# Patent

## Title
Modified microorganisms and use thereof for terpene production

## Abstract

The present invention relates to the control of gene expression by a heterologous glucose-regulated promoter, to microorganisms in which gene expression is controlled by a heterologous glucose-regulated promoter and to methods using said microorganisms for the production of terpenes during glucose-limited fed-batch fermentation.

## Background
TECHNICAL FIELD
The present invention relates to the control of gene expression by a heterologous glucose-regulated promoter, to microorganisms in which gene expression is controlled by a heterologous glucose-regulated promoter and to methods using said microorganisms for the production of terpenes during glucose-limited fed-batch fermentation.
PRIOR ART
Terpenes are found in most organisms (microorganisms, animals and plants). These compounds are made up of five carbon units called isoprene units and are classified by the number of these units present in their structure. Thus monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms respectively. Sesquiterpenes, for example, are widely found in the plant kingdom. Many monoterpene, sesquiterpene and diterpene molecules are known for their flavor and fragrance properties and their cosmetic, medicinal and antimicrobial effects. Over 300 sesquiterpene hydrocarbons and 3,000 sesquiterpenoids have been identified and many new structures are identified each year. Plant extracts obtained by different means such as steam distillation or solvent extraction are used as source of terpenes. Terpene molecules are often used as such, but in some cases chemical reactions are used to transform the terpenes into other high value molecules.
Biosynthetic production of terpenes involves enzymes called terpene synthases. There is virtually an infinity of terpene synthases present in the plant kingdom, all using acyclic terpene precursors (mainly geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) or geranyl geranyl pyrophosphate (GGPP)) but having different product profiles. Genes and cDNAs encoding terpene synthases have been cloned and the corresponding recombinant enzymes characterized. The biosynthesis of terpenes in microorganisms has been extensively studied and several methods involving modification of the microorganism metabolism in order to increase terpene production have been developed.
In order to improve sesquiterpene production, several strategies were applied to augment the intracellular pool of the acyclic terpene precursor FPP available for terpene synthases. For example in yeast, as a large part of the FPP produced goes into the sterol producing pathway, several strategies have been developed to reduce the activity of the ERG9 gene, which codes for the squalene synthase (the first committed enzyme in sterol biogenesis).
Deletion of ERG9 has been envisaged. However, mutants in which ERG9 is deleted are unable to synthesize sterols. Such transformation therefore has the disadvantage that the obtained yeast strains are not viable unless they are fed with sterols.
In order to avoid deletion of the ERG9 gene, ERG9 mutants with a reduced activity have been used, for example in WO 2010/019696. However, even if the activity of ERG9 is sufficient to avoid sterols feeding, the reduced activity of ERG9 is detrimental to the yeast growth, the growth being considerably slowed down.
Another approach was to down-regulate ERG9 by replacing its native promoter by PMET3, a yeast L-methionine regulatable promoter. This strategy has been shown to enhance sesquiterpene production in yeast (Ro et al., Nature 2006, 440:940). However, this method has the disadvantage of requiring supplementing the growth medium with various concentrations of L-methionine depending on the desired level of ERG9 activity. The cost of L-methionine and its possible consumption by the cell constitute important drawbacks of such methods. In particular, the difficulties in controlling the METS promoter activity when the repressing agent L-methionine is metabolized by the cells are evidenced in comparative Example 6 of the present application.
It is an objective of the present invention to modulate expression of enzymes consuming terpene precursors in microorganisms, for example ERG9, in a way that enables optimal growth of the microorganism as well as maximal selective reduction of the desired gene activity during terpene production and that does not require supplementing the growth medium with additional feeding such as L-methionine or sterols.
SUMMARY OF THE INVENTION
In a first aspect, the invention provides a microorganism expressing at least one terpene synthase and comprising at least one endogenous gene encoding a protein that is not a terpene synthase, characterized in that

    
    
        the native promoter of the endogenous gene has been replaced by a heterologous glucose-regulated promoter;
        said glucose-regulated promoter is induced by high glucose concentrations and repressed by low glucose concentrations; and
        said endogenous gene encodes a protein that uses an acylic terpene precursor as substrate.
    
    


In another aspect, the invention provides a glucose-limited fed batch fermentation process comprising cultivating a microorganism of the invention.
In a third aspect, the invention provides a method for producing a terpene compound comprising cultivating a microorganism of the invention in a glucose-limited fed batch fermentation process, the terpene synthase expressed by said microorganism being capable of catalysing the formation of said terpene compound.
In a fourth aspect, the present invention provides a method for increasing the production of a terpene compound in a microorganism comprising:

    a) modifying said microorganism to replace the native promoter of an endogenous gene encoding a protein that is not a terpene synthase and that uses an acyclic terpene precursor as substrate by a glucose-regulated promoter, said glucose regulated promoter being induced by high glucose concentrations and repressed by low glucose concentrations, provided that said microorganism further expresses at least one terpene synthase capable of catalysing the formation of said terpene compound; and
    b) cultivating the transformed microorganism obtained in step a) in a glucose-limited fed-batch process.





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Schematic drawing of the cloning strategy for the ERG9 promoter substitution.
FIG. 2: LacZ activity in strain SCICK10 (PMET3-lacZ) in response to different methionine concentrations, 0 mM (diamond), 1 mM (triangle) and 2 mM (circle), added respectively at 0 hours (A) and 8 hours (B) of cultivation. Arrows represent the time point of methionine addition.
FIG. 3: Ergosterol production rate (g·g biomass−1·h−1) (A) and α-(+)-santalene and E,E-farnesol production rate (g·g biomass−1·h−1) (B) in strains SCICK13 (PERG9), SCICK15 (PMET3) and SCICK12(PHXT1).



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a microorganism expressing at least one terpene synthase and comprising at least one endogenous gene encoding a protein that is not a terpene synthase wherein

    
    
        the native promoter of the endogenous gene has been replaced by a heterologous glucose-regulated promoter;
        the glucose-regulated promoter is induced by high glucose concentrations and repressed by low glucose concentrations; and
        the endogenous gene encodes a protein that uses an acylic terpene precursor as substrate.
    
    


Any microorganism that can be cultivated in a glucose-containing medium, such as bacteria or yeast, is encompassed by the present invention. Preferably, the microorganism is yeast, more preferably it is selected from yeast of the Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia and Torulopsis genera. These yeast genera have the advantage of being industrially transformable and cultivable. Even more preferably it is yeast of the Saccharomyces genus and most preferably Saccharomyces cerevisiae. 
For the purpose of the present invention, the microorganism must express at least one terpene synthase. As “terpene synthase” it is intended here any enzyme that is capable of catalysing the transformation of an acyclic terpene precursor into a terpene compound. The terpene compound can be produced as a single product or as part of a mixture of products. Preferably said terpene synthase is a monoterpene synthase (i.e. that is capable of catalysing the transformation of GPP to at least one monoterpene compound), a sesquiterpene synthase (i.e. that is capable of catalysing the transformation of FPP to a sesquiterpene compound) or a diterpene synthase (i.e. that is capable of catalysing the transformation of GGPP to a diterpene compound). Said terpene synthase must be functional, so that the microorganism is capable of catalysing the synthesis of a terpene compound from an acyclic terpene precursor using such terpene synthase. The terpene synthase can be either endogenous or exogenous and can be selected among all terpene synthases as defined above. In particular it can preferably be a monoterpene synthase, a sesquiterpene synthase or a diterpene synthase, more preferably it is a sesquiterpene synthase.
Examples of preferred sesquiterpene synthases include α-santalene synthase, patchoulol synthase, β-santalene synthase, valencene synthase, cubebol synthase, zizaene synthase, amorpha 4,11-diene synthase, humulene synthase, aristolochene synthase, bergamotene synthase, zingiberene synthase, farnesene synthase, caryophyllene synthase, isodaucene synthase, sesquithujene synthase, avermitilol synthase, eudesmol synthase, vetispiradiene synthase, longifolene synthase, cyclocopacamphene synthase, isolongifolene synthase, germacrene synthase, bicyclogermacrene synthase, bisabolol synthase, germacradienol synthase, hedycaryol synthase, barbatene synthase, epi-cedrol synthase, epi-aristolochene synthase, sesquisabinene synthase, cuprene synthase, selinene synthase, copaene synthase, macrocarpene synthase, cadinol synthase, intermedeol synthase, nerolidol synthase, muurola-3,5-diene synthase, curcumene synthase and epi-beta santalene synthase. More preferred sesquiterpene synthases include α-santalene synthase, patchoulol synthase, β-santalene synthase, valencene synthase, cubebol synthase, zizaene synthase, amorpha 4,11-diene synthase. Even more preferably, the sesquiterpene synthase is a patchoulol synthase or an α-santalene synthase. Most preferably, it is an α-santalene synthase.
Examples of preferred diterpene synthases include sclareol synthase, labdendiol synthase and taxadiene synthase. Examples of preferred monoterpene synthases include limonene synthase, pinene synthase, myrcene synthase, camphene synthase, phellandrene synthase, terpinolene synthase, ocimene synthase, linalool synthase, cineole synthase, geraniol synthase, terpinene synthase, fenchol synthase, carene synthase, sabinene synthase.
The endogenous gene of which the natural promoter is substituted is naturally present in the microorganism and encodes a protein that uses an acyclic terpene precursor as substrate. Such “acyclic terpene precursor” is intended as any precursor that is converted to terpene compounds under the action of a terpene synthase. Preferably it is geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) or geranyl geranyl pyrophosphate (GGPP). Most preferably, the acyclic terpene precursor is FPP.
As a “protein using an acyclic terpene precursor as substrate” it is intended for the purpose of the present invention any polypeptide that is capable of catalysing a reaction in which an acyclic terpene precursor is the starting material. A variety of genes having such an activity are found in microorganisms. The acyclic terpene precursor can be transformed chemically in different ways, depending on the protein that is involved in its transformation. Such proteins differ from one microorganism to the other. However, such proteins are well-known to the person skilled in the art. Examples of such proteins that can be found for example in yeast include prenyl transferases, farnesyltransferase, cis-prenyltransferase or squalene synthase.
Any gene that encodes a protein that uses such an acyclic terpene precursor as substrate can be modified for the purpose of the invention. However, said gene must not code for a terpene synthase. This ensures that modification of the gene expression enables to down-regulate the activity of said gene, while maintaining the highest possible activity of terpene synthases expressed by such microorganism.
In a preferred embodiment of the invention, the promoter-modified gene is a gene encoding a squalene synthase. For the purpose of the present invention a “squalene synthase” is defined as an enzyme capable of producing squalene from farnesyl pyrophosphate (FPP).
However, the promoter-modified gene can also be any other gene encoding for a protein that uses an acyclic terpene precursors as substrate. For example, when the microorganism is S. cerevisiae, the endogenous promoter-modified gene can be selected from genes encoding the prenyl transferases RAM1 or RAM2, the protein Coq1, the farnesyltransferase COX10, the cis-prenyltransferases SRT1 or RER2 or the squalene synthase ERG9. Most preferably it encodes ERG9. All of these specific enzymes are endogenous in Saccharomyces cerevisiae and use FPP as substrate.
The genetic modification of the gene consists in replacing its native promoter by a glucose-regulated promoter that is induced by high glucose concentrations and repressed by low glucose concentrations. For the purpose of the present invention, high glucose concentrations are defined as glucose concentrations of at least 1 gram of glucose per liter of culture medium and low glucose concentration as glucose concentrations below 1 gram of glucose per liter of culture medium. Preferably high glucose concentrations are defined as glucose concentrations of at least 10 grams of glucose per liter of culture medium and low glucose concentration as glucose concentrations below 1 gram of glucose per liter of culture medium.
The activity of the heterologous promoter is regulated by the concentration of the glucose present in the medium in which the microorganism is cultivated. These concentrations are particularly compatible with the use of the genes of the present invention in microorganisms cultivated in glucose-limited fed-batch fermentation processes, in which such high concentration of glucose are present in the growth medium during the batch phase, thus allowing for high activity of the regulated gene and rapid increase in biomass. Conversely, the promoter provides a reduced level of the regulated protein at low, growth-limiting glucose concentrations, allowing more acyclic terpene precursor, as defined in any of the above-described embodiments, to be utilized for the production of terpene compounds. Concentrations of glucose of less than 1 g per liter of culture medium are compatible with the low amounts of glucose present in the culture medium during the feeding phase of the fed-batch fermentation process because during that phase the glucose feeding is such that all the glucose fed to the medium is consumed by the microorganism, so that the concentration in the medium is close to 0 g/l.
The glucose-regulated promoter is heterologous. For the purpose of the present invention, a “heterologous promoter” means that in the modified organism such promoter regulates the expression of a gene that it does not regulate in the native microorganism. The promoter can originate from the same microorganism or from another microorganism. In the case where it originates from the same microorganism, it can be responsible for the regulation of the expression of any other gene of said microorganism. In a preferred embodiment, the glucose-regulated promoter originates from the same microorganism or from an organism having a similar philogeny. In a more preferred embodiment, the glucose-regulated promoter originates from the same microorganism.
Preferred promoters for use in the present invention are promoters that are found naturally in the microorganism in which the gene is present, but which regulate the activity of a different gene. More preferably, the promoter is found in yeast, even more preferably in a yeast of the Saccharomyces genus and most preferably in Saccharomyces cerevisiae. Most preferred promoters for use in the present invention are the hexose transporters 1 (HXT1), 3 (HXT3) and 4 (HXT4). Preferably, it is HXT1. Such promoters are extensively described in J Biol Chem 1999, 274:15350-15359.
The microorganism can optionally have undergone any other modification of its metabolism, and in particular of its terpene production metabolism. For example its native pathway leading to the formation of the acyclic terpene precursor can have been the object of genetic engineering in order to increase the amount of acyclic terpene precursor produced. As another example, a heterologous pathway leading to the production of such acyclic terpene precursor can have been introduced by genetic engineering into said microorganism. These genetic modifications may include but are not limited to: overexpression of a farnesyl diphosphate synthase (for example ERG20 in S. cerevisiae), overexpression of the catalytic domain of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), overexpression of upc2-1, a semi-dominant mutant allele that enhances the activity of UPC2, a global transcription factor regulating sterol biosynthesis in S. cerevisiae, deletion of the genes encoding lipid phosphatases active on farnesyl diphosphate (suh as Lpp1 and Dpp1 in S. cerevisiae), deletion of the NADPH-dependent glutamate dehydrogenase encoded by GDH1 and simultaneous overexpression of the NADH-dependent glutamate dehydrogenase encoded by GDH2.
The microorganisms of the present invention are particularly advantageously cultivated using a glucose-limited fed-batch fermentation process. A glucose-limited fed-batch fermentation process comprising cultivating a microorganism of the invention, as defined in any of the above-described embodiments is therefore another object of the present invention.
Glucose-limited fed-batch fermentation processes are well-known to the person skilled in the art, so that it needs not be explained extensively in the present description. In any case, examples of conditions that can be applied for such type of fermentation are described in full details in the Examples of the present application. For the purpose of the present invention, a glucose-limited fed-batch fermentation process is intended as any fermentation process in which the microorganism is first grown in a batch phase in the presence of high, non-limiting concentrations of glucose, thus allowing rapid biomass formation, this phase being followed by a feeding phase in which the biomass is cultivated in the presence of low, growth-limiting glucose concentrations. Preferably, the concentration of glucose in the culture medium used in the batch phase is of at least 1 g/l, more preferably at least 10 g/l whereas the concentration of glucose in the culture medium used in the feeding phase is of less than 1 g/l.
The microorganism of the present invention, as defined in any of the above-described embodiments is also advantageously used in a method for producing a terpene compound comprising cultivating a microorganism of the invention in a glucose-limited fed batch fermentation process, as defined above. In such method the terpene synthase expressed by the microorganism is capable of catalysing the formation of the terpene compound that is intended to be produced. Such terpene synthase is as defined above in any embodiment of the invention. Most preferably, it is a sesquiterpene compound. Examples of sesquiterpene compounds include α-santalene, patchoulol, β-santalene, valencene, cubebol, zizaene, amorpha 4,11-diene, humulene, aristolochene, bergamotene, zingiberene, farnesene, caryophyllene, isodaucene, sesquithujene, avermitilol, eudesmol, vetispiradiene, longifolene, cyclocopacamphene, isolongifolene, germacrene, bicyclogermacrene, bisabolol, germacradienol, hedycaryol, barbatene, epi-cedrol, epi-aristolochene, sesquisabinene, cuprene, selinene, copaene, macrocarpene, cadinol, intermedeol, nerolidol, muurola-3,5-diene, curcumene and epi-beta santalene. More preferred sesquiterpene include α-santalene, patchoulol, β-santalene, valencene, cubebol, zizaene, amorpha 4,11-diene. Even more preferably, the sesquiterpene is patchoulol or α-santalene. Most preferably, it is α-santalene.
Examples of preferred diterpenes include sclareol, labdendiol and taxadiene. Examples of preferred monoterpenes include limonene, pinene, myrcene, camphene, phellandrene, terpinolene, ocimene, linalool, cineole, geraniol, terpinene, fenchol, careen and sabinene.
In a preferred embodiment of the invention, the method for producing a terpene compound comprises the following steps:

    a) transforming a microorganism to replace the native promoter of an endogenous gene encoding a protein that uses an acyclic terpene precursor as substrate by a glucose-regulated promoter, said glucose regulated promoter being induced by high glucose concentrations and repressed by low glucose concentrations, provided that said gene does not encode a terpene synthase and provided that said microorganism further expresses at least one terpene synthase capable of catalysing the formation of said terpene compound;
    b) cultivating the transformed microorganism obtained in step a) in a glucose-limited fed-batch fermentation process, so that the microorganism produces the terpene compound; and
    c) optionally isolating the terpene compound produced in step b)


In another embodiment, the invention provides for a method for increasing the production of a terpene compound in a microorganism comprising:

    a) transforming said microorganism to replace the native promoter of an endogenous gene encoding a protein that uses an acyclic terpene precursor as substrate by a glucose-regulated promoter, said glucose regulated promoter being induced by high glucose concentrations and repressed by low glucose concentrations, provided that said gene does not encode a terpene synthase and provided that said microorganism further expresses at least one terpene synthase capable of catalysing the formation of said terpene compound; and
    b) cultivating the transformed microorganism obtained in step a) in a glucose-limited fed-batch fermentation process so that the microorganism produces the terpene compound.


In such method, the microorganism, the terpene compound, the gene, the promoter and the terpene synthase are all as defined in any of the above-described embodiment of the invention.
EXAMPLES
Example 1
ERG9 Promoter Replacement by the MET3 Promoter (Comparative Example)
The MET3 promoter was amplified from genomic DNA of S. cerevisiae CEN.PK 113-7D (MATa MAL2-8c SUC2 ura3-52) (BMC Genomics 2010 11:723) using the primers 1 and 2 (SEQ ID NO:1 and SEQ ID NO:2). The PCR conditions were in accordance with the Expand High Fidelity standard conditions (Roche Applied Science, Mannheim, Germany). Subsequently, the PCR fragments were digested by the restriction enzymes SpeI and SacII. In parallel, pUG6 plasmid (Nucleic Acids Res 1996, 24:2519-2524) was digested by the same couple of restriction enzymes. The DNA fragments were separated by gel electrophoresis and gel purified using the QIAEX1 II Gel extraction kit (Qiagen, Hilden, Germany). In vitro ligation of the digested plasmid with the digested PCR products was performed as the standard procedure given for T4 DNA ligase (Roche Applied Science). The resulting ligation mixture was used to transform chemically competent E. coli cells (DH5α) (Gene 1990, 96:23-28). Transformants were selected on LB medium supplemented with ampicillin (50 mg/L). The plasmid obtained was named pIP007.
The ERG9 gene is designated as YHR190W within the genome of CEN.PK 113-7D, as published in BMC Genomics 2010 11:723. The amino acid sequence of ERG9 encoded by this nucleic acid is published in GenBank, accession number: AAA34597.
In order to replace the native ERG9 promoter by the MET3 promoter, fusion PCR and a bipartite gene targeting method (Genome Res 1997, 7:1174-1183) were used. Four fragments were separately amplified before fusing them together in pairs. First, two fragments containing the MET3 promoter and the KanMX selection cassette were amplified from pIP007 in two separate, but overlapping fragments using the two couples of primers: primers 3 and 4 (SEQ ID NO:3 and 4) (Fragment A (SEQ ID NO:35)) and primers 5 and 6 (SEQ ID NO:5 and 6) (Fragment B (SEQ ID NO:36)). Furthermore, 500 by upstream of the ERG9 promoter in the genome of S. cerevisiae CEN.PK 113-7D (BMC Genomics 2010 11:723) were amplified using primers 7 and 8 (SEQ ID NO:7 and 8) (Fragment C (SEQ ID NO:37)). The first 500 by of the ERG9 ORF were as well amplified using primers 9 and 10 (SEQ ID NO:9 and 10) (Fragment D (SEQ ID NO:38)). The resulting four PCR fragments were gel-purified using the High Pure PCR Product Purification kit (Roche Applied Science) and subsequently fused together in pairs using fusion PCR. Fused fragments A and C (SEQ ID NO:39) were obtained after a fusion PCR using primers 4 and 7 (SEQ ID NO:4 and 7) while fused fragments B and D (SEQ ID NO:40) were obtained after a fusion PCR using primers 5 and 10 (SEQ ID NO:5 and 10). The two final fusion PCR fragments were gel purified with the High Pure PCR Product Purification kit.
S. cerevisiae strain CEN.PK113-5D (BMC Genomics 2010 11:723) was transformed with the obtained fusion PCR fragments to result in strain YIP-M0-04 (MATa MAL2-8c SUC2 ura3-52 PERG9Δ::kanMX-PMET3). The loxP flanked kanMX cassette in strain YIP-M0-04 was then excised with the help of the Cre recombinase expression plasmid pSH47 as described by Güldener et al. (Nucleic Acids Res. 1996, 24:2519-2524).
To prevent the conversion of the isoprenoid precursor farnesyl pyrophosphate (FPP) into farnesol, the FPP phosphatase LPP1 (GenBank NM_001180811.1) was deleted in the YIP-M0-04 strain using a bipartite gene targeting strategy (J Biol Chem. 1999, 274:14831-14837; Genome Res 1997, 7:1174-1183). The 5′ and 3′ region of the LPP1 gene were amplified by PCR using primer pairs 11/12 (SEQ ID NO:11 and 12) (fragment 1 (SEQ ID NO:41)) and 13/14 (SEQ ID NO:13 and 14) (fragment 2 (SEQ ID NO:42)), respectively, and genomic DNA of CEN.PK113-5D as template (BMC Genomics. 2010 11:723). The 5′ and the 3′ part of the kanMX cassette were amplified from plasmid pUG6 using primer pairs 15/16 (SEQ ID NO:15 and 16) (fragment 3 (SEQ ID NO:43) and 17/18 (SEQ ID NO:17 and 18) (fragment 4 (SEQ ID NO:44)), respectively. Complementary primer tails allowed for the combination of fragments 1 and 3 (SEQ ID NO:45) by fusion PCR. Likewise, fragments 2 and 4 (SEQ ID NO:46) were fused to each other. Cells were transformed with both fusion PCR fragments and integration of the kanMX cassette at the LPP1 locus was tested by PCR using primers 19 and 20 (SEQ ID NO:19 and 20). Subsequent excision of the kanMX cassette led to formation of strain SCICK00 (MATa MAL2-8c SUC2 ura3-52 dpp1Δ::loxP PERG9Δ::loxP-PMET3).
Example 2
Construction of a PMET3 Reporter Strain
To construct an integrative plasmid carrying the reporter gene LacZ under the control of the MET3 promoter, the latter was amplified from genomic DNA of S. cerevisiae CEN.PK 113-7D using the primers 1′ and 2′ (SEQ ID NO:33 and 34), restricted with HindIII/NotI and ligated into HindIII/NotI cut pSF011 plasmid resulting in pSF011-PMET3 (Yeast 2010, 27: 955-964). A yeast strain carrying a genomic integration of LacZ under the control of the MET3 promoter was constructed by transforming CEN.PK 113-7D with the NcoI restricted integrative plasmid pSF011-PMET3 resulting in strain SCICK10 (MATa MAL2-8c SUC2 ura3-52::pSF011-PMET3).
Example 3
ERG9 Promoter Substitution by the HXT1 Promoter
The strain described above was used for further promoter substitutions using the strategy depicted in FIG. 1.
To replace the ERG9 controlling MET3 promoter in SCICK00, the ERG9 promoter and the HXT1 promoter were amplified from CEN.PK113-5D genomic DNA by PCR using primer pairs 21/22 (SEQ ID NO:21 and 22) and 23/24 (SEQ ID NO:23 and 24), respectively. In addition the kanMX cassette was amplified in a PCR reaction containing primers 25 and 26 (SEQ ID NO:25 and 26). The marker cassette was combined with either of the two promoters by fusion PCR and the resulting fragments were amplified once more using primers 27 and 28 (SEQ ID NO:27 and 28) in order to extend the flanking regions for genomic integration (ERG9 fusion (SEQ ID NO:47) and HXT1 fusion fragments (SEQ ID NO:48). After successful fusion, the fragments were cloned into pJET1.2 (Fermentas GMBH) and sequenced. Transformation of SCICK00 with these fusion PCR fragments and subsequent excision of the kanMX cassette resulted in strains SCICK01 (MATa MAL2-8c SUC2 ura3-52 dpp1Δ::loxP PERG9Δ::loxP-PHXT1) and SCICK03 (MATa MAL2-8c SUC2 ura3-52 dpp1Δ::loxP PERG9Δ::loxP-PERG9).
Example 4
Construction of an (+)-α-Santalene and tHMG1 Yeast Expression Vector
To construct the (+)-α-santalene expression vector, the santalene synthase cDNA was amplified by PCR from plasmid Cont2B-27-pET101 (WO 2009109597, GenBank accession number: HQ452480), using primers 29 and 30 (SEQ ID NO:29 and 30), cut with NotI/PacI and ligated into NotI/PacI restricted vector pSP-G1 (Yeast 2010, 27:954-965). Subsequently, tHMG1 was PCR amplified using genomic DNA of S. cerevisiae CEN.PK113-5D as template and primers 31 and 32 (SEQ ID NO:31 and 32), cut with BamHI/NheI and ligated into the same vector after restriction with BamHI and NheI (Nature 2006, 440:940-943). This resulted in formation of the expression plasmid pICK01. pICK01 was then transformed in SCICK00, SCICK01 and SCICK03 resulting in strains SCICK12 (MATa MAL2-8c SUC2 ura3-52 dpp1Δ::loxP PERG9Δ::loxP-PHXT1), SCICK13 (MATa MAL2-8c SUC2 ura3-52 dpp1Δ::loxP PERG9Δ::loxP-PERG9) and SCICK15 (MATa MAL2-8c SUC2 ura3-52 dpp1Δ::loxP PERG9Δ::loxP-PMET3), respectively.
Example 5
Glucose-Limited Fed Batch Cultivation
For batch cultivations, a previously described (Yeast 1992, 8:501-517) mineral salts medium was used consisting of the following: (NH4)2SO4 (5 g/l); KH2PO4 (3 g/l); MgSO4.7H2O (0.50 g/l); Antifoam 289 (Sigma-Aldrich) (0.050 ml/l); trace metals (1 ml/l) and vitamins 1.0 ml/l. The trace metal solution consisted of the following: EDTA (sodium salt) (15.0 g/l); ZnSO4.7H2O (0.45 g/l); MnCl2.2H2O (1 g/l); CoCl2.6H2O (0.3 g/l); CuSO4.5H2O (0.3 g/l); Na2MoO4.2H2O (0.4 g/l); CaCl2.2H2O (0.45 g/l); FeSO4.7H2O (0.3 g/l); H3BO3 (0.1 g/l) and KI (0.10 g/l). The pH of the trace metal solution was adjusted to 4.0 with 2M NaOH prior to heat sterilization. The vitamin solution consisted of the following: biotin (0.05 g/l); p-amino benzoic acid (0.2 g/l); nicotinic acid (1 g/l); Ca-pantothenate (1 g/l); pyridoxine-HCl (1 g/l); thiamine-HCl (1 g/l) and myo-inositol (25 g/l). The pH of the vitamin solution was adjusted to 6.5 with 2M NaOH. The vitamin solution was filter sterilized and stored at 4° C. This medium was supplemented with 30 g/l glucose. The feed composition used for fed-batch cultivation had the same composition as described above, but the (NH4)2SO4; KH2PO4; MgSO4.7H2O, vitamin solution, and trace metal solution, concentration were increased 10 times. The glucose concentration was 200 g/l.
The seed cultures for the cultivations were grown at 30° C. in 500 ml shake flasks containing 100 ml of culture with agitation in an orbital shaker at 100 rpm. Per-cultures were used to inoculate the fermentors to a final dry weight of 1 mg/l.
The aerobic fed-batch process was performed in 2.5 liter Applikon vessels (Applikon, Schiedam, the Netherlands) with a working volume of 1.0 liter. Agitation at 600 rpm was maintained using an integrated magnetic stirrer (DasGip, Julich, Germany) and the temperature kept at 30° C. The rate of aeration was set to 0.61/min. The pH of the medium was maintained at 5.0 by automatic addition of 2 N KOH during the batch phase and 10% NH4OH during the feed phase. The temperature, agitation, gassing, pH and composition of the off-gas were monitored and controlled using the DasGip monitoring and control system. Dissolved oxygen concentration was monitored with a polarographic oxygen electrode (Mettler Toledo, Columbus, Ohio, USA) and kept above 30% via stirrer speed and gas flow rate using the DasGip control system. The effluent gas from the fermentation was analyzed for real-time determination of oxygen and CO2 concentrations by DasGip fedbatch Pro® gas analysis systems with the off gas analyzer GA4 based on zirconium dioxide and two-beam infrared sensor.
The fed-batch cultures were initiated as batch culture using 30 g/l glucose, feeding with fresh medium commenced only after residual ethanol produced from the glucose consumption phase was completely depleted. A feed strategy was designed keeping the volumetric growth rate constant. An exponential feed rate v(t) (1/h) was calculated according to the following equation:




  
    v
    ⁡
    
      (
      t
      )
    
  
  =
  
    
      
        
          Y
          xs
        
        ⁢
        
          μ
          0
        
      
      
        
          s
          f
        
        -
        
          s
          0
        
      
    
    ⁢
    
      x
      0
    
    ⁢
    
      V
      0
    
    ⁢
    
      exp
      ⁡
      
        (
        
          
            μ
            0
          
          ⁢
          t
        
        )
      
    
  




where x0, s0 and V0 were the biomass density (g DCW/l), the substrate concentration (g/l) and the reactor volume (1) at the start of the fed-batch process, Yxs was the respiratory yield coefficient (g glucose/g DCW); sf was the concentration of the growth limiting substrate (g glucose/l) in the reservoir; μ0 was the specific growth rate (h−1) during the feed phase and t the feeding time expressed in hours. According to the equation above, the feed was increased exponentially with a specific feed rate of 0.06 h−1. Correct feed rate addition was obtained programming the fermentor fb-pro software (DasGip) and controlled using the DasGip control system. Under the feeding conditions, the glucose concentrations in the fermentor are maintained under 1 g/L allowing a repression of ERG9 expression. An organic layer of dodecane (Sigma-Aldrich St. Louis, Mo.) was added aseptically to a final volume of 10% (v/v) immediately before starting the feed.

Example 6
Functional Characterization of the MET3 Promoter (Comparative Example)
The level of repression of the MET3 promoter throughout batch cultivation was determined by fusing PMET3 to the LacZ reporter gene followed by measuring the β-galactosidase activity as described earlier (Yeast 2009, 26:545-551). In previous studies, PMET3-LacZ exhibited different levels of expression depending on the concentration of methionine supplied (Curr Microbiol 2002, 45:37-40). In order to evaluate the correct supply of methionine required to reduce the LacZ expression, strain SCICK10 was cultivated in shake flasks without L-methionine, with 1 mM and 2 mM L-methionine as initial concentration, and with 1 mM and 2 mM L-methionine added when the cultures had reached an OD600 of 0.6. The concentrations of the inhibitor were chosen based on the amounts previously used for ERG9 repression (Biotechnol Bioeng 2007, 993:666-677). As shown in FIG. 2A, the β-galactosidase activity was constant in the cultures not containing L-methionine. In the cultures that contained L-methionine from the beginning, LacZ activity was initially very low. However, at about mid-exponential phase, it started to increase and rapidly reached the levels measured in the non-repressed culture. When L-methionine was added at a later stage, LacZ activity dropped to a lower level in the culture pulsed with 2 mM L-methionine than in the culture pulsed with 1 mM L-methionine, but in both cases it started to increase again towards the end of the cultivation (FIG. 2B).
These results demonstrate the difficulties in controlling the MET3 promoter activity when the repressing agent is metabolized by the cells. We therefore tested, if D-methionine or 2-hydroxy-4-(methylthio)butyric acid could serve as L-methionine analogues to repress the PMET3 promoter, because they may not be metabolized by yeast or metabolized to a lesser extent. At concentrations of up to 4 mM in the medium neither of the two compounds had the capability to reduce PMET3 activity (data not shown). These results confirm the interest of finding an alternative solution to the regulation of gene activity using the MET3 promoter.
Example 7
Evaluation of the HXT1 Promoter to Control ERG9 Expression in Fed Batch Fermentation
Three strains were constructed. Strain SCICK13 in which ERG9 is under the control of its native promoter served as a reference strain. In strains SCICK15 and SCICK12, the ERG9 promoter had been replaced by the MET3 and HXT1 promoter, respectively. In order to maintain ERG9 repressed in the SCICK15 culture during the fermentation, L-methionine was added at regular interval every 6 hours to a final concentration of 2 mM.
All strains carried a deletion in the phosphatase encoding LPP1 gene to reduce the loss of FPP to farnesol (J Biol Chem 1999, 274:14831-14837). tHMG1 and (+)-α-santalene synthase were expressed from a high copy plasmid under control of the PGK1 and TEF1 promoters, respectively.
The physiological characterization of the strains was completed in aerobic glucose limited fed batch culture. A fed batch in situ product removal (ISPR) reactor mode consisting in an aqueous/dodecane two phase partitioning system was chosen to evaluate the (+)-α-santalene production capacity of these strains engineered to accumulate FPP. (+)-α-santalene production during the course of fermentation was determined as described previously in Biotechnol Bioeng 2010, 106:86-96. Samples from the organic layer were centrifuged 5 minutes at 5000 g and the supernatants were analyzed by gas chromatography-mass spectrometry (GC/MS) with a DSQ II single quadrupole mass spectrometer (Thermo Scientific, Waltham, Mass.). Analytes from 1 μl sample were separated on a SLB-5 ms capillary column (15 m, 0.25 mm i.d., 0.25 μm film thickness; Supelco, Bellefonte, Pa., USA) using helium as carrier gas at a flow rate of 1.2 ml min−1. A split/splitless injector was used in the splitless mode. The initial oven temperature was 80° C. and the injector temperature was 250° C. The oven temperature was increased to 120° C. at a rate of 10° C./min and subsequently increased to 160° C. at a rate of 3° C./min. The oven temperature was finally increased to 270° C. at a rate of 10° C./min and held for 5 minutes at this temperature. Full mass spectra were generated by scanning m/z range within 40-500 for metabolite identification. Quantification of (+)-α-santalene and E,E-farnesol was carried out using standard curves.
For the extraction of sterols, a previously described method (Biotechnol Bioeng 2010, 106:86-96) was used with minor modifications. Known volumes of fermentation broth were harvested by centrifuging at 5,000 rpm for 10 minutes. The cell pellet was washed twice with distilled water and the cell suspension was centrifuged for another 10 minutes at 5,000 rpm. The cell pellet was resuspended in 4 ml of 0.2 N HCl and heated in a water bath at 85° C. for 1 h and then allowed to cool to room temperature. After centrifugation for 10 minutes at 5,000 rpm and removal of the supernatant, the cell pellet was resuspended in 2 nil methanol containing 0.2% (w/v) pyrogallol and 1 ml 4 N KOH and transferred to a 14 ml glass vial sealed with a PTFE lined screw cap, heated again for 2 hours in a water bath at 85° C. for saponification and then cooled to room temperature. Sterols were extracted by addition of 5 ml heptane followed by vigorous mixing for 2 minutes. After 2 hours, the n-heptane layer was transferred to a new glass vial for HPLC analyses. Quantitative determination of total ergosterol was carried out by isocratic high-performance liquid chromatography (UltiMate® 3000 Nano, Dionex) with a reverse phase Develosil column (C30-UG-5; Nomura Chemicals, Aichi, Japan) at 40° C. using 70% MeOH as the mobile phase at a flow rate of 1 nil min−1. Ergosterol concentration was measured with a UV-visible light absorbance detector set at 280 nm (Photodiode Array Detector, Dionex). The amount of ergosterol was determined with Dionex Chromeleon® software using absolute calibration curves.
The effect of the above mentioned strategies for down-regulation of ERG9 was evaluated by comparing the obtained yield of (+)-α-santalene during the feed phase. Samples were taken from the dodecane phase, analysed by GC-MS and quantified using a standard curve with different dilutions of a (+)-α-santalene standard. All transformed strains were able to synthesize (+)-α-santalene. Two major chromatographic peaks were detected in all the strains transformed with the expression vector corresponding in retention time and mass spectra to those of (+)-α-santalene and E,E-farnesol. Cultivation was started as batch with 30 g/l glucose. After complete glucose consumption and after residual ethanol produced during the glucose consumption phase was completely depleted, the organic layer was added to the fermentor and the production phase was started by initiating a feed of fresh concentrated substrate with exponential kinetics for a total feed period of 36 hours. Within the first 30 hours of feed the culture metabolism was completely respiratory characterized by complete oxidation of glucose with biomass and carbon dioxide as the major products and complete absence of fermentation products, while the respiratory coefficients remained close to 1 for all strains. After that a limitation phase occurred where yeast growth was no longer consistent with the feeding profile determining a shift towards a fermentative metabolism resulting in accumulation of glucose and ethanol production. To examine the effect of ERG9 repression on the sterol pathway the total cellular sterol content was measured. Engineered strains exhibited significant changes in the level of ergosterol production. A lower sterol content was observed in the two PERG9 replacement mutants compared to the strain bearing the original ERG9 promoter. The lowest sterol content was observed in strain SCICK12 (PHXT1) (microorganism according to the invention) with a 11-fold reduction while the MET3 promoter (strain SCICK15) led to a modest 3-fold decrease (FIG. 3A).
To establish if the lower sterol content reflected an increased availability of FPP precursor for sesquiterpene conversion, product accumulation in the organic layer was measured. A linear correlation was observed between the reduction in ergosterol content and the increase in (+)-α-santalene production. Different levels of ERG9 repression diverted FPP towards (+)-α-santalene formation, redirecting the flux distribution from the sterol pathway to sesquiterpene production. Strain SCICK12 (PHXT1) was the best (+)-α-santalene producer with a 3.4-fold and 2.3-fold improvement in (+)-α-santalene productivity compared to SCICK13 (PERG9) and SCICK15 (PMET3), respectively (FIG. 3B). These results show that the advantages of the present invention are two-fold when compared to the use of the MET3 promoter, which is already known from the prior art. Indeed, firstly the cultivation method is simplified because it is not necessary to feed the culture medium with methionine and secondly the production rate of the terpene compound is increased.

## Claims

1. A method for producing a terpene compound comprising cultivating a Saccharomyces cerevisiae yeast expressing at least one terpene synthase and comprising at least one endogenous gene encoding squalene synthase ERG9,
wherein
i) the native promoter of the endogenous gene has been replaced by a hexose transporter 1 (HTX1) promoter;
ii) the HTX1 promoter is induced by high glucose concentrations and repressed by low glucose concentrations; and
iii) the endogenous gene encodes a protein that uses an acrylic terpene precursor selected from the group consisting of geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) or geranyl geranyl pyrophosphate (GGPP) as a substrate;

in a glucose-limited fed batch fermentation process, the terpene synthase expressed by said yeast being capable of catalysing the formation of said terpene compound,
wherein said terpene compound is selected from the group consisting of α-santalene, patchoulol, β-santalene, valencene, cubebol, zizaene, amorpha 4,11-diene, humulene, aristolochene, bergamotene, zingiberene, farnesene, caryophyllene, isodaucene, sesquithujene, avermitilol, eudesmol, vetispiradiene, longifolene, cyclocopacamphene, isolongifolene, germacrene, bicyclogermacrene, bisabolol, germacradienol, hedycaryol, barbatene, epi-cedrol, epi-aristolochene, sesquisabinene, cuprene, selinene, copaene, macrocarpene, cadinol, intermedeol, nerolidol, muurola-3,5-diene, curcumene and epi-beta santalene.

2. The method of claim 1, wherein the acyclic terpene precursor is FPP.

3. The method of claim 1, wherein the HTX1 promoter is induced by glucose concentrations of at least 1 gram per liter of culture medium and repressed by glucose concentrations of less than 1 gram per liter of culture medium.

4. A method for increasing the production of a terpene compound in a Saccharomyces cerevisiae yeast comprising
a) transforming the yeast to replace a native promoter of an endogenous gene encoding a protein that uses an acyclic terpene precursor selected from the group consisting of geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) or geranyl geranyl pyrophosphate (GGPP) as a substrate by hexose transporter 1 (HTX1) promoter, said HTX1 promoter being induced by high glucose concentrations and repressed by low glucose concentrations, provided that said endogenous gene does not encode a terpene synthase, but encodes a protein selected from the group consisting of the prenyl transferases RAM1 and RAM2, the hexaprenyl diphosphate synthase Coq1, the famesyltransferase COX10, the cisprenyltranferases SRT1 and RER2, and the squalene synthase ERG9, and provided that said yeast further expresses at least one terpene synthase capable of catalysing the formation of said terpene compound,
wherein said terpene compound is selected from the group consisting of α-santalene, patchoulol, β-santalene, valencene, cubebol, zizaene, amorpha 4,11-diene, humulene, aristolochene, bergamotene, zingiberene, farnesene, caryophyllene, isodaucene, sesquithujene, avermitilol, eudesmol, vetispiradiene, longifolene, cyclocopacamphene, isolongifolene, germacrene, bicyclogermacrene, bisabolol, germacradienol, hedycaryol, barbatene, epi-cedrol, epi-aristolochene, sesquisabinene, cuprene, selinene, copaene, macrocarpene, cadinol, intermedeol, nerolidol, muurola-3,5-diene, curcumene and epi-beta santalene; and
b) cultivating the transformed yeast obtained in step a) in a glucose-limited fed-batch fermentation process, so that the yeast produces the terpene compound;
and
c) optionally isolating the terpene compound produced in step b).

5. The method of claim 4, wherein the terpene compound is a sesquiterpene compound and wherein the terpene synthase is a sesquiterpene synthase.

6. The method of claim 1, wherein the terpene compound is a sesquiterpene compound and wherein the terpene synthase is a sesquiterpene synthase.

7. The method of claim 1, wherein the terpene compound is selected from the group consisting of α-santalene, patchoulol, β-santalene, valencene, cubebol, zizaene, and amorpha 4,11-diene.

8. The method of claim 4, wherein the terpene compound is selected from the group consisting of α-santalene, patchoulol, β-santalene, valencene, cubebol, zizaene, and amorpha 4,11-diene.

9. The method of claim 4, wherein the acyclic terpene precursor is FPP.

10. The method of claim 4, wherein the endogenous gene comprises a sequence encoding squalene synthase ERG9.

11. The method of claim 1, further comprising isolating the terpene compound produced by the yeast in the fermentation process.

12. The method of claim 1, wherein the terpene compound is α-santalene or β-santalene.

13. The method of claim 4, wherein the terpene compound is α-santalene or β-santalene.