Reducing the toxicity of terpenes and increasing their production potential in microorganisms

Abstract

The present invention relates to novel methods for increasing the resistance of microbial host cells to toxic substances, such as terpenes and alcohols, and other membrane-disrupting substances, and to modified organisms having such increased resistance compared to unmodified organisms.

Description

[Technical Field]

[0001] The present invention relates to novel methods for increasing the resistance of microbial host cells to toxic substances, such as terpenes and alcohols, and other membrane-disrupting substances, and to modified organisms having such increased resistance compared to unmodified organisms. [Background technology]

[0002] Isoprenol belongs to the class of naturally occurring terpenoid compounds (Withers and Keasling, 2006). 3-Methyl-3-buten-1-ol is the basis for the chemical production of citral, menthol, and other flavor compounds, which also belong to the terpenoid class. Citral is continuously used for the synthesis of vitamins A and E and several carotenoids. Isoprenol has also been discussed as a leading nutraceutical for longevity (Pandey et al., 2019). In recent years, companies such as Amyris and Isobionics have introduced terpenoid products, such as artemisinic acid, valencene, and nootkatone, synthesized through biotechnological fermentation processes. These companies are currently developing biological production platforms to further expand their product portfolios in the fragrance and flavor business (Janssen, 2015), thus challenging chemical synthesis.

[0003] Biotechnological production of terpenoid compounds in microorganisms relies on the natural precursor isopentenyl diphosphate (IPP), from which isoprenol can be obtained by simple dephosphorylation. To date, bioengineering efforts have focused on increasing the intracellular concentration of the isoprenol precursor IPP. In the model organism Escherichia coli, this was achieved by introducing an additional metabolic pathway, the DXP pathway, which generates IPP, resulting in a product titer of 61 mg / L (Liu et al., 2014). When considering a mixture of prenol and isoprenol as the product, titers of up to 1 g / L are currently possible (Kang et al., 2017). To date, the toxic intermediate IPP has been identified as a major bottleneck in these processes (George et al., 2018; Kang et al., 2019). Current research projects are attempting to develop a unified process in which isopentenol can be obtained from hydrolyzed polysaccharides derived from biomass (Wang et al., 2019).

[0004] A key problem in the biotechnological production of terpenoids is their toxicity to microorganisms (Brennan et al., 2015), and therefore a problem that all economically viable bioprocesses must face. This problem can be overcome by using a two-phase production system as disclosed in the international patent application published as WO 2015 / 002528 and by evolutionarily engineering the producing strains to a relatively high tolerance.

[0005] The production of monoterpene esters in microorganisms has also been demonstrated. When geraniol was produced in Escherichia coli, the chloramphenicol acetyltransferase gene was observed to mediate the formation of geranyl acetate (Liu et al. Biotechnol Biofuels (2016) 9:58). The use of more specific enzymes has been shown to offer the following advantages: monoterpene alcohols such as geraniol, as well as linalool, an acyclic monoterpene found in the floral scent of many plants, are highly toxic to microorganisms, while their esters are often much less toxic. The toxicity of monoterpene alcohols often leads to the cessation of growth and / or production, resulting in very low product titers. Chacon et al. have shown that expression of RhAAT in Escherichia coli engineered to produce geraniol resulted in the formation of geranyl acetate at substantially increased levels compared to the levels of geraniol produced in the absence of RhAAT (Chacon, MG, et al. Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli. Microb Cell Fact 18, 105 (2019); https: / / doi.org / 10.1186 / s12934-019-1130-0; WO 2019 / 092388). For that reason, in situ esterification of monoterpene alcohols such as geraniol has been promoted as a means to detoxify the product and thus increase terpene production. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2015 / 002528 [Patent Document 2] International Publication No. 2019 / 092388

Non-licensed literature

[0007]

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Non-licensed Document 5

[0008] The problem to be solved was to develop host cells that are tolerant to terpenoids and / or other toxic substances, such as host cells that are relatively suitable for isoprenol bioproduction, and methods for increasing the tolerance to terpenoids and / or other toxic substances. Surprisingly, several novel and unexpected modifications to the host cell have been found to result in broad-spectrum tolerance to terpenes and other substances. [Means for solving the problem]

[0009] The present invention discloses novel methods for increasing the resistance of microbial host cells to toxic substances, such as terpenes and alcohols, and other membrane-disrupting substances, and host cells having such increased resistance compared to unmodified host cells.

[0010] The toxicity of the terpenes menthol, geraniol, citral, and isoprenol was tested using unmodified forms of Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Rhodobacter sphaeroides. All exhibited toxic effects, but geraniol and citral were particularly degraded, making them unsuitable for our genetic engineering approach. To determine modifications useful for increasing microbial resistance to these and similar toxic substances, cells of the E. coli strain MG1655 were constantly grown in the presence of 60 mM isoprenol in a manner that did not kill the cells but allowed for adaptation and mutation. Subsequently, to increase the selective pressure, the concentration was increased from an initial 60 mM (10 mM above the 50% inhibitory dose, EC50) to 80 mM isoprenol after 80 generations. Wild-type E. coli cells were unable to grow under this concentration regimen, but the adapted E. coli strains did, demonstrating even faster growth at reduced isoprenol concentrations compared to the parental E. coli. Over the course of more than 220 generations, isolates were generated for detailed analysis. Isolates from three parallel cultures (isolates A-C) and from seven different time points in evolution (T1-T7) were analyzed for modifications responsible for increased resistance. After in-depth analysis, the modifications deemed most promising were isolated and introduced into E. coli wild-type and knockout cells.

[0011] These modifications could be used to reduce the growth inhibitory effects of many substances as disclosed herein on host cells. Thus, the present invention discloses methods for reducing terpene toxicity and increasing production capacity in microorganisms, and host cells with such improved characteristics. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the structural formulas of the following substances: A: isoprenol (3-methyl-3-butan-1-ol), B: isobutanol, C: prenol, D: geraniol, and E: vanillin. [Figure 2] FIG. 1 shows the growth of isolated strains at the isobutanol concentration at which growth is inhibited by 50% (EC50) (65 mM). [Figure 3] FIG. 1 shows the growth of isolated strains at the prenol concentration at which growth is inhibited by 50% (EC50) (40 mM). [Figure 4A] Figure 1. Overview of mutation occurrence during evolution experiments. A: Total mutation frequency and time point of first occurrence in culture of mutations. [Figure 4B] Figure 1. Overview of mutation occurrence during evolution experiment. B: Mutation persistence in adapted strains. Persistence is the mutation frequency corrected for the point of occurrence in the evolution experiment. [Figure 5-1] A: Hypothetical regulatory elements in the yghB promoter region. The upper strand and the lower complementary sequence are shown. The black box indicates the start of the open reading frame (ORF) and the initiating methionine (Met). The untranslated region (UTR) upstream of this is shown. Possible regulatory motifs are predicted in this region: a direct repeat upstream of the -35 region, and an inverted repeat downstream of the -35 region. Black arrows indicate motifs in the region of deletion, indicated by the checkered box, and an inverted motif. Transcription factor binding may act as a repressor to inhibit transcription. B: Consensus sequences of hypothetical binding motifs (van Helden, Andre and Collado-Vides, 1998). [Figure 5-2]C: A magnified view of the promoter portion shown in A: The deletion upstream of the yghB ORF (dark gray bar) and annotation of the putative regulatory motif (gray arrow) are shown in more detail. D: Sequence changes in the promoter region of yghB in adapted strains. The upper strand represents the wild-type sequence (P_yghB wt), and the lower strand represents the sequence with the deletion (P_yghB del.). The checkered box represents the deletion that changed the wild-type promoter sequence to that shown at the bottom. The black box indicates the start of the open reading frame (ORF) and the initiating methionine (Met). The UTR upstream of this is shown. [Figure 6] Figure 1 shows significantly differentially expressed transcripts compared to wild type. Significantly differentially expressed genes (P<0.05) for different DE algorithms in all three biological samples. (A) Significantly over-expressed transcripts (log2>1.35), (B) significantly down-regulated transcripts (log2<-2.7). [Figure 7] FIG. 1 shows the relative fitness (μ strain / μ wt ) of mutant rob H48fs-expressing strains at 50 mM isoprenol. [Figure 8] Figure 10 shows the relative fitness (μ strain / μ wt) of the combined rob and marC knockout strain expressing mutant robH48fs with 0 μM IPTG induction at 50 mM isoprenol. [Figure 9] A: Screening results for butanol toxicity at different butanol concentrations and growth rates for the original strain at various concentrations and the adapted strain at 7.5 g / L. The abbreviation Mut T6 A defines herein the T6 generation mutant strain of isolate A as described above. B: Evaluation of growth rates of different engineered strains with 5 g / L butanol. The wild-type growth rate in this assay was 0.25 1 / h. yghB and rob H48fs were expressed from leaky IPTG-inducible promoters without induction. [Figure 10]A: Evaluation of vanillin tolerance. E. coli wild-type strains were grown at different vanillin concentrations. At 1 g / L vanillin, the growth rate of adapted strains was also tested. Mut T6 A defines the T6 generation mutant strain of isolate A as described above herein. B: Evaluation of growth rate of different engineered strains with 1.5 g / L vanillin. The wild-type growth rate in this assay was 0.23 1 / h. yghB and robH were expressed from leaky IPTG-inducible promoters without induction. [Figure 11] FIG. 1 shows the relative fitness (μ strains / μ wt) of ΔrraA at 50 mM isoprenol in strain BW25113. DETAILED DESCRIPTION OF THE INVENTION

[0013] The terms "essentially," "about," "approximately," "substantially," and the like, in association with an attribute or value, also specifically define the attribute or value exactly, respectively. The term "substantially," in the context of the same functional activity or substantially the same function, refers to a difference in function, preferably within 20%, more preferably within 10%, and most preferably within 5% or less, compared to the reference function. In the context of a formulation or composition, the term "substantially" (e.g., "a composition consisting essentially of compound X") can be used herein to mean that the formulation or composition substantially contains the reference compound having a given effect, and does not contain additional compounds having such effect, or contains the maximum amount that does not exhibit a measurable or related effect of such compounds. The term "about," in the context of a given numerical value or range, specifically refers to a value or range that is within 20%, within 10%, or within 5% of the given value or range. As used herein, the term "comprising" also encompasses the term "consisting of."

[0014] The term "isolated" means that a material is substantially free from at least one other component with which it is naturally associated in its original environment. For example, a naturally-occurring polynucleotide, polypeptide, or enzyme present in a living animal is not isolated, but the same polynucleotide, polypeptide, or enzyme separated from some or all of the coexisting materials in the natural system is isolated. As a further example, an isolated nucleic acid (e.g., a DNA or RNA molecule) is one that is not immediately contiguous with 5' or 3' flanking sequences that are normally immediately contiguous with it when present in the naturally occurring genome of the organism from which it originates. Such a polynucleotide could be part of a vector, integrated into the genome of a cell of an unrelated genetic background (or into the genome of a cell with an essentially similar genetic background, but at a location different from where it naturally occurs), or produced by PCR amplification or restriction enzyme digestion; alternatively, an RNA molecule produced by in vitro transcription, and / or such a polynucleotide, polypeptide, or enzyme, can be part of a composition and further isolated, such that such vector or composition is not part of its natural environment.

[0015] "Purified" means that the material is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, or at least about 98% or 99% pure. Preferably, "purified" means that the material is in a 100% pure state.

[0016] A "synthetic" or "artificial" compound is produced by in vitro chemical or enzymatic synthesis. This term includes, but is not limited to, mutant nucleic acids made with optimal codon usage for a host organism, such as a yeast cell host or other expression host of choice, or mutant protein sequences with amino acid modifications (e.g., substitutions) compared to the wild-type protein sequence, for example, to optimize the properties of the polypeptide.

[0017] The term "non-naturally occurring" refers to a (poly)nucleotide, amino acid, (poly)peptide, enzyme, protein, cell, organism, or other substance that is not found in its original environment or source, although it may have originally been derived from its original environment or source and subsequently reproduced by other means. Such a non-naturally occurring (poly)nucleotide, amino acid, (poly)peptide, enzyme, protein, cell, organism, or other substance may be structurally and / or functionally similar to or identical to its naturally occurring counterpart.

[0018] The terms "native" (or "wild-type" or "endogenous") cell or organism and "native" (or wild-type or endogenous) polynucleotide or polypeptide refer to a cell or organism as found in nature, and a polynucleotide or polypeptide of interest as found in a cell in its natural form and genetic environment (i.e., without any human intervention), respectively.

[0019] The term "heterologous" (or exogenous or foreign or recombinant) polypeptide is defined herein as follows: (a) A polypeptide that is not native to the host cell. Such heterologous polypeptide protein sequences are synthetic, non-naturally occurring, "man-made" protein sequences; (b) a polypeptide native to a host cell, but in which structural modifications (e.g., deletions, substitutions, and / or insertions) are contained as a result of manipulation of the host cell's DNA by recombinant DNA techniques to alter the native polypeptide; or (c) A polypeptide that is native to a host cell, the expression of which is quantitatively altered or from a different genomic location than in the native host cell as a result of manipulation of the host cell's DNA by recombinant DNA techniques (e.g., a stronger promoter).

[0020] The above descriptions (b) and (c) refer to a sequence that is in its native form but is not naturally expressed by the cells used for production. The produced polypeptide is therefore more accurately defined as a "recombinantly expressed endogenous polypeptide," which does not contradict the above definition, but reflects the special situation in which the protein sequence is not synthesized or engineered, but is the manner in which the polypeptide molecule is produced.

[0021] Similarly, the term "heterologous" (or exogenous or foreign or recombinant) polynucleotide means: (a) a polynucleotide that is not native to the host cell; (b) a polynucleotide native to a host cell, but containing structural modifications (e.g., deletions, substitutions, and / or insertions) as a result of manipulation of the host cell's DNA by recombinant DNA techniques to alter the native polynucleotide; (c) a polynucleotide native to a host cell, the expression of which has been quantitatively altered as a result of the manipulation of regulatory elements of the polynucleotide by recombinant DNA techniques (e.g., a stronger promoter); or (d) A polynucleotide that is native to a host cell as a result of genetic manipulation by recombinant DNA techniques but is not integrated into its natural genetic environment.

[0022] With respect to two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous" is used to characterize the two or more polynucleotide sequences or two or more amino acid sequences as not occurring in nature in that particular combination with each other.

[0023] The terms "polynucleotide," "nucleic acid sequence," "nucleotide sequence," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein and refer to a polymeric, unbranched form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or a combination of both.

[0024] For nucleotide sequences (e.g., consensus sequences), IUPAC nucleotide nomenclature (Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1984). "Nomenclature for Incompletely Specified Bases in Nucleic Acid Sequences") was used, including the following nucleotide and nucleotide ambiguity definitions that are important to the present invention: A, adenine; C, cytosine; G, guanine; T, thymine; K, guanine or thymine; R, adenine or guanine; W, adenine or thymine; M, adenine or cytosine; Y, cytosine or thymine; D, not cytosine; N, any nucleotide.

[0025] Additionally, the notation "N(3-5)" means that the indicated consensus position can have any of 3 to 5 (N) nucleotides. For example, the consensus sequence "AWN(4-6)" represents three possible variants: AWNNNN, AWNNNNN, and AWNNNNNN, containing either 4, 5, or 6 nucleotides at the termini.

[0026] The terms "regulatory element" and "regulatory sequence" are all used interchangeably herein and should be interpreted broadly to mean a regulatory nucleic acid sequence capable of affecting the expression of a sequence associated therewith, including, but not limited to, the expression of a polynucleotide encoding a polypeptide. Regulatory elements or sequences include any nucleotide sequence that has a function or purpose, individually and / or within a particular arrangement or group of other elements or sequences within an arrangement. Examples of regulatory sequences include, but are not limited to, leader or signal sequences (such as 5'-UTRs), initiation signals, propeptide sequences, promoters, enhancers, silencers, polyadenylation sequences, ribosome binding sites (RBSs, Shine-Dalgarno sequences), termination signals, terminators, 3'-UTRs, and combinations thereof. Regulatory elements or sequences can be native (i.e., derived from the same gene) or foreign (i.e., derived from different genes) with respect to each other or to the nucleotide sequence to be expressed.

[0027] The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences.

[0028] Nucleic acids and polypeptides can be modified to include tags or domains. Tags can be used for various purposes, including detection, purification, solubilization, or immobilization, and can include, for example, biotin, fluorophores, epitopes, mating factors, or regulatory sequences. Domains can be of any size and can provide a desired function (e.g., conferring increased stability, solubility, activity, or simplifying purification), and can include, for example, binding domains, signal sequences, promoter sequences, regulatory sequences, N-terminal extensions, or C30-terminal extensions. Combinations of tags and / or domains can also be used.

[0029] The term "fusion protein" refers to two or more polypeptides linked together by any means known in the art, including chemical synthesis or splicing of encoding nucleic acids by recombinant genetic engineering.

[0030] Methods for modifying nucleic acids to introduce changes into encoded proteins Gene editing Gene editing or genome editing can be performed by using various techniques such as "gene shuffling" or "directed evolution," which consists of repeated DNA shuffling followed by appropriate screening and / or selection, in which DNA is inserted, replaced, or removed from the genome, to generate mutants of nucleic acids or portions thereof that encode proteins with altered biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Patent Nos. 5,811,238 and 6,395,547), or by "T-DNA activation" tagging, in which the resulting transgenic organisms exhibit dominant phenotypes due to altered expression of genes adjacent to the introduced promoter (Hayashi et al. Science (1992) 1350-1353), or "TILLING" (Targeted Induced Local Lesions Induced in the Genome). TILLING refers to a type of genetic engineering that can be obtained by TILLING (Genomes) and is a mutagenesis technique useful for creating and / or identifying nucleic acids that encode proteins with altered expression and / or activity. TILLING also allows for the selection of organisms that carry such mutations. Methods for TILLING are well known in the art (reviewed by McCallum et al., (2000) Nat Biotechnol 18: 455-457; Stemple (2004) Nat Rev Genet 5(2): 145-50). Another technique uses artificially engineered nucleases, such as zinc finger nucleases, transcription activator-like effector nucleases (TALENs), CRISPR / Cas systems, and engineered meganucleases (such as engineered homing endonucleases) (Esvelt, KM.; Wang, HH. (2013), Mol Syst Biol 9 (1): 641; Tan, WS. et al. (2012), Adv Genet 80: 37-97; Puchta, H.; Fauser, F. (2013), Int. J. Dev. Biol 57: 629-637).

[0031] Mutagenesis DNAs and the proteins they encode can be modified using a variety of techniques known in molecular biology to generate mutant proteins or enzymes with new or altered properties, such as random PCR mutagenesis (see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA 89:5467-5471); or combinatorial multiple cassette mutagenesis (see, e.g., Crameri (1995) Biotechniques 18:194-196).

[0032] Alternatively, nucleic acids (e.g., genes) can be reassembled after random or "stochastic" fragmentation, see, e.g., U.S. Patent Nos. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; and 5,605,793.

[0033] Alternatively, modifications, additions, or deletions are introduced by error-prone PCR, shuffling, site-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis (phage-assisted progressive evolution, in vivo progressive evolution), cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphorothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiation-induced mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer generation, and / or combinations of these and other methods.

[0034] Alternatively, "gene site saturation mutagenesis" or "GSSM" includes methods that use degenerate oligonucleotide primers to introduce point mutations into a polynucleotide, as described in detail in U.S. Pat. Nos. 6,171,820 and 6,764,835.

[0035] Alternatively, synthetic ligation reassembly (SLR) involves methods for non-stochastically ligating oligonucleotide building blocks together (eg, as disclosed in US Pat. No. 6,537,776).

[0036] Alternatively, Tailored multi-site combinatorial assembly ("TMSCA") is a method for generating multiple progeny polynucleotides with different combinations of mutations at multiple sites in a single reaction by using at least two mutagenic non-overlapping oligonucleotide primers (as described in PCT Publication No. WO 2009 / 018449).

[0037] Sequence alignments can be generated using a number of software tools, such as: - Needleman and Wunsch algorithm - Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-453. This algorithm is implemented, for example, in the "NEEDLE" program, which performs a global alignment of two sequences. The NEEDLE program is included, for example, within the European Molecular Biology Open Software Suite (EMBOSS).

[0038] - EMBOSS: a collection of various programs: The European Molecular Biology Open Software Suite (EMBOSS), Trends in Genetics 16 (6), 276 (2000).

[0039] - BLOSUM (BLOcks SUbstitution Matrix): Typically generated based on alignment of conserved regions (e.g., protein domains) (Henikoff S, Henikoff JG: Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the USA. 1992 Nov 15;89(22):10915-9). One of many BLOSUMs is "BLOSUM62", which is often the "default" setting for many programs when aligning protein sequences.

[0040] - BLAST (Basic Local Alignment Search Tool) consists of several individual programs (BlastP, BlastN, ...) that are primarily used to search for similar sequences in large sequence databases. BLAST programs also produce local alignments. Typically, the "BLAST" interface provided by NCBI (National Center for Biotechnology Information) is used, as is an improved version ("BLAST2"). "Original" BLAST:Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410;BLAST2:Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.

[0041] Enzyme variants can be defined by their sequence identity compared to the parent enzyme. Sequence identity is usually provided as "% sequence identity" or "% identity." In the first step, to determine the percent identity between two amino acid sequences, a pairwise sequence alignment is made between the two sequences, with the two sequences aligned over their entire lengths (i.e., pairwise global alignment). The alignment is generated using a program that implements the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, pp. 443-453), preferably by using the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with the program's default parameters (gapopen=10.0, gapextend=0.5, and matrix=EBLOSUM62). For the purposes of the present invention, the preferred alignment is the one that allows the highest sequence identity to be determined.

[0042] The following examples are intended to illustrate two types of nucleotide sequences, but the same calculations apply to protein sequences: Seq A: AAGATACTG Length: 9 bases Seq B: GATCTGA Length: 7 bases. Therefore, the shorter sequence is sequence B.

[0043] Generating a pairwise global alignment showing both sequences over their full length yields: The "|" symbol in the alignment indicates identical residues (meaning bases for DNA or amino acids for proteins). The number of identical residues is 6.

[0044] The "-" symbol in the alignment indicates a gap. The number of gaps introduced by the alignment within Seq B is 1. The number of gaps introduced by the alignment at the edge of Seq B is 2, and at the edge of Seq A is 1. The alignment length, showing sequences aligned over their entire length, is 10.

[0045] Generating a pairwise alignment showing a shorter sequence over its entire length according to the present invention results in the following: TIFF0007814310000002.tif20138

[0046] Generating a pairwise alignment showing sequence A over its entire length according to the present invention results in the following: TIFF0007814310000003.tif18138

[0047] Generating a pairwise alignment showing sequence B over its entire length according to the present invention results in the following: TIFF0007814310000004.tif18138

[0048] The alignment length showing the shorter sequence over its entire length is 8 (there is one gap, which is included in the alignment length of the shorter sequence). Therefore, the alignment length showing Seq A over its entire length is 9 (meaning that Seq A is the sequence of the present invention). Therefore, the alignment length showing Seq B over its entire length is 8 (meaning that Seq B is a sequence of the present invention).

[0049] After aligning the two sequences, in a second step, an identity value is determined from the resulting alignment. For the purposes of this description, percent identity is calculated as follows: % identity = (identical residues / length of the alignment region representing the shorter sequence over its entire length) * 100. That is, the sequence identity associated with the comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the alignment region representing the shorter sequence over its entire length. This value is multiplied by 100 to give the "% identity." Following the example provided above, the % identity is: (6 / 8) * 100 = 75%.

[0050] The santalene synthase variants can have an amino acid sequence that is at least n percent identical to the amino acid sequence of the respective parent polypeptide molecule, compared to the full-length polypeptide sequence, where n is an integer between 50 and 100, preferably 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

[0051] Santarene synthase variants can be defined by their sequence similarity compared to the parent enzyme. Sequence similarity is usually provided as "% sequence similarity" or "% similarity." To calculate sequence similarity in the first step, a sequence alignment must be made as described above. In the second step, the percentage similarity must be calculated, while the percentage sequence similarity takes into account that a defined set of amino acids share similar properties, for example, due to their size, their hydrophobicity, their charge, or other characteristics. Herein, the replacement of an amino acid with a similar amino acid is referred to as a "conservative mutation." Enzyme variants containing conservative mutations are likely to have minimal impact on protein folding, thereby substantially maintaining certain enzymatic properties compared to those of the parent enzyme.

[0052] For the determination of % similarity according to the present invention, the following applies, for example, according to the BLOSUM62 matrix as used by the "NEEDLE" program (as mentioned above), which is one of the most used amino acid similarity matrices for database searching and sequence alignment. Amino acid A is similar to amino acid S Amino acid D is similar to amino acid E;N Amino acid E is similar to amino acids D; K; and Q Amino acid F is similar to amino acid W;Y Amino acid H is similar to amino acids N and Y Amino acid I is similar to amino acids L; M; V Amino acid K is similar to amino acids E; Q; and R Amino acid L is similar to amino acids I;M;V Amino acid M is similar to amino acids I;L;V The amino acid N is similar to the amino acids D; H; and S. The amino acid Q is similar to the amino acids E; K; and R. Amino acid R is similar to amino acids K and Q The amino acid S is similar to the amino acids A, N, and T. The amino acid T is similar to the amino acid S Amino acid V is similar to amino acids I;L;M Amino acid W is similar to amino acid F;Y The amino acid Y is similar to the amino acids F; H; and W.

[0053] Conservative amino acid substitutions can occur throughout the entire polypeptide sequence of a functional protein, such as an enzyme. In one embodiment, such mutations do not occur in the functional domain of the enzyme. In one embodiment, conservative mutations do not occur in the catalytic center of the enzyme. Therefore, according to this description, the following percentage similarity calculation applies: % Similarity = [(Identical Residues + Similar Residues) / Length of Alignment Region Representing the Shorter Sequence Over Its Full Length] * 100. That is, sequence similarity associated with a comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues plus the number of similar residues by the length of the alignment region representing the shorter sequence over its full length. This value is multiplied by 100 to give the "% Similarity."

[0054] Variant enzymes containing conservative mutations that are at least m% similar to their respective parent sequences compared to the full-length polypeptide sequence, where m is an integer between 50 and 100, preferably 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99, are expected to have essentially unchanged enzymatic properties (such as enzymatic activity).

[0055] As used herein, a "construct," "gene construct," or "expression cassette" (used interchangeably) is a DNA molecule composed of at least one sequence to be expressed operably linked (at least to a promoter) to one or more regulatory sequences as described herein. Typically, an expression cassette contains three elements: a promoter sequence, an open reading frame, and a 3' untranslated region (which in eukaryotes usually includes a polyadenylation site). Additional regulatory elements include transcriptional and translational enhancers. Intron sequences can also be added to the 5' untranslated region (UTR) or within the coding sequence to increase the amount of mature message accumulated in the cytoplasm. Those skilled in the art are familiar with the genetic elements that must be present in an expression cassette to be successfully expressed. Preferably, at least part of the DNA or arrangement of genetic elements forming the expression cassette is artificial. The expression cassette can be part of a vector or can be integrated into the genome of a host cell and replicated along with the genome of the host cell. Expression cassettes can increase or decrease expression of DNA and / or proteins of interest.

[0056] The terms "introduction" or "transformation," as referred to herein, encompass the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for the transfer. That is, as used herein, the term "transformation" is independent of vectors, shuttle systems, or host cells and relates not only to transforming polynucleotide transfer methods known in the art (see, e.g., Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but also encompasses any additional type of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

[0057] The term "recombinant organism" refers to a eukaryotic organism (yeast, fungus, algae, plant, animal) or prokaryotic microorganism (e.g., bacterium) that has been genetically altered, modified, or engineered, thereby exhibiting an altered, modified, or different genotype when compared to the wild-type organism from which it is derived. Preferably, a "recombinant organism" contains an exogenous nucleic acid. "Recombinant organism," "genetically modified organism," and "transgenic organism" are used interchangeably herein. The exogenous nucleic acid can be located on an extrachromosomal piece of DNA (such as a plasmid) or can be integrated into the chromosomal DNA of the organism. In the case of a recombinant eukaryotic organism, it is understood to mean that the nucleic acid used is not present in or derived from the genome of the organism, or is present in the genome of the organism but not at its natural locus in the genome of the organism, and that the nucleic acid can be expressed under the control of one or more endogenous and / or exogenous regulatory elements.

[0058] By definition, the term "terpene" includes only hydrocarbons composed of carbon and hydrogen, and terpene compounds. The term "terpene compounds" refers to terpenes and terpenes that contain additional functional groups, thereby producing derivatives such as alcohols, aldehydes, ketones, and acids, but also includes related compounds such as the four-carbon (C4) alcohols butanol and isobutanol or the eight-carbon aldehyde vanillin. Typical terpene compounds are: - alcohols having four carbon atoms (C4), such as butanol and isobutanol; - Compounds with five carbon atoms (C5), such as, but not limited to, the hemiterpene isoprene and the hemiterpenoids prenol and isovaleric acid; - 7 or 8 carbon phenolic aldehydes, such as but not limited to vanillin; - compounds having 10 carbon atoms (C10) that are terpenes or are derived from terpenes, or compounds derived from C10 terpenes, including, but not limited to, monoterpenes and monoterpenoids such as geraniol, terpineol, limonene, myrcene, linalool or pinene; - compounds having 15 carbon atoms (C15) that are terpenes or are derived from terpenes, or compounds derived from C15 terpenes, including, but not limited to, sesquiterpenes and sesquiterpenoids such as humulene, farnesene, farnesol; and - a compound having 20 carbon atoms (C20), 25 carbon atoms (C25), 30 carbon atoms (C30), 35 carbon atoms (C35) or 40 carbon atoms (C40) that is a terpene or is derived from a terpene, or a compound derived from a C20, C25, C30, C35 or C40 terpene.

[0059] In one embodiment, the terpene compound is understood to be a terpene; a terpene containing one or more additional functional groups, thereby resulting in a derivative such as an alcohol, an aldehyde, a ketone, or an acid; a C4 alcohol, preferably butanol or isobutanol; or vanillin or isovanillin. Preferably, the terpene compound is a terpene having 5, 10, or 15 carbon atoms or a compound derived therefrom.

[0060] With respect to monoterpene compounds, the C10 compound geranyl diphosphate (GPP) is the immediate precursor in the formation of monoterpenes, which involves a series of sequential reactions including hydrolysis, cyclization, and oxidation-reduction.

[0061] There are two main types of monoterpenes: acyclic (or straight-chain) and cyclic, which can be monocyclic or bicyclic. Acyclic monoterpenes, such as cis-α-ocimene and β-myrcene, are 2,6-dimethyloctane derivatives. Typical monocyclic monoterpenes, such as limonene and cymene, are generally cyclohexane derivatives with isopropyl substituents, usually containing variable double bond moieties. α-Pinene and β-pinene, on the other hand, are common types of bicyclic monoterpenes.

[0062] As used herein, "terpene alcohol" means a terpene compound containing an alcohol group as a functional group. Many examples are known in the art. As used herein, "monoterpene alcohol" means a monoterpene (C10) containing an alcohol group as a functional group. Monoterpene alcohols are well described in the art. As used herein, "sesquiterpene alcohol" means a sesquiterpene (C15) containing an alcohol group as a functional group. Sesquiterpene alcohols are well known in the art.

[0063] The terpene alcohol, eg, a monoterpene or sesquiterpene alcohol, can be a primary, secondary, or tertiary alcohol, as known in the art. Preferred primary alcohols are geraniol, citronellol, and lavandulol, and preferred secondary alcohols are borneol, isoborneol, fenchol, verbenol, carveol, and menthol. Also preferred are nerolidol, santalol, cubebol, patchoulol, bisabolol, germacrene D-ol, and hedicariol. Diterpene alcohols such as sclareol may also be used in the methods of the present invention.

[0064] Acyclic monoterpene alcohols, or monoterpenols, as they are sometimes referred to in literature, are 2,6-dimethyloctane derivatives that contain variable double bond moieties and hydroxyl functional groups.The most important substances of this class are linalool, geraniol, nerol, citronellol, myrcenol, and dihydromyrcenol.Since ancient times, they have been used in perfumery due to their pleasant olfactory properties.In one embodiment, the modified organisms or methods of the present invention can also be used to produce them.

[0065] By definition, an ester is a compound derived from an acid (organic or inorganic) in which at least one -OH (hydroxyl) group has been replaced by an -O-alkyl (alkoxy) group. Thus, a "terpene ester" is a terpene alcohol in which at least one -OH (hydroxyl) group has been replaced by an -O-alkyl (alkoxy) group.

[0066] As used herein, "monoterpene ester" refers to an ester derived from a monoterpene alcohol. This term includes esters derived from primary, secondary, or tertiary monoterpene alcohols, as defined herein.

[0067] As used herein, "sesquiterpene ester" refers to an ester derived from a sesquiterpene alcohol, which term includes esters derived from primary, secondary, or tertiary sesquiterpene alcohols, as defined herein.

[0068] The present invention is directed to modified organisms having improved tolerance to one or more terpene compounds, wherein the modified organism has one or more changes compared to a wild-type modified organism selected from the group consisting of: i. the absence, inactivation or reduced abundance of protein of SEQ ID NO: 2 or a homolog thereof and the absence, inactivation or reduced abundance of protein of SEQ ID NO: 3 or a homolog thereof and the presence of a mutant protein of protein of SEQ ID NO: 2 or a homolog thereof, in the presence of a terpene compound, wherein the mutant protein of protein of SEQ ID NO: 2 or a homolog thereof shares only the first 54, 53, 52, 51, 50, 49, 48 or 47 amino acids, in preferred order, with the protein of SEQ ID NO: 2 or a homolog thereof in an unmodified organism; ii. absence, inactivation or reduced abundance of the protein of SEQ ID NO: 2 or a homologue thereof in the presence of a terpene compound; iii. absence, inactivation or reduced abundance of the protein of SEQ ID NO: 3 or a homolog thereof in the presence of a terpene compound; iv. the absence of the protein of SEQ ID NO: 2 or a homolog thereof and the presence of a mutant protein of the protein of SEQ ID NO: 2 or a homolog thereof, wherein the mutant protein of the protein of SEQ ID NO: 2 or a homolog thereof has a mutation at a position corresponding to position 48 of SEQ ID NO: 2, in the presence of a terpene compound; v. the presence of a mutant protein of the protein of SEQ ID NO: 2 or a homolog thereof in the presence of a terpene compound, wherein the mutant protein of the protein of SEQ ID NO: 2 or a homolog thereof has a mutation at a position corresponding to position 48 of SEQ ID NO: 2; vi. an increased level or increased activity of the protein of SEQ ID NO: 1 or a homolog thereof in the presence of terpene compounds compared to an unmodified organism, preferably wherein the endogenous gene for the homolog of SEQ ID NO: 1 has been deleted and wherein the recombinant expression of the gene encoding SEQ ID NO: 1 or a variant thereof is present, and even more preferably wherein the recombinant expression of the gene encoding SEQ ID NO: 1 or a variant thereof is under a low to medium strength promoter or other control element; vii. the presence of a mutant protein of the protein of SEQ ID NO: 4 or a homolog thereof in the presence of a terpene compound, wherein the mutant protein of the protein of SEQ ID NO: 4 or a homolog thereof has a mutation at a position corresponding to position 74 of SEQ ID NO: 4; viii. The presence of a mutant protein of the protein of SEQ ID NO: 5 or a homolog thereof in the presence of a terpene compound, preferably the mutant protein of the protein of SEQ ID NO: 5 or a homolog thereof having (a) a mutation at a position corresponding to position 291 of SEQ ID NO: 5 and / or (b) a mutation at a position corresponding to position 274 or later of SEQ ID NO: 5, wherein the mutant protein has a shorter presence than the protein of SEQ ID NO: 5 or a homolog thereof, or the absence, inactivation or reduced abundance of the protein of SEQ ID NO: 5; ix. the presence, in the presence of a terpene compound, of a mutant protein of the protein of SEQ ID NO: 6 or a homolog thereof, wherein the mutant protein of the protein of SEQ ID NO: 6 or a homolog thereof has a mutation at a position corresponding to position 96 of SEQ ID NO: 6 (preferably, the mutation is a substitution of valine with glutamic acid) and / or a mutation at a position corresponding to position 67 of SEQ ID NO: 6, preferably a substitution of glycine with serine; x. absence, inactivation, or reduced abundance of the protein of SEQ ID NO: 6 or a homolog thereof in the presence of a terpene compound; xi. the absence, inactivation, or reduced abundance of a modified protein of SEQ ID NO: 8 or a homolog thereof, preferably the protein of SEQ ID NO: 8 or a homolog thereof, in the presence of a terpene compound; xii. The absence, inactivation, or reduced abundance of a modified protein of SEQ ID NO: 9 or a homolog thereof, preferably the protein of SEQ ID NO: 9 or a homolog thereof, in the presence of a terpene compound; xiii. absence, inactivation, increased activity or decreased abundance of a modified protein of SEQ ID NO: 7 or a homolog thereof, preferably the protein of SEQ ID NO: 7 or a homolog thereof, in the presence of a terpene compound; xiv. Any combination of i-xiii above; At this time, resistance is improved compared to the unmodified organism.

[0069] Preferably, the modified organisms are utilized in a method for the production of terpene esters, preferably monoterpene esters, from terpene compounds, preferably monoterpene alcohols.

[0070] Modified organisms according to the present invention can be produced based on conventional methods for mutating organisms and / or standard genetic and molecular biology techniques generally known in the art (e.g., as described in Sambrook, J., and Russell, DW "Molecular Cloning: A Laboratory Manual" 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001); and F.M. Ausubel et al., eds., "Current protocols in molecular biology", John Wiley and Sons, Inc., New York (1987), and later supplements thereto, and including techniques such as CRISPR / CAS, etc.).

[0071] The modified organism can be any cell selected from a bacterial cell, a yeast cell, a fungal cell, an algae cell or a cyanobacterial cell, a non-human animal cell or a mammalian cell, or a plant cell. Specifically, the modified organism can be selected from any one of the following organisms:

[0072] bacteria The bacterial modified organism can be, for example, selected from the group consisting of Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Amycolatopsis, Rhodobacter, Pseudomonas, Paracoccus, or Lactococcus.

[0073] Gram-positive: Bacillus, Streptomyces, etc. Useful Gram-positive bacterial modified organisms include, but are not limited to, Bacillus cells, such as Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus Jautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus Prokaryotic organisms include Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. Most preferably, the prokaryotic organism is a Bacillus cell, preferably a Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, or Bacillus lentus bacillus cell.

[0074] Some other preferred bacteria include strains of the order Actinomycetales, preferably of the genus Streptomyces, preferably Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382), Streptomyces lividans or Streptomyces murinus or Streptoverticillum verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include strains of the genus Myxococcus, such as M. virescens.

[0075] Gram-negative: Escherichia coli, Pseudomonas, Rhodobacter, Paracoccus Preferred Gram-negative bacteria are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11), Rhodobacter capsulatus or Rhodobacter sphaeroides, Paracoccus carotinifaciens or Paracoccus zeaxanthinifaciens.

[0076] fungi Aspergillus, Fusarium, Trichoderma The modified organism may be a fungal cell. As used herein, "fungi" includes the Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota phyla, as well as the Oomycota and Deuteromycotina subdivisions, and all mitosporic fungi. Representative groups of the Ascomycota include, for example, Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts listed below. Examples of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of the Chytridiomycota include, for example, Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of the Oomycota include, for example, Saprolegniomycetous aquatic fungi (water molds), such as Achlya. Examples of vegetative spore-forming fungi include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of the Zygomycota include, for example, Rhizopus and Mucor.

[0077] Some preferred fungi include strains belonging to the subdivision Deuteromycotina, class Hyphomycetes, such as the genera Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, and the like. resii, Myrothecium verrucana (IFO 6113), Verticillium alboatrum, Verticillium dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli or Dressclera halodes.

[0078] Other preferred fungi include strains belonging to the subdivision Basidiomycotina, class Basidiomycetes, such as the genera Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (formerly called Polyporus), such as T. versicolor (e.g. PR4 28-A).

[0079] Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, such as the genus Rhizopus or Mucor, in particular Mucor hiemalis. Yeasts such as the following can also be used in the present invention:

[0080] The fungal modified organism may be a yeast cell. As used herein, yeast includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Ascosporogenous yeast is divided into the Spermophthoraceae and Saccharomycetaceae families. The latter is composed of four subfamilies: Schizosaccharomycoideae (e.g., Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., Kluyveromyces, Pichia, and Saccharomyces). Basidiospore-forming yeasts include the genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the Fungi Imperfecti are divided into two families: Sporobolomycetaceae (eg, Sporobolomyces and Bullera) and Cryptococcaceae (eg, Candida).

[0081] eukaryotes Eukaryotic modified organisms further include, but are not limited to, non-human animal cells, non-human mammalian cells, avian cells, reptilian cells, insect cells, or plant cells. In a preferred embodiment, the modified organism is a modified organism selected from the following group:

[0082] (a) bacterial cells of the group of Gram-negative bacteria, such as Rhodobacter (e.g., Rhodobacter sphaeroides or Rhodobacter capsulatus), Paracoccus (e.g., P. carotinifaciens, P. zeaxanthinifaciens), Escherichia or Pseudomonas;

[0083] (b) bacterial cells selected from the group of Gram-positive bacteria, such as Bacillus, Corynebacterium, Brevibacterium, Amycolatopis, etc.;

[0084] (c) a fungal cell selected from the group consisting of Aspergillus, Blakeslea, Peniciliium, Phaffia (Xanthophyllomyces), Pichia, Saccharomyces, Kluyveromyces, Yarrowia, and Hansenula;

[0085] (d) a transgenic plant cell or a culture comprising a transgenic plant cell, wherein the cell is from a transgenic plant selected from Arabidopsis spp., Nicotiana spp., Chichorum intybus, Lacuca sativa, Mentha spp., Artemisia annua, tuber-forming plants, oil crops (e.g., Brassica spp. or Brassica napus), fruit-producing flowering plants (angiosperms) (including, but not limited to, strawberry or raspberry plants), and trees; or

[0086] (e) A culture comprising a transgenic mushroom or transgenic mushroom cells, wherein the microorganism is selected from the genera Schizophyllum, Agaricus, and Pleurotisi.

[0087] More preferred modified organisms derived from living organisms are modified organisms derived from microorganisms belonging to the genera Escherichia, Saccharomyces, Pichia, Rhodobacter, Pseudomonas, or Paracoccus (e.g., Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens), such as E. coli, S. cerevisae, Rhodobacter sphaeroides, Rhodobacter capsulatus, or Amycolatopis species. Even more preferred are those of the species genus Streptococcus sp.

[0088] Particularly preferred are modified Rhodobacter organisms selected from the group Rhodobacter capsulatus and Rhodobacter sphaeroides, or Escherichia coli.

[0089] A further aspect of the invention relates to a mutant protein selected from the group consisting of: i. A mutant variant of the protein set forth as SEQ ID NO: 2 or a homologue thereof, which variant comprises only the first 54, 53, 52, 51, 50, 49, 48 or 47 amino acids from the N-terminus of the protein, in order of preference, having the protein of SEQ ID NO: 2 or its homologue in an unmodified organism. ii. A mutant variant of the protein set forth in SEQ ID NO: 2 or a homolog thereof, having a mutation at a position corresponding to position 48 of SEQ ID NO: 2; iii. A mutant variant of the protein set forth in SEQ ID NO: 4 or a homolog thereof, having a mutation at a position corresponding to position 74 of SEQ ID NO: 4; iv. A mutant variant of the protein of SEQ ID NO: 5 or a homolog thereof, having (a) a mutation at a position corresponding to position 291 of SEQ ID NO: 5 and / or (b) a mutation at a position corresponding to position 274 or higher of SEQ ID NO: 5, wherein the mutant protein is shorter than the protein of SEQ ID NO: 5 or a homolog thereof; v. A mutant variant of the protein of SEQ ID NO: 6 or a homolog thereof, having a mutation at position corresponding to position 96 of SEQ ID NO: 6, preferably a mutation replacing valine with glutamic acid, and / or a mutation at position corresponding to position 67 of SEQ ID NO: 6, preferably a mutation replacing glycine with serine.

[0090] Further embodiments of the present invention relate to any nucleic acid encoding a mutant protein of the invention, an expression cassette comprising a nucleic acid encoding a mutant protein of the invention, a vector comprising a nucleic acid encoding a mutant protein of the invention, a host cell comprising a nucleic acid encoding a mutant protein of the invention and a recombinant non-human organism comprising a mutant protein of the invention.

[0091] In one preferred embodiment, the modified organisms or mutant proteins of the invention are used in the production of one or more terpene compounds and / or one or more terpene esters.

[0092] The method of the present invention for producing terpene compounds and / or terpene esters preferably comprises the following steps: (a) culturing a modified organism of the present invention under suitable conditions; and (b) obtaining the terpene compounds and / or terpene esters from the modified organism of step (a). In another preferred embodiment, modified organisms are suitable for carrying out the methods of the present invention.

[0093] For example, modified organisms can be used in the method for preparing monoterpene esters, and the method comprises the step of esterifying monoterpene alcohols into monoterpene esters in the presence of alcohol acyltransferase.For this purpose, modified organisms preferably heterologously express desired alcohol acyltransferase.When monoterpene alcohols are linalool, geraniol, α-terpineol, γ-terpineol, lavandulol, fenchol, perillyl alcohol, menthol or verbenol, and when it is desired to produce monoterpene esters, it is preferred that any of these or their mixtures are used as the substrate for alcohol acyltransferase.When organisms preferably contain one or more alcohol acyltransferases suitable for producing monoterpene esters, the monoterpene alcohol substrate can be produced by modified organisms and / or can be exogenously added to modified organisms.

[0094] A further aspect of the present invention is a method for increasing the tolerance of a modified organism to one or more terpene compounds compared to an unmodified organism, the method comprising the steps of producing a modified organism of the present invention and optionally maintaining the modified organism.

[0095] In a preferred embodiment, the present invention is a method for producing one or more terpene compounds using an organism, the method comprising the steps of generating a modified organism of the present invention, maintaining the modified organism in the presence of terpene compounds under conditions suitable for the modified organism to grow and produce the one or more terpene compounds, and optionally isolating the one or more terpene compounds from the modified organism.

[0096] In a preferred embodiment, the methods and modified organisms of the present invention are directed to the production of one or more terpene compounds, wherein at least one terpene compound is a C4 and C5 alcohol.

[0097] The method, use or modified organism of the present invention, wherein the terpene compound has a logP value of 2.0 or less, preferably 1.5 or less, and / or a solubility in water under standard conditions of at least 1.0 g / L, preferably 1.5 g / L or more.

[0098] A preferred embodiment of the present invention is directed to a method, use, mutant protein or modified organism of the invention, wherein tolerance to isoprenol, prenol, butanol, isobutanol, vanillin, geraniol and / or citral (preferably both geranial and neral), preferably to isoprenol, prenol, butanol, isobutanol and / or vanillin, is increased compared to an unmodified organism.

[0099] A further embodiment is the method, use or modified organism of the invention, wherein the modified organism comprises (a) a knockout or deletion, knockout of part or all of the gene encoding the protein of SEQ ID NO: 3 or a homologue thereof, or (b) a deletion of part or all of the gene encoding the protein of SEQ ID NO: 2 or a homologue thereof, or (c) the presence of a mutant protein of SEQ ID NO: 2 or a homologue thereof in the presence of a terpene compound, wherein the mutant protein of SEQ ID NO: 2 or a homologue thereof shares only the first 50, 49, 48, even more preferably only the first 47 amino acids from the N-terminus with the protein of SEQ ID NO: 2 or a homologue thereof from an unmodified organism, or any combination of (a)-(c).

[0100] In yet another embodiment, any of the methods of the invention comprises downregulating expression of a gene encoding the protein of SEQ ID NO: 6 or a homolog thereof, deleting a gene encoding the protein of SEQ ID NO: 6 or a homolog thereof, or knocking out a gene encoding the protein of SEQ ID NO: 6 or a homolog thereof.

[0101] In a preferred embodiment, the present invention is directed to a method for increasing the tolerance of a modified organism to vanillin compared to an unmodified organism, the method comprising the step of expressing or producing in the modified organism a DNA sequence encoding a protein that shares only the first 54, 53, 52, 51, 50, 49, 48 or 47 amino acids, in order of preference, with the protein of SEQ ID NO: 2, wherein the modified organism has the further characteristic that the proteins of SEQ ID NO: 1 and / or 2 or homologs thereof are absent, inactive or substantially reduced. Further encompassed by the present invention is the use of the deregulated protein of SEQ ID NO: 2 or a homolog thereof to increase the growth of a modified organism in the presence of a terpene.

[0102] The mutant or deregulated protein of SEQ ID NO: 2, or a homolog thereof, in a preferred embodiment has a mutation of the histidine residue corresponding to position 48 of SEQ ID NO: 2, which results in a frameshift, preferably a frameshift that shortens the resulting protein compared to the protein of SEQ ID NO: 2.

[0103] In another preferred embodiment, any of the sequences of SEQ ID NOs: 1-9 is mutated to carry the mutations as shown in Table 3 for the respective protein. Furthermore, the present invention provides: (i) for heterologous reconstitution of terpene biosynthetic pathways; (ii) for producing industrial products, preferably flavors or fragrances, biofuels, insecticides, insect repellents or antimicrobial agents; (iii) for producing aliphatic and / or aromatic monoterpene esters from monoterpene alcohols, preferably tertiary monoterpene alcohols, This includes the use of modified organisms or mutant proteins of the invention.

[0104] The present invention further relates to the use of a modified organism or mutant protein of the invention, a nucleic acid of the invention, a vector or gene construct of the invention, a host cell of the invention, or a transgenic non-human organism of the invention (i) for heterologous reconstitution of a terpene biosynthetic pathway; (ii) for producing industrial products, preferably flavors or fragrances, biofuels, insecticides, insect repellents or antimicrobial agents; (iii) for producing aliphatic and / or aromatic monoterpene esters from monoterpene alcohols, preferably from tertiary monoterpene alcohols; or (iv) for detoxifying monoterpene alcohols during fermentation, thereby increasing monoterpene production by said fermentation.

[0105] The present invention also provides (i) for heterologous reconstitution of terpene biosynthetic pathways; (ii) for producing industrial products, preferably flavors or fragrances, biofuels, insecticides, insect repellents or antimicrobial agents; (iii) for producing aliphatic and / or aromatic monoterpene esters from monoterpene alcohols, preferably tertiary monoterpene alcohols; (iv) detoxifying monoterpene alcohols in a mixture of modified organisms of the present invention and microorganisms, such as bacteria or fungi (e.g., yeast), thereby increasing monoterpene production by the mixture of microorganisms; It also relates to the use of a modified organism or mutant protein of the invention, a nucleic acid of the invention, a vector or gene construct of the invention, a host cell of the invention, or a transgenic non-human organism of the invention.

[0106] Preferred tertiary monoterpene alcohols include, but are not limited to, linalool (S-linalool and / or R-linalool), α-terpineol, fenchol, γ-terpineol, p-cymen-8-ol, p-menth-3-en-1-ol, p-menth-8-en-1-ol, 4-carvomenthol, and 4-thuyanol.

[0107] One aspect of the present invention is a method for producing monoterpenes according to the method of the present invention and the modified organism of the present invention, and esterifying them into monoterpene esters to produce monoterpene esters.Such esterification can be carried out in parallel, for example, in the same modified organism of the present invention with improved monoterpene production capacity, or in a subsequent step using an esterification enzyme in the same or different cells or extracts or isolated, or by chemical esterification, preferably after isolation and purification of monoterpenes.The monoterpene esters produced according to this method of the present invention can be used as they are, for example, as flavors or fragrances, as insect repellents, as insecticides, or as antimicrobial agents; can also be used to produce biofuels, or can be used as starting materials for other compounds, for example, other flavors or fragrances. [Example]

[0108] 1. Results 1.1 Examination of the mode of adaptation in adapted strains At the end of adaptive evolution to isoprenol, several strains were isolated that displayed increased tolerance to isoprenol. Tolerance to isoprenol was confirmed using an established toxicity assay of growth in M9 medium containing isoprenol in sealed 250 mL baffled flasks. Chemicals with similar properties were tested to investigate the mode of action of the tolerance trait.

[0109] Because chemicals with solvent-like properties often interfere with membrane function, a standard assay, propidium iodide staining, was used to determine cell membrane properties under isoprenol stress in evolutionarily adapted strains.

[0110] 1.1.1 Resistance to different chemicals To assess the limits of the resistance mechanism, we tested resistance to three additional chemicals: prenol, a biological isomer of isoprenol; isobutanol, a branched alcohol; and geraniol, a monoterpene. TIFF0007814310000005.tif56165

[0111] After establishing a 50% inhibitory concentration of approximately 65 mM, we tested the tolerance of isoprenol to the final adapted strains, which were also used for sequencing. All strains show increased tolerance to isoprenol. The tolerance mechanism is not limited to isoprenol; isobutanol was also well tolerated. Among the adapted strains, the strain isolated from culture A showed the highest tolerance, while the strain isolated from culture C showed a relatively small increase in growth rate. Isobutanol has very similar physicochemical properties, i.e., similar log P values ​​and solubility in water.

[0112] In the next set of experiments, we systematically determined the prenol tolerance of wild-type strains and found that the 50% inhibitory prenol concentration was 40 mM. Despite the structural similarity between prenol and isoprenol, only isolate A showed increased tolerance to prenol. Isolate C did not differ from wild-type tolerance, and isolate B even had a reduced growth rate at 40 mM prenol. The different resistance of the different strains isolated could suggest different genotypes despite the same isoprenol tolerance phenotype.

[0113] Finally, the monoterpene compound geraniol was tested. All of the isolated adapted strains showed greatly increased sensitivity to geraniol. Not only do traits that increase isoprenol tolerance fail to protect against geraniol toxicity, but the mechanism appears to make the compound even more toxic. Because we found no evidence of isoprenol degradation in the adapted strains, it is unlikely that geraniol is gradually degraded by these strains, and thus toxic degradation products could accumulate. Rather, the resistance mechanism must alter the structure of cellular components so that they become more sensitive to the toxic effects of geraniol.

[0114] 1.1.2 Membrane permeability of adapted strains With a log P close to 1, isoprenol may exert its toxic effect by increasing membrane permeability (Heipieper et al., 1994). To test this, propidium iodide (PI) staining was used. Propidium iodide staining is a dead / live stain, and because dead cells usually have defective cell membranes, the stain can cross the membrane and intercalate into the cell's DNA. This means that propidium iodide staining is suitable for detecting cell membrane damage.

[0115] Untreated wild-type cells had approximately 1.4 × 10 3 The median PI-mediated fluorescence of 1.3 × 10 isoprenolic acid (PI-mediated fluorescence) was 1.3 × 10, and when cells were treated with the disinfectant Bacillol AF, used as a positive control for the staining procedure, prior to staining, the median fluorescence increased by 100-fold. E. coli cells incubated with 50 mM isoprenol (i.e., an intermediate isoprenol concentration at which cells still grew) exhibited a fluorescence intensity of approximately 1.3 × 10, which lies between the intensity of viable cells and that of bacillol-treated dead cells. 4 Since this population is still actively growing, this means that isoprenol does indeed compromise cell membrane integrity, but not enough to terminate growth.

[0116] Interestingly, isoprenol treatment resulted in a unimodal shift to higher PI staining. Isoprenol could, in principle, also increase the killing of viable bacteria, which would result in a bimodal division of isoprenol-treated cells into "live" and "dead" staining. This is another indication that isoprenol destabilizes the cell membrane.

[0117] Next, we investigated how the adapted strains responded to isoprenol treatment. Isolates A–C showed 2.2–3.4 × 10 3 The treated cells had a reduced median PI fluorescence intensity of 1000 s. However, PI fluorescence remained slightly increased compared to wild-type untreated cells. This implies that evolutionarily adapted cells have developed mechanisms to cope with membrane stress and, in part, restore membrane integrity, thus reducing permeability to PI staining.

[0118] 1.2 DNA sequencing of target strains To understand the genetic basis of the observed adaptation mechanisms, i.e., tolerance to isoprenol, isobutanol, and prenol and reduced membrane permeability under isoprenol stress, several strains were isolated from adaptive evolution experiments and sequenced.

[0119] Strains were isolated after 32 to 226 generations at isoprenol concentrations ranging from 64 to 80 mM, as listed in Table 1. Low-temperature cultures of each of the three evolutionary cultures were streaked onto LB agar containing isoprenol. The five largest strains were then evaluated for their growth in M9 with isoprenol, and the fastest cultures were saved and used for sequencing. In addition to the adapted strains, one wild-type culture was prepared for sequencing.

[0120] [Table 1]

[0121] 1.2.1 Mutations identified in evolutionary experiments The mutations identified in the experiment are listed in Table 2. The E. coli MG 1655 wild-type strain exists in different mutants (Freddolino, Amini, and Tavazoie, 2012). Our wild-type mutants have a reconstructed gatC gene, which is part of the galactitol PTS, and a functional glrR glycerol-3-phosphate repressor. In addition, there is variation in the repeat REP321j.

[0122] [Table 2] TIFF0007814310000008.tif43161

[0123] To give an overview of the time course of mutation acquisition and their location in the genome, the results are presented diagrammatically in Figure 4A. It can be seen that the number of mutations steadily increases from the beginning of the experiment, but some mutations appear only to be lost again. Mutations appear not to be restricted to specific loci but appear to be spread throughout the genome.

[0124] The majority of mutations are present at a relatively low frequency, less than 10%, compared to all sequenced genomes (Figure 4). There are four types of mutations that are present at a higher frequency. This becomes clear when calculating the "persistence" of each mutation, i.e., the frequency normalized to the number of time points it remains in during the evolution experiment. This means that if a mutation occurs at the start and remains in all cultures, persistence will be 100%. If a mutation occurs midway through the experiment but is not lost, the total frequency will be 50%, but persistence will be 100%. As a result, mutations identified in the wild type have 100% persistence, but those genes can be excluded from the analysis. The four mutations with high persistence are fabF F74C, marC M35stop, and P yghb Delta-35 and rob H48frameshift.

[0125] 1.2.2 fabF F74C and marC The highly persistent mutation fabF F74C was previously described in a previous mutation experiment screening for 1-butanol (Haeyoung and Jihee, 2010). FabF encodes β-ketoacyl-ACP synthase II and is part of fatty acid biosynthesis. This mutation increases the concentration of cis-vaccenic acid compared to wild-type FabF activity.

[0126] Disrupted versions of marC were previously identified in adaptive evolution experiments of E. coli EcNR1 against isobutanol (Minty et al., 2011). marC is a conserved membrane protein, and deletion of this protein resulted in an isobutanol-tolerant phenotype. The most frequent mutation in the marC gene present in our evolution experiments was the introduction of a stop codon after M35, leaving only approximately 15% of the native protein. This mutation is thought to abolish the function of the marC gene, although the truncated version may still have a tolerance benefit.

[0127] 1.2.3 rob The next most prominent target is a mutation in the rob gene. The rob gene is a constitutively expressed regulatory element, and the regulon is shared with the marA / soxS regulatory element (Rosenberg et al., 2003; Griffith et al., 2009). The regulon is involved in antibiotic resistance, superoxide resistance, and tolerance to organic solvents (Aono, 1998). Overexpression of rob confers resistance to cyclohexane and n-hexane, and deletion confers sensitivity to these compounds (White et al., 1997). Two mutations in our sequencing results introduced premature stop codons after G273 and Y103, and the most frequent mutation introduced a frameshift after H48. The H48 frameshift mutation disrupts the protein in its helix-turn-helix domain, the part where the protein interacts with its DNA-binding site, potentially rendering it inactive (source: https: / / www.rcsb.org / pdb / protein / P0ACI0).

[0128] 1.2.4 P yghb Delta-35 The last mutation with a high frequency is in the intergenic region between metC and yghB. metC belongs to the methionine biosynthesis pathway, and yghB is a transmembrane protein involved in temperature and antibiotic resistance (Kumar and Doerrler, 2014). yghB belongs to the DedA protein family in E. coli, and double deletion of yghB and yqjA (also belonging to the DedA family) results in temperature sensitivity, but not mdfA (Na + -K + / H + This can be restored by overexpression of the antiporter.

[0129] To date, the regulator of yghB is unknown, but computational evidence suggests that σ 70This suggests that the yghB gene is regulated by a housekeeping sigma factor. Sequence analysis of the mutants reveals that the deletion eliminates the upstream portion of position -35 in the wild type. This may result in the loss of a binding site for the repressible promoter, thereby deregulating the expression of the yghB gene and increasing the yghB-mRNA concentration. See Figure 5C.

[0130] 1.2.5 Genotype correlations All other mutations have much lower persistence during the experiment, but some target genes (such as plsX gene) appear to have a relatively high frequency.If mutations occur in the same gene in different samples, it can be assumed that they have the same phenotypic effect, and therefore may have the same effect on gene functionality.In order to identify the gene set and how gene target correlates with genotype, we simplified the data set by only considering target genes, and performed principal component analysis without distinguishing between different target gene mutations.

[0131] Principal component analysis (PCA) was performed. The largest influence on the first most significant loading vector was observed for the previously identified targets, fabF, rob, and P. yghb and marC. The second component defines the genotype consisting of plsX, rraA, and gltA. As expected, the phenotype at the end of the experiment (T7) is dominated by the first component.

[0132] 1.2.6 PCA Component 2 Genotype Interestingly, a second genotype component was strongly present in culture A at T4 and T5. This genotype consists of mutations in the plsX gene, which is part of the phospholipid biosynthesis pathway, the ribonuclease inhibitor rraA, and the citrate synthase gltA. The mutation in the plsX gene may be an adaptation similar to the fabF mutation, which alters the fatty acid composition of the cell. Although plsX does not belong to the canonical phospholipid pathway, it shares homology with the alternative pathway present in Staphylococcus aureus (S. aureus) (Yao and Rock, 2013). Given that the alternative and canonical pathways have different preferences for different fatty acids, this mutation may alter the fatty acid composition of the cell membrane.

[0133] Two other mutations in this genotype may be related to more pleiotropic effects of isoprenol on cells, such as energy metabolism and protein synthesis. Mutations in gltA affect the allosteric response of citrate synthase to the inhibitory action of NADH (Duckworth et al., 2013), thus deregulating the TCA cycle and affecting energy metabolism. Two independent mutations in the ribonuclease E inhibitor rraA also appear. Loss of function in this gene would have an effect on tRNA and rRNA processing, but would also render mRNA more unstable. Indeed, V96E appears to be at a rather conserved residue (Monzingo et al., 2003).

[0134] 1.2.7 Other mutations Two frequent mutations in the third component, trkH and iscR, may be a response to ion loss due to membrane stress by isoprenol (Heipieper et al., 1994). iscR is an iron-sulfur cluster regulator, and this mutation may differentially regulate iron-sulfur cluster biogenesis. Increased potassium uptake is a known adaptation in Pseudomonas putida P8 to solvent stress (Heipieper et al., 1994), and a similar mechanism may be manifested in mutations in potassium ion transporters (Cao et al., 2011).

[0135] In addition to mutations in fatty acid metabolism genes at fabF and plsX, one additional mutation was found once in the plsB gene, which is required for phospholipid biosynthesis. yfgO is another membrane protein target of mutation, in addition to marC and yghB.

[0136] Interestingly, two of the last three isolates (T7 B and C) display independent mutations in the frmR repressor, which regulates formaldehyde metabolism. Formaldehyde sensitivity was previously tested and no differences were found between isoprenol and high and low residual formaldehyde concentrations, which may correspond to long-term acclimation, in which case formaldehyde accumulation is important. Interestingly, strains with mutations in the frmR gene have a relatively high sensitivity to prenol.

[0137] 1.3 RNA sequencing-based analysis of isoprenol stress responses Although the genotype of the adapted strains determines how they respond to isoprenol stress, secondary effects not directly apparent from the genotype may arise due to a combination of physiological changes and regulatory responses in the strain. These secondary effects are ultimately important targets for strain engineering. To identify them, we used RNA sequencing of the three final adapted strains to compare the transcriptomes of the adapted strains to those of the wild type in response to isoprenol stress.

[0138] In this analysis, we used three standard algorithms for identifying differential expression (DE): cuffdiff, edgeR, and DESeq2. The algorithms differ in four key respects. First, raw read data typically need to be corrected; this correction is primarily due to variable sequencing depth across replicates. Second, the underlying statistical model for counting is the beta-negative binomial model assumed in cuffdiff, while edgeR and DESeq2 assume a negative binomial distribution. Thus, the algorithms differ in how the distribution parameters are estimated. Because the parameters of each distribution are typically sparse (e.g., three biological replicates per sample), they cannot be directly estimated from a single data point and must be inferred from the entire data set. Finally, different significance tests can be used to identify DE.

[0139] 1.3.1 Differential expression of adapted strains compared to wild type Because the genotypes of the three adapted strains are very similar, we reasoned that the strongest transcriptome changes should occur in all three strains. The top 10 up- and down-regulated genes in all three algorithms used are shown in (Figure 6).

[0140] Due to the nature of transcriptome data, downregulation is relatively difficult to confirm, as it also depends on the quality and high coverage of the alignment. In the upregulated gene set, we observe a strong overexpression of the yghB transcript. This corresponds well with the genomic data of adapted strains, as the yghB promoter is deregulated by the deletion of the -35 region. We now find significant changes in genes upstream of the deleted metC.

[0141] Other targets from the upregulation are the ala-ala peptide exporter alaE, the outer membrane porin ompF, the valine biosynthesis genes ilvG and ilvM, and the yahO gene involved in UV and X-ray resistance. Other highly expressed genes were significant in only one of the three algorithms and therefore are not considered valid targets.

[0142] 1.3.2 Differential responses of lipid biosynthesis and the rob regulon Since many of the mutations in the experiment were targeted in fatty acid biosynthesis, we suspected that there might be differential regulation in the corresponding pathways. All fatty acid synthesis genes are upregulated compared to the wild type, most notably fabH, fabB, and clsB. Interestingly, only psd, which catalyzes a key step in phospholipid synthesis, is downregulated. However, for psd alone, this downregulation is significant for all cultures and all algorithms, while fabB upregulation is only significant for the edgeR algorithm.

[0143] Sequencing data identified the rob regulator as one of the four most important mutation targets. The exact effect of this mutation was unclear from genetic data alone, and we hypothesized that the mutation had a deleterious effect, and that because rob acts as an activator, this would decrease expression of genes in the rob regulon.

[0144] The results show that, with the exception of acnA, aldA, and fumC, all genes belonging to the rob regulon (as designated by ecocyc.org) are downregulated compared to the wild type. This supports the hypothesis that the observed rob mutation has a deleterious effect and that loss of this activator leads to subsequent downregulation of regulon genes. Because the rob regulon overlaps with the Sox and Mar regulons, genes that appear upregulated may be under even stronger control of other regulatory factors. The strongest downregulation occurs in parts of the AcrAB-TolC multidrug efflux pump miniprotein acrZ and the acid-inducible protein inaA.

[0145] 1.3.3 Differential expression between adapted strains Finally, we considered how the different mutants differed from each other in their transcriptome responses to isoprenol stress. As a comprehensive approach to assessing differences across all three datasets at once, we performed PCA analysis of the differential expression data of each isolate compared to the wild type. This analysis showed that the differential expression of frmRAB in the three mutant strains was consistent across all three algorithms. As shown above, only mutant strains 7B and 7C harbor mutations in the frmR regulator, which correlate with increased prenol sensitivity. As an example, we examined the differential expression between strains calculated by the edgeR algorithm in relation to the differential expression in the three isolates. Indeed, the expression of frmRAB did not differ between strains B and C, but frmRAB was upregulated in strains B and C compared to strain A. This data suggests that both mutations in frmR have the same effect, namely deregulation of the frmRAB operon, resulting in constitutive expression or upregulation compared to wild type and strain A.

[0146] 1.4 Reconstruction of mutations 1.4.1 Keio knockout strain We began investigating the mutations found in the final phenotype by testing knockout strains of the most promising gene targets from the readily available Keio collection. The Keio collection is implemented in the BW25113 background, which we subsequently used as a reference for resistance testing when using strains from the Keio collection. Compared to MG1655, the BW25113 strain is auxotrophic for arabinose and rhamnose. Since glucose was the only carbon source in our growth assays, this should not affect the physiology of resistance.

[0147] Wild-type BW25113 appears to have a slightly higher growth rate under isoprenol stress than MG1655, but this difference is not significant. Knocking out the regulator rob slightly increases the growth rate, but this difference is not significant. Keio strains with deleted marC exhibit a significantly increased growth rate, consistent with the results obtained in a previous study on isobutanol stress (Minty et al., 2011). We hypothesized that mutations found upstream of the yghB gene increase gene expression, and conversely, deletion of this gene should have a negative effect on tolerance. Indeed, we observe a reduced growth rate under isoprenol stress in yghB-deficient strains from the Keio collection.

[0148] 1.4.2 yghB reconfiguration To increase the tolerance to terpenes by increasing the expression of yghB, an expression vector for yghB was constructed by Gibson cloning. The plasmid has a pUC-derived ori, i.e., a high-copy plasmid (Hoschek, Buhler, and Schmid, 2017). Expression was achieved by P trc1OThe yghB gene is regulated by the promoter, which is derived from the highly expressed trp promoter and the lacUV5 promoter and contains a single lac1O operator for lacI expression (Brosius, Erfle, and Storella, 1985). To control transcription, the plasmid carries a single copy of the lacI inhibitor. The expression plasmid was confirmed by colony PCR and sequencing. In the host, the plasmid can be selected via ampicillin or chloramphenicol resistance and contains the IPTG-inducible Ptrc1O promoter used for expression of yghB.

[0149] yghB overexpression in a wild-type background As described in a previous report, yghB mRNA levels were upregulated approximately 14-fold; therefore, we hypothesized that additional expression of yghB from an overexpression plasmid in MG1655 wild-type strains might result in mutant-like expression levels of yghB and restore the resistance phenotype. Full induction with 100 μM IPTG was found to reduce growth compared to the empty vector control strain. yghB-overexpressing strains without induction or with low induction of 10 μM IPTG exhibit a small but insignificant increase in fitness.

[0150] 1.4.2.1 yghB overexpression in a marC knockout background Strong overexpression of yghB in a wild-type background did not have a positive fitness benefit. In the mutant strains in the evolution experiments, yghB mutations did not occur in isolation but concomitantly with other mutations. Because single mutations can have negative fitness effects and positive fitness effects only in conjunction with other mutations (Minty et al., 2011), we wanted to test yghB overexpression in the context of the mutation with the strongest fitness effect to date: the marC knockout. To this end, we introduced a yghB expression plasmid into a ΔmarC strain from the Keio collection. While the marC knockout exhibits a significant fitness increase, this does not affect the fitness effect of yghB induction. Minimal induction with 10 μM IPTG slightly reduces fitness compared to the ΔmarC strain, and strong induction with 100 μM IPTG reduces the fitness of the marC knockout to below that of the wild-type strain. Thus far, our data indicate that yghB overexpression has only a negative effect on fitness. We hypothesized that our expression plasmid might produce functional YghB and complement a yghB knockout strain.

[0151] 1.4.2.2 yghB overexpression in a yghB knockout background Finally, the yghB overexpression plasmid was transformed into a ΔyghB strain from the Keio collection. Knockout of yghB reduces fitness by approximately 30%. Knockout strains complemented with the yghB overexpression plasmid exhibit diverse responses to isoprenol stress. Without induction, the fitness of the complemented strain slightly exceeds that of the wild-type, but this fitness increase is not significant. Mild induction with 3–10 μM IPTG results in isoprenol tolerance similar to that of the reference strain. As in previous experiments, strong induction with 50 μM again reduces tolerance. The yghB plasmid can complement the yghB-deficient strain, albeit only under a narrow induction regime.

[0152] Hypothesized basis for yghB dysregulation While planning the CRISPR gRNA construct, we noticed that the deleted portion of the promoter upstream of the -35 region contained a sequence motif that overlaps and directly repeats the -35 region (including a 1-bp exchange) and is completely repeated on the opposite strand downstream of the -35 region (Figure 5A). Using the TOMTOM tool of the MEME suite (Gupta et al., 2007), we identified the fatty acid degradation regulator FadR and the cAMP receptor protein CRP as possible regulators with similar binding motifs; among common prokaryotic motifs, the Bacillus subtilis NatR regulator has the most similar binding motif.

[0153] 1.4.3 Knockout complementation using mutant proteins Although we initially hypothesized that the mutations found in the marC and rob genes might result in loss of function, it is unclear whether the mutant proteins retain some of their functions or have different functionality, thereby having a positive effect on isoprenol tolerance. To investigate this, we expressed the corresponding mutant proteins in a knockout background.

[0154] 1.4.3.1 marC The most frequent mutation in the marC gene introduces a stop codon after methionine at position 35 (M35stop), thereby significantly truncating the protein after the first transmembrane domain. A plasmid for expression of a version of marC with a stop after methionine at position 35 was constructed using standard methods. The plasmid is based on a pUC background, can be selected via ampicillin or chloramphenicol resistance, and contains the IPTG-inducible Ptrc1O promoter used for expression of marC M35stop.

[0155] As described hereinabove, the marC knockout alone has increased tolerance to isoprenol. Complementation of the knockout with the IPTG-inducible marC M35stop protein did not further increase tolerance to isoprenol.

[0156] 1.4.3.2 rob The transcriptional regulator rob is mutated by a frameshift at histidine 48, resulting in a truncated protein 107 amino acids long. While the protein-binding HTH motif may be intact, the remainder of the protein shares little similarity with the original protein. To test whether such a frameshifted version might have any effect when overexpressed in a knockout background, a plasmid for overexpression of robH48fs was constructed using standard methods. The plasmid is based on a pUC background, can be selected via ampicillin or chloramphenicol resistance, and contains the IPTG-inducible Ptrc1O promoter used for expression of robH48fs.

[0157] Knockout of the rob gene results in only a slight increase in resistance to isoprenol. 8 shows that introduction of a plasmid containing rob H48fs further increases resistance. This effect is lost again when the protein is strongly induced, which is thought to be due to the additional metabolic burden of protein expression. Although the mutant Rob protein may retain its DNA-binding ability due to the intact HTH motif, its regulatory function may be altered due to the deletion of the C-terminal interaction domain.

[0158] 1.4.4 Double knockout tolerance testing After examining E. coli strains with single rearranged mutations, we wanted to investigate the possible epistatic effects of multiple gene mutations. To this end, we replaced the kanamycin resistance cassette in the rob knockout by FLP recombination. This facilitates the introduction of additional knockouts using kanamycin resistance cassettes.

[0159] We investigated the combined effect of a double knockout of rob and marC. The combination of both knockouts resulted in a strain with increased tolerance to isoprenol, but the fitness increase relative to the wild-type strain was smaller than with a single knockout of marC. It is possible that knockout of rob alone does not reconstitute the actual mutation, and as shown above, the mutant Rob H48fs protein alters regulation to benefit isoprenol tolerance. To test this, we introduced a plasmid expressing the mutant Rob H48fs protein into the double knockout of rob and marC. Indeed, with the plasmid expressing the mutant protein, tolerance is slightly increased compared to the marC knockout alone.

[0160] 1.5 Broad applicability of increased resistance in the host cells and methods of the present invention 1.5.1 Butanol Butanol is another substance known to have toxic effects on microorganisms. Demonstrating the broad applicability of the host cells and methods of the present invention, mutant strains exhibiting all four major mutations showed increased tolerance to 7.5 g / L butanol (the literature value for 50% inhibitory concentration) (Figure 9A).

[0161] Experimental EC in our experimental setting 50Since we found that the yghB concentration was close to 5 g / L, we subsequently tested the most relevant mutations at this concentration (Figure 9B). Similarly, for isoprenol tolerance, we find that knocking out yghB reduces tolerance to butanol. Complementing the yghB knockout with a yghB expression plasmid expressing yghB under leaky expression conditions (0 μM IPTG) increases relative fitness by approximately 11%. As expected from isoprenol tolerance, knocking out marC increases tolerance to butanol by 32%, and interestingly, the rob knockout also increases tolerance by approximately 25%. Tolerance is further increased to 34% by leaky expression of rob H48frameshift in the Δrob background. The highest tolerance to butanol, accompanied by a 41% increase in growth rate, can be observed in the double knockout of rob and marC complemented with rob H48fs expression.

[0162] 1.5.2 Vanillin Vanillin is a commercially interesting substance with some similarity to terpenes, which also has negative effects on many microorganisms. To test the potential application of resistance mechanisms to this product, we systematically evaluated the growth rate of wild-type E. coli MG1655 using vanillin (FIG. 10A). At the concentration regimes tested, we did not find complete growth inhibition, only a reduction of the wild-type growth rate by up to one-third. At an intermediate vanillin concentration of 1 g / L, an isoprenol-adapted mutant strain (isolate A at generation 6, MutT6A) exhibits a significantly increased growth rate.

[0163] With vanillin, 50% growth inhibition is achieved at 1-1.5 g / L. For comparison, we tested significant mutations at a vanillin concentration of 1.5 g / L (see Figure 10B). In contrast to the other tested chemicals, the yghB knockout has a positive effect on vanillin tolerance, increasing growth rate by 18%. Complementing this strain with additional yghB expression only has a minor effect of an additional 3% faster growth. Unexpectedly, knocking out marC has no significant positive effect under vanillin stress. Similarly, knocking out rob results in only a minor increase of 8%. However, when the rob knockout is complemented with a mutant version of rob H48fs, tolerance increases to 36% compared to the wild type. Adding a marC knockout to this strain again reduces tolerance to a 28% growth rate increase.

[0164] 1.6 Screening targets from RNA-Seq experiments Our RNA-Seq analysis of isoprenol stress on the adapted strain revealed a list of target genes that were significantly up- and down-regulated in the adapted strain compared to the wild type. The down-regulated phenotypes could, in principle, be mimicked by knockout strains. To this end, we tested strains from the Keio knockout library for their isoprenol tolerance.

[0165] Of the five target genes glgS, rraA, menA, cspL and flu, only the rraA knockout showed a significantly increased growth rate with 50 mM isoprenol compared to the wild type.

[0166] 2 Discussion 2.1 Mutations discovered in adaptive evolution experiments We initially identified a set of 22 mutations that arose during evolution. Of these 22, four target genes and mutations were highly stable and persistent from the time of their emergence in the evolution experiment. A literature search revealed that the rob and yghB mutations had not previously been mentioned in solvent or alcohol tolerance. The fabF mutation had previously been identified in butanol tolerance (Jeong et al., 2012).

[0167] Although marC deletion mutants have been studied in the context of isobutanol tolerance (Minty et al., 2011), it was unclear whether truncated proteins such as MarC M35stop confer additional resistance effects. Our experiments revealed that expression of mutant MarC M35stop does not confer additional resistance to isoprenol. Therefore, the marC mutation likely functions as a gene deletion, and our evolution experiments did not reveal a novel resistance mechanism.

[0168] We also isolated three strains containing mutations in the rraA gene, and these mutations may also have deleterious effects on protein function, as RNA-Seq data showed strong downregulation of the rraA gene in adapted strains. Indeed, studies revealed that the rraA knockout strain had increased isoprenol tolerance.

[0169] In another embodiment, novel plsX mutants found in four of the isolated strains are useful for increasing tolerance to toxic substances such as terpenes in the host cells and methods of the invention. A different plsX mutant, PlsX E216G, has been found in isobutanol-tolerant evolution (Minty et al., 2011), but its mechanism and action are unknown.

[0170] [Table 3]

[0171] 2.2 yghB promoter mutation Genomic analysis revealed a 15-bp deletion upstream of the yghB gene, close to the -35 region, in all final isolates. Further investigation by RNA-Seq showed that expression of the yghB gene was significantly upregulated by 14-fold in all adapted strains compared to the wild type. Close examination of the yghB promoter sequence indicated that the -35 region may be flanked by two repeated sequence motifs. Interestingly, the upstream repeat of the motif was deleted in the mutant strain. The structure of this motif suggests the repressive action of a possible DNA-binding factor. Deletion of the putative regulatory factor binding site may lead to deregulation of the promoter, resulting in increased average expression of yghB. Because this motif has not been described in the literature, the role of the putative repressor remains unclear.

[0172] It is possible that yghB expression is repressed under isoprenol stress in the wild type, and this repression is relieved in the mutant. However, yghB is highly expressed in the wild type; approximately sixfold higher than the median expression. Another hypothesis could be that yghB expression is only heterologously repressed, and that heterologous repression in this subpopulation is relieved by promoter deletion mutations. In this case, studying yghB promoter activity using fluorescence microscopy under isoprenol stress would be suggested.

[0173] The initial approach to reconstituting this mutation was to construct an overexpression plasmid. However, expression of yghB in a wild-type background and only strong induction resulted in a reduced growth rate. When yghB was expressed without induction (i.e., dependent on leaky expression of the plasmid), it was able to improve resistance in the ΔyghB background. This suggests that yghB has a nonlinear, limited effect on resistance; i.e., yghB expression is beneficial only in a finely regulated expression regime, and expression levels can exceed this regime when a high-copy plasmid containing a strong expression promoter is used. Complementation of ΔyghB with leaky yghB expression increased tolerance to isoprenol, butanol, and vanillin. However, knockout of yghB also improved tolerance under vanillin stress.

[0174] 2.3 rob mutation Among the isolated strains, three distinct mutations in the rob gene were identified. Two mutations cause truncation of the protein after G273 and Y103, and the most common mutation causes a frameshift after H48, resulting in a 107-aa protein. Deletion of the rob gene had only a moderate effect on resistance to isoprenol. Complementation of a rob knockout strain with the mutant rob H48 frameshift significantly increases resistance to isoprenol. This knockout strain with rob H48 fs also develops high resistance to vanillin and butanol. The mutant Rob H48 fs contains part of the HTH DNA-binding motif; therefore, the protein is still able to bind DNA but has lost its ability to respond to molecular cues using its C-terminal receiver domain (Griffith et al., 2009).

[0175] 2.4 Combinatorial Actions During the evolutionary process, the acquisition of new mutations is often assisted by so-called epistatic effects, i.e., the fitness benefit of two mutations combined exceeds the sum (or product) of the individual fitness benefits. We found additive fitness benefits for isoprenol and butanol tolerance in the combination of marC and rob knockout with Rob H48fs expression, but this combination did not exhibit any synergistic effects. In the case of vanillin toxicity, the addition of a marC knockout to a Δrob rob H48fs strain reduced fitness, providing evidence of a negative epistatic interaction.

[0176] 2.5 RNA-Seq-derived knockout targets In our RNA-Seq experiments comparing expression of adapted versus wild-type strains under isoprenol stress, we identified a set of genes that were highly up- and down-regulated in the adapted strain. If differential regulation is beneficial for resistance, molecular engineering of up- and down-regulation could mimic this effect. This is clearly the case for the up-regulation of yghB, as shown above. Extreme down-regulation of a target gene in an adapted strain could theoretically be achieved by knocking out the target gene.

[0177] To this end, we tested a set of available knockout strains for their isoprenol tolerance. We identified the rraA gene as a targeted knockout gene beneficial for isoprenol tolerance. In addition to downregulation in the mutant strain, we also found two mutations in the initial isolates of the evolution experiment. Because the knockout strain achieved a positive resistance effect, we hypothesize that the amino acid exchanges V96-E and G67-S in the mutant RraA protein may have a negative effect on in vivo RraA function. The G67-S mutation is adjacent to a structural β-sheet element that is also present in other species. The more frequent V96-E mutation is at a highly conserved valine residue and may be crucial for RraA function (Monzingo et al., 2003). As an inhibitor of RNase E, the absence of rraA pleiotropically reduces mRNA transcript levels (Lee et al., 2003).

[0178] 2.6 Extension of positive mutations to additional chemicals The host cells and methods of the present invention achieve increased isoprenol tolerance in microorganisms such as E. coli. Furthermore, the host cells and methods of the present invention increase the tolerance of microorganisms to additional chemicals. The host cells of the present invention have relatively high tolerance to butanol and also isobutanol, and have been applied to other alcohols or aldehydes with C4 and C5 units.

[0179] We also tested the adapted strains for tolerance to the monoterpene compound geraniol and found that these strains had increased sensitivity to geraniol. We reasoned that our resistance mechanism applies only to compounds with similar physicochemical properties. Therefore, we did not test citral or menthol, both of which have lower solubility in water and higher logP values ​​than geraniol. As a result, we attempted to determine the critical physicochemical properties at which the resistance mechanism operates and selected vanillin, which has a logP value intermediate between isoprenol and geraniol. We found that vanillin tolerance can be achieved with the host cells and methods of our invention, with the exception that marC did not play a role in vanillin tolerance. Expression of mutant rob H48 fs in a Δrob background had a strong positive effect on tolerance. yghB also plays a role in vanillin tolerance, but in a manner different from that for C4 and C5 alcohols. Knockout of yghB had a negative effect on isoprenol and butanol tolerance, while it had a positive effect on vanillin tolerance.

[0180] Clearly, a single resistance mechanism will not be applicable to a wide range of compounds with highly variable physicochemical properties. However, when the same intracellular target (e.g., the cell membrane) is involved, the same gene may still be involved in the resistance mechanism, albeit in a different manner. In one embodiment of the present invention, the intracellular target of the toxic compound is the same, and resistance can be achieved by finely regulated expression and function of the genes disclosed in the present invention. This means that for membrane stress-inducing compounds, one membrane gene may need to be overexpressed or downregulated depending on the exact physical properties, but in each case, the same target gene can be utilized in one embodiment of the present invention. In another embodiment, the toolbox approach also presents targets for directed evolution approaches. Genes involved in specific resistance mechanisms can be amplified using an error-prone PCR approach and selected for their benefit to resistance.

[0181] [Table 3B] We have published some of these results (see Babel and Kromer 2020).

[0182] 3 Experimental Materials and Methods 3.1 Strains, plasmids and primers

[0183] [Table 4]

[0184] [Table 5]

[0185] [Table 6]

[0186] [Table 7] TIFF0007814310000015.tif255166 TIFF0007814310000016.tif64169

[0187] 3.2 Strain and plasmid construction 3.2.1 Expression plasmids Knockout strains were constructed by amplification of a resistance cassette with a 25-bp overlap from the corresponding Keio strain (primers 3+4, 5+6, and 7+8). PCR products carrying the homologous 25-bp sequence and kanamycin resistance were used to transform E. coli MG1655 using standard procedures (Baba et al., 2006). For the overexpression plasmids, the target gene was amplified containing 25 bp of homology to the pAH030 overexpression plasmid. The plasmid was linearized using the SpeI restriction site, and the PCR product containing the gene of interest was inserted using Gibson assembly (Gibson et al., 2009).

[0188] 3.2.2 Knockout strains Knockout strains were prepared using DNA fragments isolated from the corresponding Keio Collection strains. Recombination was performed using a standard RED / ET kit (Genebridges Red / ET Kit, 2019).

[0189] 3.3 Microbial cultivation 3.3.1 Chemically defined media For E. coli growth, M9 medium (Green and Sambrook, 2012) was used. M9 10x was adjusted to pH 7.

[0190] [Table 8]

[0191] [Table 9]

[0192] [Table 10]

[0193] 3.4 Culture scheme and conditions 3.4.1 Shake flask-based evolution The E. coli was incubated at a temperature of 37°C. Microorganisms were streaked onto suitable chemically defined media and grown at the optimal temperature. Once colony formation was observed, 10 mL of chemically defined media was inoculated with the colony and incubated in a 100 mL baffled flask at a shaking speed of 200 rpm in an Infors HT Multitron (Bottmingen, Switzerland) or Ecotron (25 mm shaking distance). From this culture, an overnight culture of 25 mL of medium in a 250 mL baffled flask was inoculated and incubated for 16 hours to ensure that the culture was in mid-exponential phase the following day.

[0194] The following day, 25 mL of medium in a 250 mL baffled flask with a Teflon-lined screw cap was inoculated to an OD of 0.2 and incubated at 200 rpm. Terpenoid stress was added to the specified concentration. Before the cell culture reached stationary phase, a portion of the culture was transferred to fresh medium in a new flask containing terpenoids. The average culture growth rate was determined by comparing the initial OD with the OD of the culture before subculture. Before passaging, 600 μL of cell culture was removed and mixed with 600 μL of 50% v / v glycerol solution. Samples were stored at −80°C.

[0195] [Table 11]

[0196] 3.5 Evaluation of growth data Growth rates were determined by natural logarithmically transforming the OD values ​​for each experiment. For linear growth, a line was fitted to the data to determine the slope, which equals the growth rate. Growth rates were determined separately for each flask, and the growth rate for each condition is given as the mean and standard deviation of three biological replicates.

[0197] 50% growth rate (MIC 50The compound concentration at 50% growth rate was estimated using linear interpolation of two adjacent data points. MICs were calculated using the MIC formula. 50 The standard error of was calculated by error propagation.

[0198] 3.6 Propidium iodide staining For propidium iodide staining, cells were grown in a 250 mL baffled, sealed shake flask under the appropriate isoprenol concentration for 5 hours. A 2 mL sample from each condition was taken and resuspended in the same volume of 0.85% NaCl solution. As a negative control, a wild-type sample was incubated with Basilol AF for 5 minutes. After washing the negative control, the sample was diluted with 1.5 × 10 7 The cells were diluted to a concentration of 1.5 x 10 cells / mL and 50 μL of SYTO 9 stock solution (in DMSO) and 6 mM propidium iodide stock solution (in DMSO) were added to give a final concentration of 5 μM SYTO 9 and 6 μM PI. SYTO 9 staining was used as a positive stain to distinguish cells from debris in the sample. Samples were incubated at room temperature for at least 20 minutes. Samples were diluted to 1.5 x 10 cells / mL before measurement. 6 The cells were diluted to a final concentration of 1000 cells / mL. Samples were measured using a Beckman Coulter CytoFLEX flow cytometer. Propidium iodide staining was detected using a 488 nm laser and a 610 / 20 BP filter for excitation, while SYTO 9 was measured using a 524 / 40 BP filter and the same excitation wavelengths.

[0199] 3.7 DNA and RNA sequencing 3.7.1 DNA sequencing 3.7.1.1 Sample preparation Selected strains were grown overnight in 5 mL LB medium supplemented with 60 mM isoprenol (for mutant strains). Genomic DNA was isolated using standard methods.

[0200] 3.7.1.2 Library preparation and sequencing 1. DNA Fragmentation Using Covaris (Desired Fragment Size: 300 bp) 2. Sample purification using a MinElute column (Qiagen), eluted in 20 μL EB buffer 3. Illumina library construction: Prepare indexed Illumina libraries using the Ovation Rapid DR Multiplex System 1-96 (NuGEN) according to the manufacturer's instructions. 4. Library amplification and size selection Libraries were amplified for 13 cycles using MyTaq (Bioline) and standard Illumina primers. Size selection was performed on the Pippin Prep system (Sage Science) using a range of 300–500 bp. 5. Final library purification step and DNA library quality control via BioAnalyzer and Qubit 6. Sequencing was performed on an Illumina NextSeq 500 / 550 (Illumina) with a 2 × 150 bp read length according to the manufacturer's instructions.

[0201] 3.7.1.3 Data analysis 1. Pre-processing of readings: Separation of all libraries for each sequencing lane using Illumina bcl2fastq 2.17.1.14 software (folder "RAW"): One or two mismatches or N in the barcode reads were allowed if the barcode distance between all libraries on the lane allowed. Clipping of sequencing adapter residues from all raw reads (folder "AdapterClipped") Reads with a final length of less than 20 bases were discarded. Quality trimming of adapter-clipped Illumina reads (folder "QualityTrimmed") Removal of readings containing N Trimming of reads at the 3' end to obtain a minimum average Phred quality score of 20 over a window of 10 bases Reads with a final length of less than 20 bases were discarded. Generate FastQC reports for all FASTQ files Generate read_counts.xlsx, which contains all read counts for all samples at a glance

[0202] 2. Alignment and variant discovery Alignment of quality trimmed reads to the reference genome using BWA-MEM version 0.7.12 (http: / / bio-bwa.sourceforge.net / ) (folder "Alignments"): One alignment file per sample in BAM format, sorted by coordinates Markup of PCR and optical duplicate reads using Picard v1.92 MarkDuplicates (http: / / picard.sourceforge.net / ) Variant discovery and sample genotyping using Freebayes v1.0.2-16 (https: / / github.com / ekg / freebayes#readme) (folder "VariantAnalysis / [reference] / Freebayes") Reads with more than two mismatches were excluded. MNP and complex variants were excluded Ploidy was set to 1.

[0203] 3.7.2 RNA sequencing 3.7.2.1 Sample preparation The wild-type and three final mutant strains were grown in biological triplicates (25 mL sealed flasks) with 50 mM isoprenol to an OD of 1.0, as described above. Subsequently, 10 mL of cell culture was vacuum filtered using a Supor® 800 Grid filter with a 0.8 μM pore size. The filter containing the cells was placed in a 15 mL Falcon tube containing 700 μL PGTX solution and immediately frozen in liquid nitrogen. Samples were stored at -80°C until further processing.

[0204] To extract RNA, the samples were incubated in a water bath at 65°C for 15 minutes with occasional vortexing, followed by incubation on ice for 5 minutes. 700 μL of chloroform was then added and incubated at room temperature for 10 minutes. The samples were centrifuged for 15 minutes, the upper aqueous phase was transferred to a new vial, and an equal volume of chloroform was added. After mixing, the samples were centrifuged for an additional 15 minutes. The upper aqueous phase (approximately 500 μL) was transferred to a new vial and mixed with an equal volume of isopropanol. The mixture was then incubated overnight at -20°C.

[0205] The next day, the mixture was centrifuged at 12,000 g at 4°C for 30 minutes in a centrifuge cleaned with RNaseZAP. The supernatant was removed, and the pellet was carefully washed with 1 mL of 70% v / v ethanol solution (without resuspending the pellet). After a further centrifugation step at 12,000 g at 4°C for 5 minutes, the pellet was air-dried for approximately 15 minutes under a clean bench. Finally, the RNA was resuspended in RNase-free water (40 μL). Sample concentrations were measured using Nanodrop. After sample treatment with the Turbo DNA-free kit (Invitrogen), concentrations were measured again, and the samples were examined on a 1.5% agarose non-denaturing gel in TAE. Samples were stained using EZ-Vision Three stain.

[0206] 3.7.2.2 Library preparation and sequencing 1. Total RNA quality control check via Bioanalyzer 2. rRNA depletion using the Ribo-Zero Bacterial rRNA Removal Kit (Illumina) according to the manufacturer's instructions 3. First-strand cDNA synthesis: NEBNext RNA First-Strand Synthesis Module (New England Biolabs) was used according to the manual. 4. Second-strand synthesis: NEBNext RNA Second-Strand Synthesis Module (New England Biolabs) was used according to the manual. 5. cDNA Purification and Concentration: The cDNA from step 5 was purified using a MinElute column (Qiagen) and eluted in 20 μL EB buffer. 6. Illumina Library Construction The Encore Rapid DR Multiplex system (Nugen) was used for library preparation according to the manual. 7. Library amplification and size selection Libraries were amplified in a volume of 100 μL using MyTaq (Bioline) and standard Illumina primers for 12 cycles. Size selection was performed on a preparative agarose gel selecting for fragments between 300 and 500 bp. 8. RNA library quality control was performed via Bioanalyzer and Qubit. 9. Sequencing on Illumina NextSeq500 / 550 (1 × 75 bp) according to the manufacturer's instructions.

[0207] 3.7.2.3 Data analysis 1. Data preprocessing Separation of all libraries for each sequencing lane using Illumina bcl2fastq 2.17.1.14 software (folder "RAW"): One or two mismatches or N in the barcode reads were allowed if the barcode distance between all libraries on the lane allowed. Clipping of sequencing adapter residues from all raw reads (folder "AdapterClipped") Reads with a final length of less than 20 bases were discarded. Filtering rRNA sequences using RiboPicker 0.4.3 (http: / / ribopicker.sourceforge.net / ) (folder "RiboPicker") Generate read_counts.xls, which contains all read counts for all samples at a glance Generate FastQC reports for all FASTQ files

[0208] 2. Differential Expression Analysis Alignment against reference (folder "Alignments") using STAR 2.4. (https: / / github.com / alexdobin / STAR / releases) Post-alignment filtering of reads aligning to rRNA or tRNA regions (folder "Alignments") Counting of TopHat aligned reads using htseq-count (http: / / www-huber.embl.de / users / anders / HTSeq / ) (folder "Alignments") Differential expression analysis using edgeR 3.2.3 (http: / / www.bioconductor.org / packages / release / bioc / html / edgeR.html), DESeq 1.12.0 (http: / / bioconductor.org / packages / release / bioc / html / DESeq.html) and cuffdiff 2.1.1 (http: / / cufflinks.cbcb.umd.edu) (folder "ExpressionAnalysis", subfolders "edgeR", "DESeq" and "cuffdiff"): Raw p-values ​​from statistical tests were adjusted for multiple testing by the Benjamini-Hochberg false discovery rate (FDR) method. We have published some of these results (see Babel and Kromer 2020).

[0209] 4 References TIFF0007814310000021.tif249170TIFF0007814310000022.tif255170TIFF0007814310000023.tif188169

[0210] Further aspects of the invention Preferably, the growth rate in the presence of a toxic substance such as a terpene is increased by 5%, 10%, or 15%, more preferably by 20%, 25%, 30%, 35%, 40%, 45%, or 50% or more compared to a control (i.e., an unmodified organism).

[0211] More preferably, the growth rate in the presence of a toxic substance such as a terpene is improved by 1.1 fold, 1.2 fold, 1.25 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.75 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold.

[0212] Modifications corresponding to the E. coli modifications of the invention are particularly useful in the methods and modified organisms of the invention, preferably corresponding to the disclosed modifications in genes encoding proteins as provided in SEQ ID NOs: 1-9, or those with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity thereto.

[0213] Unless otherwise specified, it should be understood that the terms used herein are in accordance with the conventional usage by those skilled in the relevant technical field.In addition to the definitions of terms provided herein, the definitions of common terms in molecular biology can also be found in Rieger et al., 1991 Glossary of genetics: classical and molecular, 5th Ed., Berlin: Springer-Verlag; and Current Protocols in Molecular Biology, FM Ausubel et al., Eds., Current Protocols (a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.) (1998 supplement).

[0214] As used herein and in the claims, it should be understood that "a" or "an" can mean one or more, depending on the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell is available. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0215] Standard techniques for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, etc., for cloning, DNA isolation, amplification and purification, as well as various separation techniques, are well known and commonly employed by those skilled in the art.Many standard techniques are described in M. Green & J. Sambrook (2012) Molecular Cloning: a laboratory manual, 4th Edition, Cold Spring Harbor Laboratory Press, CSH, New York; Ausubel et al., Current Protocols in Molecular Biology, Wiley Online Library; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY; Wu (eds.) 1993 Meth. Enzymol. 218, Part I; Wu (eds.) 1979 Meth. Enzymol. 68; Wu et al., (eds.) 1983 Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) 1980 Meth. Enzymol. 65; Miller (eds.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Old and Primrose, These techniques are described in 1981 Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (ed.), 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.), 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender, 1979 Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York.

[0216] Unless otherwise specified herein, the abbreviations and nomenclature, when used, are considered to be standard in the art and commonly used in professional journals such as those cited herein.

[0217] Introduction of a DNA construct or vector into a host cell can be carried out using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection-mediated or DEAE-dextran-mediated transfection or transfection using recombinant phage viruses), DNA precipitation by incubation with calcium phosphate, high-velocity bombardment of DNA-coated microparticles, and protoplast fusion. General transformation techniques are known in the art (see, for example, Current Protocols in Molecular Biology, FM Ausubel et al. (eds.) Chapter 9, 1987; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989; and Campbell et al., Curr. Genet. 16:53-56, 1989, each of which is incorporated herein by reference in its entirety, particularly with regard to transformation methods).Expression of heterologous polypeptides in Trichoderma is described in U.S. Pat. Nos. 6,022,725; 6,268,328; 7,262,041; WO 2005 / 001036; Harkki et al., Enzyme Microb. Technol. 13:227-233, 1991; Harkki et al., Bio Technol 7:596-603, 1989; European Patent Application Publication Nos. 244,234; 215,594; and Nevalainen et al., "The Molecular Biology of Trichoderma and Its Application to the Expression of Both Homologous and Heterologous Genes," in Molecular Industrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY pp. 129-148, 1992 (each of which is incorporated herein by reference in its entirety, particularly with respect to transformation and expression methods). Regarding transformation of Aspergillus strains, reference is also made to Cao et al., Sd. 9:991-1001, 2000; European Patent Application Publication No. 238023; and Yelton et al., Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (each of which is incorporated herein by reference in its entirety, particularly with respect to transformation methods). The introduced nucleic acid may be integrated into chromosomal DNA or maintained as an extrachromosomal replicating sequence.

[0218] In one embodiment, the present invention relates to isolated genes and / or proteins encoded by them that transmit increased resistance to terpene compounds, preferably monoterpene compounds, to organisms or host cells. Also included are mutants of the genes and proteins, as well as variants and nucleic acids that hybridize to the nucleic acids described herein in such capacity, where these variants and hybridizing sequences of the present invention transmit to organisms or host cells a protective effect against terpene compounds that is at least substantially the same as the protective effect of the nucleic acids of the present invention.

[0219] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and tail) as well as intervening sequences (introns) between individual coding segments (exons). Typically, it is a segment of DNA that contains the genetic information that is passed from parent to offspring and contributes to the phenotype of an organism. The influence of a gene on the form and function of an organism is mediated through transcription into RNA (tRNA, rRNA, mRNA, non-coding RNA) and, in the case of mRNA, through translation into peptides and proteins.

[0220] The term "hybridization," as defined herein, refers to the process by which substantially complementary nucleotide sequences anneal to each other. The hybridization process can occur entirely in solution, i.e., both complementary nucleic acids are in solution. The hybridization process can also occur when one complementary nucleic acid is immobilized on a matrix such as magnetic beads, Sepharose beads, or any other resin. The hybridization process can also occur when one complementary nucleic acid is immobilized on a solid support such as a nitrocellulose membrane or nylon membrane, or on a siliceous glass support, for example, by photolithography (the latter known as a nucleic acid array, microarray, or nucleic acid chip). To allow hybridization to occur, nucleic acid molecules are generally thermally or chemically denatured to melt the double strand into two single strands and / or to remove hairpins or other secondary structures from single-stranded nucleic acids.

[0221] The term "stringency" refers to the conditions under which hybridization occurs. Hybridization stringency is affected by conditions such as temperature, salt concentration, ionic strength, and hybridization buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. Moderate stringency conditions are when the temperature is 20°C lower than Tm, and high stringency conditions are when the temperature is 10°C lower than Tm. High stringency hybridization conditions are typically used to isolate hybridizable sequences with high sequence similarity to the target nucleic acid sequence. However, due to the degeneracy of the genetic code, nucleic acids may encode substantially identical polypeptides despite diverging sequences. Therefore, moderate stringency hybridization conditions may be necessary in some cases to identify such nucleic acid molecules.

[0222] "Tm" is the temperature at which 50% of a target sequence hybridizes to a perfectly matched probe at a defined ionic strength and pH. Tm depends on the solution conditions and the base composition and length of the probe. For example, longer sequences hybridize specifically at higher temperatures. Maximum hybridization rates are achieved at temperatures approximately 16°C to as much as 32°C below Tm. The presence of monovalent cations in the hybridization solution reduces the electrostatic repulsion between two nucleic acid strands, thereby facilitating hybrid formation; this effect is observed at sodium concentrations up to 0.4M (although this effect is negligible at higher concentrations). Formamide lowers the melting temperatures of DNA-DNA and DNA-RNA duplexes by 0.6-0.7°C per 1% formamide; the addition of 50% formamide allows hybridization to occur at 30-45°C, but the hybridization rate will be slower. Base pair mismatches reduce the hybridization rate and the thermal stability of the duplex. On average, for large probes, T decreases by about 1°C per 1% of base mismatches. Depending on the type of hybrid, T can be calculated using the following equation: DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984): Tm = 81.5°C + 16.6 × log[Na+]a + 0.41 × %[G / Cb] - 500 × [Lc] - 1 - 0.61 × % formamide DNA-RNA or RNA-RNA hybrids: Tm=79.8+18.5(log10[Na+]a)+0.58(%G / Cb)+11.8(%G / Cb)2-820 / Lc Oligo-DNA or oligo-RNAd hybrids: For less than 20 nucleotides: Tm=2(ln) For 20-35 nucleotides: Tm = 22 + 1.46 (ln) For a or other monovalent cations, it is only accurate in the range of 0.01 to 0.4 M. b Accurate for %GC only in the range of 30%–75%. c L = length of the duplex in base pairs. d Oligo: oligonucleotide; ln: effective length of primer = 2 × (number of G / C) + (number of A / T).

[0223] Non-specific binding can be controlled using any one of a number of known techniques, such as blocking the membrane with a protein-containing solution, adding heterologous RNA, DNA, and SDS to the hybridization buffer, and treating with RNase.For unrelated probes, a series of hybridizations can be performed by changing one of the following: (i) gradually decreasing the annealing temperature (e.g., from 68°C to 42°C) or (ii) gradually decreasing the formamide concentration (e.g., from 50% to 0%).Those skilled in the art are aware of various parameters that can be changed during hybridization and will maintain or change the stringency conditions.

[0224] In addition to hybridization conditions, hybridization specificity typically also depends on the function of post-hybridization washing. To remove background from nonspecific hybridization, samples are washed with a dilute salt solution. Important factors for such washing include the ionic strength and temperature of the final washing solution: the lower the salt concentration and the higher the washing temperature, the higher the stringency of the wash. Washing conditions are typically performed at or below the hybridization stringency. Positive hybridization produces a signal at least twice the background signal. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as described above. Higher or lower stringency conditions can also be selected. Those skilled in the art are aware of various parameters that can be changed during washing and will maintain or change the stringency conditions.

[0225] For example, typical high stringency hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 1xSSC at 65°C or in 1xSSC and 50% formamide at 42°C, followed by washing in 0.3xSSC at 65°C. Examples of moderate stringency hybridization conditions for DNA hybrids longer than 50 nucleotides include hybridization in 4xSSC at 50°C or in 6xSSC and 50% formamide at 40°C, followed by washing in 2xSSC at 50°C. The hybrid length is the expected length for the hybridizing nucleic acid. When hybridizing nucleic acids of known sequence, the hybrid length can be determined by aligning the sequences and identifying the conserved regions described herein. 1x SSC is 0.15M NaCl and 15mM sodium citrate; hybridization and wash solutions may further contain 5x Denhardt's reagent, 0.5-1.0% SDS, 100µg / mL denatured fragmented salmon sperm DNA, and 0.5% sodium pyrophosphate. Another example of high stringency conditions is hybridization in 0.1x SSC containing 0.1% SDS and optionally 5x Denhardt's reagent, 100µg / mL denatured fragmented salmon sperm DNA, and 0.5% sodium pyrophosphate at 65°C, followed by washing in 0.3x SSC at 65°C.

[0226] For purposes of defining stringency levels, reference may be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989 and annually revised).

[0227] "Recombinant" (or transgenic), with respect to a cell or organism, means that the cell or organism contains an exogenous polynucleotide introduced by means of genetic techniques, and with respect to the polynucleotide, means either: (a) the sequence of a polynucleotide or a portion thereof; or (b) one or more gene control sequences (e.g., promoters) operably linked to the polynucleotide; or (c) Both (a) and (b) means all those constructs resulting from genetic / recombinant DNA techniques in which the genes are not located in or modified in their wild-type genetic environment.

[0228] It will be further noted that the terms "isolated nucleic acid" or "isolated polypeptide" can in some instances be considered synonymous with "recombinant nucleic acid" or "recombinant polypeptide," respectively, and refer to a nucleic acid or polypeptide that is not located in its natural genetic or cellular environment and / or has been modified by recombinant methods, respectively. An isolated nucleic acid sequence or isolated nucleic acid molecule is one that is not in its native environment or in proximity to its native nucleic acid, but is physically and functionally linked to other nucleic acid sequences or molecules and is found as part of a nucleic acid construct, vector sequence, or chromosome. Typically, isolated nucleic acids are obtained in laboratory conditions by isolating RNA from cells and converting it into copy DNA (cDNA).

[0229] A "parent" (or "reference" or "template") of a nucleic acid, protein, enzyme, or organism (also referred to as a "parent nucleic acid," "reference nucleic acid," "template nucleic acid," "parent protein," "reference protein," "template protein," "parent enzyme," "reference enzyme," "template enzyme," "parent organism," "reference organism," or "template organism") is the starting point for introducing changes that result in "variants" of the parent (e.g., by introducing one or more nucleic acid or amino acid substitutions). Thus, terms such as "enzyme variant" or "sequence variant" or "variant protein" are used to distinguish modified or mutant sequences, proteins, enzymes, or organisms from the parent sequence, protein, enzyme, or organism from which the respective variant sequence, protein, enzyme, or organism is derived. Thus, a parent sequence, protein, enzyme, or organism includes wild-type sequences, proteins, enzymes, or organisms, as well as variants of the wild-type sequence, protein, enzyme, or organism that are used to develop further variants. Variant proteins or enzymes differ to a certain extent in their amino acid sequence from the parent proteins or enzymes; however, the variants retain at least the functional properties (e.g., enzymatic properties) of their respective parents. In one embodiment, the enzymatic properties are improved in the variant enzymes when compared to their respective parent enzymes. In one embodiment, the variant enzymes have at least the same enzymatic activity when compared to their respective parent enzymes, or the variant enzymes have increased enzymatic activity when compared to their respective parent enzymes.

[0230] In describing variants, the nomenclature described as follows is used: abbreviations for single amino acids used within the present invention follow the commonly accepted IUPAC one-letter or three-letter amino acid abbreviations. Although the following definitions describe variants in the context of amino acid changes, nucleic acids can be similarly modified (e.g., by nucleotide substitution, deletion, and / or insertion).

[0231] "Substitutions" are described by giving the original amino acid followed by the number of its position in the amino acid sequence, followed by the substituted amino acid. For example, substitution of histidine at position 120 with alanine is described as "His120Ala" or "H120A."

[0232] A "deletion" is the original amino acid followed by the number of its position in the amino acid sequence, followed by * Thus, the deletion of glycine at position 150 is described as "Gly150 * " or "G150 * Alternatively, the deletion may be indicated by, for example, "deletion of D183 and G184."

[0233] An "insertion" is described by giving the original amino acid followed by the number of the position in the amino acid sequence, followed by the original amino acid and the additional amino acid. For example, the insertion of a lysine at position 180 next to a glycine would be described as "Gly180GlyLys" or "G180GK." If more than one amino acid residue is inserted (e.g., Lys and Ala after Gly180), this can be indicated as Gly180GlyLysAla or G180GKA. If the substitution and insertion occur at the same position, this can be designated as S99SD+S99A or for short S99AD.

[0234] It is clear that degeneracy in nomenclature occurs when an amino acid residue identical to an existing amino acid residue is inserted, e.g., if a glycine is inserted after the glycine in the above example, this would be indicated by G180GG.

[0235] Variants containing multiple changes are separated by "+", e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple changes may be separated by spaces or commas (e.g., R170Y G195E or R170Y, G195E, respectively).

[0236] Where different changes can be introduced at a single position, the different changes are separated by commas, e.g., "Arg170Tyr, Glu" denotes the substitution of arginine at position 170 with tyrosine or glutamic acid. Alternatively, the different changes or optional substitutions can be indicated in parentheses (e.g., Arg170[Tyr, Gly] or Arg170{Tyr, Gly} or, in short, R170[Y,G] or R170{Y,G}).

[0237] A variant can contain one or more changes of any of the same type, e.g., all substitutions or a combination of substitutions, deletions, and / or insertions. Changes can be introduced into a nucleic acid or amino acid sequence.

[0238] In one embodiment, the sequence variant (i.e., amino acid sequence variant or nucleic acid sequence variant) contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 or more changes.

[0239] Variants include nucleic acids and polypeptides having about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 1-9, 10 or 10-1820, respectively.

[0240] For the substitution of an amino acid in a base sequence selected from any of the sequences of SEQ ID NOs: 1-9, regardless of the presence of that amino acid in others of these sequences, the following conditions apply, where letters indicate L-amino acids using common abbreviations and numbers in parentheses indicate the priority of the substitution (higher numbers indicate higher priority): A can be replaced by any amino acid selected from S(1), C(0), G(0), T(0) or V(0). C can be replaced by A(0). D can be replaced by any amino acid selected from E(2), N(1), Q(0) or S(0). E can be replaced by any amino acid selected from D(2), Q(2), K(1), H(0), N(0), R(0) or S(0). F can be replaced by any amino acid selected from Y(3), W(1), I(0), L(0) or M(0). G can be replaced by any amino acid selected from A(0), N(0) or S(0). H can be replaced by any amino acid selected from Y(2), N(1), E(0), Q(0) or R(0). I can be replaced by any amino acid selected from V(3), L(2), M(1) or F(0). K can be replaced by any amino acid selected from R(2), E(1), Q(1), N(0) or S(0). L can be replaced by any amino acid selected from I(2), M(2), V(1) or F(0). M can be replaced by any amino acid selected from L(2), I(1), V(1), F(0) or Q(0). N can be replaced by any amino acid selected from D(1), H(1), S(1), E(0), G(0), K(0), Q(0), R(0) or T(0). Q can be replaced by any amino acid selected from E (2), K (1), R (1), D (0), H (0), M (0), N (0) or S (0).R can be replaced by any amino acid selected from K(2), Q(1), E(0), H(0) or N(0). S can be replaced by any amino acid selected from A(1), N(1), T(1), D(0), E(0), G(0), K(0) or Q(0). T can be replaced by any amino acid selected from S(1), A(0), N(0) or V(0). V can be replaced by any amino acid selected from I(3), L(1), M(1), A(0) or T(0). W can be replaced by any amino acid selected from Y(2) or F(1). Y can be replaced by any amino acid selected from F(3), H(2) or W(2).

[0241] Nucleic acids and polypeptides can be modified to include tags or domains. Tags can be used for various purposes, including detection, purification, solubilization, or immobilization, and can include, for example, biotin, fluorophores, epitopes, mating factors, or regulatory sequences. Domains can be of any size and can provide a desired function (e.g., conferring increased stability, solubility, activity, or simplifying purification), and can include, for example, binding domains, signal sequences, promoter sequences, regulatory sequences, N-terminal extensions, or C30-terminal extensions. Combinations of tags and / or domains can also be used.

[0242] "Enzyme activity" means at least one catalytic function exerted by an enzyme. In one embodiment, enzyme activity is expressed in units of molecules of substrate converted per milligram of enzyme (specific activity) or per molecule of enzyme per minute (molecular activity).

[0243] Sequence alignment is preferably performed using the Needleman and Wunsch algorithm. Needleman and Wunsch algorithm: Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-453. This algorithm is implemented, for example, in the "NEEDLE" program, which performs a global alignment of two sequences. The NEEDLE program is included, for example, in the European Molecular Biology Open Software Suite (EMBOSS), a collection of various programs: The European Molecular Biology Open Software Suite (EMBOSS), Trends in Genetics 16 (6), 276 (2000).

[0244] A number of techniques are known for targeted modifications in the genome of an organism. The technique known as CRIPR or CRISPR / CAS is the most widely known: CRISPR (clustered regularly interspaced short palindromic repeats) technology can be used to modify the genome of a target organism, for example, to introduce any given DNA fragment into almost any site in the genome, to replace a portion of the genome with a desired sequence, or to precisely delete a given region in the genome of a target organism, allowing for unprecedented precision in genome engineering.

[0245] The CRISPR system was initially identified as an adaptive defense mechanism in bacteria belonging to the genus Streptococcus (WO 2007 / 025097). These bacterial CRISPR systems rely on a guide RNA (gRNA) in a complex with a cleavage protein to direct the degradation of complementary sequences present in invading viral DNA. The application of CRISPR systems for genetic manipulation in various eukaryotic organisms has been demonstrated (WO 2013 / 141680; WO 2013 / 176772; WO 2014 / 093595). Cas9, the first protein identified in the CRISPR / Cas system, is a large monomeric DNA nuclease that is guided to DNA target sequences flanked by a protospacer adjacent motif (PAM) sequence motif by a complex of two non-coding RNAs: the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA). Synthetic RNA chimeras (single guide RNAs or sgRNAs) created by fusing crRNA with tracrRNA have also been shown to be equally functional (WO 2013 / 176772). CRISPR systems from other sources containing DNA nucleases different from Cas9 (such as Cpf1, C2c1p, or C2c3p) have been described to have the same functionality (WO 2016 / 0205711, WO 2016 / 205749). Other authors have described systems in which the nuclease is guided by a DNA molecule rather than an RNA molecule. Such a system is, for example, the AGO system as disclosed in U.S. Patent Application Publication No. 2016 / 0046963.

[0246] Several research groups have found that CRISPR cutting properties can be used to destroy target regions in the genome of almost any organism with unprecedented ease.In recent years, it has been found that by providing repair template, it can be edited at almost any site and with almost any desired sequence in the genome, making CRISPR a powerful gene editing tool (International Publication Nos. 2014 / 150624, 2014 / 204728).Repair template is called donor nucleic acid, and contains the sequence complementary to target region at 3' and 5' end, which allows homologous recombination in each template after introducing double-strand break in target nucleic acid by each nuclease.

[0247] The main limitation of selecting target region in a given genome is the necessity of the presence of PAM sequence motifs near the region where CRISPR-associated nuclease introduces double-strand break.However, various CRISPR systems recognize different PAM sequence motifs.This allows the most suitable CRISPR system to be selected for each target region.In addition, the AGO system does not require any PAM sequence motifs.

[0248] This technology can be applied to change gene expression in any organism, for example, by replacing the promoter upstream of the target gene with a promoter of different strength or specificity. Other methods disclosed in the prior art describe fusing an activating or repressing transcription factor to a nuclease, a minus CRISPR nuclease protein. Such fusion proteins can be expressed in the target organism together with one or more guide nucleic acids that guide the transcription factor portion of the fusion protein to any desired promoter in the target organism (WO 2014 / 099744; WO 2014 / 099750). Gene knockout can be easily achieved by introducing point mutations or deletions into the respective target genes, for example, by introducing non-homologous end-joining (NHEJ), which usually leads to gene disruption (WO 2013 / 176772).

[0249] "Modified organisms" are organisms that have been modified, isolated, selected, and / or domesticated by human intervention and that differ from organisms that existed or continue to exist in the wild. Modified organisms include recombinant organisms and host cells as defined herein, but also include mutant organisms that do not use gene editing or further recombinant elements, for example, CRISPR technology used to create mutant organisms.

[0250] "Host cell" The host cell, also referred to as the host organism, can be any cell selected from a bacterial cell, a yeast cell, a fungus, an algae, or a cyanobacterial cell, a non-human animal or mammalian cell, or a plant cell. Those skilled in the art are familiar with the genetic elements that must be present on the genetic construct in order to successfully transform, select, and propagate a host cell containing the sequence of interest. In one embodiment, host cell or host organism are used interchangeably.

[0251] Typical host cells or modified organisms are Gram-positive bacteria: Bacillus, Streptomyces, and the like. Useful Gram-positive bacteria include, but are not limited to, Bacillus cells, such as Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus iautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus Prokaryotic organisms include Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. Most preferably, the prokaryotic organism is a Bacillus cell, preferably a Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, or Bacillus lentus bacillus cell.Some other preferred bacteria include strains of the order Actinomycetales, preferably of the genus Streptomyces, preferably Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382), Streptomyces lividans or Streptomyces murinus or Streptoverticillum verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include strains of the genus Myxococcus, such as M. virescens.

[0252] Further exemplary host cells or modified organisms are Gram-negative: Escherichia coli, Pseudomonas; preferred Gram-negative bacteria are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11).

[0253] Further exemplary host cells or modified organisms are fungi such as Aspergillus, Fusarium, and Tricoderma. The microorganism may be a fungal cell. As used herein, "fungi" includes Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and Deuteromycotina, and all mitosporic fungi. Representative groups of the Ascomycota include, for example, the genera Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeasts listed below. Examples of the Basidiomycota include mushrooms, rusts, and smuts. Representative groups of the Chytridiomycota include, for example, Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi. Representative groups of the phylum Oomycota include, for example, Saprolegniomycetous aquatic fungi (water molds), such as Achlya. Examples of vegetative spore-forming fungi include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of the phylum Zygomycota include, for example, Rhizopus and Mucor.

[0254] Some preferred fungi include strains belonging to the subdivision Deuteromycotina, class Hyphomycetes, such as the genera Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii, and the like. resii, Myrothecium verrucana (IFO 6113), Verticillium alboatrum, Verticillium dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli or Dressclera halodes.

[0255] Other preferred fungi include strains belonging to the subdivision Basidiomycotina, class Basidiomycetes, such as the genera Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (formerly called Polyporus), such as T. versicolor (e.g. PR4 28-A).

[0256] Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, such as the genus Rhizopus or Mucor, in particular Mucor hiemalis.

[0257] Another typical host cell or modified organism is yeast. Such as Pichia species or Saccharomyces species. Fungal host cells can be yeast cells. As used herein, "yeast" includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Ascosporogenous yeast is divided into Spermophthoraceae and Saccharomycetaceae families. The latter is composed of four subfamilies: Schizosaccharomycoideae (e.g., Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., Kluyveromyces, Pichia, and Saccharomyces). Basidiospore-forming yeasts include the genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the Fungi Imperfecti are divided into two families: Sporobolomycetaceae (eg, Sporobolomyces and Bullera) and Cryptococcaceae (eg, Candida).

[0258] Exemplary host cells or modified organisms are also eukaryotic organisms such as non-human animals, non-human mammals, birds, reptiles, insects, plants, yeast, fungi or plants. In one embodiment, the modified organism is a prokaryotic microorganism. Preferably, the host organism or modified organism according to the present invention may be a Gram-positive or Gram-negative prokaryotic microorganism.

[0259] Useful Gram-positive prokaryotic microorganisms include, but are not limited to, Bacillus cells, such as Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus iautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus Prokaryotic organisms include Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. Most preferably, the prokaryotic organism is a Bacillus cell, preferably a Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, or Bacillus lentus bacillus cell.Some other preferred bacteria include strains of the order Actinomycetales, preferably of the genus Streptomyces, preferably Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus (IFO 12382), Streptomyces lividans or Streptomyces murinus or Streptoverticillum verticillium ssp. verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include strains of the genus Myxococcus, such as M. virescens.

[0260] Further exemplary prokaryotes are gram-negative: Escherichia coli, Pseudomonas; preferred gram-negative prokaryotic microorganisms are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC 15958) or Pseudomonas fluorescens (NRRL B-11). Most preferably, the prokaryotic microorganism is Escherichia coli.

[0261] The terms "increase," "improve," or "enhance," in the context of reducing sensitivity to and increasing growth in the presence of toxic substances such as terpenes, are interchangeable and, in the applied sense, will mean an increase of at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, or 40%, as defined herein, compared to a control.

[0262] Cultivation of microorganisms often requires that the cells be cultured in a medium containing a carbon source, a nitrogen source, and various nutrient sources required for the growth of the cells, including, but not limited to, amino acids, vitamins, minerals, etc. The fermentation medium can be a minimal medium as described in WO 98 / 37179, or the fermentation medium can be a complex medium containing complex nitrogen and carbon sources, where the complex nitrogen source may be partially hydrolyzed, as described in WO 2004 / 003216.

[0263] That is, the fermentation medium contains components necessary for the growth of the cultured microorganism. In one embodiment, the fermentation medium contains one or more components selected from the group consisting of a nitrogen source, a phosphorus source, a sulfur source, and salts, and optionally one or more additional components selected from the group consisting of vitamins, amino acids, minerals, and trace elements, such as micronutrients. In one embodiment, the fermentation medium also contains a carbon source. Such components are generally well known in the art (see, e.g., Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, 1989 Cold Spring Harbor, NY; Talbot, Molecular and Cellular Biology of Filamentous Fungi: A Practical Approach, Oxford University Press, 2001; Kinghom and Turner, Applied Molecular Genetics of Filamentous Fungi, Cambridge University Press, 1992; and Bacillus (Biotechnology Handbooks) by Colin R. Harwood, Plenum Press, 1989). Culture conditions for a given cell type can also be found in the scientific literature and / or from cell sources such as the American Type Culture Collection (ATCC) and the Fungal Genetics Stock Center.

[0264] As nitrogen sources, inorganic and organic nitrogen compounds can be used, both individually and in combination. Suitable organic nitrogen sources include, but are not limited to, protein-containing substances such as extracts derived from microbial, animal, or plant cells, including, but not limited to, plant protein preparations, soybean flour, corn flour, pea flour, corn gluten, cotton flour, peanut flour, potato flour, meat and casein, gelatin, whey, fish meal, yeast protein, yeast extract, tryptone, peptone, bactotryptone, bactopeptone, waste products derived from the processing of microbial cells, plants, meat, or animal bodies, and combinations thereof. Inorganic nitrogen sources include, but are not limited to, ammonium, nitrate, and nitrite, and combinations thereof. In one embodiment, the fermentation medium contains a nitrogen source, wherein the nitrogen source is a complex nitrogen source, a defined nitrogen source, or a combination thereof. In one embodiment, the complex nitrogen source is selected from the group consisting of plant proteins, including but not limited to potato protein, soy protein, corn protein, peanut protein, cotton protein, and / or pea protein, casein, tryptone, peptone, and yeast extract, and combinations thereof. In one embodiment, the defined nitrogen source is selected from the group consisting of ammonia, ammonium, ammonium salts (e.g., ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, ammonium acetate), urea, nitric acid, nitrates, nitrite, and amino acids (including but not limited to glutamic acid), and combinations thereof.

[0265] In one embodiment, the fermentation medium further comprises at least one carbon source. The carbon source can be a complex carbon source, a defined carbon source, or a combination thereof. Various sugars and sugar-containing substances are suitable carbon sources, and sugars may be present at various stages of polymerization. Complex carbon sources include, but are not limited to, molasses, corn steep liquor, cane sugar, dextrin, starch, starch hydrolysates, and cellulose hydrolysates, as well as combinations thereof. Defined carbon sources include, but are not limited to, carbohydrates, organic acids, and alcohols. In one embodiment, defined carbon sources include, but are not limited to, glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, lactose, gluconate, acetate, propionate, lactic acid, formate, malate, citric acid, fumarate, glycerol, inositol, mannitol, and sorbitol, as well as combinations thereof. In one embodiment, the defined carbon source is provided in the form of a syrup, which may contain up to 20%, up to 10%, or up to 5% impurities. In one embodiment, the carbon source is sugar beet syrup, sugar cane syrup, or corn syrup (including but not limited to high fructose corn syrup). Complex carbon sources include, but are not limited to, molasses, corn steep liquor, dextrin, and starch, or combinations thereof. In a preferred embodiment, the defined carbon source includes, but is not limited to, glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, dextrin, lactose, gluconate, or combinations thereof.

[0266] In one embodiment, the fermentation medium also includes a phosphorus source, including but not limited to, phosphate, and / or a sulfur source, including but not limited to, sulfate. In one embodiment, the fermentation medium also includes a salt. In one embodiment, the fermentation medium includes one or more inorganic salts, including but not limited to, alkali metal salts, alkaline earth metal salts, phosphates, and sulfates. In one embodiment, the one or more salts include, but are not limited to, NaCl, KH2PO4, MgSO4, CaCl2, FeCl3, MgCl2, MnCl2, ZnSO4, Na2MoO4, and CuSO4. In one embodiment, the fermentation medium also includes one or more vitamins, including but not limited to, thiamine chloride, biotin, and vitamin B12. In one embodiment, the fermentation medium also includes trace elements, including but not limited to, Fe, Mg, Mn, Co, and Ni. In one embodiment, the fermentation medium includes one or more salt cations selected from the group consisting of Na, K, Ca, Mg, Mn, Fe, Co, Cu, and Ni. In one embodiment, the fermentation medium includes one or more divalent or trivalent cations, including but not limited to Ca and Mg. In one embodiment, the fermentation medium also includes an antifoaming agent.

[0267] In one embodiment, the fermentation medium also contains a selection agent, including but not limited to, an antibiotic (including but not limited to, ampicillin, tetracycline, kanamycin, hygromycin, bleomycin, chloramphenicol, streptomycin, or phleomycin) or a herbicide to which the selectable marker of the cells provides resistance.

[0268] Fermentation can be carried out as a batch, repeated batch, fed-batch, repeated fed-batch, or continuous fermentation process. In a fed-batch process, before the start of fermentation, no or only a portion of a compound containing one or more structural and / or catalytic elements, such as a carbon source or nitrogen source, is added to the medium, and all or the remaining portion of the compound containing one or more structural and / or catalytic elements, respectively, is supplied during the fermentation process. The compounds selected for supply can be supplied to the fermentation process together or separately from each other. In a repeated fed-batch or continuous fermentation process, a complete starting medium is additionally supplied during fermentation. The starting medium can be supplied together with or separately from the feed. In a repeated fed-batch process, a portion of the fermentation broth containing biomass is removed at regular time intervals, while in a continuous process, a portion of the fermentation broth is continuously removed. Thereby, the fermentation process is replenished with a portion of fresh medium corresponding to the amount of fermentation broth removed.

[0269] Many cell cultures incorporate a carbon source, such as glucose, as a substrate feed in the cell culture during fermentation. Thus, in one embodiment, the method for culturing a microorganism includes a feed comprising a carbon source. The carbon source-containing feed can include a defined carbon source or a complex carbon source, or a mixture thereof, as described in detail herein.

[0270] Fermentation time, pH, conductivity, temperature, or other specific fermentation conditions can be adapted according to standard conditions known in the art, In one embodiment, the fermentation conditions are adjusted to obtain maximum yield of the protein of interest. In one embodiment, the temperature of the fermentation broth during fermentation is between 30°C and 45°C. In one embodiment, the pH of the fermentation medium is adjusted to pH 6.5-9. In one embodiment, the conductivity of the fermentation medium, after pH adjustment, is between 0.1 and 100 mS / cm. In one embodiment, the fermentation time is from 1 to 200 hours.

[0271] In one embodiment, the fermentation is carried out while stirring and / or shaking the fermentation medium, hi one embodiment, the fermentation is carried out while stirring the fermentation medium at 50 to 2000 rpm.

[0272] In one embodiment, oxygen is added to the fermentation medium during cultivation, including but not limited to by agitation (stirring and / or agitation) or aeration (including but not limited to aeration with 0-3 bar air or oxygen). In one embodiment, the fermentation is carried out under oxygen saturation.

[0273] In one embodiment, the fermentation medium and methods using the fermentation medium are for industrial-scale fermentation, hi one embodiment, the fermentation medium of the present description may be useful for any fermentation having at least 20 liters, at least 50 liters, at least 300 liters, or at least 1000 liters of culture medium.

[0274] In one embodiment, the fermentation method is for the production of a protein of interest in relatively high yields, including, but not limited to, the protein of interest expressed in an amount of at least 2 g protein (dry matter) / kg raw fermentation medium, at least 3 g protein (dry matter) / kg raw fermentation medium, at least 5 g protein (dry matter) / kg raw fermentation medium, at least 10 g protein (dry matter) / kg raw fermentation medium, or at least 20 g protein (dry matter) / kg raw fermentation medium.

[0275] Tolerance should be understood as the ability of organisms to carry out their normal functions at a substantial level (for example, organisms grow at normal or somewhat reduced speed).Depending on their toxicity and dosage, toxic substances such as terpenes can cause substantial reduction in growth or stop growth, or even kill organisms.Improved tolerance to toxic substances such as terpenes will allow organisms to perform better at dosages that usually have a more severe effect on organisms.

[0276] In a preferred embodiment, a homologue of protein X is one or more proteins that correspond in function and / or sequence to protein X in an organism other than the organism in which protein X is originally found.

[0277] The activity of a target protein should be understood as the normal biological function of the protein. Inactivation should be understood as the activity not being present at the same normal level, but being substantially lower or completely absent. The presence of a target protein at a normal level is also required for normal biological function. If the presence of a target protein is substantially reduced, the biological function and therefore the overall activity will be reduced. If the target protein is absent (for example, because the gene encoding it is made non-functional, partially or completely deleted, knocked out, or its expression is prevented), the biological function will be abolished relatively quickly or slowly in the organism, or will no longer exist.

[0278] In a preferred embodiment, the terpene compound is preferably a C4 or C5 alcohol, a substance having a logP value of 2.0 or less, preferably 1.5 or less, as shown in Figure 1, and / or a solubility in water of at least 1.0 g / L, preferably 1.5 g / L or more, and / or any of these compounds: isoprenol, prenol, butanol, isobutanol, vanillin. In another embodiment, the terpene compounds include geraniol, citral, (-)-carvone, linalool, farnesol, limonene, and menthol.

[0279] In a preferred embodiment, an organism having increased tolerance to terpenes and / or useful in the methods of the invention comprises a protein that shares the first 47 amino acids with the protein of SEQ ID NO:2 or a homolog of SEQ ID NO:2 in that organism, but does not share any substantial identity beyond the amino acid corresponding to position 48 of SEQ ID NO:2, or is truncated compared to an unmodified homolog of SEQ ID NO:2 or SEQ ID NO:2 in the portion following the amino acids corresponding to positions 1-47 of SEQ ID NO:2. The present invention provides the following: 1. A modified organism having improved tolerance to one or more terpene compounds, wherein the modified organism has one or more changes compared to a wild-type modified organism selected from the group consisting of: i. the absence, inactivation or reduced abundance of protein of SEQ ID NO: 2 or a homolog thereof, and the absence, inactivation or reduced abundance of protein of SEQ ID NO: 3 or a homolog thereof, and the presence of a mutant protein of protein of SEQ ID NO: 2 or a homolog thereof, in the presence of one or more terpene compounds, wherein the mutant protein of protein of SEQ ID NO: 2 or a homolog thereof shares only the first 47 amino acids with the protein of SEQ ID NO: 2 or a homolog thereof in an unmodified organism; ii. absence, inactivation or reduced abundance of the protein of SEQ ID NO:2 or a homologue thereof in the presence of one or more terpene compounds; iii. absence, inactivation or reduced abundance of the protein of SEQ ID NO: 3 or a homologue thereof in the presence of one or more terpene compounds; iv. the absence of the protein of SEQ ID NO: 2 or a homolog thereof, and the presence of a mutant protein of the protein of SEQ ID NO: 2 or a homolog thereof, in the presence of one or more terpene compounds, wherein the mutant protein of the protein of SEQ ID NO: 2 or a homolog thereof has a mutation at a position corresponding to position 48 of SEQ ID NO: 2; v. the presence of a mutant protein of the protein of SEQ ID NO: 2 or a homolog thereof in the presence of one or more terpene compounds, wherein the mutant protein of the protein of SEQ ID NO: 2 or a homolog thereof has a mutation at a position corresponding to position 48 of SEQ ID NO: 2; vi. An increased level or increased activity of the protein of SEQ ID NO: 1 or a homolog thereof in the presence of one or more terpene compounds compared to an unmodified organism, preferably wherein the endogenous gene for the homolog of SEQ ID NO: 1 has been deleted and wherein the recombinant expression of the gene encoding SEQ ID NO: 1 or a variant thereof is present, and even more preferably wherein the recombinant expression of the gene encoding SEQ ID NO: 1 or a variant thereof is under a low to medium strength promoter or other control element; vii. the presence of a mutant protein of the protein of SEQ ID NO: 4 or a homolog thereof, in the presence of one or more terpene compounds, wherein the mutant protein of the protein of SEQ ID NO: 4 or a homolog thereof has a mutation at a position corresponding to position 74 of SEQ ID NO: 4; viii. The presence of a mutant protein of the protein of SEQ ID NO: 5 or a homolog thereof in the presence of one or more terpene compounds, preferably the mutant protein of the protein of SEQ ID NO: 5 or a homolog thereof having (a) a mutation at a position corresponding to position 291 of SEQ ID NO: 5 and / or (b) a mutation at a position corresponding to position 274 or later of SEQ ID NO: 5, wherein the mutant protein is shorter than the protein of SEQ ID NO: 5 or a homolog thereof, or the absence, inactivation or reduced abundance of the protein of SEQ ID NO: 5; ix. the presence of a mutant protein of SEQ ID NO: 6 or a homolog thereof in the presence of one or more terpene compounds, wherein the mutant protein of SEQ ID NO: 6 or a homolog thereof has a mutation at position 96 of SEQ ID NO: 6, preferably a substitution of valine with glutamic acid, and / or a mutation at position 67 of SEQ ID NO: 6, preferably a substitution of glycine with serine; x. absence, inactivation, or reduced abundance of the protein of SEQ ID NO: 6 or a homolog thereof in the presence of one or more terpene compounds; xi. the absence, inactivation, or reduced abundance of a modified protein of SEQ ID NO: 8 or a homolog thereof, preferably the protein of SEQ ID NO: 8 or a homolog thereof, in the presence of one or more terpene compounds; xii. The absence, inactivation, or reduced abundance of a modified protein of SEQ ID NO: 9 or a homolog thereof, preferably the protein of SEQ ID NO: 9 or a homolog thereof, in the presence of one or more terpene compounds; xiii. absence, inactivation, increased activity or decreased abundance of a modified protein of SEQ ID NO: 7 or a homolog thereof, preferably a protein of SEQ ID NO: 7 or a homolog thereof, in the presence of one or more terpene compounds; xiv. Any combination of the above i to xiii. 2. A method for increasing the tolerance of a modified organism to one or more terpene compounds compared to an unmodified organism, comprising the steps of producing a modified organism described in 1 above, and optionally maintaining the modified organism. 3. A method for producing one or more terpene compounds using an organism, comprising the steps of: creating a modified organism described in 1 above; maintaining the modified organism in the presence of one or more terpene compounds under conditions suitable for the modified organism to grow and produce the one or more terpene compounds; and optionally separating the one or more terpene compounds from the modified organism. 4. The method of claim 2 or 3, wherein the modified organism comprises, in the presence of one or more terpene compounds, (a) a knockout or deletion, knockout, of a portion or the entire gene encoding the protein of SEQ ID NO: 3 or a homolog thereof, or (b) a deletion of a portion or the entire gene encoding the protein of SEQ ID NO: 2 or a homolog thereof, or (c) the presence of a mutant protein of SEQ ID NO: 2 or a homolog thereof, wherein the mutant protein of SEQ ID NO: 2 or a homolog thereof shares only the first 47 amino acids with the protein of SEQ ID NO: 2 or a homolog thereof in an unmodified organism, or any combination of (a) to (c). 5. A method according to any one of 2 to 4 above, comprising the step of downregulating the expression of a gene encoding the protein of SEQ ID NO: 6 or a homolog thereof, deleting the gene encoding the protein of SEQ ID NO: 6 or a homolog thereof, or knocking out the gene encoding the protein of SEQ ID NO: 6 or a homolog thereof. 6. A method for producing monoterpene esters, comprising producing one or more monoterpenes according to any of the methods described in 3 to 5 above, and esterifying at least one monoterpene to a monoterpene ester, and optionally separating the one or more monoterpene esters. 7. A method for increasing the tolerance of a modified organism to vanillin compared to an unmodified organism, comprising expressing or producing in the modified organism a DNA sequence encoding a protein that shares only the first 47 amino acids with the protein of SEQ ID NO:2, wherein the modified organism has the additional characteristic that the proteins of SEQ ID NO:1 and / or 2 or homologs thereof are absent, inactive, or substantially reduced. 8. Use of a deregulated protein of SEQ ID NO: 2 or a homolog thereof to increase the growth of a modified organism in the presence of one or more terpene compounds, preferably one or more terpenes or one or more terpene esters. 9. A modified organism, method or use according to any one of 1 to 8 above, wherein the mutant or deregulated protein of SEQ ID NO: 2 or a homologue thereof has a mutation of the histidine residue corresponding to position 48 of SEQ ID NO: 2, which results in a frameshift, preferably a frameshift that shortens the resulting protein compared to the protein of SEQ ID NO: 2. 10. A modified organism, method or use according to any one of 1 to 9 above, wherein any of the sequences of SEQ ID NOs: 1 to 9 is mutated to carry the mutations shown in Table 3 for the respective proteins. 11. A method, use, mutant protein or modified organism according to any one of 1 to 10 above, wherein the tolerance to isoprenol, prenol, butanol, isobutanol, vanillin, geraniol, santalene, valencene, sclareol, artemisinic alcohol, artemisinic acid and / or citral is increased compared to an unmodified organism. 12. A method, use, mutant protein or modified organism according to any one of 1 to 11 above, wherein the tolerance to isoprenol, prenol, butanol, isobutanol and / or vanillin is increased compared to an unmodified organism. 13. A method, use or modified organism according to any one of 1 to 12 above, wherein at least one terpene compound has a logP value of 2.0 or less, preferably 1.5 or less. 14. A method, use or modified organism according to any one of 1 to 13 above, wherein the solubility of the at least one terpene compound in water is at least 1.0 g / L, preferably 1.5 g / L or more. 15. A method, use or modified organism according to any one of 1 to 14 above, wherein at least one terpene compound is a monoterpene alcohol or a C4 and C5 alcohol.

Claims

1. A composition for the production of one or more terpene compounds comprising a modified E. coli, wherein the modified E. coli is capable of: The absence, inactivation, or reduced abundance of the protein of SEQ ID NO: 3 or a homolog thereof in the presence of one or more terpene compounds compared to unmodified E. coli; and Knockout or deletion of part or the whole of the gene encoding the protein of SEQ ID NO: 3 or its homologue The composition has one or more changes compared to the wild-type modified E. coli selected from the group consisting of: and has improved resistance to one or more terpene compounds compared to the unmodified E. coli.

2. A method for the production of one or more terpene compounds using an organism, comprising the steps of: generating a modified Escherichia coli having improved tolerance to one or more terpene compounds, the modified Escherichia coli comprising a knockout or deletion of part or all of a gene encoding the protein of SEQ ID NO: 3 or a homologue thereof; and maintaining the modified E. coli in the presence of one or more terpene compounds under conditions suitable for the modified E. coli to grow and produce the one or more terpene compounds. A method comprising:

3. isolating one or more terpene compounds from the modified E. coli.

3. The method of claim 2, further comprising:

4. Production of one or more monoterpenes according to the method of claim 2, and Esterifying at least one monoterpene to a monoterpene ester.

1. A method for producing monoterpene esters, comprising:

5. isolating the monoterpene esters.

5. The method of claim 4, further comprising:

6. The composition of claim 1, wherein the sequence of the gene encoding the protein of SEQ ID NO: 3 has been mutated to have an I135-stop or M35-stop mutation, or a frameshift at the stop codon.

7. The composition of claim 1 or 6, wherein the resistance of the modified E. coli to isoprenol, prenol, geraniol, santalene, valencene, sclareol, artemisinic alcohol, artemisinic acid and / or citral is increased compared to unmodified E. coli.

8. 8. The composition of any one of claims 1, 6 and 7, wherein at least one terpene compound has a logP value of 2.0 or less or 1.5 or less.

9. 9. The composition of any one of claims 1 and 6 to 8, wherein the solubility of the at least one terpene compound in water is at least 1.0 g / L or 1.5 g / L or more.

10. 10. The composition of any one of claims 1 and 6 to 9, wherein at least one terpene compound is a monoterpene alcohol.

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