PRODUCTION OF TERPENES AND TERPENOIDS

Abstract

The present invention relates to a nucleic acid construct comprising a nucleic acid molecule encoding a protein involved in the biosynthesis of a terpenoid or a precursor thereof, wherein said nucleic acid molecule is operably linked to a derepressible promoter.

Claims

1. A nucleic acid construct comprising a nucleic acid molecule encoding a protein involved in the biosynthesis of a terpenoid or a precursor thereof, wherein said nucleic acid molecule is operably linked to a derepressible promoter.

2. The nucleic acid construct according to claim 1, wherein the protein involved in the biosynthesis of a terpenoid or a precursor thereof is selected from the group consisting of geranylgeranyl diphosphate synthases or taxadiene synthases.

3. The nucleic acid construct according to claim 1, wherein the derpressible promoter is selected from the group consisting of CAT1 promoter, FDH1 promoter, FLD1 promoter, PEX5 promoter, DAK1 promoter, FGH1 promoter, GTH1 promoter, G1 promoter, G2 promoter, G3 promoter, G4 promoter, G5 promoter, G6 promoter, FMD promoter and a functional variant thereof.

4. The nucleic acid construct according to claim 1, wherein the promoter is an orthologous promoter.

5. The nucleic acid construct according to claim 1, wherein the derepressible promoter is linked to a second promoter forming a bidirectional promoter or a bidirectional derepressible promoter.

6. The nucleic acid construct according to claim 5, wherein the second promoter is a constitutive, derepressible or inducible promoter.

7. The nucleic acid construct according to claim 6, wherein the constitutive promoter is selected from the group consisting of a GAP promoter, PGCW14 promoter, TEF1 promoter, TPI promoter, PGK1 promoter or a histone promoter.

8. The nucleic acid construct according to claim 6, wherein the inducible promoter is selected from the group consisting of a AOX1 promoter, promoters of the methanol utilization (MUT) pathway, AOX2, DAS1, DAS2, FLD1, GTH1, PEX8 and PHO89/NSP.

9. The nucleic acid construct according to claim 5, wherein the bidirectional promoter comprises a combination of a GAP promoter, a CAT1 promoter, a PGCW14 promoter, a TEF1 promoter, a TPI promoter, a PGK1 promoter or a histone promoter, a promoter of the methanol utilization (MUT) pathway, a FDH1 promoter, a FLD1 promoter, a PEX5 promoter, a DAK1 promoter, a FGH1 promoter, a GTH1 promoter, a G1 promoter, a G2 promoter, a G3 promoter, a G4 promoter, a G5 promoter, a G6 promoter or a FMD promoter.

10. The nucleic acid construct according to claim 9, wherein the promoter of the methanol utilization (MUT) pathway is selected from the group consisting of an AOX1 promoter, an AOX2 promoter, a DAS1 promoter, a DAS2 promoter, a FLD1 promoter, a GTH1 promoter, a PEX8 promoter or a PHO89/NSP promoter.

11. The nucleic acid construct according to claim 5, wherein the second promoter is operably linked to a second nucleic acid molecule encoding a second protein involved in the biosynthesis of a terpenoid or a precursor thereof.

12. The nucleic acid construct according to claim 3, wherein the derepressible promoter is operably linked to a nucleic acid molecule encoding for a geranylgeranyl diphosphate synthase.

13. The nucleic acid construct according to claim 3, wherein the CAT1 promoter is operably linked to a nucleic acid molecule encoding for a geranylgeranyl diphosphate synthase.

14. The nucleic acid construct according to claim 1, wherein the nucleic acid molecule encoding the protein involved in the biosynthesis of a terpenoid or a precursor thereof comprises a terminator sequence at its 3 end.

15. A vector comprising a nucleic acid construct according to claim 1.

16. A host cell comprising a nucleic acid construct according to claim 1.

17. The host cell according to claim 16, wherein said host cell is a yeast cell.

18. The host cell according to claim 16, wherein said host cell is a methylotrophic yeast cell.

19. The host cell according to claim 16, wherein the methylotrophic yeast cell is selected from the group of Pichia pastoris, Hansenula polymorpha, Candida boidinii, Komagataella pastoris, Komagataella phaffii, Komagataella populi, Komagataella pseudopastoris, Komagataella ulmi and Komagataella sp. 11-1192.

20. A method for producing a terpenoid or a precursor thereof comprising the step of cultivating a host cell according to claim 16.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0029] FIG. 1 shows a nucleic acid construct according to the present invention wherein a bidirectional promoter (BDP) is inserted between GGPPS and TDS (see FIG. 1). The arrows indicate the orientation of the promoters of the BDP and the transcription direction of GGPPS and TDS.

[0030] FIG. 2 shows nucleotide sequences of bidirectional promoters.

[0031] FIG. 3 shows taxadiene (first dedicated precursor of taxol) production in P. pastoris using bidirectional promoters. The graph shows that there is a 50-fold difference in taxadiene yields depending on the promoter used. The indicated BDPs were cloned between the enzymes TDS and GGPPS and transformed into P. pastoris. The strains were cultivated in shake flasks with a dodecane overlay and induced with methanol as described in the example. Taxadiene yields were determined by GC-MS. Mean values and standard deviation of biological triplicates shown.

[0032] FIG. 4 shows the production of taxadiene under different cultivation conditions using a P. pastoris strain harboring TDS-pGAP|pCAT1-GGPPS. The use of a cultivation medium comprising 3% glycerol resulted in the production of up to 9.4 mg/l taxadiene. The strains were cultivated for 60 h on the glycerol concentrations indicated.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention relates to a nucleic acid construct comprising a nucleic acid molecule encoding a protein involved in the biosynthesis of a terpenoid or a precursor thereof, wherein said nucleic acid molecule is operably linked to a derepressible promoter.

[0034] It turned surprisingly out that polypeptides and proteins involved in the biosynthesis of a terpenoid or a precursor thereof show high enzymatic activity if these polypeptides and proteins are expressed in a host cell using a derepressible promoter. In contrast thereto, the expression of these polypeptides and proteins using solely inducible or constitutive promoters operably linked to the respective nucleic acid molecules resulted in a significantly lower enzymatic activity, whereby this enzymatic activity is determined by measuring the production of the terpenoid or a precursor thereof.

[0035] Nucleic acid construct, as used herein, refers to any nucleic acid molecule such as cDNA, genomic DNA, synthetic DNA, semi synthetic DNA and RNA.

[0036] A protein involved in the biosynthesis of a terpenoid or a precursor thereof, as used herein, refers to proteins and polypeptides which are part of the biosynthetic pathways leading to terpenoids or precursors of the final compound. These proteins are either enzymatically active or influence directly the activity of enzymes involved in these pathways.

[0037] Terpenoids, as used herein, refers to a large and diverse class of organic molecules derived from five-carbon isoprenoid units assembled and modified in a variety of ways and classified in groups based on the number of isoprenoid units used in group members. The term terpenoids includes therefore also hemiterpenoids, monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, triterpenoids, tetraterpenoids and polyterpenoids.

[0038] The term terpenoid precursor refers to any molecule that is used by organisms in the biosynthesis of terpenoids. Terpenoid precursor molecules can be any isoprenoid substrate molecule of terpene synthases such as peranylpyrophosphate, farnesylpyrophosphate or geranylgeranylpyrophosphate, and/or initial products made by terpene synthases such as amorphadiene, taxadiene, hopene, limonene (Degenhardt J et al. Phytochemistry 70(2009):1621-37).

[0039] Operably linked, as used herein, means that the promoter of the present invention is fused to nucleic acid molecule encoding a protein involved in the biosynthesis of a terpenoid or a precursor thereof to be able to regulate and influence the transcription of said nucleic acid molecule into RNA which thereafter is translated into the protein involved in the biosynthesis of a terpenoid or a precursor thereof.

[0040] As used herein, the term promoter refers to a nucleic acid sequence that is generally located upstream of a gene (i.e., towards the 5 end of a gene) and is necessary to initiate and drive transcription of the gene. A promoter may permit proper activation or repression of a gene that it controls. A promoter includes a core promoter, which is the minimal portion of the promoter required to properly initiate transcription and can also include regulatory elements such as transcription factor binding sites. The regulatory elements may promote transcription or inhibit transcription. Regulatory elements in the promoter can be binding sites for transcriptional activators or transcriptional repressors. A promoter can be constitutive, inducible or derepressible. The promoters of the present invention are preferably operable in yeast cells, in particular in methylotrophic yeast cells such as Pichia pastoris. These promoters are therefore preferably derived/obtained/isolated from yeast cells, in particular in methylotrophic yeast cells such as Pichia pastoris or are viral promoters which are functional in yeasts or synthetic promoters active in yeasts.

[0041] A constitutive promoter refers to one that is always active and/or constantly directs transcription of a gene above a basal level of transcription.

[0042] An inducible promoter is one which is capable of being induced by a molecule or a factor added to the cell or expressed in the cell. An inducible promoter may still produce a basal level of transcription in the absence of induction, but induction typically leads to significantly more production of the protein.

[0043] A derepressible promoter, as used herein, refers to a promoter that is substantially less active in prescence of a repressing compound. By changing the environment, repression is alleviated from the derepressible promoter and transcription rate increases. For instance, for some derepressible promoters glucose or glycerol can be used. Such promoters are repressed in the presence of glucose or glycerol and start expression once glucose or glycerol in the media is depleted.

[0044] According to a preferred embodiment of the present invention the protein involved in the biosynthesis of a terpenoid or a precursor thereof is selected from the group consisting of geranylgeranyl diphosphate synthases (GGPPS) and taxadiene synthases (TDS).

[0045] The protein involved in the biosynthesis of a terpenoid or a precursor thereof is particularly preferred geranylgeranyl diphosphate synthase (GGPPS).

[0046] According to a further preferred embodiment of the present invention the derepressible promoter is selected from the group consisting of CAT1 promoter, FDH1 promoter, FLD1 promoter, PEX5 promoter, DAK1 promoter, FGH1 promoter, GTH1 promoter, G1 promoter, G2 promoter, G3 promoter, G4 promoter, G5 promoter, G6 promoter, FMD promoter and a functional variant thereof, whereby a CAT1 promoter is particularly preferred.

[0047] A functional variant of a promoter, as used herein, refers to a promoter or a functional fragment thereof containing changes in relation to the wild-type promoter sequence which affect one or more nucleotides of the sequence. These nucleotides may be deleted, added and/or substituted, while maintaining at least substantially promoter function. The promoter function of functional promoter variants or fragments can be tested by operably linking a promoter variant or fragment to a nucleic acid molecule encoding a protein and evaluation the expression rate of the expressed protein or the transcription rate. Variant promoters can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant promoter or a portion thereof.

[0048] Functional variants of promoters are at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, identical to the wild-type promoter sequence.

[0049] Identical, as used herein, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms. It is particularly preferred to use BLAST and BLAST 2.0 algorithms (see e.g. Altschul et al. J. MoI. Biol. 215(1990): 403-410 and Altschul et al. Nucleic Acids Res. 25(1977): 3389-3402) using standard or default parameters. For amino acid sequences, the BLASTP program (see http://blast.ncbi.nlm.nih.gov/Blast.cgi) uses as defaults a wordlength (W) of 6, an expectation (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89(1989):10915) using Gap Costs Existance:11 Extension:1.

[0050] Functional variants of promoters include also functional fragments of promoters. The functional fragments of the promoters of the present invention retain at least substantially the promoter function of the entire promoter from which they are derived from. A functional fragment of a promoter may comprise at least 30%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, of the length of the entire promoter. A functional fragment of a promoter may comprise at least 100 consecutive bp, preferably at least 150 consecutive bp, more preferably at least 200 consecutive bp, more preferably at least 300 consecutive bp, more preferably at least 400 consecutive bp, more preferably at least 500 consecutive bp, of a wild type promoter.

[0051] The CAT1 promoter preferably comprises or consists of 100 to 500, 200 to 500, 300 to 500, 400 to 500 or 500 consecutive nucleotides of following nucleic acid sequence (see Vogl T et al., ACS Synth. Biol. 5(2016):172-186) (SEQ ID No. 1):

TABLE-US-00001 TAATCGAACTCCGAATGCGGTTCTCCTGTAACCTTAATTGTAGCATAGAT CACTTAAATAAACTCATGGCCTGACATCTGTACACGTTCTTATTGGTCTT TTAGCAATCTTGAAGTCTTTCTATTGTTCCGGTCGGCATTACCTAATAAA TTCGAATCGAGATTGCTAGTACCTGATATCATATGAAGTAATCATCACAT GCAAGTTCCATGATACCCTCTACTAATGGAATTGAACAAAGTTTAAGCTT CTCGCACGAGACCGAATCCATACTATGCACCCCTCAAAGTTGGGATTAGT CAGGAAAGCTGAGCAATTAACTTCCCTCGATTGGCCTGGACTTTTCGCTT AGCCTGCCGCAATCGGTAAGTTTCATTATCCCAGCGGGGTGATAGCCTCT GTTGCTCATCAGGCCAAAATCATATATAAGCTGTAGACCCAGCACTTCAA TTACTTGAAATTCACCATAACACTTGCTCTAGTCAAGACTTACAATTAAA

[0052] The FDH1 promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 2):

TABLE-US-00002 tagatggttatcttgaatggtatttgtaaggattgatctcgaaggttgta tatagtcgtgccgtgcaagtggaggagaatgaaagaagatgtaagaattc tggcccttgcacctgatcgcgaaggtggaaatggcagaaggatcagcctg gacgaagcaaccagttccaactgctaagtaaagaagatgctagacgaagg agacttcagaggtgaaaagtttgcaagaagagagctgcgggaaataaatt ttcaatttaaggacttgagtgcgtccatattcgtgtacgtgtccaactgt tttccattacctaagaaaaacataaagattaaaaagataaacccaatcgg gaaactttagcgtgccgtttcggattccgaaaaacttttggagcgccaga tgactatggaaagaggagtgtaccaaaatggcaagtcgggggctactcac cggatagccaatacattctctaggaaccagggatgaatccaggtttttgt tgtcacggtaggtcaagcattcacttcttaggaatatctcgttgaaagct acttgaaatcccattgggtgcggaaccagcttctaattaaatagttcgat gatgttctctaagtgggactctacggctcaaacttctacacagcatcatc ttagtagtcccttcccaaaacaccattctaggtttcggaacgtaacgaaa caatgttcctctcttcacattgggccgttactctagccttccgaagaacc aataaaagggaccggctgaaacgggtgtggaaactcctgtccagtttatg gcaaaggctacagaaatcccaatcttgtcgggatgttgctcctcccaaac gccatattgtactgcagttggtgcgcattttagggaaaatttaccccaga tgtcctgattttcgagggctacccccaactccctgtgcttatacttagtc taattctattcagtgtgctgacctacacgtaatgatgtcgtaacccagtt aaatggccgaaaaactatttaagtaagtttatttctcctccagatgagac tctccttcttttctccgctagttatcaaactataaacctattttacctca aatacctccaacatcacccacttaaaca

[0053] The FLD1 promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 3):

TABLE-US-00003 tgtgaatatcaagaattgtatgaacaagcaaagttggagctttgagcgat gtatttatatgagtagtgaaatcctgattgcgatcaggtaaggctctaaa aatcgatgatggtcccgaattctttgataggctaaggacttcctcatcgg gcagttcgaaggaagaaggggcatgagccctgcgaaaccatatgaggaag ggagatagaagcagaagattatccttcgggagcaagtctttccagcccgc atcttgtgattggatgatagttttaactaaggaaagagtgcgacatccgt tgtgtagtaatcatgcatacgtctattattctctctagttacccaactct gttatctcactaattcatggaatgccctccaggtagatactacaacgatt caatagtactgcaacacacagatgagattagtttagtttcccataatgag aattcagagtacaagaacaatctagtagccataagcaaggttcaccctct cctgtttttatcctataggcggcatatccagatatatcgactacctcagc tccgttggataactaccattagcaccgtgccagagattcctgca

[0054] The PEX5 promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 4):

TABLE-US-00004 tccaaaccaaacggtctagcaaaaacgataactttaaagaacttttcaat tggttttgtacactaccaccggtttactacctctgccttcggttcttctc ctcacatttttcgcaactgggatagcgtagcctaaagtgtcacatgctcg ctgctcacattccctacacaacagagattgtcagcagaggaaattgagct ccaccattcaacacttgtggatttatgatagtctgtgctatcagctctct tttttttgttgctgtagaatttaccgtgctagcaaccttttaaactttgt ttagctctccttccctcttccattcatctgtttcggtccgatccgtctct ggtcatctcctccgcattttttttttaccgttagcgataggggtcagatc aattcaatcagttttggcaagggtatttaaaggtggcgaaatccccctcc gtttgttgaacacatccaactattctcaacccaaccatctaactaatcgt a

[0055] The DAK1 promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 5):

TABLE-US-00005 tgtcatctgctgatgctgtgagggagaaagaagtaggggtgatacatggt ttataggcaaagcatgtttgtttcagatcaaagattagcgtttcaaagtt gtggaaaagtgaccatgcaacaatatgcaacacattcggattatctgata agtttcaaagctactaagtaagcccgtttcaagtctccagaccgacatct gccatccagtgattttcttagtcctgaaaaatacgatgtgtaaacataaa ccacaaagatcggcctccgaggttgaacccttacgaaagagacatctggt agcgccaatgccaaaaaaaaatcacaccagaaggacaattcccttccccc ccagcccattaaagcttaccatttcctattccaatacgttccatagaggg catcgctcggctcattttcgcgtgggtcatactagagcggctagctagtc ggctgtttgagctctctaatcgaggggtaaggatgtctaatatgtcataa tggctcactatataaagaacccgcttgctcaaccttcgactcctttcccg atcctttgcttgttgcttcttcttttataacaggaaacaaaggaatttat acactttaa

[0056] The FGH1 promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 6):

TABLE-US-00006 atgtcatcaattactacttcaatcttcaaggtaacagctgaaatccaaag ttttgggggaaagctagtcaaacttcaacacaagtccgatgagacgaaga ctgacatggatgtgaacgtctaccttccagctcaattctttgccaatgga gccaagggaaaatcattaccagttctactttatttgagtggtctgacttg cactcccaacaatgcctcagagaaggcattttggcaaccatatgcaaata agtacggttttgctgtggttttcccggatacttcacccagagggctcaac atcgaaggagagcacgactcttatgattttggatccggtgccgggttcta cgtggatgccactactgagaaatggaaggataattatagaatgtacagtt atgttaactcggaattgctacccaaattgcaggctgacttcccaattcta aactttgacaatatttcaatcacgggccactccatgggaggttacggagc tttacagttattcttgagaaacccgggaaaattcaagtcggtttccgcat tttctccaatctccaaccccactaaagccccatggggtgagaagtgcttc tctggatacctgggacaggacaagtccacttggactcagtacgacccaac cgaattgattggaaaataccaaggcccctcagattccagcattttgattc acgttggaaagagtgattcgttctacttcaaggaccaccagctgctacct gagaacttcttgaaggcttcagagaactctgtgttcaagggaaaagtgga cttgaacttggtagatggctatgaccattcttactactttatctcttcat tcacagacgttcatgctgctcaccatgcaaagtatttggggttaaactag

[0057] The G1 (GTH1) promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 7):

TABLE-US-00007 ccccaaacatttgctccccctagtctccagggaaatgtaaaatatactgc taatagaaaacagtaagacgctcagttgtcaggataattacgttcgactg tagtaaaacaggaatctgtattgttagaaagaacgagagttttttacggc gccgccatattgggccgtgtgaaaacagcttgaaaccccactactttcaa aggttctgttgctatacacgaaccatgtttaaccaacctcgcttttgact tgactgaagtcatcggttaacaatcaagtaccctagtctgtctgaatgct cctttccatattcagtaggtgtttcttgcacttttgcatgcactgcggaa gaattagccaatagcgcgtttcatatgcgcttttaccccctcttttgtca agcgcaaaatgcctgtaagatttggtgggggtgtgagccgttagctgaag tacaacaggctaattccctgaaaaaactgcagatagacttcaagatctca gggattcccactatttggtattctgatatgtttttcctgatatgcatcaa aactctaatctaaaacctgaatctccgctatttttttttttttttgatga ccccgttttcgtgacaaattaatttccaacggggtcttgtccggataaga gaattttgtttgattatccgttcggataaatggacgcctgctccatattt ttccggttattaccccacctggaagtgcccagaattttccggggattacg gataatacggtggtctggattaattaatacgccaagtcttacattttgtt gcagtctcgtgcgagtatgtgcaataataaacaagatgagccaatttatt ggattagttgcagcttgaccccgccatagctaggcatagccaagtgctat gggtgttagatgatgcacttggatgcagtgagttttggagtataaaagat ccttaaaattccaccctt

[0058] The G3 promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 8):

TABLE-US-00008 cagcaatccagtaaccttttctgaatagcagagccttaactaaaataatg gccagggtaaaaaattcgaaatttgacaccaaaaataaagacttgtcgtt ataagtcttaacaaagtccgcaattttggagctaacggtggcggttgctg ggatattcaataatggtagaatgttgctgcgggtatatgacagagcgtga aacacactgaacaaggtaaatggaacaacagcaattgcaatatgggggag gatagtcaagaacaaagcagcaatggcaaagtactgaatattctccaaag ccaaaaggtccagtggtttcaacgacaaagtcttgttggtatagctttgg aacaaaaggacaccgaaagactcgacagcgcccacaaatacagcgttgta gaagaacgaattgattgctccagagcttctaatagtcagaagatacccca aacctccgagcaacgttagcacatgacctaagaaccaggcgaagtgaaga gtctggaataacgacacccagtcagtttttcctgagctcctggtgggatt ggtagaagcatttgatttgcttggagtggttttatttgaagatggtgttg aagccattgttgctaaagagtcggagttttgcttttagggtttgttaagc aaaggaggaaaaactgcgccgtttgaagtcccaggtagtttcgcgtgtga ggccagccagggaaagcttccttcggtacttttttttcttttgcaggttc cggacggattaagcttcgggttatgaggggggcggtagccaattccggac acaatattgcgtcgcagctagtcaccccgccataaatatacgcaggattg aggtaataacatcgatagtcttagtaattaatacaattcagtggcgaatt tggcaacatgacgtaaggcccactgttgtctataaaaggggatgaatttt catgtttttgaggcctcccggacaatttattgaactcaa

[0059] The G4 promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 9):

TABLE-US-00009 tggactgttcaatttgaagtcgatgctgacgatgtcaagagagatgctca attatatttgtcatttgctggttacactggaaacgctacttttgttggcg gaaactctaccagtttggccgtccatgtaaacgatgtcgttctgggccgt gaccgtttcaacacgaacataaccaatgacaaatccacttacaggtctag ttcatatggaggcaattggtaccttacttctttggatgtcccaagtgggg ctttaacgtctggtactaacaatgtctcgtttgtcactacaaactccgag gtaaataaaggattcttgtgggattctctcaagtttgtttggaagttgta acaggtttataagcatatcgtgcgcttgtccacaattgaatcatttattg ttgcgagatacatgaacaaagtgtgaactgggacccattactacaattcc cacgcaaccgttgtttcaaagcccatattttttgacaattgtttcgttac acccccagtttgatgtacatcgcttgcaatgatgtgtgtcccggagtatt ttccatattcagcttgaattcgtatactcaaccaatatctgggggtatac ttttatgtaacctatacaaatcaactatactatttcacctttcgaccatc atctcccatcttgttaagttttgcttcctatatccctgaccctgacatca cccatgattccgctcaacggttctcctctacatcgtccctcttttggaga gggtgttcagtttgacattcaaattaccccccgccatcacgcgcaaccga gaccgcacccccgaattttcacaaattaccccacaccctatactccacca ctatgagggttattagaactgatcacgtataaataccaccgcaagttccc aagggatcgtgttcttcttctccaattgcaatcatatttctgactctttc tagttcagattaattcctttacacttgcttttttcccttacctttatcc

[0060] The G6 promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 10):

TABLE-US-00010 ccagaccagcagtttaactacgcaaatccacaggaatttctacatcacaa taccaatggtaataccacgacgtcaaggaatggaaacgacgacttggagg aagacttcgtcaacctcttgcggagtacccgaggctaagacaataagaag aaaaaaaaagaaaagcggtgggggagggattattaaataaggattatgta accccagggtaccgttctatacatatttaaggattatttaggacaatcga tgaaatcggcatcaaactggatgggagtatagtgtccggataatcggata aatcatcttgcgaggagccgcttggttggttggtgagaggagtgaaatat gtgtctcctcacccaagaatcgcgatatcagcaccctgtgggggacacta ttggcctccctcccaaaccttcgatgtggtagtgctttattatattgatt acattgattacatagctaaaccctgcctggttgcaagttgagctccgaat tccaatattagtaaaatgcctgcaagataacctcggtatggcgtccgacc ccgcttaattattttaactcctttccaacgaggacttcgtaatttttgat tagggagttgagaaacggggggtcttgatacctcctcgatttcagatccc accccctctcagtcccaagtgggacccccctcggccgtgaaatgcgcgca ctttagtttttttcgcatgtaaacgccggtgtccgtcaattaaaagtcgc agactagggtgaactttaccatttttgtcgcactccgtctcctcggaata ggggtgtagtaattctgcagtagtgcaatttttaccccgccaaggggggg cgaaaagagacgacctcatcacgcattctccagtcgctctctacgcctac agcaccgacgtagttaactttctcccatatataaagcaattgccattccc ctgaaaactttaacctctgctttttcttgatttttccttgcccaaagaaa ag

TABLE-US-00011 Gene identifier (P. Promoter pastoris GS115 strain) Genbank Acc. No. G7 PAS_chr1-4_0570 NC_012963.1 G8 PAS_chr1-3_0165 NC_012963.1

[0061] The FMD promoter preferably comprises or consists of the following nucleic acid sequence (SEQ ID No. 11):

TABLE-US-00012 aatgtatctaaacgcaaactccgagctggaaaaatgttaccggcgatgcg cggacaatttagaggcggcgatcaagaaacacctgctgggcgagcagtct ggagcacagtcttcgatgggcccgagatcccaccgcgttcctgggtaccg ggacgtgaggcagcgcgacatccatcaaatataccaggcgccaaccgagt ctctcggaaaacagcttctggatatcttccgctggcggcgcaacgacgaa taatagtccctggaggtgacggaatatatatgtgtggagggtaaatctga cagggtgtagcaaaggtaatattttcctaaaacatgcaatcggctgcccc gcAacgggaaaaagaatgactttggcactcttcaccagagtggggtgtcc cgctcgtgtgtgcaaataggctcccactggtcaccccggattttgcagaa aaacagcaagttccggggtgtctcactggtgtccgccaataagaggagcc ggcaggcacggagtctacatcaagctgtctccgatacactcgactaccat ccgggtctctcagagaggggaatggcactataaataccgcctccttgcgc tctctgccttcatcaatcaaatc

[0062] The promoter comprised in the nucleic acid construct of the present invention can be an orthologous promoter.

[0063] The promoters used in the construction of the nucleic acid construct of the present invention can be of the same

[0064] Orthologous promoter, as defined herein, is a promoter derived from another organism, preferably from another yeast strain or species. Such promoters are derived from the same precursor promoter and have similar biological and/or biochemical characteristics.

[0065] According to a particularly preferred embodiment of the present invention the derepressible promoter is linked to a second promoter forming a bidirectional promoter.

[0066] Bidirectional promoters are able of directing transcription in both the forward and reverse orientations. A bidirectional promoter can direct the transcription of two transcripts placed in either orientation (i.e., downstream or upstream) of the promoter simultaneously (e.g., the sense and antisense strands of a gene). In other words, a bidirectional promoter can direct transcription from either strand of the promoter region. The use of bidirectional promoters enables co-expression of two genes by placing them in opposing orientations and placing a bidirectional promoter in between them (see FIG. 1 and EP 2 862 933). The two promoters within the bidirectional promoter may be separated by a linker comprising or consisting of 1 to 500, preferably 1 to 300, more preferably 1 to 200, more preferably 1 to 100, more preferably 1 to 50, nucleotides.

[0067] The second promoter of the bidirectional promoter can be a constitutive, derepressible or inducible promoter. Hence, the bidirectional promoter of the present invention comprises a derepressible promoter and constitutive or inducible promoter in inverse orientation.

[0068] The constitutive promoter is preferably selected from the group consisting of a GAP promoter, PGCW14 promoter, TEF1 promoter, TPI promoter, PGK1 promoter or a histone promoter (see e.g. Vogl T et al. (ACS Synth. Biol. 5(2016):172-186)).

[0069] The inducible promoter is preferably selected from the group consisting of promoters of the methanol utilization (MUT) pathway, preferably selected from the group consisting of AOX1 promoter, AOX2 promoter, DAS1 promoter, DAS2 promoter, FLD1 promoter, GTH1 promoter, PEX8 promoter or PHO89/NSP promoter (see e.g. Vogl T et al. (ACS Synth. Biol. 5(2016):172-186)).

[0070] According to a preferred embodiment of the present invention the bidirectional promoter comprises a combination of the aforementioned promoters preferably a combination of two promoters selected from the group consisting of a GAP promoter, a CAT1 promoter, a PGCW14 promoter, a TEF1 promoter, a TPI promoter, a PGK1 promoter, a histone promoter, a promoter of the methanol utilization (MUT) pathway, preferably a AOX1 promoter, a AOX2 promoter, a DAS1 promoter, a DAS2 promoter, a FLD1 promoter, a GTH1 promoter, a PEX8 promoter or a PHO89/NSP promoter, a FDH1 promoter, a FLD1 promoter, a PEX5 promoter, a DAK1 promoter, a FGH1 promoter, a GTH1 promoter, a G1 promoter, a G2 promoter, a G3 promoter, a G4 promoter, a G5 promoter or a G6 promoter.

[0071] Particularly preferred is a bidirectional promoter comprising a CAT1 promoter in combination with a GAP promoter or a promoter of the methanol utilization (MUT) pathway, preferably a AOX1 promoter, or two CAT1 promoters without any other promoter.

[0072] The order of the various promoters within the bidirectional can be any whereby particularly preferred are GAP-CAT1 and AOX1-CAT1 promoters.

[0073] According to a further preferred embodiment of the present invention the second promoter is operably linked to a second nucleic acid molecule encoding a second protein involved in the biosynthesis of a terpenoid or a precursor thereof.

[0074] Proteins involved in the biosynthesis of a terpenoids or precursors thereof and nucleic acid molecules encoding said proteins are known in the art. These proteins are also involved in the biosynthesis of terpenoid precursor molecules (i.e. any isoprenoid substrate molecule) and include terpene synthases such as peranylpyrophosphate, farnesylpyrophosphate or geranylgeranylpyrophosphate, and/or initial products made by terpene synthases such as amorphadiene, taxadiene, hopene, limonene (see e.g. Degenhardt J et al. Phytochemistry 70(2009):1621-37).

[0075] According to a particular preferred embodiment of the present invention the CAT1 promoter is operably linked to a nucleic acid molecule encoding for a geranylgeranyl diphosphate synthase.

[0076] It turned out that a CAT1 promoter controlling the expression of geranylgeranyl diphosphate synthase allows obtaining high product yields.

[0077] In order to stop transcription of a nucleic acid molecule into mRNA and to release the nascent transcript it is advantageous to provide terminator sequence at the 3 end of a coding region to be transcribed. Hence, the nucleic acid molecule of the present invention encoding the protein involved in the biosynthesis of a terpenoid or a precursor thereof comprises preferably a terminator sequence at its 3 end.

[0078] Another aspect of the present invention relates to a vector comprising a nucleic acid construct according to the present invention.

[0079] The vector of the present invention can be used to deliver the nucleic acid construct of the invention into a host cell, for instance.

[0080] A further aspect of the present invention relates to a host cell comprising a nucleic acid construct or a vector according to the present invention.

[0081] The nucleic acid construct and the vector of the present invention can be part of a host cell. The host cell can harbor these molecules for cloning purposes and/or for expressing the coding regions/genes present in these nucleic acid molecules. Depending on the host cell the nucleic acid construct and the vector of the present invention may comprise additional elements like antibiotic resistance genes and genetic markers.

[0082] The host cell of the present invention is preferably a yeast cell, preferably a methylotrophic yeast cell.

[0083] According to a preferred embodiment of the present invention the methylotrophic yeast cell is selected from the group of Pichia pastoris, Hansenula polymorpha (Ogataea polymorpha), Candida boidinii, Komagataella pastoris, Komagataella phaffii, Komagataella populi, Komagataella pseudopastoris, Komagataella ulmi and Komagataella sp. 11-1192.

[0084] Another aspect of the present invention relates to a method for producing a terpenoid or a precursor thereof comprising the step of cultivating a host cell according to the present invention.

[0085] The host cell of the present invention comprises a nucleic acid construct comprising a nucleic acid molecule encoding a protein involved in the biosynthesis of a terpenoid or a precursor thereof which is operably linked to a derepressible promoter. In order to express the aforementioned protein derepressible conditions have to be used. These conditions can vary and depend on the derepressible promoter to be used.

[0086] The present invention is further illustrated in the following examples, however, without being restricted thereto.

Example

[0087] Materials and Methods

[0088] Plasmids

[0089] Codon optimized GGPPS (geranylgeranyl diphosphate synthase) and TDS (taxadiene synthase) genes were used for taxadiene production in P. pastoris. The genes were synthesized as double stranded DNA fragments with suitable overhangs for Gibson assembly.

TABLE-US-00013 TABLEA Entryvectors SEQID Name Sequence No. p_aox1_syn-swai- cagatcgggaacactgaaaaatacacagttattattcatttaa 12 das1tt-3prime-gib atgacccttgtgactgacactttgggagtc aox1tt-5prime- caggcaaatggcattctgacatcctcttgagcggccgcacggg 13 noti-das1tt- aagtctttacagttttagttaggag 5prime-gib intarg4-sbfi- gtagatatttataccattctgcgagaaggtcccctgcagggac 14 das1tt-3prime-gib ccttgtgactgacactttgggagtc gblock- atgtttgatttcaatgagtacatgaagtctaaggccgttgcag 15 ggpps_opttv- ttgatgcagctctggataaggctatcccactggagtacccaga aox1tt-gib aaagatccacgaatctatgagatactctttgcttgcaggtgga aagagagttagacctgctctttgcattgctgcttgtgagttgg ttggaggttctcaagaccttgctatgccaactgcttgcgccat ggagatgattcacactatgtctttgattcatgatgatttgcct tgcatggataacgacgacttccgtcgtggtaagcctaccaacc acaaggttttcggtgaggacaccgctgttttggccggtgacgc tctgttatctttcgctttcgaacacattgccgtcgcaacctct aagaccgtgccttcagacagaacccttagagttatttcagagc tgggtaagaccattggttctcaaggattggtcggaggacaagt tgttgatattacttctgaaggtgacgcaaacgttgacctgaag actttggaatggattcacatccacaaaactgccgtcttattgg agtgttctgtcgtctctggaggaatcttgggaggagctaccga ggacgagattgctagaattagaagatacgctcgttgcgttgga ttgttattccaggttgttgacgatatccttgacgtcactaagt cctccgaagaattgggtaaaactgctggtaaagaccttcttac tgacaaggcaacctaccctaagttgatgggtctggaaaaagct aaggagttcgcagctgagttggcaactcgtgctaaggaggaat tgtcatcttttgatcaaatcaaggccgctccattgttgggatt ggcagactatatcgcctttagacaaaactgatcaagaggatgt cagaatgccatttgcctg tds-bmri-stuffer- cttggaagtaccagtagaagaggacatagcacccagtggcgcg 16 gib ccgacctctgttgcctctttgttggac ggpps-bmri- cttagacttcatgtactcattgaaatcaaacatcagtcccagt 17 stuffer-gib gagctcttaagctggaagagccaatctcttgaaag gblock-tds_opttv- atgtcctcttctactggtacttccaaggttgtctctgaaactt 18 part1 cctctaccatcgttgacgacatcccaagattgtcagccaacta ccacggtgacttgtggcaccacaatgttatccagactttggaa actcctttcagagaatcttccacttatcaggagagagctgacg agctggtcgtcaagatcaaggacatgttcaacgctctgggtga cggagacatctccccttctgcatacgatactgcttgggtggcc cgtcttgccactatttcttccgacggttctgaaaagcctagat tccctcaggctcttaattgggtcttcaataaccaattgcaaga tggttcctggggaattgaatcccacttctctctttgtgacaga cttttgaacactaccaattctgttatcgcactgtctgtgtgga aaaccggtcattcccaggtccaacaaggtgctgagttcattgc tgagaacttgagacttcttaacgaggaagacgagctttcccct gattttcagatcattttcccagctttgcttcaaaaagctaaag cattgggtattaacttgccttacgacttgcctttcattaagta tttgtcaaccaccagagaagctcgtttaaccgacgtctccgct gcagccgataacattccagcaaacatgttgaacgcccttgagg gtttggaggaagttattgactggaacaagattatgagattcca gtccaaggacggttctttcctttcttctccagcctctaccgcc tgcgttttgatgaacactggagatgagaagtgttttacttttc tgaacaacttgttggataaatttggtggttgcgttccatgtat gtattcaatcgacctgttggagagattatcattggtggataac atcgaacacttgggaatcggtcgtcacttcaagcaagaaatta agggagctttggactatgtctacagacactggtctgagagagg tattggttggggtcgtgattctttagtccctgacctgaacacc actgctttgggtttgagaactcttagaatgcacggttacaatg tttcttctgacgttttgaacaacttcaaggatgaaaacggtag atttttctcctctgccggtcaaactcatgtcgagctgagatct gttgtcaacttgttccgtgcttctgatttggcattcccagatg aaagagctatggacgacgctagaaagtttg gblock-tds_opttv- aagagctatggacgacgctagaaagtttgcagagccttacttg 19 part2-das1tt-gib agagaagctctggccactaagatttctactaacactaaacttt tcaaggagatcgagtacgttgtcgaatacccttggcacatgtc tattccacgtcttgaagctagatcttacatcgattcttacgat gacaactacgtttggcagcgtaagactttatacagaatgccat cactttcaaactcaaagtgcttggaattggctaaactggactt caacattgttcagtccttgcatcaggaggagttgaagttgttg actagatggtggaaggaatcaggtatggccgatattaacttca ccagacaccgtgttgctgaggtttacttctcctccgcaacctt tgagccagagtattctgctactagaatcgctttcactaaaatt ggttgcttacaagtcttgttcgatgacatggctgatatcttcg ctactcttgacgagcttaagtctttcactgagggagttaagcg ttgggacacttccttgttacacgaaattccagaatgtatgcag acttgtttcaaagtctggttcaagttgatggaggaggttaata acgatgttgttaaggtgcaaggtagagatatgttggctcacat tcgtaagccttgggagttatacttcaactgttatgttcaagag agagagtggcttgaggctggttacattccaacttttgaggaat acttgaagacttacgctatctcagtcggtttgggtccttgcac tttacaacctatcctgttgatgggtgagttagtcaaggacgac gttgttgaaaaagttcactatccttctaacatgttcgaattgg tgtctttgtcttggagattgactaacgacactaagacctacca agcagagaaggctcgtggacaacaagcctctggtattgcttgt tacatgaaagacaaccctggtgctaccgaggaagacgctatta agcacatttgtagagttgtcgaccgtgctcttaaggaagcatc atttgaatacttcaagccatccaacgatattccaatgggttgt aagtctttcattttcaacttaagactgtgcgttcaaattttct ataagttcattgacggttacggtatcgcaaacgaagagattaa agattacattcgtaaggtctacattgacccaattcaagtctaa acgggaagtctttacagttttagttaggag seqggpps_tvopt- tctaactctctttccacctgcaag 20 118..141rev seqtds_opttv- cagctctctcctgataagtggaag 21 146..169rev seqtds_opttv- gatgaacactggagatgagaagtg 22 783..806fwd

[0090] The GGPPS and TDS genes were cloned in opposite orientation to insert bidirectional promoters (BDPs) in between them (see FIG. 1).

[0091] To facilitate cloning at first an intermediate vector providing two different transcription terminators (TAOX1 and TDAS1) in opposite orientation separated by a NotI restriction site was generated. If two genes (such as GGPPS and TDS) should be co-expressed, this vector can be used for insertion. Two different cloning vectors were prepared: pPpT4_S-DAS1TT-NotI-AOX1TT and pPpT4mutZeoMlyI-intArg4-DAS1TT-NotI-AOX1TT. The former is based on the pPpT4_S vector reported by Ntsaari et al. (PLoS One 7(2012):e39720): following NotI and SwaI digestion and purification of the backbone a PCR product of the TDAS1 bearing overhangs to the vector (primers: P_AOX1_Syn-SwaI-DAS1TT-3prime-Gib and AOX1TT-5prime-NotI-DAS1TT-5prime-Gib) was cloned by Gibson assembly (Gibson D G et al. Nat Methods 6(2009):343-5). The latter vector contained in addition a sequence to target specific genomic integration (intArg4) and a mutated MlyI site in the Zeocin resistance gene (silent mutation). This vector was generated by digesting the pPpT4mutZeoMlyI-intArg4-bidi-dTOM-eGFP-BmrIstuffer vector (see US 2015/0011407) with SbfI and NotI and inserting a PCR product containing the respective overhangs (primers: intARG4-SbfI-DAS1TT-3prime-Gib and AOX1TT-5prime-NotI-DAS1TT-5prime-Gib) by Gibson assembly.

[0092] An entry vector containing the GGPPS and TDS genes separated by a stuffer/placeholder fragment was generated. This vector for taxadiene coexpression was generated by using P. pastoris codon optimized GGPPS and TDS genes. The genes were provided as synthetic double stranded fragments (gBlocks by Integrated DNA Technologies) with overhangs for Gibson assembly (gBlock-GGPPS_optTV-AOX1TT-Gib, gBlock-TDS_optTV-Part1 and gBlock-TDS_optTV-Part2-DAS1TT-Gib). A stuffer fragment with complementary overhangs was amplified using primers TDS-BmrI-stuffer-Gib and GGPPS-BmrI-stuffer-Gib. The four fragments were mixed in equimolar ratios with the NotI digested pPpT4mutZeoMlyI-intArg4-DAS1TT-NotI-AOX1TT backbone and joined by Gibson assembly. This vector was named pPpT4mutZeoMlyI-intArg4-DAS1TT-AOX1TT-TDS_optTV-GGPPS_optTV-BmrIstuffer.

[0093] Finally the stuffer fragment was cut out by BmrI digestion and the BDPs cloned in by Gibson assembly. The primers used for amplification are provided in Table B.

TABLE-US-00014 TABLEB SEQID Name Sequence No. TDS-pDAS2-Gib cttggaagtaccagtagaagaggacatttttgatgtttgatagtt 23 tgataagagtgaac GGPPS-pDAS1- gacttcatgtactcattgaaatcaaacatTTTGTTCGATTATTCT 24 Gib CCAGATAAAATCAAC TDS-pDAS1-Gib cttggaagtaccagtagaagaggacatTTTGTTCGATTATTCTCC 25 AGATAAAATCAAC GGPPS-pDAS2- gacttcatgtactcattgaaatcaaacatttttgatgtttgatag 26 Gib tttgataagagtg TDS-HHT2-Gib cttggaagtaccagtagaagaggacatTTTTACTACGATAGACAC 27 AAGAAGAAGCAG GGPPS-HHF2- gacttcatgtactcattgaaatcaaacatATTTATTGATTATTTG 28 Gib TTTATGGGTGAGTC TDS-HHF2-Gib cttggaagtaccagtagaagaggacatATTTATTGATTATTTGTT 29 TATGGGTGAGTC GGPPS-HHT2- gacttcatgtactcattgaaatcaaacatTTTTACTACGATAGAC 30 Gib ACAAGAAGAAGCAG TDS-AOX1-Gib cttggaagtaccagtagaagaggacatCGTTTCGAATAATTAGTT 31 GTTTTTTGATC GGPPS-CAT1- gacttcatgtactcattgaaatcaaacatTTTAATTGTAAGTCTT 32 Gib GACTAGAGCAAGTG TDS-CAT1-Gib cttggaagtaccagtagaagaggacatTTTAATTGTAAGTCTTGA 33 CTAGAGCAAGTG GGPPS-AOX1- gacttcatgtactcattgaaatcaaacatCGTTTCGAATAATTAG 34 Gib TTGTTTTTTGATC TDS-GAP-Gib cttggaagtaccagtagaagaggacatTGTGTTTTGATAGTTGTT 35 CAATTGATTG GGPPS-GAP-Gib gacttcatgtactcattgaaatcaaacatTGTGTTTTGATAGTTG 36 TTCAATTGATTG pGAP-pCAT1- gacgaggacaccaagacatttctacaaaaaTAATCGAACTCCGAA 37 Gib TGCGGTTCTC TDS-HTA1 cttggaagtaccagtagaagaggacatTGTTGTAGTTTTAATATA 38 GTTTGAGTATG GGPPS-HTB1 gacttcatgtactcattgaaatcaaacatTTTGATTTGTTTAGGT 39 AACTTGAACTGGATG

[0094] The primer combinations for the amplification of the promoters are listed in Table C.

TABLE-US-00015 TABLEC Bidirectional promoter Primer1 Primer2 DAS2-DAS1 TDS-pDAS2-Gib GGPPS-pDAS1-Gib DAS1-DAS2 TDS-pDAS1-Gib GGPPS-pDAS2-Gib DAS2-d8-DAS1-d2d5 TDS-pDAS2-Gib GGPPS-pDAS1-Gib shBDP-28fwd TDS-HHT2-Gib GGPPS-HHF2-Gib shBDP-28rev TDS-HHF2-Gib GGPPS-HHT2-Gib AOX1-CAT1 TDS-AOX1-Gib GGPPS-CAT1-Gib CAT1-AOX1 TDS-CAT1-Gib GGPPS-AOX1-Gib AOX1-GAP TDS-AOX1-Gib GGPPS-GAP-Gib GAP-AOX1 TDS-GAP-Gib GGPPS-AOX1-Gib GAP-CAT1 TDS-GAP-Gib GGPPS-CAT1-Gib CAT1-GAP TDS-CAT1-Gib GGPPS-GAP-Gib HTA1-HTB1 TDS-HTA1 GGPPS-HTB1 HHT2-HHF2 TDS-HHT2-Gib GGPPS-HHF2-Gib

[0095] The nucleotide sequences of the bidirectional promoters (BDPs) obtained with the primers of Table B and used herein are depicted in FIG. 2.

[0096] Strains, Cultivation Conditions and Measurements

[0097] Pichia pastoris strain CBS7435 was used as host for transformation. Transformations of P. pastoris cells were performed with SwaI linearized plasmids following the condensed protocol by Lin-Cereghino et al. (Biotechniques 38(2005):44, 46, 48).

[0098] Taxadiene producing strains were cultivated in shake flasks in 50 ml buffered yeast peptone glycerol media (BYPG; 1% glycerol, 20 g/l peptone, 10 g/l yeast extract, 200 mM potassium phosphate buffer pH 6). A dodecane overlay of 10% of the volume (e.g. 5 ml) was added when the cultivation was started. In case methanol induction was performed, only 25 ml BYPG media were used and grown for 60 h, subsequently 25 ml BYPM2 media were added (1% (v/v) methanol). Methanol to 0.5% (v/v) was again added after 12, 24, 48 h and the shake flasks harvested after 72 h. For methanol induction, the dodecane overlay was added after growth on glycerol for 60 h together with the BMM2 addition. Selected strains were also cultivated on 2% and 3% BYPG media and harvested after 60 h.

[0099] The dodecane overlay was harvested by centrifugation at 3220 g for 25 min at 4 C. and analyzed by mass spectrometry for taxadiene contents (using a calibration curve based on peak areas comparison to a taxadiene standard curve).

[0100] Results

[0101] Diterpenoids are GGPP (geranylgeranyl diphosphate) derivatives. GGPP is produced by geranylgeranyl diphosphate synthase (GGPPS). The diterpenoid, taxadiene, is generated from mevalonate pathway products by two enzymatic steps: GGPPS and taxadiene synthase (TDS). The taxadiene production can be transcriptionally influenced by using differently regulated promoters (see FIG. 3), whereby bidirectional promoters (BDPs) have been using exemplarily in this example. The promoters featured similarly high expression levels, but combinations of different regulatory profiles on each side (constitutive, inducible and derepressed/derepressible activity). The yields obtained from P. pastoris strains transformed with plasmids bearing these BDPs spanned a 50-fold range.

[0102] P. pastoris strains expressing only TDS and GGPPS from a BDP reached yields comparable to a heavily engineered S. cerevisiae strain (6.2 mg/l mg/L vs. 8.7 mg/l; Engels B et al. Metab Eng 10(2008):201-6). Even in shake flasks the yields could be further improved by adapting the cultivation conditions, reaching 9.4 mg/l (FIG. 3).

[0103] This shows that the regulation of the expression of GGPPS is a key factor for high yields. Inducible or constitutive regulation suggested in literature resulted in 5- to 50-fold lower yields than derepressed regulation (activation when a repressing carbon source is depleted). Constitutive expression of the GGPPS appeared even lethal resulting in no taxadiene production at all.

[0104] These results suggest that host cells like P. pastoris alongside the flux optimization/transcriptional fine-tuning strategies outlined here, are a production platform for terpenoids such as Taxol precursors. Here, the methylotrophic yeast Pichia pastoris was used for controlled, balanced expression of terpenoid pathway genes, exemplified by the production of a diterpene, the Taxol precursor taxadiene. Unexpectedly, by transformation of a single plasmid into P. pastoris, higher taxadiene yields than in a highly engineered comparable S. cerevisiae strain (Engels B et al.) were obtained. Surprisingly, expression of GGPPS under derepressed conditions turned out to be a key factor for product high yields.