the nucleotide sequence and produc and kits thereof. ing diterpenoids. The diTPS can contain a bifunctional class If II dTPS. BACKGROUND 0011. Also provided is a method for producing one or 0006 Conifers produce a diverse array of diterpenoids as more diterpenoids by contacting (E.E.E)-geranylgeranyl major oleoresin components that play a role in the chemical diphosphate (GGPP) with at least one polypeptide having a defense against herbivores and pathogens, such as bark diterpene synthase (diTPS) activity under conditions effec beetles and their associated fungi (Trapp et al. (2001) Annu. tive to produce diterpenoids. Optionally, the diterpenoids pro Rev. Plant Physiol. Plant Mol. Biol., 52:689-724; Keeling et duced can be isolated. The diTPS can contain a bifunctional al. (2006) Phytochemistry, 67:2415-2423: Keeling et al. class I/II diTPS. (2006) New Phytol., 170:657-675). Oleoresin diterpenoids 0012. Also provided herein is a method for producing are also used as large-volume, renewable raw material for the diterpenoids by contacting (E.E.E)-geranylgeranyl diphos production of a Suite of industrial resins and coatings, and phate (GGPP) with at least one polypeptide having a diter other bioproducts (Bohlmann et al. (2008) Plant J., 54.656 pene synthase (diTPS) activity under conditions effective to 669; Hillwig et al. (2011) Biopolymer, 95:71-76). There is a produce diterpenoids and optionally, isolating the produced need for alternative methods for producing diterpenoids. diterpenoids. 0013. In one example, provided herein is an isolated SUMMARY nucleic acid molecule containing a sequence of nucleotides encoding a bifunctional class I/II cis-abienol synthase (CAS) 0007 Provided herein are methods for producing diterpe polypeptide or an active fragment, wherein the encoded noids. Also provided herein are nucleic acids encoding diter polypeptide or active fragment comprises a class II active site pene synthase (diTPS), diTPS amino acid sequences, and comprising a DxDD motif and a class I active site comprising vectors, hosts containing diTPS and that can be used to pro a DDXXD motif, and the encoded polypeptide or active frag duce diterpenoids. More specifically, the methods provided ment catalyzes the formation of cis-abienol from gera herein relate to the production of bicyclic tertiary diterpene nylgeranyl diphosphate (GGPP). For example, the isolated alcohols such as for example cis-abienol and the production nucleic acid molecule has a sequence of nucleotides encoding of diterpene olefins such as for example abietadiene, levopi a cis-abienol synthase (CAS) polypeptide set forth in SEQID maradiene, palustradiene or neoabietadiene. The production NO:7, an active fragment thereof, or a sequence that exhibits US 2013/0224809 A1 Aug. 29, 2013 at least 75% sequence identity to SEQID NO:7, wherein the tional class I/II synthase is a cis-abienol synthase (CAS) encoded polypeptide or active fragment catalyzes the forma polypeptide or active fragment that catalyzes the formation of tion of cis-abienol from geranylgeranyldiphosphate (GGPP). cis-abienol from geranylgeranyl diphosphate (GGPP). The sequence of nucleotides that encodes a CAS polypeptide 0018 For example, in the above provided methods, the exhibits at least 80%, 85%, 90%, 95% or 98% sequence bifunctional class I/II synthase is a CAS polypeptide or active identity to SEQID NO:7. In some examples, the sequence of fragment that contains the sequence of amino acids set forth nucleotides encodes a polypeptide that has the sequence of in SEQID NO:7, an active fragment thereof, or a sequence amino acids set forth in SEQID NO:7 or an active fragment that exhibits at least 75% sequence identity to SEQID NO:7, thereof. In other examples, the sequence of nucleotides wherein the encoded polypeptide or active fragment catalyzes encodes a polypeptide that is the sequence of amino acids set the formation of cis-abienol from geranylgeranyl diphos forth in SEQID NO:7 or an active fragment thereof.