PRODUCTION OF SESQUITERPENE PRODUCTS AND RELATED MOLECULES PRIORITY

This application claims the benefit of U.S. Provisional Application No. 62/396,804 filed on September 19, 2016, which is incorporated herein by reference in its entirety.

BACKGROUND OF INVENTION

Field of the invention

This disclosure relates to genetic engineering and recombinant host cells useful in producing sesquiterpene products by increasing production and/or accumulation of sesquiterpene products through epi-isozizaene synthase, thujopsene terpene synthase, and/or longifolene synthase. The recombinant host cells provided by the invention generally have higher metabolic flux through the mevalonate biochemical pathway, and can comprise additional recombinant expression constructs encoding epi-isozizaene synthase, thujopsene terpene synthase, longifolene synthase and other enzymes useful for increasing sesquiterpene products downstream of the mevalonate pathway, particularly epi-isozizaene, thujopsene, and/or longifolene.

Background of the related art

Epi-isozizaene, thujopsene, longifolene, barbatene and related molecules comprise a large class of biologically derived organic molecules produced only in plants or bacterium, and only in small quantities. Epi-isozizaene, thujopsene, longifolene, barbatene and related molecules are derived from the fifteen-carbon precursor famesyl pyrophosphate (FPP). FPP serves as precursor in the biosynthesis of a number of biologically and commercially important molecules including valencene, squalene, ubiquinone, sterol, heme A and dolichol. There is a need for efficient production of large quantities of epi-isozizaene, thujopsene, longifolene, barbatene and related molecules.

SUMMARY OF INVENTION

The present invention comprises methods for increased production of epi-isozizaene, thujopsene, longifolene and related molecules, advantageously in recombinant host cells resulting from overexpression of epi-isozizaene synthase, thujopsene terpene synthase, longifolene synthase and increasing production of farnesyl pyrophosphate (FPP) and other mevalonate pathway precursors. In particular, the invention relates to methods for increasing the production and/or accumulation of epi-isozizaene, thujopsene, longifolene, barbatene in recombinant host cells. In one aspect, the invention relates to a method for producing a sesquiterpene product or sesquiterpenoid in a recombinant host cell, the method comprising the steps of: culturing a recombinant host cell comprising one or more recombinant nucleic acids encoding heterologous enzymes for producing the sesquiterpene product or sesquiterpenoid under conditions wherein the sesquiterpene product or sesquiterpenoid is produced, wherein the recombinant host cell has reduced expression or activity of an endogenous squalene synthase. In an embodiment of the invention, the one or more recombinant nucleic acids encoding heterologous enzymes for producing the sesquiterpene product or sesquiterpenoid is epi- isozizaene synthase, thujopsene terpene synthase, or longifolene synthase. In other embodiments, the epi-isozizaene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:08, the thujopsene terpene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:06, and the longifolene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:07. In another embodiment, the recombinant host cell is genetically engineered to reduce activity of the endogenous squalene synthase.

In a second aspect, the invention relates to a recombinant cell for producing a sesquiterpene product or sesquiterpenoid genetically engineered to have reduced expression or activity of an endogenous squalene synthase, and further comprising one or more recombinant expression constructs encoding heterologous enzymes for producing the sesquiterpene product or sesquiterpenoid. In an embodiment of the invention, the one or more recombinant nucleic acids encoding heterologous enzymes for producing the sesquiterpene product or sesquiterpenoid is epi-isozizaene synthase, thujopsene terpene synthase, or longifolene synthase. In other embodiments, the epi-isozizaene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 08, the thujopsene terpene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:06, and the longifolene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:07. In another embodiment, the recombinant host cell is genetically engineered to reduce activity of the endogenous squalene synthase.

In a third aspect, the invention relates to a fuel composition comprising one or more of the sesquiterpene products or sesquiterpenoids. In an embodiment, the sesquiterpene product or sesquiterpenoid is epi-isozizaene, longifolene, thujopsene and/or barbatene. Embodiment 1. A method for producing a sesquiterpene product or sesquiterpenoid in a recombinant host cell, the method comprising the steps of: culturing a recombinant host cell comprising one or more recombinant nucleic acids encoding heterologous enzymes for producing the sesquiterpene product or sesquiterpenoid under conditions wherein the sesquiterpene product or sesquiterpenoid is produced, wherein the recombinant host cell has reduced expression or activity of an endogenous squalene synthase.

Embodiment 2. The method of embodiment 1, wherein the one or more recombinant nucleic acids encoding heterologous enzymes for producing the sesquiterpene product or sesquiterpenoid is epi-isozizaene synthase, thujopsene terpene synthase, or longifolene synthase.

Embodiment 3. The method of embodiment 2, wherein the epi-isozizaene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:01, the thujopsene terpene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, and the longifolene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:04 or SEQ ID NO:05. Embodiment 4. The method of embodiment 2, wherein the epi-isozizaene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:08, the thujopsene terpene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:06, and the longifolene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:07.

Embodiment 5. The method of embodiment 1, wherein the recombinant host cell is genetically engineered to reduce expression of an enzyme having squalene synthase activity. Embodiment 6. The method of embodiment 1, wherein the recombinant host cell is genetically engineered to reduce activity of the endogenous squalene synthase.

Embodiment 7. The method of embodiment 1, wherein the recombinant host cell is genetically engineered to reduce expression of an enzyme having geranylgeranyl diphosphate synthase activity.

Embodiment 8. The method of embodiment 5, wherein the enzyme having

geranylgeranyl diphosphate synthase activity is encoded by ERG20.

Embodiment 9. The method of embodiment 1, wherein the endogenous squalene synthase is encoded by ERG9. Embodiment 10. The method of embodiment 1, wherein the reduced expression of endogenous squalene synthase is caused: (a) by introducing a recombinant genetic construct into the cell, and wherein the squalene synthase is operably linked to a messenger RNA destabilizing motif; or (b) by introducing a recombinant genetic construct into the cell, and wherein the squalene synthase is operably linked to a weak promoter.

Embodiment 11. The method of embodiment 1, wherein the reduced activity of endogenous squalene synthase is caused by introducing a recombinant genetic construct into the cell comprising a mutant squalene synthase gene, and wherein the mutant squalene synthase gene encodes an enzyme less active than the endogenous wild type squalene synthase.

Embodiment 12. The method of embodiment 1, wherein the recombinant host cell further comprises a truncated version of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR) comprising the catalytically active carboxyl terminal portion thereof.

Embodiment 13. The method of embodiment 1, wherein the recombinant host cell is a eukaryotic cell or a prokaryotic cell.

Embodiment 14. The method of embodiment 13, wherein the eukaryotic cell is a mammalian cell, a plant cell, a fungal cell or a yeast cell.

Embodiment 15. The method of embodiment 14, wherein the eukaryotic cell is a yeast cell.

Embodiment 16. The method of embodiment 15, wherein the yeast cell is a yeast of species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Candida boidinii, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Ashbya gossypii, Arxula adeninivorans, Cyberlindnera jadinii, Candida albicans, Rhodotorula sp, Sporobolomyces sp, or Rhodosporidium sp.

Embodiment 17. The method of embodiment 16, wherein the yeast cell is a

Saccharomyces cerevisiae cell.

Embodiment 18. The method of embodiment 1, wherein the sesquiterpene product or sesquiterpenoid is epi-isozizaene, longifolene, thujopsene, barbatene, or a by-product thereof. Embodiment 19. A method for producing epi-isozizaene, thujopsene, longifolene and/or barbatene from a bioconversion reaction, comprising:

(a) growing a recombinant host cell in a culture medium, under conditions in which epi-isozizaene synthase, thujopsene terpene synthase, and/or longifolene synthase is produced in the host, wherein the host comprises a gene encoding epi-isozizaene synthase, thujopsene terpene synthase, and/or longifolene synthase polypeptides capable of converting FPP to a sesquiterpene product and wherein the gene encoding epi-isozizaene synthase, thujopsene terpene synthase, and/or longifolene synthase is expressed in the host;

(b) contacting the host with FPP in a reaction buffer to produce a sesquiterpene product; and

(c) purifying the sesquiterpene product.

Embodiment 20. The method of embodiment 19, wherein the epi-isozizaene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:01, the thujopsene terpene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, and the longifolene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:04 or SEQ ID NO:05. Embodiment 21. The method of embodiment 19, wherein the epi-isozizaene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:08, the thujopsene terpene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:06, and the longifolene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:07.

Embodiment 22. The method of embodiment 19, wherein the recombinant host cell is a eukaryotic cell or a prokaryotic cell.

Embodiment 23. The method of embodiment 22, wherein the eukaryotic cell is a mammalian cell, a plant cell, a fungal cell, or a yeast cell.

Embodiment 24. The method of embodiment 23, wherein the eukaryotic cell is a yeast cell.

Embodiment 25. The method of embodiment 24, wherein the yeast cell is a yeast of species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Candida boidinii, Hansenula polymorpha, Kluyveromyces lactis,

Kluvermyces marxianus, Pichia pastoris, Ashbya gossypii, Arxula adeninivorans,

Cyberlindnera jadinii, Candida albicans, Rhodotorula sp, Sporobolomyces sp, or

Rhodosporidium sp.

Embodiment 26. The method of claim 25, wherein the yeast cell is a Saccharomyces cerevisiae cell.

Embodiment 27. A recombinant cell for producing a sesquiterpene product or sesquiterpenoid genetically engineered to have reduced expression or activity of an endogenous squalene synthase, and further comprising one or more recombinant expression constructs encoding heterologous enzymes for producing the sesquiterpene product or sesquiterpenoid.

Embodiment 28. The recombinant cell of embodiment 17, wherein the one or more recombinant nucleic acids encodes an epi-isozizaene synthase, a thujopsene terpene synthase, or a longifolene synthase.

Embodiment 29. The recombinant cell of embodiment 27, wherein the epi-isozizaene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:01, the thujopsene terpene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, and the longifolene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:04 or SEQ ID NO:05.

Embodiment 30. The recombinant cell of embodiment 27, wherein the epi-isozizaene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:08, the thujopsene terpene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:06, and the longifolene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:07.

Embodiment 31. The recombinant cell of embodiment 27, wherein the cell is genetically engineered to have reduced activity of an endogenous squalene synthase.

Embodiment 32. The recombinant cell of embodiment 27, wherein the cell is genetically engineered to have reduced expression of an endogenous squalene synthase.

Embodiment 33. The recombinant cell of embodiment 27, wherein the cell is genetically engineered to have reduced expression of an endogenous enzyme having geranylgeranyl diphosphate synthase activity.

Embodiment 34. The recombinant cell of embodiment 33, wherein the endogenous enzyme having geranylgeranyl diphosphate synthase activity is ERG20.

Embodiment 35. The recombinant cell of embodiment 27, wherein the endogenous squalene synthase is ERG9.

Embodiment 36. The recombinant cell of embodiment 27, wherein the reduced expression of the endogenous squalene synthase is caused by introducing into the cell a recombinant genetic construct expressing a messenger RNA destabilizing motif specific to the squalene synthase. Embodiment 37. The recombinant cell of embodiment 27, wherein the reduced expression of endogenous squalene synthase is caused by introducing into the cell a recombinant genetic construct wherein the squalene synthase is operably linked by the construct to a weak promoter.

Embodiment 38. The recombinant cell of embodiment 27 further comprising a truncated version of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR) comprising the catalytically active carboxyl terminal portion thereof.

Embodiment 39. The recombinant cell of embodiment 27, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

Embodiment 40. The recombinant cell of embodiment 39, wherein the eukaryotic cell is a mammalian cell, a plant cell, a fungal cell or a yeast cell.

Embodiment 41. The recombinant cell of embodiment 39, wherein the eukaryotic cell is a yeast cell.

Embodiment 42. The recombinant cell of embodiment 41, wherein the yeast cell is a yeast of species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Candida boidinii, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Ashbya gossypii, Arxula adeninivorans, Cyberlindnera jadinii, Candida albicans, Rhodotorula sp, Sporobolomyces sp, or Rhodosporidium sp.

Embodiment 43. The recombinant cell of embodiment 41, wherein the yeast cell is a Saccharomyces cerevisiae cell.

Embodiment 44. The recombinant cell of embodiment 27, wherein the sesquiterpene product or sesquiterpenoid is epi-isozizaene, longifolene, thujopsene, barbatene , or a byproduct thereof.

Embodiment 45. A fuel composition comprising one or more of the sesquiterpene products or sesquiterpenoids of embodiment 1 or embodiment 19 or claim 27.

Embodiment 46. The fuel composition of embodiment 45, wherein the sesquiterpene product or sesquiterpenoid is epi-isozizaene, longifolene, thujopsene, barbatene , or a byproduct thereof .

Embodiment 47. The fuel composition of embodiment 45, wherein the composition further comprises at least one fuel additive.

Embodiment 48. The fuel composition of embodiment 45, wherein the fuel additive is an oxygenate, an antioxidant, a thermal stability improver, a stabilizer, a cold flow improver, a combustion improver, an anti-foam, an anti-haze additive, a corrosion inhibitor, a lubricity improver, an icing inhibitor, an injector cleanliness additive, a smoke suppressant, a drag reducing additive, a metal deactivator, a dispersant, a detergent, a de-emulsifier, a dye, a marker, a static dissipater, a biocide, or combinations thereof.

In other embodiments, the recombinant host cell is a eukaryotic cell or a prokaryotic cell. The recombinant host cell can be from a genus such as Agaricus, Anastrepta,

Aristolochia, Aspergillus, Bacillus, Bazzania, Candida, Clavularia, Corynebacterium, Dacrydium, Escherichia, Ferula, Fusarium/Gibberella, Juniperus, Kluyveromyces,

Laetiporus, Lentinus, Mylia, Nasutitermes, Panaxa, Phaffla, Phanerochaete, Picea, Pichia, Pinus, Physcomitrella, Pogostemon, Reboulia, Rhodoturula, Saccharomyces, Sarcophyton, Schistostephium, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces or Yarrowia.

In an embodiment, the recombinant host cell is a eukaryotic cell and is a mammalian cell, a plant cell, a fungal cell or a yeast cell. In some embodiments, the host cell can be

Anastrepta orcadensis, Arxula adeninivorans, Ashbya gossypii, Aristolochia indica, Bazzania tridens, Candida albicans, Candida boidinii, Candida glabrata, Clavularia inflata var.

luzoniana, Clavularia inflate, Clavularia viridis, Cyberlindnera jadinii, Dacrydium cupressinum, Ferula galbaniflua, Hansenula polymorpha, Juniperus communis,

Kluyveromyces lactis, Kluvermyces marxianus, Mylia nuda, Mylia taylorii, Nasutitermes ephratae, Nasutitermes rippertii, Panaxa ginseng, Pichia pastoris, Pinus longifolia, Pogostemon cablin, Reboulia hemishpaerica, Saccharomyces cerevisiae, Sarcophyton acutangulum, Schizosaccharomyces pombe, Xanthophyllomyces dendrorhous, or Yarrowia lipolytica.

In a further embodiment, the eukaryotic cell is a yeast cell, and the yeast cell is

Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Candida boidinii, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Ashbya gossypii, Arxula adeninivorans, Cyberlindnera jadinii, Candida albicans,

Rhodotorula sp, Sporobolomyces sp, or Rhodosporidium sp. In a particular embodiment, the yeast cell is Saccharomyces cerevisiae, and includes epi-isozizaene synthase, thujopsene terpene synthase, and/or longifolene synthase.

The invention described here relates to recombinant host cells genetically engineered to produce of epi-isozizaene, thujopsene, longifolene, barbatene and related molecules. The recombinant host cells can have increased mevalonate production and/or have higher metabolic flux through the mevalonate biochemical pathway, and can also comprise additional recombinant expression constructs encoding enzymes useful for increasing products of the mevalonate pathway. Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the structure of epi-isozizaene.

Figure 2 shows the biosynthetic pathway engineered to produce epi-isozizaene.

Figure 3 shows the nucleic acid sequence of the codon optimized EIZS gene (SEQ ID

NO:01).

Figure 4 shows the plasmid expressing the epi-isozizaene synthase gene (EIZS).

Figure 5 shows a GC-FID chromatogram from an epi-isozizaene producing strain, showing the product profile of the purified sample.

Figure 6 shows the structure of 3-thujopsene.

Figure 7 shows the biosynthetic pathway engineered to produce 3-thujopsene.

Figure 8 shows the nucleic acid sequence of the wild type At5g44630 gene (SEQ ID NO:02).

Figure 9 shows the nucleic acid sequence of the codon optimized At5g44630 gene (SEQ ID NO:03).

Figure 10 shows the plasmids expressing the wild type and codon optimized thujopsene terpene synthase gene A t5g44630.

Figure 11 shows a GC-FID chromatogram from a thujopsene producing strain, showing the product profile in fermentor broth extract, distillate, and purified samples.

Figure 12 shows the structure of (+)-longifolene.

Figure 13 shows the biosynthetic pathway engineered to produce (+)-longifolene.

Figure 14 PsTPS3 shows the nucleic acid sequence of the wild-type PsTPS3 {Pinus sylvestris longifolene synthase) gene (SEQ ID NO:04).

Figure 15 shows the nucleic acid sequence of the codon optimized PsTPS3 gene (SEQ ID NO: 05).

Figure 16 shows the plasmids expressing the wild type and codon optimized P. sylvestris longifolene synthase gene.

Figure 17 shows GC-FID chromatogram for a longifolene producing strain, showing the product profile in fermentor broth, distillate, and purified samples. DETAILED DESCRIPTION

All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and PCR techniques. See, for example, techniques as described in Maniatis et al., 1989, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, New York; Ausubel et al, 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et al, 1990, Academic Press, San Diego, CA).

Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "nucleic acid" means one or more nucleic acids.

It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

As used herein, the terms "or" and "and/or" is utilized to describe multiple components in combination or exclusive of one another. For example, "x, y, and/or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x and (y or z)," or "x or y or z." In some embodiments, "and/or" is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group. As used herein, the terms "polynucleotide," "nucleotide," "oligonucleotide," and "nucleic acid" can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.

As used herein, the term "sesquiterpene," "sesquiterpene product" or

"sesquiterpenoid" shall be taken to include molecules in which at least part of the molecule is derived from a prenyl pyrophosphate, such as farnesyl pyrophosphate (FPP), isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), etc.

As used herein, the terms "host cell," "microorganism," "microorganism host," "microorganism host cell," "recombinant host," and "recombinant host cell" can be used interchangeably.

Regarding sequence identity between nucleotide and amino acid sequences as set forth herein, and as would be understood by the skilled worker, a high level of sequence identity indicates likelihood that a first sequence is derived from a second sequence. Amino acid sequence identity requires identical amino acid sequences between two aligned sequences. Thus, a candidate sequence sharing 70% amino acid identity with a reference sequence requires that, following alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity according to the present invention is determined by aid of computer analysis, such as, without limitations, the ClustalW computer alignment program (Higgins et al, 1994, Nucleic Acids Res. 22: 4673-4680), and the default parameters suggested therein. The ClustalW software is available from as a ClustalW WWW Service at the European Bioinformatics Institute at www.ebi.ac.uk/clustalw. Using this program with its default settings, the mature (bioactive) part of a query and a reference polypeptide are aligned. The number of fully conserved residues are counted and divided by the length of the reference polypeptide. The ClustalW algorithm can similarly be used to align nucleotide sequences. Sequence identities can be calculated in a similar way as indicated for amino acid sequences. In certain embodiments, the cell of the present invention comprises a nucleic acid sequence encoding modified, heterologous and additional enzymatic components of terpene and terpenoid biosynthetic pathways, as defined herein.

The methods of the invention can be used, for example, for large-scale production of epi-isozizaene, thujopsene, and/or longifolene and related molecules by a recombinant host cell, as described for the methods of the invention. As shown in the examples that follow, the methods of the invention can be used to produce recombinant host cells with increased metabolic flux through the pathway of interest and efficient production of epi-isozizaene, thujopsene, and/or longifolene or other related molecule of interest at unexpectedly higher levels in a recombinant host cell.

Mevalonate Pathway

In some embodiments, the invention relates to engineered recombinant host cells having altered activity or expression of endogenous mevalonate pathway genes. For example, the recombinant host cells can have altered activity or expression of endogenous enzyme having geranylgeranyl diphosphate synthase activity. In particular embodiments, when a wild type host cell expresses an enzyme with geranylgeranyl diphosphate synthase activity, then the host recombinant host cells of the invention preferably have altered activity of said enzyme with geranylgeranyl diphosphate synthase activity. A non-limiting example of this is the microorganism S. cerevisiae and the endogenous enzyme encoded by the ERG20 gene. In other embodiments of the invention, the wild type host cells do not express any enzyme with geranylgeranyl diphosphate synthase activity. In an embodiment, the host cells preferably have reduced activity of geranylgeranyl diphosphate synthase. Said reduced activity results in production or accumulation or both of FPP, and thus, the host cells of the invention are useful in methods for accumulating and producing FPP, as well as compounds having FPP as a precursor, and for producing increased amounts of epi-isozizaene, thujopsene, and/or longifolene.

The geranylgeranyl diphosphate synthase can be any of the geranylgeranyl pyrophosphate synthases described herein. In general, the recombinant host cells as provided by the invention have been genetically engineered in order to reduce the activity of geranylgeranyl diphosphate synthase.

A recombinant host cell having reduced activity of geranylgeranyl diphosphate synthase activity according to the invention can have an activity of geranylgeranyl diphosphate synthase, which is about 80%, about 50%, about 30%, for example in the range of 10 to 50% of the activity of geranylgeranyl diphosphate synthase in a similar cell having wild type geranylgeranyl diphosphate synthase activity. It is in general important that the recombinant host cell retains at least some geranylgeranyl diphosphate synthase activity, since this is essential for most cells. Geranylgeranyl diphosphate synthase activity can be greatly reduced without significantly impairing cell viability. Recombinant host cells with greatly reduced geranylgeranyl diphosphate synthase activity can have a somewhat slower growth rate than corresponding wild type cells. Thus, it is preferred that recombinant host cells of the invention have a growth rate which is at least 50% of the growth of a similar cell having wild type geranylgeranyl diphosphate synthase activity. In certain embodiments of the invention the recombinant host cell having reduced activity of an enzyme with squalene synthase activity according to the invention has an activity of said enzyme, which is at the most 80%, preferably at the most 50%, such as at the most 30%, for example in the range of 10 to 50% of the activity of said enzyme in a similar host cell having a wild type enzyme with squalene synthase activity. It is in general important that recombinant host cells retain at least some squalene synthase, since this is essential for most host cells. Squalene synthase activity can be greatly reduced without significantly impairing cell viability. Recombinant host cells with greatly reduced activity can have a somewhat slower growth rate than corresponding wild type cells. Thus it is preferred that the recombinant host cells of the invention have a growth rate which is at least 50% of the growth of a similar cell having a wild enzyme with squalene synthase activity. In other embodiments of the invention, recombinant host cells have altered activity, expression or localization of HMG-CoA synthase. According to the invention, HMG-CoA synthase, can have an activity, which is at least 150%, preferably at the least 200%, such as at least 300% or more of the activity of HMG-CoA synthase in a similar host cell having wild type HMG- CoA synthase activity. In some embodiments, the HMG-CoA synthase can be truncated so that it is more soluble in the cytoplasm.

Activity of mevalonate pathway enzymes can be altered in a number of different ways. In certain embodiments, the wild type promoter of a gene encoding a mevalonate pathway enzyme can be exchanged for a strong promoter, such as any of the strong promoters described herein. Accordingly, the recombinant cell can comprise an ORF encoding a mevalonate pathway enzyme under the control of a strong promoter. In general, cells of the invention can contain one ORF encoding the mevalonate pathway enzymes endogenous to the recombinant host cell, ensuring that the overall levels of the mevalonate pathway enzymes are increased.

In an embodiment of the invention, the promoter sequence can be a strong constitutive promoter or a strong inducible promoter. A strong constitutive promoter or a strong inducible promoter according to the present invention is a promoter, which directs only an increased level of transcription in the recombinant host cell. In particular, the strong constitutive promoter or the strong inducible promoter sequence directs expression of an ORF encoding a target protein at an expression level which is significantly higher than the expression level obtained with the wild type target protein promoter. The strong constitutive promoter or the strong inducible promoter sequence can direct expression of the ORF encoding a target protein at an expression level, which is at least 125%, at least 150%, at least 200%, or at least 400% or more of the expression level obtained with the wild target protein. Respective promoters are known in the art, some non-limiting examples of strong constitutive or strong inducible promoters include, but are not limited to, the AOX1, GAL1, PGK, FDH, FLD, CUP, TDH3, TEF1, TPI1, ADH1 or TEF2 promoters.

In another embodiment, the promoter sequence can be a weak promoter. A weak promoter according to the present invention is a promoter, which directs only a low level of transcription in the recombinant host cell. In particular, the weak promoter sequence directs expression of an ORF encoding a target protein at an expression level which is significantly lower than the expression level obtained with the wild type target protein promoter. The weak promoter sequence can direct expression of the ORF encoding a target protein at an expression level, which is at most 70%, or at most 60%, or at most 50%, or at most 40%, or less of the expression level obtained with the wild target protein. Respective promoters are known in the art, some non-limiting examples of weak promoters include, but are not limited to, the CYC-1 promoter or the KEX-2 promoter.

In other embodiments, alternatively or simultaneously, the recombinant host cell can comprise a heterologous insert sequence, which increases the expression of mRNA encoding a mevalonate pathway enzyme. In particular embodiments, the heterologous nucleic acid insert sequence can be positioned between the promoter sequence and the ORF encoding a mevalonate pathway enzyme.

In particular embodiments of the invention, the recombinant host cell can also have inactivated and/or no endogenous enzyme activity for molecules downstream of FPP in the wildtype pathway of the recombinant host cell. This can for example be accomplished by: a) deletion of the entire gene encoding downstream endogenous enzymes; or b) deletion of the entire coding region encoding downstream endogenous enzymes; or c) deletion of part of the gene encoding downstream enzymes leading to a total loss of the endogenous enzyme's activity.

In embodiments of the invention where a recombinant host cell has no endogenous farnesyl pyrophosphate synthase activity, in advantageous embodiments:

a) recombinant host cells are cultivated in the presence of ergosterol; or

b) recombinant host cells comprise a heterologous nucleic acid encoding an enzyme with farnesyl pyrophosphate synthase.

In other embodiments, epi-isozizaene, thujopsene, longifolene and/or barbatene and other sesquiterpene products can be produced artificially by incubating FPP in vitro with heterologously expressed epi-isozizaene synthase, thujopsene terpene synthase, and/or longifolene synthase. For example, the invention further provides a method for producing epi-isozizaene, thujopsene, longifolene and/or barbatene from a bioconversion reaction, comprising:

a) growing a recombinant host cell in a culture medium, under conditions in which epi- isozizaene synthase, thujopsene terpene synthase, and/or longifolene synthase is produced in the host, wherein the host comprises a gene encoding epi-isozizaene synthase, thujopsene terpene synthase, and/or longifolene synthase polypeptides capable of converting FPP to a sesquiterpene product;

wherein the gene encoding epi-isozizaene synthase, thujopsene terpene synthase, and/or longifolene synthase is expressed in the host;

b) contacting the host with FPP in a reaction buffer to produce a sesquiterpene product; and

c) purifying the sesquiterpene product.

In some aspects of the method for producing the sesquiterpene product from a bioconversion reaction, the epi-isozizaene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:01 , the thujopsene terpene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:02 or SEQ ID NO:03, and the longifolene synthase comprises a nucleic acid sequence having at least 85% identity to the nucleic acid sequence set forth in SEQ ID NO:04 or SEQ ID NO:05. As listed in the table 2 below, the gene sequences disclosed in SEQ ID NOS: 1, 3 and 5 encode the amino acid sequences of SEQ ID NOS: 8, 6 and 7, respectively. In other embodiments, the epi-isozizaene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:08, the thujopsene terpene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:06, and the longifolene synthase comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:07.

In an embodiment, in order to maximize production of farnesyl pyrophosphate (FPP), mutants of the ERG9 gene of Saccharomyces cerevisiae can be used (see U. S. Patent Nos. 8,481,286, 8,609,371 and 8,753,842, which are herein incorporated by reference in their entirety). The ERG9 gene encodes squalene synthase. These mutants have reduced, but not eliminated, squalene synthase activity. As such, they allow sufficient production of squalene and subsequent sterols to allow growth, but are sufficiently reduced in activity to allow accumulation of FPP and overproduction of terpenes. This can be done by using defective squalene synthase genes, which when expressed, result in reduced cellular squalene synthase activity rather than downregulating the transcription of a normally active squalene synthase enzyme. This results in a recombinant host cell with reduced squalene synthase activity independent of the activity of a repressor.

In an alternative embodiment, the ERG9 gene can have at least one change that occurs in the coding region for the wild-type ERG9 gene and its flanking sequences, including the sequences both upstream and downstream from the coding region. The change in the ERG9 gene can result in reduced ERG9 squalene synthase activity, even though the specific activity may potentially be unaltered. The reduction of the activity of the squalene synthase enzyme can occur through one or more of the following mechanisms: (1) reduction in transcription so that less mRNA that can be translated into squalene synthase enzyme is generated; (2) reduction of mRNA stability, again reducing translation; and (3) reduction of enzyme stability brought about by an increased rate of protein degeneration in vivo. In other words, either: (1) the specific activity of the resulting squalene synthase enzyme is reduced through at least one change in the amino acid sequence of the enzyme expressed from the nucleic acid molecule; or (2) the in vivo activity of the enzyme is reduced through a reduction in transcription, a reduction in translation, or a reduction of enzyme stability.

Host and recombinant cells

Host and recombinant host cells provided herein can be any cell suitable for protein expression {i.e., expression of heterologous genes) including, but not limited to, eukaryotic cells, prokaryotic cells, yeast cells, fungal cells, mammalian cells, plant cells, microbial cells and bacterial cells. Furthermore, cells according to the invention meet one or more of the following criteria: said cells should be able grow rapidly in large fermenters and should produce small organic molecules in an efficient way. Furthermore, a host cell is a cell that can be genetically engineered according to the invention to produce a recombinant host cell, which is a cell wherein a nucleic acid has been disabled (by deletion or otherwise), or substituted (for example, by homologous recombination at a genetic locus to change the phenotype of the cell, inter alia, to produce reduced expression of a cellular enzyme or any gene of interest), or a heterologous nucleic acid, inter alia, encoding an enzyme or enzymes to confer a novel or enhanced phenotype on the cell has been introduced.

In further and particular embodiments, recombinant host cells are yeast cells that are of yeast species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Candida albicans, Arxula adeninivorans, Candida boidinii, Hansenula polymorpha, Kluyveromyces lacti, Pichia pas tor is , Rhodotorula sp, Sporobolomyces sp, and Rhodosporidium sp.. Yeasts are known in the art to be useful as host cells for genetic engineering and recombinant protein expression. Yeast of different species differ in productivity and with respect to their capabilities to process and modify proteins and to secrete metabolic products thereof. The different 'platforms' of types of yeast make them better suited for different industrial applications. In general, yeasts and fungi are excellent host cells to be used with the present invention. They offer a desired ease of genetic manipulation and rapid growth to high cell densities on inexpensive media. As eukaryotes, they are able to perform protein

modifications like glycosylation (addition of sugars), thus they can produce even complex foreign proteins that are identical or very similar to native products from plant or mammalian sources.

In other embodiments, the host cell for genetic engineering as set forth herein can be a microalgal cell such as a cell from Chlamydomonas, Chlorella or Prototheca species. In other embodiment, the host cell can be a cell of a filamentous fungus, for example,

Aspergillus species. In other embodiments, the host cell can be a plant cell. In yet additional embodiments, the host cell can be a mammalian cell, such as a human, feline, porcine, simian, canine, murine, such as rat or mouse, or rabbit cell. The host cell can also be a CHO, CHO-K1, HEI193T, HEK293, COS, PC12, HiB5, RN33b, BHK cell. In other embodiments, the host cell can be a prokaryotic cell, such as a bacterial cell, including, but not limited to E. coli or cells of Corynebacterium, Bacillus, Pseudomonas and Streptomyces species.

Additional Aspects of Recombinant Cells

In certain embodiments, the invention provides recombinant host cells comprising a heterologous nucleic acid sequence encoding a dual function enzyme, wherein the dual function enzyme is an acetoacetyl-CoA thiolase and a HMG-CoA reductase, including, but not limited to, the mvaE gene encoded by E. faecalis or a functional homologue thereof. In addition to the heterologous nucleic acid sequence encoding a dual function enzyme, the recombinant host cell also can also comprise a heterologous nucleic acid sequence encoding a 3-hydroxy-3-methyl-glutaryl coenzyme A synthase (HMGS), including but not limited to, mvaS gene encoded by E. faecalis or a functional homologue thereof.

In yet further embodiments, the invention provides recombinant cells comprising a recombinant expression construct encoding a truncated version of 3-hydroxy-3-methyl- glutaryl coenzyme A reductase (HMGR) comprising the catalytically active carboxyl terminal portion thereof. In additional or alternative embodiments, said recombinant host cell comprises a heterologous nucleic acid sequence encoding a dual function enzyme as set forth herein, wherein said cell produces and/or accumulates enhanced metabolites in the mevalonate pathway, in particular mevalonate, including inter alia expression of heterologous HMGS. In further additional or alternative embodiments, said recombinant host cell is a yeast cell that is genetically engineered for reduced ERG9 expression or activity.

In additional specific embodiments, the invention provides methods and recombinant host cells for producing FPP, particular wherein production and/or accumulation of FPP is enhanced, wherein FPP is obtained in advantageously greater yields by culturing a recombinant host cell that has been genetically engineered for reduced expression of farnesyl diphosphate synthase activity, geranylgeranyl diphosphate synthase activity and/or the activity of an enzyme having both farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase activity, and wherein said recombinant cell further comprises a recombinant expression construct encoding a heterologous FPP synthase.

Compositions

In certain aspects, the invention generally relates to fuels produced by the methods, recombinant host cells, and sesquiterpenes produced herein. In some embodiments, the invention generally relates to methods for manufacturing fuels including, providing a sesquiterpene composition comprising epi-isozizaene, thujopsene, longifolene and/or barbatene generated by recombinant host cells from substrates including glucose, sucrose, fructose, other reducing sugars, cellobiose, cellulose, hemicellulose, lignocellulose, lignin, methane, and/or CO ₂ . Fuel compositions are contemplated herein that can include one or more of epi-isozizaene, thujopsene, longifolene, barbatene and related molecules and mixtures thereof. Further, fuel compositions contemplated herein may include one or more by-products of epi-isozizaene, thujopsene, and longifolene production as either major or minor fuel components.

As used herein, the term "by-product" refers to a chemical compound produced in conjunction with the production of an intended sesquiterpene. Further, a "by-product of thujopsene" refers to any other sesquiterpene or related chemical entity produced during the production of thujopsene, as contemplated herein. Similarly, a "by-product of epi- isozizaene" and a "by-product of longifolene" refer to any other sesquiterpenes or related chemical entities that are produced during the production of epi-isozizaene and longifolene, respectively. As used herein, the terms "fuel" and "fuel composition," which may be used interchangeably herein, refer to compositions including one or more sesquiterpenes, and/or one or more hydrocarbons, and/or one or more alcohols, and/or one or more fatty esters or a mixture thereof. In some embodiments, liquid hydrocarbons are used. Fuel can be used to power internal combustion engines such as reciprocating engines (e.g. , gasoline engines and diesel engines), Wankel engines, jet engines, rocket engines, missile engines and gas turbine engines. In some embodiments, fuel typically comprises a mixture of hydrocarbons such as alkanes, cycloalkanes and aromatic hydrocarbons. In other embodiments, fuel refers to a composition comprising epi-isozizaene, thujopsene, longifolene and/or barbatene.

As used herein, the term "fuel compound" refers to any compound or a mixture of compounds that are used to formulate a fuel composition. There can also be "major fuel components" and "minor fuel components." A major fuel component is present in a fuel composition by at least 50% by volume, and a minor fuel component is present in a fuel composition by less than 50%. Fuel additives can also be included and represent minor fuel components.

As used herein, the term "fuel additive" refers to chemical components added to fuels to alter the properties of the fuel (e.g., to improve engine performance, fuel handling, fuel stability, or for contaminant control). Types of additives include, but are not limited to, antioxidants, thermal stability improvers, cetane improvers, stabilizers, cold flow improvers, combustion improvers, anti-foams, anti-haze additives, corrosion inhibitors, lubricity improvers, icing inhibitors, injector cleanliness additives, smoke suppressants, drag reducing additives, metal deactivators, dispersants, detergents, demulsifiers, dyes, markers, static dissipaters, biocides and combinations thereof.

In embodiments, a fuel mixture can comprise greater than 75%, greater than 80%, greater than 85%, greater than 90% or greater than 95% iso-zizaene along with additional sesquiterpenes. In other embodiments, a fuel mixture can comprise greater than 75%, greater than 80%, greater than 85%, greater than 90% or greater than 95% longifolene along with additional sesquiterpenes. In yet other embodiments, a fuel mixture can comprise greater than 10%, greater than 15%, greater than 20% or greater than 25% thujopsene along with additional sesquiterpenes. In yet another embodiment, a fuel mixture can comprise greater than 15%, greater than 20%, greater than 25% or greater than 30% barbatene along with additional sesquiterpenes.

In an embodiment, a fuel mixture comprising iso-zizaene, longifolene and/or thujopsene mixture can be dimerized to generate a lubricant mixture. Iso-zizaene, longifolene and/or thujopsene can be dimerized thermally or in the presence of a

homogeneous or heterogeneous catalyst. In certain embodiments, the catalyst can be an acid catalyst including, but not limited to, zeolites, aluminosilicates, clays, or cation exchange resins.

In other embodiments, antioxidants including phenolics can be added to iso-zizaene, longifolene and/or thujopsene to increase the storage stability of the hydrocarbon. In one embodiment, iso-zizaene, longifolene, and/or thujopsene can be hydrogenated in the presence of a catalyst under a hydrogen atmosphere to obtain longifolane or a mixture of saturated sesquiterpenes. In some embodiments, the hydrogenation catalyst can have at least one metal selected from Ni, Cu, Pd, Pt, and Ru. In other embodiments, the hydrogenation can be conducted in acetic acid. In an embodiment, the unsaturated fuel has a density of 0.94 g/mL at 20°C, a net heat of combustion (NHOC) of >142 kBtu/gal, a flashpoint of 88°C, a -20°C dynamic viscosity of 53.1 cP, a 40°C dynamic viscosity of 5.82 cP, and a glass transition temperature of -98°C. In embodiments the hydrogenated hydrocarbon mixture can have a density of 0.918 g/mL, a volumetric NHOC of 138-142 kBtu/gal, a -20°C dynamic viscosity of 70 cP, a 40°C dynamic viscosity of 6.6 cP, and a glass transition temperature of -97°C. In another embodiment, the unsaturated fuel has a density of 0.94 g/mL, a volumetric net heat of combustion of 147.4 kBtu/gal, a flashpoint of 98°C, a -20°C dynamic viscosity of 28.2 cP, a 40°C dynamic viscosity of 3.93 cP, and a glass transition temperature of -94°C. In some embodiments, the saturated sesquiterpene mixture has a density of 0.929 g/mL, a volumetric net heat of combustion of 141.9 kBtu/gal, a -20°C dynamic viscosity of 42.9 cP, a 40°C dynamic viscosity of 4.3 cP, and a glass transition temperature of -94°C. In yet another embodiment, the unsaturated fuel has a density of 0.93 g/mL, a volumetric net heat of combustion of 144 kBtu/gal, a flashpoint of 98°C, a -20°C dynamic viscosity of 34.9 cP, a 40°C dynamic viscosity of 3.93 cP, and a glass transition temperature of -91°C. In certain embodiments, the saturated sesquiterpene mixture has a density of 0.901 g/mL, a volumetric net heat of combustion of 138-140 kBtu/gal, a -20°C dynamic viscosity of 46.9 cP, a 40°C dynamic viscosity of 4.9 cP, and a glass transition temperature of -97°C.

In other embodiments, iso-zizaene, longifolene, thujopsene or saturated sesquiterpene mixtures can be isomerized with an acid catalyst for the purposes of decreasing the viscosity, increasing the density and net heat of combustion, or increasing the cetane number. In certain embodiments, the product of the isomerization reaction is a diamondoid structure. In some embodiments, the isomerized mixture is purified by fractional distillation. In some embodiments, the fuels can be pure sesquiterpenes or prepared by selective fractional distillation of sesquiterpene mixtures (density >0.90 g/mL, NHOC > 137,000 btu/gal, cetane number >30). In yet other embodiments, the fuels can be generated by blending sesquiterpene mixtures with known cetane enhancers or antioxidants for fuels. In embodiments, the fuels can be generated by blending sesquiterpene fuels with petroleum- based fuels including JP-10, RJ-4, JP-8, JP-5, F-76, Diesel #2, Jet A, and/or any renewable fuel. In other embodiments, the sesquiterpene fuel mixtures can be blended with high cetane fuels derived via a Fischer-Tropsch process or Alcohol-to-Jet (ATJ) process to generate fuels with cetane numbers in the range of 40-50. In other embodiments, the sesquiterpene fuel mixtures can be blended with nitrate esters or other cetane enhancers in low concentration to yield fuels with increased cetane numbers. Contemplated fuel compositions include those exemplified by Table 1.

Table 1. Contemplated fuel compositions.

Table 2. Nucleic acid and protein sequences.

Sequence of the ATGCACGCCTTCCCACATGGTACTACTGCTACTCCAACTGCTATTGCTGTTCC

ACCATCTTTGAGATTGCCAGTTATTGAAGCTGCTTTCCCAAGACAATTGCATC

codon optimized CATATTGGCCAAAGTTGCAAGAAACTACTAGAACCTGGTTGTTGGAAAAAAGA

TTGATGCCAGCTGATAAGGTTGAAGAATATGCTGATGGTTTGTGCTACACTGA EIZS gene.

TTTGATGGCTGGTTATTACTTGGGTGCTCCAGATGAAGTTTTACAAGCTATTG SEQ ID NO:01 CAGATTACTCTGCCTGGTTTTTTGTTTGGGATGATAGACACGATAGAGATATC

GTTCATGGTAGAGCTGGTGCTTGGAGAAGATTGAGAGGTTTGTTGCATACTGC TTTGGATTCTCCAGGTGATCACTTGCATCATGAAGATACTTTGGTTGCTGGTT TCGCTGATTCCGTTAGAAGATTATATGCTTTCTTGCCAGCTACTTGGAATGCT AGATTTGCTAGACATTTCCACACTGTTATC GAAGCT T AC GAC AGAGAAT T C CA TAACAGAACTAGAGGTATAGTTCCAGGTGTCGAAGAATATTTGGAATTGAGAA GATTAACCTTCGCCCATTGGATTTGGACTGATTTGTTGGAACCATCTTCTGGT TGTGAATTGCCAGATGCTGTTAGAAAACATCCAGCTTATAGAAGAGCTGCCTT GTTGTCTCAAGAATTTGCTGCTTGGTACAACGATTTGTGCTCTTTGCCAAAAG AAATTGCCGGTGATGAAGTTCACAACTTGGGTATTTCTTTGATCACCCATCAT TCCTTGACTTTGGAAGAAGCTATTGGTGAAGTTAGAAGAAGAGTAGAAGAATG CATCACCGAATTCTTGGCTGTTGAAAGAGATGCATTGAGATTCGCTGATGAAT TGGCTGATGGTACTGTTAGAGGTAAAGAATTGTCTGGTGCAGTTAGAGCTAAT GTCGGTAATATGAGAAACTGGTTCTCTTCCGTTTACTGGTTCCATCATGAATC CGGTAGATATATGGTTGATTCTTGGGATGACAGATCTACTCCACCATACGTTA ACAATGAAGCAGCTGGTGAAAAGTAA Sequence of the MHAFPHGTTATPTAIAVPPSLRLPVIEAAFPRQLHPYWPKLQETTRTWLLEKR LMPADKVEEYADGLCYTDLMAGYYLGAPDEVLQAIADYSAWFFVWDDRHDRDI EIZS protein. VHGRAGAWRRLRGLLHTALDSPGDHLHHEDTLVAGFADSVRRLYAFLPATWNA SEQ ID NO:08 RFARHFHTVIEAYDREFHNRTRGIVPGVEEYLELRRLTFAHWIWTDLLEPSSG

CELPDAVRKHPAYRRAALLSQEFAAWYNDLCSLPKEIAGDEVHNLGISLITHH SLTLEEAIGEVRRRVEECITEFLAVERDALRFADELADGTVRGKELSGAVRAN VGNMRNWFSSVYWFHHESGRYMVDSWDDRSTPPYVNNEAAGEK

Sequence of the wild ATGGAAGCATTAGGAAACTTTGATTACGAAAGCTACACCAATTTTACAAAATT

GCCATCCTCCCAATGGGGTGATCAGTTCCTCAAGTTTTCCATTGCTGATTCGG

type At5g44630 ATTTTGATGTCCTTGAAAGAGAGATTGAAGTACTAAAGCCTAAAGTAAGAGAG gene. AACATATTCGTGTCGTCTTCCACAGACAAAGACGCGATGAAAAAGACAATTCT

TTCTATTCATTTTCTGGACAGTCTTGGTCTCTCTTATCATTTTGAGAAGGAAA

SEQ ID NO:02 TCGAAGAGAGCCTAAAACATGCTTTCGAGAAGATAGAAGATTTGATCGCTGAT

GAAAATAAATTGCACACAATCTCCACCATCTTCCGAGTTTTCAGGACATACGG TTACTACATGTCTTCGGATGTTTTCAAGATATTCAAAGGAGACGATGGTAAAT TCAAGGAAAGTTTAATAGAAGATGTCAAGGGTATGCTAAGCTTCTACGAAGCA GTGCACTATGGGACAACGACAGATCATATATTGGATGAAGCTTTAAGCTTCAC ATTGAACCACTTGGAGTCACTAGCTACAGGTCGTAGAGCAAGCCCACCACATA TTTCAAAGCTTATACAAAATGCTCTTCACATACCTCAGCACCGAAACATCCAA GCGTTGGTTGCAAGGGAGTATATCTCGTTCTATGAACACGAAGAGGACCACGA CGAAACACTTCTCAAGCTAGCTAAGCTCAATTTCAAGTTCTTGCAGCTTCATT ACTTCCAAGAATTAAAAACCATCACAATGTGGTGGACGAAATTAGACCATACA TCAAACCTTCCACCAAACTTCAGAGAGAGAACTGTAGAGACATGGTTTGCAGC ATTGATGATGTACTTCGAGCCACAATTTTCACTTGGGAGAATTATGTCGGCTA AGTTATACTTAGTAATAACATTTCTAGACGACGCTTGCGATACTTATGGTTCA ATTTCTGAAGTTGAAAGCCTGGCCGATTGTTTGGAAAGATGGGATCCAGATTA CATGGAAAATCTTCAAGGTCACATGAAGACTGCCTTCAAATTTGTGATGTATC TTTTTAAAGAGTATGAAGAAATACTAAGGTCACAAGGAAGATCCTTTGTGTTG GAGAAAATGATAGAAGAGTTCAAGATTATTGCTAGGAAAAACCTCGAACTTGT CAAATGGGCACGTGGAGGTCATGTTCCTAGCTTTGATGAGTATATAGAGTCTG GTGGAGCCGAGATTGGTACATATGCAACCATAGCATGTTCCATCATGGGACTT GGAGAGATTGGTAAGAAGGAAGCTTTTGAGTGGCTAATATCTAGACCAAAGCT CGTTCGGATTTTAGGTGCAAAGACCCGTCTCATGGATGACATAGCCGACTTTG AGGAGGACATGGAAAAAGGTTACACTGCAAATGCACTCAACTATTACATGAAC GAACACGGAGTTACAAAAGAAGAAGCCAGTAGAGAACTTGAAAAGATGAATGG AGATATGAACAAGATCGTAAACGAAGAATGCTTGAAGATAACTACCATGCCAC GCCGAATTCTTATGCAATCCGTCAATTATGCACGTTCATTGGATGTTCTCTAT ACCGCGGATGATGTTTACAACCACCGCGAAGGAAAACTCAAAGAGTATATGAG GCTTTTGCTCGTAGATCCTATACTTCTTTAG

Sequence of the wild MEALGNFDYESYTNFTKLPSSQWGDQFLKFSIADSDFDVLEREIEVLKPKVRE

NIFVSSSTDKDAMKKTILSIHFLDSLGLSYHFEKEIEESLKHAFEKIEDLIAD

type At5g44630 ENKLHTISTIFRVFRTYGYYMSSDVFKIFKGDDGKFKESLIEDVKGMLSFYEA protein. VHFGTTTDHILDEALSFTLNHLESLATGRRASPPHISKLIQNALHIPQHRNIQ

ALVAREYISFYEHEEDHDETLLKLAKLNFKFLQLHYFQELKTITMWWTKLDHT

SEQ ID NO:06 SNLPPNFRERTVETWFAALMMYFEPQFSLGRIMSAKLYLVITFLDDACDTYGS

ISEVESLADCLERWDPDYMENLQGHMKTAFKFVMYLFKEYEEILRSQGRSFVL EKMIEEFKI IARKNLELVKWARGGHVPSFDEYIESGGAEIGTYATIACSIMGL GEIGKKEAFEWLISRPKLVRILGAKTRLMDDIADFEEDMEKGYTANALNYYMN EHGVTKEEASRELEKMNGDMNKIVNEECLKITTMPRRILMQSVNYARSLDVLY TADDVYNHREGKLKEYMRLLLVDPILL

Sequence of the ATGGAAGCTTTGGGTAACTTCGACTACGAATCTTACACTAACTTCACCAAGTT

GCCATCTTCTCAATGGGGTGATCAATTCTTGAAGTTCTCCATTGCTGATTCCG

codon optimized ATTTCGATGTCTTGGAAAGAGAAATCGAAGTCTTGAAGCCAAAGGTCAGAGAA At5g44630 gene. AACATCTTCGTTTCTTCATCCACTGATAAGGACGCTATGAAGAAAACCATCTT

GTCCATCCATTTCTTGGACTCATTGGGTTTGTCTTACCACTTCGAAAAAGAAA SEQ ID NO:03 T T GAAGAAT C C T T GAAGC AC GC C T T C GAAAAGAT T GAAGAT T T GAT T GC T GAC GAAAACAAGTTGCATACCATCTCCACTATCTTCAGAGTTTTCAGAACTTACGG TTACTACATGTCCTCCGATGTTTTCAAGATTTTCAAGGGTGATGACGGTAAAT TCAAAGAATCATTGATCGAAGATGTCAAGGGTATGTTGTCTTTCTACGAAGCT GTTCATTACGGTACTACCACCGATCATATTTTGGATGAAGCTTTGTCTTTCAC CTTGAACCACTTGGAATCTTTGGCTACTGGTAGAAGAGCTTCTCCACCACATA T T T C CAAGTT GAT T CAAAAC GC C T T GC AT AT C C C ACAAC AC AGAAAT AT T C AA GC T T T GGT T GC C AGAGAAT AT AT CTCATTCTAC GAAC AC GAAGAAGAT C AC GA CGAAACT T T GT T GAAAT T GGCCAAGT T GAAT T T CAAGT T CT T GCAAT T ACACT ACTTCCAAGAATTGAAAACCATTACCATGTGGTGGACCAAGTTGGATCATACT TCTAATTTGCCACCAAACTTCAGAGAAAGAACTGTTGAAACTTGGTTTGCTGC CTTGATGATGTACTTCGAACCACAATTTTCTTTGGGTAGAATCATGTCCGCTA AGTTGTACTTGGTTATCACCTTCTTGGATGATGCTTGTGATACCTACGGTTCC ATTTCTGAAGTTGAATCTTTAGCCGACTGTTTGGAAAGATGGGATCCAGATTA TATGGAAAACTTGCAAGGTCATATGAAGACCGCCTTTAAGTTCGTTATGTACT TGTTTAAAGAATACGAAGAAATCTTGAGATCCCAAGGTAGATCCTTCGTTTTG GAAAAGAT GAT AGAAGAAT T CAAGAT TAT C GC C AGAAAGAAT T TGGAAT T GGT TAAGTGGGCTAGAGGTGGTCATGTTCCATCTTTCGATGAATATATTGAATCCG GTGGTGCCGAAATTGGTACTTATGCTACTATTGCTTGCTCCATTATGGGTTTG GGTGAAATCGGTAAGAAAGAAGCTTTCGAATGGTTGATCTCTAGACCAAAGTT GGTTAGAATTTTGGGTGCTAAGACCAGATTGATGGATGATATTGCCGACTTTG AA GAAGAT ATGGAAAAGGGTTATACCGCTAACGCTTTGAACTACTACATGAAC GAACAT GGT GT CACCAAAGAAGAAGCT TCAAGA GAAT T GGAAAAAAT GAACGG T GAC AT GAAC AAGAT C GT C AAC GAAGAAT GC T TAAAGAT T AC C AC C AT GC C T A GAAGAATCTTGATGCAATCTGTTAACTACGCCAGATCTTTGGATGTCTTGTAT ACT GCT GAT GAT GT T T ACAACCACAGAGAAGGT AAAT T GAAAGAAT AT AT GAG ATTGTTGTTGGTCGACCCAATTTTGTTGTAA

Sequence of the ATGGCTCAAATTTCTATAGGTGCACCACTATCTGCCGAGGTGAACGGAGCCTG

C AT C AAC AC T C AT C AT C AT GGAAAT C T GT GGGAC GAC T AT T T C AT AC AAT C T C

wild-type PsTPS3 TTAAGTCGCCTTATGAGGCACCTGAATGCCATGAACGCTGTGAAAAGATGATT gene. GAAGAAGTGAAGCATTTACTTTTGAGTGAGATGAGAGATGGCAACGATGATTT

AATCAAACGTCTCCAGATGGTTGACATTTTTGAATGTCTAGGAATTGATCGGC

SEQ ID NO:04 ACTTTCACCATGAAATACAAGCTGCTCTTGATTACGTGTACAGATATTGGAAC

GAGCTGGAAGGCATCGGTGTTGGAACAAGAGATTCCCTCACCAAAGATCTGTA TGCTACCGGTTTGGGATTTCGGGCTCTCCGACTCCATCGATATAATGTATCCT CAGCTGTCTTGGAGAATTTCAAGAACGAAAATGGGCTGTTCTTCCACAGTTCC GCGGTTCAAGAAGAAGAAGTGAGATGCATGTTGACGTTACTTAGGGCTTCAGA AATTTCATTTCCCGGAGAAAAGGTGATGGACGAGGCAAAGGCATTCGCAACAG AATATCTAAACCAACTTTTGACGAGAGTGGATATAACGGAAGTGGGTGAAAAC CTCTTAAGAGAGGTTAGGTATGCCCTAGATTTTCCTTGGTACTGCAGTGTGCC GAGATGGGAGGCTAGGAGCTTCATCGAAATATTTGGACAAAACAATTCATGGC TTAAGT C AAC TAT GAAC AAAAAAGT T TTAGAGTTGGC T AAAT TGGACTT C AAT ATTCTGCAATCCGCACATCAAAGAGAGCTACAGCTTCTCTCAAGGTGGTGGTC ACAATCGGATATAGAGAAGCAGAATTTCTACCGGAAGCGTCACGTGGAATTTT ACTTTTGGATGGTTATAGGCACGTTCGAACCGGAGTTTTCGAGCAGCAGAATT GC AT T C GC AAAAAT T GC GAC AC T GAT GAC T AT C C T AGAT GAT C T C TAT GAT AC T CACGGAACGT T GGAAC AAC T AAAAAT CT T CACAGAAGCAGT CAAACGAT GGG ATCTTTCATTACAAGACCGTCTTCCAGACTACATAAAGATTACTCTGGAATTC T T C T T C AAC AC AT C C AAT GAAT T GAAT GC C GAAGT T GC T AAAAT GC AAGAACG GGATATGTCAGCCTACATACGAAAAGCAGGCTGGGAACGATACATTGAAGGGT ATATGCAAGAGTCCGAATGGATGGCGGCTCGACATGTCCCTACGTTTGACGAT TACATGAAGAATGGCAAACGCAGCTCTGGAATGTGTATACTAAATTTGTATTC GCTTCTCTTAATGGGGCAACTTGTACCGGACAACATTCTGGAGCAAATACACC TTCCATCCAAGATCCATGAGCTTGTGGAATTGACGGCCAGACTGGTCGACGAC TCAAAGGATTTCCAGGCGAAGAAGGATGGTGGGGAGTTTGCTTCAGGTACAGA GTGCTACTTGAAAGAGAAGCCTGAATGTACAGAGGAAGATGCAATGAATCATC TCATTGGGCTCCTCAATCTGACAGCGATGGAATTAAATTGGGAATTTGTAAAA CATGACGGTGTGGCGCTGTGTCTCAAGAAGTTCGTCTTCGAAGTTGCACGAGG TCTCCGATTCATCTACAAATACAGAGACGGCTTTGACTATTCCAACGAGGAGA TGAAAAGCCAGATAACCAAAATCCTTATCGATCAAGTGCCCATCTGA

Sequence of the MAQISIGAPLSAEVNGACINTHHHGNLWDDYFIQSLKSPYEAPECHERCEKMI

EEVKHLLLSEMRDGNDDLIKRLQMVDIFECLGIDRHFHHEIQAALDYVYRYWN

wild-type PsTPS3 ELEGIGVGTRDSLTKDLYATGLGFRALRLHRYNVSSAVLENFKNENGLFFHSS

AVQEEEVRCMLTLLRASEISFPGEKVMDEAKAFATEYLNQLLTRVDITEVGEN

protein.

LLREVRYALDFPWYCSVPRWEARSFIEIFGQNNSWLKSTMNKKVLELAKLDFN

SEQ ID NO:07 ILQSAHQRELQLLSRWWSQSDIEKQNFYRKRHVEFYFWMVIGTFEPEFSSSRI

AFAKIATLMTILDDLYDTHGTLEQLKIFTEAVKRWDLSLQDRLPDYIKITLEF FFNTSNELNAEVAKMQERDMSAYIRKAGWERYIEGYMQESEWMAARHVPTFDD YMKNGKRSSGMCILNLYSLLLMGQLVPDNILEQIHLPSKIHELVELTARLVDD SKDFQAKKDGGEFASGTECYLKEKPECTEEDAMNHLIGLLNLTAMELNWEFVK HDGVALCLKKFVFEVARGLRFIYKYRDGFDYSNEEMKSQITKILIDQVPI

Sequence of the ATGGCCCAAATTTCTATTGGTGCTCCATTGTCTGCTGAAGTTAATGGTGCTTG

TATTAACACCCATCATCATGGTAATTTGTGGGACGATTACTTCATCCAATCTT

codon optimized TGAAGTCTCCATACGAAGCTCCAGAATGTCACGAAAGATGTGAAAAGATGATC

GAAGAAGTCAAGCACTTGTTGTTGTCCGAAATGAGAGATGGTAACGACGATTT

PsTPS3 gene.

GATCAAGAGATTGCAAATGGTCGATATCTTCGAATGCTTGGGTATCGATAGAC SEQ ID NO:05 ATTTCCATCACGAAATTCAAGCTGCTTTGGATTACGTTTACAGATACTGGAAT

GAATTGGAAGGTATCGGTGTTGGTACTAGAGATTCTTTGACTAAGGACTTGTA TGCTACTGGTTTGGGTTTTAGAGCTTTGAGATTGCACAGATACAACGTTTCTT CAGCCGTTTTGGAAAACTTCAAGAACGAAAACGGTTTGTTCTTCCATTCCTCT GCTGTTCAAGAAGAAGAAGTTAGATGTATGTTGACCTTGTTGAGAGCCTCCGA AATTTCATTTCCAGGTGAAAAGGTTATGGATGAAGCTAAGGCTTTTGCTACCG AATACTTGAATCAATTATTGACCAGAGTCGACATCACCGAAGTTGGTGAAAAT TTGTTAAGAGAAGTCAGATACGCCTTGGATTTTCCATGGTACTGTTCTGTTCC AAGATGGGAAGCTAGATCCTTCATTGAAATTTTCGGTCAAAACAACTCCTGGT TGAAGTCCACTATGAACAAGAAGGTTTTGGAATTGGCCAAGTTGGATTTCAAC ATCTTGCAATCTGCTCACCAAAGAGAATTACAATTATTGTCTAGATGGTGGTC CCAATCCGATATCGAAAAGCAAAATTTCTACAGAAAGAGACACGTCGAATTCT ACTTTTGGATGGTTATCGGTACTTTCGAACCAGAATTCTCCTCATCTAGAATT GCTTTCGCTAAGATTGCTACCTTGATGACCATTTTGGATGACTTGTACGATAC TCACGGTACATTGGAACAATTGAAGATTTTCACCGAAGCCGTTAAGAGATGGG ATTTGTCTTTACAAGATAGATTGCCAGACTACATCAAGATCACCTTGGAATTT TTCTTCAACACCTCCAACGAATTGAACGCTGAAGTTGCTAAAATGCAAGAAAG AGATATGTCCGCCTACATTAGAAAAGCTGGTTGGGAAAGATATATCGAAGGTT ACATGCAAGAATCCGAATGGATGGCTGCTAGACATGTTCCAACTTTTGATGAC TACATGAAGAACGGTAAGAGATCTTCTGGTATGTGCATCTTGAACTTGTATTC TTTGTTGTTGATGGGTCAATTGGTCCCAGACAACATTTTGGAACAAATCCATT TGCCATCCAAGATCCACGAATTGGTTGAATTGACTGCTAGATTGGTTGATGAC TCCAAGGATTTCCAAGCTAAAAAGGATGGTGGTGAATTTGCTTCTGGTACTGA ATGCTACTTGAAAGAAAAGCCAGAATGCACTGAAGAAGATGCCATGAATCATT TGATCGGTTTGTTGAATTTGACCGCCATGGAATTGAACTGGGAATTCGTAAAA CATGATGGTGTTGCTTTGTGCTTGAAGAAGTTCGTTTTCGAAGTTGCAAGAGG TTTAAGATTCATCTACAAGTACAGAGATGGTTTCGACTACTCTAACGAAGAAA TGAAGTCTCAAATCACCAAGATTTTGATCGACCAAGTCCCAATCTAA

EXAMPLES The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only and are not taken as limiting the invention. In particular, the examples demonstrate the effective production of epi-isozizaene, thujopsene, and longifolene.

EXAMPLE 1. PRODUCTION OF EPI-ISOZIZAENE

A. Generating an epi-isozizaene producing strain

To produce epi-isozizaene (see Figures 1 and 2), the plasmid pAlx68-37 containing the codon optimized EIZS gene (SEQ ID NO:01 ; Figure 3), was transformed into ALX11- 30.1 (ura3, trpl, erg9def25, HMG2cat/TRPl::rDNA, dppl, sue) strain of Saccharomyces cerevisiae using a lithium acetate yeast transformation kit (Sigma- Aldrich). ALX11 -30.1 is derived from CALI5-1 (ura3, leu2, his3, trpl, Aerg9::HIS3, HMG2cat/TRPl::rDNA, dppl, sue) (Takahashi, 2007) by three engineering steps. First, the leu2 mutation was restored to wild type by transformation of aLEU2 gene fragment into CALI5-1 to create ALX7-95. Next, the erg9def25 mutant gene was introduced into ALX7-95 to restore ERG9 protein production, allowing the strain to grow in the absence of feeding ergosterol. Finally, the wild type HIS 3 gene was introduced to make the strain prototrophic for histidine. This strain is designated ALXl l-30.

B. Production of epi-isozizaene

Transformants with plasmid pAlx68-37 (plasmid shown in Figure 4) were selected on SDE-ura medium (0.67 % Bacto yeast nitrogen base without amino acids, 2% glucose, 0.14 % yeast synthetic drop-out medium supplement without uracil, and 40 mg/L ergosterol for strains carrying the Aerg9::HIS3 mutation). Colonies were picked and screened for epi- isozizaene production using a microculture assay in 96 deep well plates. Transformant yeast colonies were inoculated into individual wells of 96-well microtiter plates filled with 200 of SDE. The plate was grown for two to three days at 28 °C. After growth to saturation, 10 from each well was used to inoculate a 96 deep well plate containing 300 of medium suitable for growth and epi-isozizaene production. After three days of growth and production, epi-isozizaene was extracted first by introducing 250 μΐ. of acetone and vortexing, followed by addition of 500 μΐ. of w-hexane and vortexing. After phase separation, the plate was sealed with aluminum tape and placed on the sample tray of a gas chromatography autosampler. A one microliter sample was injected into the GC. The acetone and hexane used for extraction were each spiked with internal standards to aid in quantitation of the samples. The extracted samples were analyzed by gas chromatography and the amount of epi-isozizaene was calculated from the peak area.

Production of epi-isozizaene was performed in a 15-L fermentation tank (New Brunswick Bioflow 110). Eight liters of fermentation medium was prepared and autoclaved. The medium was composed of glucose, 20 g/L; (NH ₄ ) ₂ S0 ₄ , 20 g/L; KH ₂ P0 ₄ , 28 g/L;

MgS0 ₄ ^» 7H ₂ 0, 12 g/L; yeast extract, 5 g/L; soybean oil, 100 mL/L; Foam Blast® Fl 11-GF (Emerald Foam Materials®, WY, USA), 5 mL/L. Trace metal and vitamin solutions were prepared separately and filter-sterilized before addition to the fermentation batch medium. The final concentrations of the trace metals in the fermentation batch medium were:

FeS0 ₄ ^» 7H ₂ 0, 206 mg/L; ZnS0 ₄ ^» 7H ₂ 0, 5.8 mg/L; CuS0 ₄ ^» 7H ₂ 0, 1.6 mg/L; NaMo ^» 2H ₂ 0, 4.8 mg/L; MnCl ₂ , 2.6 mg/L; CoCl ₂ ^» 6H ₂ 0, 4.8 mg/L. The final concentrations of the vitamins in the fermentation batch medium are: thiamine-HCl, 1.8 mg/L; calcium pantothenate, 1.8 mg/L; biotin, 0.5 mg/L; inositol, 9 mg/L; pyridoxine-HCl, 1.8 mg/L.

Yeast inoculum for the production fermentation was prepared by propagation of yeast from small starter vials. A sufficient quantity of yeast was generated to enable rapid fermentation in large production fermenters. Seed banks used for inoculation of the seed shake flask were stored at -80 °C with 25% (v/v) glycerol as a cryoprotectant. The seed for a main fermentation was initiated with a shake flask culture inoculated with one or more cryovials (2 mL) obtained from the seed bank. The cryovials were thawed, and the contents were added to the shake flask containing 250 mL of SD-THUL medium in a 1.0 L flask. SD- THUL medium contained 20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids (Difco™, New Jersey, USA), and 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan, and uracil (Difco™, New Jersey, USA).

The shake flasks were incubated with agitation of 250 RPM and temperature of 28° C for 48 hrs. A targeted cell density of the shake flask culture for transfer to the main fermenter is optical density of -10 at 600 nm. The 250 mL shake flask inoculum was transferred to a 15L fermenter (New Brunswick) containing 8L of the fermentation batch medium. Agitation was set at 1100 RPM and aeration is set at 8 slpm (standard liter per minute). The pH of the fermenter vessel was maintained at 5.0 ± 0.1 and controlled with 28% ammonium hydroxide solution. The temperature of the production vessel was maintained at 28 ± 1°C during cultivation. Samples were taken to measure OD, glucose, and ethanol. The fermentation was run in a fed-batch mode where 50% w/w glucose feed solution was added continuously once the batch glucose level dropped to 5 g/L. The glucose feed was added at rates to minimize fermentation by-products formation. The fermentation was run for several days, and the whole broth was harvested when the cell density reached OD ₆ oo of 300. The harvested fermentation broth contained -10% cell biomass, -7% oil fraction containing epi-isozizaene, and -83% water.

C. Assessment of epi-isozizaene

To isolate the epi-isozizaene/oil fraction from the cell biomass and water in the fermentation broth, the agitation and air were turned off, and the oil was allowed to separate and float to the top. A final separation was done by removing the oil layer and centrifuging at 4,000 rpm for 20 minutes at room temperature. The oil fraction contained the epi- isozizaene, soybean oil, and fatty acids.

The epi-isozizaene was purified from the soybean oil by passage through a wiped-film evaporator (WFE, 2-inch wiped-film distillation system made by Pope Scientific, WI, USA). The oil was first run through the WFE and degassed at 1 - 2 Torr at room temperature to remove residual solvent. The epi-isozizaene was then distilled and collected by operating the WFE at 1 -2 Torr and 180°C. The flow rate was maintained at 3 - 5 mL per min.

Column chromatographic separation of epi-isozizaene was performed to remove non- sesquiterpene and oxygenated molecules. A 52 cm X 4.0 cm (i.d.) slurry column was packed with silica (200 g) and hexane. Crude material (40.3 g) was adsorbed onto the column, and the epi-isozizaene (hydrocarbon fraction) was eluted with 2% ethyl acetate in hexane (-350 mL), until the eluate contained no further material as determined by thin layer

chromatography (TLC). The oxygenated sesquiterpenes were eluted with 10% ethyl acetate in hexane (500 mL). Finally the column was stripped with 50% ethyl acetate in hexane (250 mL). Solvent was evaporated under vacuum and 27.9 g epi-isozizaene (95% GC AUC) was isolated from hydrocarbon fraction. Figure 5 shows a GC-FID chromatogram from an epi- isozizaene producing strain, showing the product profile of the purified sample.

EXAMPLE 2. PRODUCTION OF 3-THUJOPSENE

A. Generating a 3-thujopsene producing strain

To produce 3-thujopsene (see Figures 6 and 7), either the plasmid pAlx68-14.3 or pAlx68-14.4, containing either the native At5g44630 (see Figure 8) or codon optimized gene (see Figure 9), respectively, were transformed into ALX11-30.1 (ura3, trpl, erg9def25, HMG2cat/TRP 1 : :rDNA, dppl, sue) strain of Saccharomyces cerevisiae using the lithium acetate yeast transformation kit (Sigma- Aldrich). ALX11-30.1 is derived from CALI5-1 (ura3, leu2, his3, trpl, Aerg9::HIS3, HMG2cat/TRP 1 : : rDNA, dppl, sue) (Takahashi, 2007) by three engineering steps. First, the leu2 mutation was restored to wild type by

transformation of a LEU2 gene fragment into CALI5-1 to create ALX7-95. Next, the erg9def25 mutant gene was introduced into ALX7-95 to restore ERG9 protein production, allowing the strain to grow in the absence of feeding ergosterol. Finally, the wild type HIS3 gene was introduced to make the strain prototrophic for histidine. This strain is designated ALX11-30.

B. Production of 3- thujopsene

Transformants with pAlx68-14.3 or pAlx68-14.4 (see Figure 10) were selected on SDE-ura medium (0.67 % Bacto™ yeast nitrogen base without amino acids, 2 % glucose, 0.14 % yeast synthetic drop-out medium supplement without uracil). Colonies were picked and screened for thujopsene production using a microculture assay in 96 deep well plates. Transformant yeast colonies were inoculated into individual wells of 96-well microtiter plates filled with 200 of SDE. The plate was grown for two to three days at 28°C. After growth to saturation, \0 μΐ. from each well was used to inoculate a 96 deep well plate containing 300 of medium suitable for growth and thujopsene production. After three days of growth and production, thujopsene was extracted first by introducing 250 of acetone vortexing, followed by addition of 500 μΐ. of n-hexane and vortexing. After phase separation, the plate was sealed with aluminum tape and placed on the sample tray of a gas chromatography autosampler. A one microliter sample was injected into the GC. The acetone and hexane used for extraction were each spiked with internal standards to aid in quantitation of the samples. The extracted samples were analyzed by gas chromatography and the amount of thujopsene was calculated from the peak area.

Production of thujopsene was performed in a 15-L fermentation tank (New Brunswick Bioflow 110). Eight liters of fermentation medium was prepared and autoclaved. The medium was composed of glucose, 20 g/L; (NH ₄ ) ₂ S0 ₄ , 20 g/L; KH ₂ P0 ₄ , 28 g/L;

MgS0 ₄ ^» 7H ₂ 0, 12 g/L; yeast extract, 5 g/L; soybean oil, 100 mL/L; Foam Blast Fl 11-GF (Emerald Foam Materials®, WY, USA), 5 mL/L. Trace metal and vitamin solutions were prepared separately and filter-sterilized before addition to the fermentation batch medium. The final concentrations of the trace metals in the fermentation batch medium were:

FeS0 ₄ ^» 7H ₂ 0, 206 mg/L; ZnS0 ₄ ^» 7H ₂ 0, 5.8 mg/L; CuS0 ₄ ^» 7H ₂ 0, 1.6 mg/L; NaMo ^» 2H20, 4.8 mg/L; MnC^, 2.6 mg/L; CoC^eL^O, 4.8 mg/L. The final concentrations of the vitamins in the fermentation batch medium were: thiamine-HCl, 1.8 mg/L; calcium pantothenate, 1.8 mg/L; biotin, 0.5 mg/L; inositol, 9 mg/L; pyridoxine-HCl, 1.8 mg/L.

Yeast inoculum for the production fermentation was prepared by propagation of yeast from small starter vials. A sufficient quantity of yeast was generated to enable rapid fermentation in large production fermenters. Seed banks used for inoculation of the seed shake flask were stored at -80 °C with 25% (v/v) glycerol as a cryoprotectant. The seed for a main fermentation was initiated with a shake flask culture inoculated with one or more cryovials (2 mL) obtained from the seed bank. The cryovials were thawed and the contents were added to the shake flask containing 250 mL of SD-THUL medium in a 1L flask. SD- THUL medium contained 20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids (Difco™, New Jersey, USA), and 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan, and uracil (Difco™, New Jersey, USA).

The shake flasks were incubated with agitation of 250 RPM and temperature of 28° C for 48 hrs. A targeted cell density of the shake flask culture for transfer to the main fermenter is optical density of -10 at OD ₆ oo- The 250 mL shake flask inoculum was transferred to a 15L fermenter (New Brunswick) containing 8L of the fermentation batch medium. Agitation was set at 1 100 RPM and aeration was set at 8 slpm. The pH of the fermenter vessel was maintained at 5.0 ± 0.1 and controlled with 28% ammonium hydroxide solution. The temperature of the production vessel was maintained at 28 ± 1°C during cultivation. Samples were taken to measure OD, glucose, and ethanol.

The fermentation was run in a fed-batch mode where 50% w/w glucose feed solution was added continuously once the batch glucose level dropped to 5 g/L. The glucose feed was added at rates to minimize fermentation by-products formation. The fermentation was run for several days and the whole broth was harvested when the cell density reached OD ₆ oo of 300. The harvested fermentation broth contained -10% cell biomass, -7% oil fraction containing thujopsene, and -83% water.

C. Assessment of thujopsene

To isolate the thujopsene/oil fraction from the cell biomass and water in the fermentation broth, the agitation and air were turned off, and the oil was allowed to separate and float to the top. A final separation was done by removing the oil layer and centrifuging at 4,000 rpm for 20 minutes at room temperature. The oil fraction contained the thujopsene, soybean oil, and fatty acids.

The thujopsene was purified from the soybean oil by passage through a wiped-film evaporator (WFE, 2-inch wiped-film distillation system made by Pope Scientific, WI, USA). The oil was first run through the WFE and degassed at 1 - 2 Torr at room temperature to remove residual solvent. The thujopsene was then distilled and collected by operating the WFE at 1 -2 Torr and 180°C. The flow rate was maintained at 3 - 5 mL per min.

Column chromatographic separation of thujopsene was performed to remove non- sesquiterpene and oxygenated molecules. The Pope distilled material containing (-)-3- thujopsene was purified on silica gel with an isocratic eluent mixture of n-hexane and ethyl acetate (98:2). The fractions containing the product were identified by thin layer chromatography (TLC) on using mixture of hexanes and ethyl acetate (8:2) for development and KMnC>4 stain. Solvent was evaporated under vacuum and the purified thujopsene contained a-barbatene (24%), thujopsene (17%), acoradiene (11%), β-chamigrene (10%), a- cuparene (7%), and other sesquiterpenes (31%). Figure 11 shows a GC-FID chromatogram from a thujopsene producing strain, showing the product profile in fermentor broth extract, distillate and purified samples.

EXAMPLE 3. PRODUCTION OF LONGIFOLENE

A. Generating a longifolene producing strain

To produce longifolene (see Figures 12 and 13, the plasmid pAlx68-14.7 or pAlx68-

14.8, containing the wild type (see Figure 14) or codon optimized (see Figure 15) PsTPS3 gene respectively, was transformed into ALX11-30.1 (ura3, trpl, erg9def25,

HMG2catlTRP lv.rDNA, dppl, sue) strain of Saccharomyces cerevisiae using the lithium acetate yeast transformation kit (Sigma- Aldrich). ALX11-30.1 is derived from CALI5-1 (ura3, leu2, his3, trpl, Aerg9: :HIS3, HMG2catl TRP1 : DNA, dppl, sue) (Takahashi, 2007) by three engineering steps. First, the leu2 mutation was restored to wild type by

transformation of a LEU2 gene fragment into CALI5-1 to create ALX7-95. Next, the erg9def25 mutant gene was introduced into ALX7-95 to restore ERG9 protein production, allowing the strain to grow in the absence of feeding ergosterol. Finally, the wild type HIS3 gene was introduced to make the strain prototrophic for histidine. This strain is designated ALX11-30.

B. Production of longifolene Transformants with pAlx68-14.7 or pAlx68-14.8 {see Figure 16) were selected on SD-ura medium (0.67 % Bacto™ yeast nitrogen base without amino acids, 2 % glucose, 0.14 % yeast synthetic drop-out medium supplement without uracil). Colonies were picked and screened for longifolene production using a microculture assay in 96 deep well plates.

Transformant yeast colonies were inoculated into individual wells of 96-well microtiter plates filled with 300 of SDE. The plate was grown for two to three days at 28°C. After growth to saturation, 10 from each well was used to inoculate a 96 deep well plate containing 300 of medium suitable for growth and longifolene production. After three days of growth and production, longifolene was extracted first by introducing 250 of acetone vortexing, followed by addition of 500 μί of n-hexane and vortexing. After phase separation, the plate was sealed with aluminum tape and placed on the sample tray of a gas chromatography autosampler. A one microliter sample was injected into the GC. The acetone and hexane used for extraction were each spiked with internal standards to aid in quantitation of the samples. The extracted samples were analyzed by gas chromatography and the amount of longifolene was calculated from the peak area.

Production of longifolene was performed in a 15-L fermentation tank (New

Brunswick Bioflow 110). Eight liters of fermentation medium was prepared and autoclaved. The medium was composed of glucose, 20 g/L; (NH ₄ ) ₂ S0 ₄ , 20 g/L; KH ₂ P0 ₄ , 28 g/L;

MgS0 ₄ »7H ₂ 0, 12 g/L; yeast extract, 5 g/L; soybean oil, 100 mL/L; Foam Blast® Fl 11-GF (Emerald Foam Materials®, WY, USA), 5 mL/L. Trace metal and vitamin solutions were prepared separately and filter-sterilized before addition to the fermentation batch medium. The final concentrations of the trace metals in the fermentation batch medium were:

FeS0 ₄ ^» 7H ₂ 0, 206 mg/L; ZnS0 ₄ ^» 7H ₂ 0, 5.8 mg/L; CuS0 ₄ ^» 7H ₂ 0, 1.6 mg/L; NaMo ^» 2H ₂ 0, 4.8 mg/L; MnCl ₂ , 2.6 mg/L; CoCl ₂ ^» 6H ₂ 0, 4.8 mg/L. The final concentrations of the vitamins in the fermentation batch medium are: thiamine-HCl, 1.8 mg/L; calcium pantothenate, 1.8 mg/L; biotin, 0.5 mg/L; inositol, 9 mg/L; pyridoxine-HCl, 1.8 mg/L.

Yeast inoculum for the production fermentation was prepared by propagation of yeast from small starter vials. A sufficient quantity of yeast was generated to enable rapid fermentation in large production fermenters. Seed banks used for inoculation of the seed shake flask were stored at -80°C with 25% (v/v) glycerol as a cryoprotectant. The seed for a main fermentation was initiated with a shake flask culture inoculated with one or more cryovials (2 mL) obtained from the seed bank. The cryovials were thawed, and the contents were added to the shake flask containing 250 mL of SD-THUL medium in a 1.0 L flask. SD- THUL medium contained 20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids (Difco™, New Jersey, USA), and 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan, and uracil (Difco™, New Jersey, USA).

The shake flasks were incubated with agitation of 250 RPM and temperature of 28°C for 48 hrs. A targeted cell density of the shake flask culture for transfer to the main fermenter is optical density of -10 at OD ₆ oo- The 250 mL shake flask inoculum was transferred to a 15L fermenter (New Brunswick) containing 8L of the fermentation batch medium. Agitation was set at 1100 RPM, and aeration is set at 8 slpm. The pH of the fermenter vessel was maintained at 5.0 ± 0.1 and controlled with 28% ammonium hydroxide solution. The temperature of the production vessel was maintained at 28 ± 1 °C during cultivation.

Samples were taken to measure OD, glucose, and ethanol.

The fermentation was run in a fed-batch mode where 50% w/w glucose feed solution was added continuously once the batch glucose level dropped to 5 g/L. The glucose feed was added at rates to minimize fermentation by-products formation. The fermentation was run for several days and the whole broth was harvested when the cell density reached OD ₆ oo of 300. The harvested fermentation broth contained -10% cell biomass, -7% oil fraction containing longifolene, and -83% water.

C. Assessment of longifolene

To isolate the longifolene/oil fraction from the cell biomass and water in the fermentation broth, the agitation and air were turned off, and the oil was allowed to separate and float to the top. A final separation was done by removing the oil layer and centrifuging at 4,000 rpm for 20 minutes at room temperature. The oil fraction contained the longifolene, soybean oil, and fatty acids.

The longifolene was purified from the soybean oil by passage through a wiped-film evaporator (WFE, 2-inch wiped-film distillation system made by Pope Scientific, WI, USA). The oil was first is run through the WFE and degassed at 1 - 2 Torr at room temperature to remove residual solvent. The longifolene was then distilled and collected by operating the WFE at 1 - 2 Torr and 180°C. The flow rate was maintained at 3 - 5 mL per min.

Column chromatographic separation of longifolene was performed to remove non- sesquiterpene and oxygenated molecules. Column chromatographic separation from Pope distilled crude material was performed on a 24 cm X 7.0 cm (i.d.) column, slurry packed with silica (500 g) in hexane. Crude material was adsorbed onto the column, and gradient solvent system composed of hexane and ethyl acetate used for effective elution. The terpene fraction was eluted with 2% ethyl acetate in hexane, until the eluate contained no further material as determined by TLC. Solvent was evaporated under vacuum and longifolene was isolated. Longifolene constituted 81.6% of the terpene mixture. Figure 17 shows GC-FID

chromatogram for longifolene producing strain, showing the product profile in fermentor broth, distillate and purified samples.