Method for producing vanillin

Abstract

The present application relates to a novel method for producing vanillin and / or its derivatives by using an improved biocatalyst. An expression system for preparing the improved biocatalyst is also provided. In addition, novel enzyme mutants, corresponding coding sequences and vectors that can be used for the biochemical production of vanillin are also provided herein. The present invention further provides a recombinant host cell or organism that has been genetically modified to improve the expression of the biocatalyst function, and a recombinant host cell or organism for producing vanillin.

Description

Technical Field

[0001] This article provides a biochemical method for the production of vanillin and related compounds and derivatives, which includes the use of novel combinations of peptides. Furthermore, this article provides novel enzyme mutants that can be used for the biochemical production of vanillin. Background Technology

[0002] Vanillin is a crucial compound used globally in food, beverages, and pharmaceuticals. Only a small fraction of global production is derived naturally from vanilla pods; however, these natural plant sources are scarce, and production methods are laborious and slow. While complex extracts from the orchid pods of vanilla (V. planifolia) or Tahitian vanilla (V. tahitensis) offer unique and complex blends and olfactory qualities at high cost, the majority of vanillin (>99%, or 16,000 tonnes annually) is produced from petrochemical feedstocks, with a small amount still obtained from lignin waste from the paper industry. However, the market for natural “vanillin not from the bean” (NFB) is small but growing, primarily serving North America and Europe. These two markets have different regulatory requirements for natural status.

[0003] Different biochemical methods can be used to convert various raw materials, including ferulic acid, curcumin, eugenol, or isoeugenol, into vanillin. Biotechnological methods for producing vanillin have been reviewed [Gallage NJ, BL.2015. Mol Plant 8, 40-57; Kaur, B, Chakraborty, D. 2013. Appl Biochem Biotechnol 169, 1353-1372; Walton NJ, Mayer MJ, Narbad A. 2003. Phytochem 63, 505-515].

[0004] Recently, microorganisms have been engineered to convert simple carbon sources (such as sugars, like glucose) into vanillin [Hansen EH, Lindberg Moller B, Kock GR, Bünner CM, C Kristensen, Jensen OR, Okkels FT, Olsen CE, Motawia MS, Hansen J. 2009. Appl Environ Microbiol 75, 2765-2774; Ni J, Tao F, Du H, Xu P. 2015. Sci Rep 5, 1-11].

[0005] Isoeugenol is a phenylpropenoid compound found in many plants. However, its abundance appears to be lower than that of eugenol, which was traditionally obtained from eugenol via heating isomerization in the presence of a strong base. This method is no longer compatible with European regulations on natural flavorings. Nevertheless, herbal medicines rich in isoeugenol can be identified.

[0006] Isoeugenol can be converted to vanillin via a range of different biochemical methods. For example, enzymes containing non-heme iron, such as lipoxygenases, or enzymes containing iron porphyrins (such as horseradish peroxidase) have been used to oxidatively cleave isoeugenol to vanillin. Recently, it has been found that separately purified ferric chloride is as effective as non-specific heme-containing enzymes for this biotransformation [Mutti FG. 2012. Bioorg Chem Appl 2012, 1-13; Li YH, Sun ZH, Zhao LQ, Xu Y. 2005. Appl Biochem Biotechnol 125, 1-10; Mutti FG, Lara M, Kroutil M, Kroutil W. 2010. Chem Eur J 16, 14142-14148]. However, these methods are prone to forming byproducts.

[0007] Interestingly, microbial isoeugenol oxidases, including but not limited to isoeugenol monooxygenases, have been described for the conversion of isoeugenol to vanillin [Ryu JY, J Seo, S Park, JH Ahn, YChong, MJ Sadowsky, HG Hur. 2013. Biosci Biotechnol Biochem 77, 289-294; Yamada M, Y Okada, T Yoshida, T Nagasawa. 2007. Appl Microbiol Biotechnol 73, 1025-1030; Yamada M, Y Okada, T Yoshida, T Nagasawa 2008. Biotechnol Lett 30, 665-670]. Oxidation of isoeugenol with these enzymes showed the formation of vanillin and acetaldehyde as major products [Yamada M, Y Okada, TYoshida, T Nagasawa. 2007. Arch Microbiol 187, 511-517; Ryu JY, J Seo, S Park, J-HAhn, Y Chong, MJ Sadowsky, HG Hur. 2013. Biosci Biotechnol Biochem 77, 289-294]. However, the methods described therein are not for the industrial-scale production of vanillin.

[0008] For industrial processes, it is often necessary to overproduce one or more functional enzymes in recombinant bacteria such as *E. coli*. Overproduction of an enzyme in suitable bacteria provides a safe and economical source of that enzyme. Overproduction of key enzymes also helps to minimize side effects caused by endogenous enzyme activity in the host organism (e.g., enzymes that can reduce or oxidize vanillin to vanillyl alcohol or vanillic acid, respectively).

[0009] Proper folding of enzymes in the host organism is crucial for catalytic activity. Improperly folded enzymes exhibit reduced or no catalytic activity because they readily aggregate (e.g., into inclusion bodies) or degrade within the host. Various strategies can be employed to ensure proper enzyme folding in the host organism. For example, reducing expression levels in the recombinant host organism through low gene doses, or reducing transcript levels through low inducer concentrations (in the case of inducible systems) or low promoter strength can be beneficial. Low temperatures during enzyme synthesis also contribute to improved folding of recombinant proteins. Alternatively, sometimes improperly folded enzymes unfold due to strong denaturing chemicals and then refold under physiological conditions. However, such processes are both time-consuming and expensive.

[0010] In molecular biology, a large class of molecular chaperones represents proteins that assist in the covalent folding or unfolding of other macromolecular structures, as well as the assembly or disassembly of other macromolecular structures. The chaperonin proteome belongs to this large class of molecular chaperones. These chaperonins have structures resembling two donut-shaped structures stacked on top of each other to form a barrel. Each ring consists of 7, 8, or 9 subunits, depending on the organism in which the chaperonin was discovered.

[0011] Group I molecular chaperones have been found in bacteria and endosymbiotic organelles: chloroplasts and mitochondria. Group II molecular chaperones, found in eukaryotic cytosols and archaea, are difficult to characterize. The GroEL / GroES complex is a Group I molecular chaperone. It is not assumed that Group II molecular chaperones utilize GroES-type cofactors to fold their substrates.

[0012] As previously mentioned, the molecular chaperone system GroES / GroEL forms a barrel-shaped structure with cavities, which can absorb misfolded proteins for refolding at the cost of ATP [Gragerov A, E Nudler, N Komissarova, GAGaitanaris, ME Gottesman, V Nikiforov. 1992. Proc Nat Acad Sci 89, 10341-10344; Keskin O, Bahar I, Flatow D, Covell DG, Jernigan RL. 2002. Biochem 41, 491-501].

[0013] To date, no member of the major class of molecular chaperones has been investigated for the recombinant production of monooxygenases, particularly the applicability of isoeugenol monooxygenase for the enzymatic synthesis of vanillin.

[0014] Although several reports exist on the bioengineering production of natural vanillin (e.g., WO2013 / 022881, KR101163542(B1)), there remains a need to discover simpler, more efficient, and cost-effective methods for the production of natural vanillin. In particular, higher specific activity of enzymes would allow for lower catalyst loading, thus improving process performance and economics. Furthermore, enzymes requiring further refinement are needed for the biochemical production of vanillin, especially from isoeugenol as a starting material. Summary of the Invention

[0015] This invention addresses the aforementioned drawbacks associated with known systems applied to the biochemical production of vanillin to date.

[0016] The inventors of this invention unexpectedly observed that certain auxiliary peptides can be successfully used for the large-scale production of certain known, and even some novel, highly functional isoeugenol oxidases. In particular, the inventors unexpectedly discovered that co-expression with molecular chaperones GroES and GroEL significantly increased the amount of catalytically active isoeugenol monooxygenase produced in Escherichia coli.

[0017] Furthermore, the inventors of this invention were unexpectedly able to genetically modify microbial oxidases from *Pseudomonas putida*, thereby enabling them, for the first time, to be used for the conversion of isoeugenol, particularly (E)-eugenol, to vanillin in the presence of molecular oxygen.

[0018] Therefore, the inventors of this invention can unexpectedly improve the biochemical method for producing vanillin from isoeugenol by applying isoeugenol oxidase, which is described in more detail below. Attached Figure Description

[0019] Figure 1 The one-step conversion of isoeugenol to vanillin and acetaldehyde.

[0020] Figure 2 Polycistronic constructs of isoeugenol monooxygenase (IEM1) from *Pseudomonas nitroreducens* Jin1, along with molecular chaperones GroES and GroEL, were co-expressed using low-copy and high-copy plasmids. The plasmid names of the low-copy-number constructs are shown to the left of each construct. The numbers above the horizontal bars indicate the following elements: 1: RBS (strong); 2: IEM1; 3: GroES; 4: native RBS; 5: GroEL; 6: spacer; 7: RBS (medium); 8: RBS (weak).

[0021] Figure 3 First-generation catalyst based on IEM1: Product titers at different biocatalyst loadings at an unrestricted substrate concentration of pH 10.5. Symbols: vanillin (◆), remaining trans-isoeugenol (■), remaining (Z)-isoeugenol (▲). Average data from two experiments are shown.

[0022] Figure 4 Second-generation catalysts: Product titers expressed as different optical unit densities under different catalyst loadings: OD 600 =18 (vertical stripes), OD 600 =10 (horizontal stripes), OD 600 =5 (dashed line), OD 600 =2 (diagonal stripes), OD 600=1 (black). This biocatalyst was pre-grown in LB medium. IEM1: Isoeugenol monooxygenase of *Pseudomonas nitroreducens* Jin1. Co-expressed molecular chaperones are also shown. IEM1 (control) represents the activity of wild-type isoeugenol monooxygenase of *Pseudomonas nitroreducens* Jin1 expressed according to Example 1 in the absence of any molecular chaperones. This value reflects the mean endpoint measurement within 10%.

[0023] Figure 5 Using one of the first-generation catalysts (vertical stripes), second-generation catalysts (horizontal stripes), and third-generation catalysts (dashed lines), at OD 600 Product titers for small-scale reactions at a catalyst loading of 5. A negative control was obtained using empty cells (black filler). The average endpoint concentration (maximum 10% variation) from the two experiments is shown.

[0024] Figure 6 Vanillin titers obtained using single, double, and triple mutants of isoeugenol monooxygenase (IEM2) from *Pseudomonas putida* IE27. "Ref" refers to (inactive) isoeugenol monooxygenase from *Pseudomonas putida* IE27 used as a reference. Cells producing isoeugenol monooxygenase (IEM1) from *Pseudomonas nitroreducens* Jin1 were used as a positive control (C+). Cells containing the empty vector were used as a negative control (C-). Data points are the average of duplicate experiments.

[0025] Figure 7 The bioconversion of isoeugenol to vanillin under different catalyst loadings is shown as a triple-codon mutant (horizontal stripes) of isoeugenol monooxygenase (IEM2) from *Pseudomonas putida* IE27 and isoeugenol monooxygenase (IEM1) from *Pseudomonas nitroreducens* Jin1 (vertical stripes). *E. coli* cells with a dual-plasmid system for co-expressing isoeugenol monooxygenase and the molecular chaperones GroES and GroEL acted as catalysts. The isoeugenol monooxygenase from *Pseudomonas nitroreducens* Jin1 was produced by the high-copy construct, while the triple-copy mutant from *Pseudomonas putida* IE27 was produced by the low-copy construct. The average product titers from the two experiments are shown, with a deviation of less than 10%.

[0026] Figure 8Amino acid sequence alignment of IEM 1 of Pseudomonas nitroreducens Jin1 and IEM 2 of Pseudomonas putida IE27.

[0027] Figure 9 Annotations and corresponding plasmid diagram for the triple mutant of IEM2, pIEM2_C154_T222_A1318, used to express the IEM2 of Pseudomonas putida IE27.

[0028] Figure 10 The relative activities of isoeugenol monooxygenase mutants of *Pseudomonas putida* IE27, obtained by individual saturation mutagenesis at amino acid positions T52, Q74, or D440, are shown on the horizontal axis. The corresponding amino acids at these positions are indicated. The relative activity of each mutant is expressed as a fraction of the mean vanillin titer obtained under standard assay conditions with mutant “C154” (i.e., mutant a154c; corresponding to PQD). For comparison, previously identified mutants “T222” (i.e., mutant g222t, corresponding to THD) and G1318 (i.e., mutant g1318a; corresponding to TQN) are also shown. Empty cells were used as a negative control (C-).

[0029] Figure 11 : Overall scheme for expressing IEM (such as the triple mutant of isoeugenol monooxygenase IE27 described in this article), as well as the modified insertion structures of molecular chaperones GroES and GroEL.

[0030] Figure 12 The standardized product titer of vanillin obtained from E. coli strains containing different plasmid constructs under standard assay conditions. Figure 11 A universal protocol was developed for the preparation of triple mutants of isoeugenol monooxygenase IE27, as well as molecular chaperones GroES and GroEL. The plasmid constructs differ in the promoter sequences expressing molecular chaperones and optionally contain transcription terminators, denoted as +term or -term, after the ORF of isoeugenol monooxygenase. The polycistronic construct PC1_triple is shown as reference C+. Detailed Implementation

[0031] abbreviations used

[0032] bp base pairs

[0033] kb kilobase

[0034] DNA deoxyribonucleic acid

[0035] cDNA complementary DNA

[0036] DTT dithiothreitol

[0037] GC gas chromatography

[0038] IPTG isopropyl-D-thiogalactopyranoside

[0039] IEM isoeugenol monooxygenase

[0040] LB solubilized broth

[0041] MS mass spectrometer / mass spectrometer

[0042] PCR polymerase chain reaction

[0043] RBS ribosome binding site

[0044] RNA (ribonucleic acid)

[0045] mRNA messenger ribonucleic acid

[0046] miRNA

[0047] siRNA (small interfering RNA)

[0048] rRNA ribosomal RNA

[0049] tRNA transfer RNA

[0050] Pseudomonas

[0051] Specific definition

[0052] The term “isoeugenol” refers to 2-methoxy-4-(prop-1-en-1-yl)phenol (CAS Registry No.: 97-54-1) and trans- and cis-isoeugenol, or any mixture of (E)- and (Z)-isoeugenol isomers.

[0053] The terms "isoeugenol monooxygenase," "polypeptide with isoeugenol monooxygenase activity," "isoeugenol monooxygenase protein," "isoeugenol oxidase," "polypeptide with isoeugenol oxidation activity," "isoeugenol oxidase protein," or "IEM" refer to a polypeptide capable of catalyzing the synthesis of vanillin from isoeugenol in the presence of molecular oxygen in the formation of acetaldehyde, without being limited to any specific molecular mechanism of action of the enzyme. Preferably, the enzyme stereoisomerizes the (E)- or trans-isomer of isoeugenol. Vanillin is preferably obtained as the major product.

[0054] The oxidative activity of isoeugenol was determined under standard conditions, as described in more detail in the following examples: recombinant IEM expression cells, ruptured IEM expression cells, fractions or enriched or purified IEM enzymes of these cells, in the presence of molecular oxygen, at a temperature of about 20 to 30°C and in the presence of a reference substrate, preferably buffered at a pH of 8.5 to 11, preferably 9 to 10, where the reference substrate is isoeugenol, particularly trans-isoeugenol, at an initial concentration of 1 to 40 mg / ml, preferably 1 to 10 mg / ml, more preferably 3 to 7 mg / ml.

[0055] The terms “biological function,” “function,” “biological activity,” or “activity” refer to the ability of isoeugenol oxidase to catalyze the formation of vanillin from isoeugenol.

[0056] As used herein, the term "host cell" or "transformed cell" refers to a cell (or organism) that has been altered to carry at least one nucleic acid molecule, such as a recombinant gene encoding a desired protein or nucleic acid sequence, which, upon transcription, produces a polypeptide as described herein. Host cells are particularly bacterial, fungal, or plant cells. Host cells may contain recombinant genes integrated into the host cell's nuclear or organelle genome. Alternatively, the host may contain recombinant genes extrachromosomally.

[0057] "Homologous" sequences include orthologous or paralogous sequences. Methods for identifying orthologous or paralogous sequences include phylogenetic methods, sequence similarity methods, and hybridization methods known in the art and described herein.

[0058] "Paralleloids," or paralogous sequences, arise from gene replication, resulting in two or more genes with similar sequences and functions. Paralogs typically cluster together and form through gene replication within related plant species. Paralogs are identified in groups of similar genes using pairwise BLAST analysis or procedures such as CLUSTAL during phylogenetic analysis of gene families. In paralogs, the shared sequence can be identified as a sequence characteristic of the related gene and possessing a similar function.

[0059] "Orthologs," or orthologous sequences, are sequences that are similar to each other because they are found in species descended from a common ancestor. For example, plant species known to share a common ancestor contain many enzymes with similar sequences and functions. For instance, by constructing a phylogenetic tree of a gene family for a species using procedures such as CLUSTAL or BLAST, a technician can identify orthologous sequences and predict the functions of orthologs. One method for identifying or confirming similar functions between homologous sequences is by comparing transcript profiles in host cells or organisms (such as plants or microorganisms) that overexpress or lack (in gene knockout / reduction) the relevant polypeptide. A technician can understand that genes with similar transcript profiles (having more than 50% regulated common transcripts, or more than 70% regulated common transcripts, or more than 90% regulated common transcripts) will have similar functions. By causing host cells, organisms such as plants or microorganisms to produce isoeugenol monooxygenase, homologs, parahomologs, orthologs, and any other variants of the sequences described herein are expected to function in a similar manner.

[0060] The term "plant" is used interchangeably to include plant cells, including plant protoplasts, plant tissues, plant cell tissue cultures that produce regenerated plants or plant parts, or plant organs such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits, etc. Any plant can be used to implement the methods described herein.

[0061] "Capable of producing vanillin" means that a particular organism or cell naturally produces vanillin, or that it does not naturally produce vanillin but is transformed to produce vanillin before or with the nucleic acid described herein. Organisms or cells transformed to produce higher amounts of vanillin than naturally occurring organisms or cells are also included in "capable of producing vanillin".

[0062] As used herein, the terms “purified,” “substantially purified,” and “isolated” refer to a state free from other distinct compounds that are normally associated with the compounds of the present invention in their natural state. Therefore, “purified,” “substantially purified,” and “isolated” articles comprise at least 0.5%, 1%, 5%, 10%, or 20%, or at least 50% or 75% by weight of a given sample. In one embodiment, these terms refer to the compounds of the present invention comprising at least 95%, 96%, 97%, 98%, 99%, or 100% by weight of a given sample. As used herein, the terms “purified,” “substantially purified,” and “isolated,” when referring to nucleic acids or proteins, also refer to a purified or concentrated state that differs from a state naturally present in, for example, prokaryotic or eukaryotic environments, such as in bacterial or fungal cells, or in mammals, particularly humans. Any degree of purification or concentration greater than that of naturally present purification or concentration, including (1) purification from other related structures or compounds, or (2) association with structures or compounds that are normally unrelated in the said prokaryotic or eukaryotic environment, is within the meaning of “isolated.” Nucleic acids, proteins, or classes of nucleic acids or proteins described herein may be isolated or associated with structures or compounds that are otherwise unrelated in nature, according to various methods and processes known to those skilled in the art.

[0063] In the context of the specification and appended claims provided herein, unless otherwise stated, the use of “or” means “and / or”.

[0064] Similarly, the meanings of "containing", "containing", "including", and "comprise" in various tenses are interchangeable rather than restrictive.

[0065] It should be further understood that, where the term “comprising” is used in the description of various implementation schemes, those skilled in the art will understand that, in certain specific cases, the language of “substantially consisting of” or “consisting of” can be used instead to describe the implementation schemes.

[0066] The term “about” indicates a possible variation of ±25% in the value, particularly ±15%, ±10%, more particularly ±5%, ±2%, or ±1%.

[0067] The term "substantially" describes a value range of approximately 80% to 100%, such as 85% to 99.9%, particularly 90% to 99.9%, even more particularly 95% to 99.9%, or 98% to 99.9%, particularly 99% to 99.9%.

[0068] "Mainly" refers to a proportion in the range of 50%, such as in the range of 51% to 100%, particularly in the range of 75% to 99.9%; especially in the range of 85% to 98.5%, such as 95% to 99%.

[0069] In the context of this invention, "major product" means a single compound or a group of at least two compounds, such as two, three, four, five or more, particularly two or three compounds, which are "majorly" prepared by the reaction described herein and are contained in the reaction in a major proportion based on the total amount of the components of the products formed by the reaction. The proportion may be a molar proportion, a weight proportion, or preferably an area proportion calculated from the corresponding chromatograms of the reaction products based on chromatographic analysis.

[0070] In the context of this invention, "byproduct" means a single compound or a group of at least two compounds, such as two, three, four, five or more, particularly two or three compounds, which are not "mainly" prepared by the reaction described herein.

[0071] Due to the reversibility of enzymatic reactions, unless otherwise stated, this invention relates to enzymatic or biocatalytic reactions described herein in both reaction directions.

[0072] The “functional mutants” of the peptides described in this article include “functional equivalents” of such peptides as defined below.

[0073] The term "stereoisomer" specifically includes conformational isomers.

[0074] According to the present invention, it generally includes all “stereoisomeric forms” of the compounds described herein, such as structural isomers, especially stereoisomers and mixtures thereof, such as optical isomers or geometric isomers, such as E and Z isomers, and combinations thereof. If several asymmetric centers exist in a molecule, the present invention includes all combinations of different conformations of these asymmetric centers, such as enantiomer pairs.

[0075] "Stereoselectivity" describes the ability to produce a specific stereoisomer of a compound in its stereoisomeric pure form, or the ability to specifically convert a specific stereoisomer from a variety of stereoisomers using the enzymatic catalytic method described herein. More specifically, this means that the product of the invention is enriched relative to a specific stereoisomer, or that the precipitate can be depleted relative to a specific stereoisomer. This can be quantified by a purity parameter %ee calculated according to the following formula:

[0076] %ee=[X A -X B ] / [X A +X B ]*100,

[0077] Where X A and X B Mole ratio (Molenbruch) represents the ratio of stereoisomers A and B.

[0078] The “yield” and / or “conversion” of the reaction according to the invention are determined within a specified time period, such as 4, 6, 8, 10, 12, 16, 20, 24, 36 or 48 hours (during which the reaction takes place). In particular, the reaction is carried out under precisely defined conditions, such as the “standard conditions” as defined herein.

[0079] Different yield parameters (“yield” or YP / S; “specific productivity yield”; or space-time yield (STY)) are well known in the art and are measured as described in the literature.

[0080] "Yield" and "YP / S" (both expressed as the quality of products produced / the quality of materials consumed) are used as synonyms in this document.

[0081] Specific productivity yield describes the amount of product (such as vanillin) produced per hour per liter of fermentation broth per gram of biomass. Wet cell weight (WCW) describes the number of biologically active microorganisms in the biochemical reaction. This value is given as grams of product per g WCW per hour (i.e., g / gWCW). -1 h -1 Alternatively, the amount of biomass can also be expressed as the amount of dry cell weight, denoted as DCW. Furthermore, biomass concentration can be more easily determined by measuring the optical density at 600 nm (OD600) and estimating the corresponding wet or dry cell weight using experimentally determined relevant factors.

[0082] The term “fermentation production” or “fermentation” refers to the ability of microorganisms (assisted by enzyme activity contained in or produced by said microorganisms) to produce compounds in cell cultures using at least one carbon source added to incubation.

[0083] The term “fermentation broth” should be understood as referring to a liquid, particularly an aqueous solution or aqueous / organic solution, that is based on a fermentation process and has been subjected to or has been subjected to post-processing, such as that described herein.

[0084] "Enzyme-catalyzed" or "biocatalytic" methods refer to methods carried out under the catalytic action of enzymes (including enzyme mutants as defined herein). Therefore, such methods can be carried out in the presence of the enzyme in isolated (purified, enriched) or crude form, or in the presence of cellular systems, particularly natural or recombinant microbial cells containing the enzyme in its active form and possessing the catalytic transformation capabilities disclosed herein.

[0085] The terms "selective conversion" or "enhanced selectivity" generally refer to the conversion of a specific stereoisomer, such as the E-form of an unsaturated hydrocarbon, to a higher proportion or amount (compared to a molar basis) than the corresponding Z-form, at a certain point in time or during a "period" of the reaction, throughout the entire process of the reaction (i.e., between the start and end of the reaction). Specifically, the selectivity can be observed over a "period" as a conversion of 1 to 99%, 2 to 95%, 3 to 90%, 5 to 85%, 10 to 80%, 15 to 75%, 20 to 70%, 25 to 65%, 30 to 60%, or 40 to 50% corresponding to the initial amount of substrate. The higher proportion or amount can be expressed, for example, as:

[0086] -High maximum yield of isomers observed throughout the reaction process or during the period described;

[0087] - Higher relative abundance of isomers at a defined percentage substrate conversion value; and / or

[0088] -At higher conversion percentage values, the same relative content of isomers;

[0089] Each of these preferred methods is observed relative to a reference method, which is performed under otherwise identical conditions using known chemical or biochemical methods.

[0090] "E-stereoselectivity" or "E-selectivity" describes the ability, in the enzyme-catalyzed method described herein, to produce, in a specific form, a specific C=C double bond E-isomer from a variety of other isomers or a mixture of E- and Z-isomers, at a specific position of the double bond, either in a pure or substantially pure or enriched form, or to specifically or substantially specifically convert E-isomers.

[0091] The invention typically also includes all “isomeric forms” of the compounds described herein, such as structural isomers, especially stereoisomers and mixtures thereof, such as optical isomers or geometric isomers, such as E and Z isomers, and combinations thereof. If multiple asymmetric centers exist in a molecule, the invention includes all combinations of different conformations of these asymmetric centers, such as enantiomer pairs, or any mixture of stereoisomeric forms.

[0092] If this disclosure relates to features, parameters, and ranges of different priorities (including superior, non-preferred features, parameters, and ranges), then unless otherwise stated, any combination of two or more of these features, parameters, and ranges is covered in the disclosure of this invention regardless of their respective priority.

[0093] "Polycistronic" refers to nucleic acid molecules that can encode more than one polypeptide within the same nucleic acid molecule, especially mRNA or the corresponding cDNA.

[0094] "Derived from" "polycistronic" constructs or nucleic acid molecules refer to molecules that can be modified by introducing one or more identical or different regulatory sequences, such as promoters, RBSs, and / or terminators, at appropriate locations to affect or regulate the transcription and / or translation of at least one coding sequence contained in the nucleic acid molecule.

[0095] Detailed description

[0096] a. Specific embodiments of the present invention

[0097] This invention specifically relates to the following implementation schemes:

[0098] 1. An expression system for recombinantly expressing a polypeptide having isoeugenol oxidation activity, the expression system comprising a combination of at least two different nucleic acid sequences contained in at least one recombinant nucleic acid construct; wherein the expression system comprises:

[0099] a. A nucleotide sequence (A) encoding a polypeptide with enzymatic oxidation activity of isoeugenol, particularly catalyzing the formation of vanillin and acetaldehyde from isoeugenol in the presence of oxygen, especially molecular oxygen; and

[0100] b. At least one nucleotide sequence (B) encoding at least one, such as one, two or three, preferably two, accessory polypeptides, which individually or preferably synergistically assist in the functional expression of the polypeptide, particularly the correct folding of the expressed polypeptide encoded by the nucleotide sequence (A);

[0101] The expression system described therein provides co-expression of the nucleotide sequences (A) and (B).

[0102] 2. The expression system of Implementation Scheme 1 is selected from:

[0103] a. A combination of at least two, preferably independent, functional or effective nucleic acid constructs, each having the nucleic acid sequence (A) and at least one nucleic acid (B); and

[0104] b. A single nucleic acid construct having the nucleic acid sequence (A) and the at least one nucleic acid (B), which may be, for example, a nucleic acid construct or vector having multiple cloning sites, or more preferably a polycistronic nucleic acid construct, which is ultimately transcribed into a single mRNA molecule containing a coding sequence (A) for a polypeptide having enzymatic isoeugenol oxidation activity and a coding sequence (B) for an accessory polypeptide. The single nucleic acid construct having the nucleic acid sequence (A) and the at least one nucleic acid (B) may also be derived from a polycistronic nucleic acid construct. The transcription and / or translation of at least one coding sequence (A) and (B) contained in the nucleic acid molecule can be influenced or regulated, for example, by introducing one or more identical or different regulatory sequences, such as promoters, RBSs, and / or terminators, at appropriate locations, as further illustrated in the experimental section.

[0105] 3. The expression system of one of the aforementioned embodiments, wherein the nucleotide sequence (A) encodes:

[0106] a. A polypeptide having isoeugenol oxidation activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2 (i.e., IEM1, originally isolated from Pseudomonas nitroreducens Jin 1); or

[0107] b. A polypeptide having isoeugenol oxidative activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:4 (i.e., IEM2, originally isolated from Pseudomonas putida IE27), wherein the amino acid sequence is identical to that of SEQ ID NO:4. NO:4 has an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, containing at least one mutation at an amino acid sequence position selected from T52, Q74, and D440, and optionally at an amino acid sequence position selected from N120, T121, F281, M298, and L470, particularly selected from N120I, T121P, F281Q, M298K, and L470S, containing at least one (e.g., 1, 2, 3, 4, or 5) additional mutation.

[0108] 4. The expression system of embodiment 3, wherein the polypeptide having isoeugenol oxidation activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:4 is a mutant polypeptide containing at least one mutation at an amino acid sequence position selected from T52, Q74, and D440.

[0109] 5. The expression system of embodiment 4, wherein the mutation is selected from the following IEM 2 mutants:

[0110] a. Single mutants (T52X1), (Q74X2), and (D440X3)

[0111] b. Double mutants (T52X1, Q74X2), (T52X1, D440X3) and (Q74X2, D440X3) and

[0112] c. Triple mutant (T52X1, Q74X2, D440X3)

[0113] in

[0114] X1 is a mutant of P, K, or M or any other natural or non-natural, especially natural, amino acid substitution that produces a functional vanillin-producing enzyme; X1 is preferably P or M, with P being the most preferred.

[0115] X2 is H or A or any other natural or non-natural, especially natural, amino acid substitution that produces a mutant of a functional vanillin-producing enzyme; X2 is most preferably H; and;

[0116] X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y or any other natural or non-natural, especially natural, amino acid substitution, which produces a mutant of a functional vanillin-producing enzyme; X3 is preferably N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V or Y; most preferably N.

[0117] As a non-restrictive example of such a double mutant expressed by this expression system, the following can be mentioned:

[0118] (T52X1, Q74X2):

[0119] Where X1 is P, and X2 is H or A; or

[0120] Where X1 is K, and X2 is H or A; or

[0121] Where X1 is M, and X2 is H or A.

[0122] (T52X1, D440X3)

[0123] Where X1 is P, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y; or

[0124] Where X1 is K, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y; or

[0125] Where X1 is M, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y.

[0126] (Q74X2, D440X3)

[0127] Where X2 is H, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y; or

[0128] Where X2 is A, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y.

[0129] The preferred double mutant is:

[0130] (T52P, Q74H);

[0131] (T52P, D440X3) where X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V, or Y.

[0132] (Q74H, D440X3) where X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V, or Y.

[0133] As a non-restrictive example of such a triple mutant expressed by this expression system, the following can be mentioned:

[0134] (T52X1, Q74X2, D440X3)

[0135] Where X1 is P, X2 is H, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

[0136] Where X1 is K, X2 is H, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

[0137] Where X1 is M, X2 is H, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

[0138] Where X1 is P, X2 is A, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y;

[0139] Where X1 is K, X2 is A, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y;

[0140] Where X1 is M, X2 is A, and X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, or Y.

[0141] The preferred triple mutant is:

[0142] (T52P, Q74H, D440X3) where X3 is N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V or Y.

[0143] The aforementioned single, double, or triple mutants may optionally be further modified at the amino acid sequence position of SEQ ID NO:4 selected from N120, T121, F281, M298, and L470, particularly selected from mutants N120I, T121P, F281Q, M298K, and L470S by at least one, for example, 1, 2, 3, 4, or 5, preferably at least 1, 2, or 3, and most preferably one additional mutation.

[0144] 6. The expression system of one of the foregoing embodiments, wherein the at least nucleotide sequence (B) comprises nucleotide sequences (B1) and (B2), wherein

[0145] a. (B1) Encoding a polypeptide with molecular chaperone activity, the polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8 (GroEL); and

[0146] b. (B2) encodes a polypeptide with molecular chaperone activity, the polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:10 (GroES).

[0147] A non-limiting example of a preferred expression system of the present invention is the IEM 1 expression system, which comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO:11 (PC1); SEQ ID NO:12 (PC2); SEQ ID NO:13 (PC3); SEQ ID NO:14 (PC4); SEQ ID NO:15 (PC5); These systems, according to the present invention, can be used to express functional polypeptides having isoeugenol oxidation activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2 (IEM1);

[0148] Other non-limiting examples of preferred expression systems can be derived from this IEM-1 expression system of SEQ ID NO:11, 12, 13, 14 or 15 by replacing the coding sequence of IEM1 with the nucleotide sequence encoding a functional IEM2 mutant (derived from SEQ ID NO:4, as described above with respect to embodiment 5). Such IEM2 expression systems are also preferred.

[0149] 7. A polypeptide having isoeugenol oxidation activity and obtainable by recombinant expression using an expression system defined in any of the preceding embodiments.

[0150] 8. A polypeptide having isoeugenol oxidation activity and comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:4 (i.e., IEM2), wherein the amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:4 contains at least one mutation at an amino acid sequence position selected from T52, Q74, D440, and optionally contains at least one (e.g., 1, 2, 3, 4 or 5) additional mutations at an amino acid sequence position selected from N120, T121, F281, M298 and L470, particularly selected from N120I, T121P, F281Q, M298K and L470S.

[0151] 9. The polypeptide of embodiment 8, wherein the polypeptide contains at least one mutation at the amino acid sequence positions selected from T52, Q74 and D440 of SEQ ID NO:4.

[0152] 10. The polypeptide of embodiment 9, wherein the mutation is selected from:

[0153] a. Single mutants (T52X1), (Q74X2), and (D440X3)

[0154] b. Double mutants (T52X1, Q74X2), (T52X1, D440X3) and (Q74X2, D440X3) and

[0155] c. Triple mutant (T52X1, Q74X2, D440X3)

[0156] in

[0157] X1 is a mutant of P, K, or M or any other natural or non-natural, especially natural, amino acid substitution that produces a functional vanillin-producing enzyme. X1 is preferably P or M, with P being the most preferred.

[0158] X2 is H or A or any other natural or non-natural, especially natural, amino acid substitution that produces a mutant of a functional vanillin-producing enzyme, with H being the most preferred; and

[0159] X3 is N, A, C, E, F, G, H, I, K, L, M, Q, R, S, T, V, W, Y, or any other natural or non-natural, especially natural, amino acid substitution, which produces a mutant of a functional vanillin-producing enzyme; X3 is preferably N, A, C, E, F, G, H, K, L, M, Q, R, S, T, V, or Y; most preferably N.

[0160] For non-limiting examples of such double and triple mutants of the present invention, see embodiment 5 above.

[0161] The single, double, or triple mutants may optionally be further modified at the amino acid sequence position of SEQ ID NO:4 selected from N120, T121, F281, M298, and L470, particularly selected from mutants N120I, T121P, F281Q, M298K, and L470S by at least one, for example, 1, 2, 3, 4, or 5, preferably at least 1, 2, or 3, and most preferably one additional mutation.

[0162] 11. A recombinant nucleic acid comprising a nucleotide sequence encoding a polypeptide of any one of embodiments 8 to 10.

[0163] 12. A recombinant nucleic acid comprising a nucleotide sequence, or its reverse complementary sequence, having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or less than 100%, such as 99.9%, of sequence identity with SEQ ID NO:1, 3, or 5.

[0164] One related embodiment provides a nucleic acid sequence complementary to the nucleic acid sequence according to SEQ ID NO:1, 3 or 5, or a nucleic acid sequence that hybridizes under stringent conditions to at least a portion of the nucleotide sequence according to SEQ ID NO:1, 3 or 5.

[0165] Specific examples of recombinant nucleic acids are those that contain a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% or less than 100%, such as 99.9%, of SEQ ID NO:3 or 5, and that encode an IEM 2 mutant as described in Embodiment 5; or its reverse complementary sequence.

[0166] As further described herein, such recombinant nucleic acids can be contained in monocistronic or polycistronic expression vectors.

[0167] 13. A recombinant nucleic acid construct comprising the recombinant nucleic acid of embodiment 11 or embodiment 12 or its reverse complementary sequence.

[0168] 14. An expression carrier comprising:

[0169] a. A recombinant expression system according to any one of implementation schemes 1 to 6, or one of the described partial nucleic acid constructs, or its reverse complement; or

[0170] b. Nucleic acids as defined in Implementation Scheme 11 or 12; or

[0171] c. Implementation of scheme 13.

[0172] 15. The expression vector of embodiment 14, wherein the vector is a prokaryotic vector, a viral vector, a eukaryotic vector, or one or more plasmids.

[0173] The aforementioned nucleic acid molecules or vectors can be such that monocistronic mRNA or, in particular polycistronic mRNA, encoding the isoeugenol oxidase of the present invention and one or more accessory polypeptides described herein is transcribed during expression.

[0174] 16. A non-human host organism or host cell comprising a substance that is optionally stably integrated into its genome,

[0175] a. A recombinant expression system according to any one of implementation schemes 1 to 6, or one of the described partial nucleic acid constructs; or

[0176] b. The nucleic acid or its reverse complementary sequence as defined in Implementation Scheme 11 or 12; or

[0177] c. The construct of implementation scheme 13; or

[0178] d. The carrier of implementation plan 14 or 15.

[0179] 17. The non-human host organism or host cell of implementation scheme 16, which is selected from prokaryotic or eukaryotic microorganisms, plants, or cells derived therefrom.

[0180] 18. The non-human host organism or host cell of implementation scheme 17, wherein the microorganism is a bacterium or fungus, particularly yeast.

[0181] 19. The non-human host organism or host cell of embodiment 18, wherein the bacteria are selected from the genus Escherichia, particularly E. coli, and the yeast is selected from the genus Saccharomyces or the genus Pichia, particularly Saccharomyces cerevisiae or Pichia pastoris.

[0182] 20. A method for producing an isolated catalytically active polypeptide having isoeugenol oxidation activity, the method comprising co-expressing, for example substantially simultaneously and particularly simultaneously, the polypeptide having isoeugenol oxidation activity and at least one accessory polypeptide in a host cell system, each encoded by an expression system defined in any one of embodiments 1 to 6; and optionally, isolating the polypeptide having isoeugenol oxidation activity.

[0183] The term "co-expression" or the verb "co-expression" should be understood broadly, as long as it occurs in a synergistic manner leading to the functional expression of a polypeptide with isoeugenol oxidation activity (IEM), particularly the correct folding of the expressed IEM polypeptide. Simultaneous or substantially simultaneous co-expression of two polypeptides represents a non-limiting alternative. Another non-limiting alternative can be seen in the timely sequential expression of two polypeptides, which begins with the expression of the auxiliary polypeptide followed by the expression of the IEM polypeptide. Yet another non-limiting alternative might be the timely overlapping co-expression of two polypeptides, wherein only the auxiliary polypeptide is expressed in the initial stage, while both polypeptides are expressed in the overlapping stage. Those skilled in the art can develop other alternatives without inventive effort.

[0184] 21. The method of embodiment 20, wherein isoeugenol oxidase as defined in any one of embodiments 7 to 10 is prepared.

[0185] 22. The method of implementation scheme 20 or 21, wherein a non-human host organism or host cell as defined in any one of implementation schemes 16 to 19 is used for expression.

[0186] 23. A method for producing vanillin, comprising:

[0187] a. In the presence of oxygen, particularly molecular oxygen, contacting isoeugenol with a polypeptide having isoeugenol oxidizing activity as defined in any one of embodiments 7 to 10, or a polypeptide having isoeugenol oxidizing activity prepared by any one of embodiments 20 to 22, to produce vanillin and acetaldehyde; and

[0188] b. Optionally, the vanillin produced in step a is separated.

[0189] In a preferred embodiment, vanillin is isolated.

[0190] In another preferred embodiment, vanillin (and acetaldehyde) is obtained as the main product of isoeugenol oxidation treatment.

[0191] Vanillin produced in any of the methods described herein can be converted into derivatives, such as, but not limited to, hydrocarbons, esters, amides, glycosides, ethers, epoxides, aldehydes, ketones, alcohols, diols, acetals, or ketals.

[0192] Vanillin derivatives can be obtained by chemical methods, such as, but not limited to, oxidation, reduction, alkylation, acylation and / or rearrangement.

[0193] Alternatively, vanillin derivatives can be obtained using biochemical methods by contacting vanillin with an enzyme, such as, but not limited to, oxidoreductases, monooxygenases, dioxygenases, and transferases. Biochemical transformation can be performed in vitro using isolated enzymes, cell-lysing enzymes, or in vivo using whole cells.

[0194] 24. The method of embodiment 23, comprising transforming a non-human host organism or host cell with an expression system as defined in any one of embodiments 1 to 6 to express its encoding nucleotide sequences (A) and (B).

[0195] 25. The method of any one of embodiments 23 or 24, wherein isoeugenol is contacted with host cells, cell lysates of the host cells or a culture medium containing said host cells and / or with a polypeptide having isoeugenol oxidizing activity as defined in any one of embodiments 7 to 10, said polypeptide being isolated from the host cells, cell lysates or culture medium.

[0196] The isoeugenol substrate can be added to the applied reaction medium, or in another embodiment, a non-human host organism or host cell already capable of producing isoeugenol can be transformed with an expression system defined in any one of embodiments 1 to 6 to express at least one polypeptide having isoeugenol monooxygenase activity, and cultured under conditions favorable to vanillin production.

[0197] 26. The method of implementation scheme 25, wherein vanillin is produced by fermentation of the non-human host organism or host cell.

[0198] 27. The method of embodiment 25, wherein vanillin is enzymatically produced by converting isoeugenol with a separated polypeptide having isoeugenol oxidation activity as defined in any one of embodiments 7 to 10 in the presence of oxygen and optionally an additional adjuvant.

[0199] 28. The method of any one of Examples 23 to 27, further comprising, prior to step a, chemically or biochemically isomerizing eugenol to isoeugenol, and optionally, separating isoeugenol from the reaction medium.

[0200] 29. A method for preparing a mutant polypeptide with isoeugenol oxidation activity, the method comprising the following steps:

[0201] a. Select any one of the nucleic acids according to implementation plans 11 and 12;

[0202] b. Modify the selected nucleic acid to obtain at least one mutant nucleic acid;

[0203] c. Provide a host cell or single-celled organism with a mutated nucleic acid sequence to express a polypeptide encoded by that mutated nucleic acid sequence;

[0204] d. Screen for at least one mutant polypeptide that is active in the oxidation of isoeugenol;

[0205] e. Optionally, if the mutant peptide does not have the desired activity, steps a through d are repeated until a peptide with the desired activity is obtained; and,

[0206] f. Optionally, if a mutant polypeptide with the desired activity is identified in step d or e, the corresponding mutant nucleic acid is isolated.

[0207] Other forms and embodiments of the invention as described above with reference to specific preferred embodiments are described in the following sections.

[0208] b. The polypeptide applicable according to the present invention

[0209] In the context of this article, the following definitions apply:

[0210] The commonly used terms “polypeptide” or “peptide” refer to a natural or synthetic, continuous, peptide-linked linear chain or sequence of amino acid residues containing approximately 10 to more than 1,000 residues. Short-chain polypeptides with up to 30 residues are also called “oligopeptides”.

[0211] The term "protein" refers to a large molecular structure composed of one or more polypeptides. The amino acid sequence of the polypeptide represents the protein's "primary structure." The amino acid sequence also predetermines the protein's "secondary structure" by forming specific structural elements (such as α-helices and β-sheets formed within the polypeptide chain). The arrangement of multiple such secondary structural elements defines the protein's "tertiary structure," or spatial arrangement. If a protein contains more than one polypeptide chain, these chains are arranged spatially to form the protein's "quaternary structure." Proper spatial arrangement, or "folding," is a prerequisite for protein function. Denaturation or unfolding disrupts protein function. If this disruption is reversible, protein function can be restored by refolding.

[0212] The typical protein function referred to in this article is "enzyme function," which means that a protein acts as a biocatalyst on a substrate, such as a compound, and catalyzes the conversion of said substrate into a product. Enzymes can exhibit high or low levels of substrate and / or product specificity.

[0213] Therefore, the term "peptide" as used in this article to refer to a specific "activity" implicitly refers to a correctly folded protein that exhibits the indicated activity, such as a specific enzyme activity.

[0214] Therefore, unless otherwise stated, the term "polypeptide" also covers the terms "protein" and "enzyme".

[0215] Similarly, the term "peptide fragment" encompasses the terms "protein fragment" and "enzyme fragment".

[0216] The term "isolated polypeptide" refers to an amino acid sequence extracted from its natural environment by any method known in the art or a combination of such methods (including recombinant, biochemical, and synthetic methods).

[0217] A "target peptide" is an amino acid sequence that targets a protein or polypeptide to intracellular organelles (i.e., mitochondria or plastids) or to the extracellular space (secretory signal peptides). The nucleic acid sequence encoding the target peptide can be fused to the amino-terminal (e.g., N-terminus) nucleic acid sequence encoding the protein or polypeptide, or it can be used to replace the natural target peptide.

[0218] The present invention also relates to “functional equivalents” (also referred to as “analogs” or “functional mutations”) of the polypeptides specifically described herein.

[0219] For example, a “functional equivalent” refers to a polypeptide that, in a test used to determine the enzymatic oxidation activity of isoeugenol or more specifically, the IEM activity, shows an IEM activity that is at least 1 to 10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% higher or lower than that of the individual polypeptides specifically described herein.

[0220] "Functional equivalents" can also be derived from auxiliary peptides as described herein, which facilitate the functional expression of another peptide, preferably one with enzymatic activity, particularly the correct folding of the expressed peptide, such as a peptide with isoeugenol oxidation activity or especially IEM activity. Such modified auxiliary peptides can still be considered functional as long as they improve the correct expression or folding of the enzymatically active peptide relative to the expression of the peptide with the same enzymatic activity under otherwise identical conditions but in the absence of such auxiliary peptides.

[0221] According to the invention, "functional equivalents" also encompass specific mutants that have an amino acid at at least one sequence position in the amino acid sequence described herein that differs from the specifically stated amino acid, but still possess one of the aforementioned biological activities, such as enzyme activity. Thus, "functional equivalents" include mutants obtainable by the addition, substitution, particularly conserved substitution, deletion, and / or inversion of one or more, for example, 1 to 20, 1 to 15, or 5 to 10 amino acids, wherein said changes can occur at any sequence position, as long as they result in the mutant possessing the general characteristics of the invention. Functional equivalence is also particularly provided if the activity pattern qualitatively overlaps between the mutant and the unaltered polypeptide, i.e., if, for example, interactions with the same agonist or antagonist or substrate are observed, but at different rates (i.e., by EC...). 50 or IC 50 (Values ​​or any other parameters suitable in this technical field). The table below shows examples of suitable (conservative) amino acid substitutions:

[0222]

[0223] The “functional equivalents” in the above sense are also the “precursors” of the polypeptides described in this article, as well as the “functional derivatives” and “salts” of the polypeptides.

[0224] In this case, a "precursor" is a natural or synthetic precursor of a polypeptide, which may or may not have the desired biological activity.

[0225] The term "salt" as used in this invention refers to salts of the carboxyl group of the protein molecule and salts formed by the acid addition of the amino group. Salts of the carboxyl group can be produced in known ways and include inorganic salts such as sodium, calcium, ammonium, iron, and zinc salts, as well as salts formed with organic bases such as amines, such as triethanolamine, arginine, lysine, piperidine, etc. Salts formed by acid addition, such as those formed with inorganic acids such as hydrochloric acid or sulfuric acid, and salts formed with organic acids such as acetic acid and oxalic acid, are also covered by this invention.

[0226] The “functional derivatives” of the polypeptides according to the invention can also be generated using known techniques at the side groups of functional amino acids or their N-terminus or C-terminus. Such derivatives include, for example: aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, which can be obtained by reacting with ammonia or with primary or secondary amines; N-acyl derivatives of free amino groups, which are generated by reacting with acyl groups; or O-acyl derivatives of free hydroxyl groups, which are generated by reacting with acyl groups.

[0227] "Functional equivalents" naturally include polypeptides that can be obtained from other organisms as well as naturally occurring variants. For example, the area of ​​homologous sequence regions can be determined by sequence comparison, and equivalent polypeptides can be determined based on the specific parameters of this invention.

[0228] "Functional equivalents" also include "fragments" of the polypeptide according to the invention, such as single domains or sequence motifs, or truncated N-terminuses and / or C-termini, which may or may not exhibit the desired biological function. Preferably, such "fragments" at least qualitatively retain the desired biological function.

[0229] Furthermore, a “functional equivalent” is a fusion protein having one of the polypeptide sequences described herein or a functional equivalent derived therefrom, and having at least one additional functionally distinct heterologous sequence in functional N-terminal or C-terminal association (i.e., without substantial mutual functional impairment of the fusion protein portion). Non-limiting examples of such heterologous sequences are, for example, signal peptides, histidine anchors, or enzymes.

[0230] The invention also includes “functional equivalents” that are homologs of the specifically disclosed polypeptides. They have at least 60%, preferably at least 75%, particularly at least 80 or 85%, such as 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology (or identity) with the specifically disclosed amino acid sequence, calculated using the algorithm described in Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85(8), 1988, 2444-2448. The homology or identity of the homologous polypeptides according to the invention, expressed as a percentage, refers in particular to identity expressed as a percentage of amino acid residues based on the total length of one of the amino acid sequences specifically described herein.

[0231] Identity data expressed as a percentage can also be determined using BLAST alignment, the blastp (protein-protein BLAST) algorithm, or by applying the Clustal settings detailed below.

[0232] In the case of possible protein glycosylation, the “functional equivalents” according to the invention include polypeptides in deglycosylated or glycosylated forms as described herein, as well as polypeptides in modified forms that can be obtained by changing the glycosylation pattern.

[0233] Functional equivalents or homologs of the polypeptides according to the present invention can be generated by mutagenesis, for example by point mutation, lengthening or shortening of the protein, or as described in more detail below.

[0234] Such functional equivalents or homologs of the polypeptides according to the invention can be identified by screening a combined database of mutants, such as shortened mutants. For example, a database of protein variant diversity can be generated by combinatorial mutagenesis at the nucleic acid level, for example by enzymatic ligation of a mixture of synthetic oligonucleotides. Numerous methods are available for generating a database of potential homologs from degenerate oligonucleotide sequences. The chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer, and the synthesized gene can then be ligated into a suitable expression vector. The use of degenerate genomes makes it possible to provide all sequences in a mixture that encode the desired set of potential protein sequences. Methods for synthesizing degenerate oligonucleotides are known to those skilled in the art.

[0235] Several techniques are known for screening gene products from combinatorial databases generated by point mutations or shortening, and for screening cDNA libraries containing gene products with selected properties. These techniques can be applied to rapidly screen gene libraries generated by combinatorial mutagenesis of homologs according to the present invention. The most commonly used high-throughput analysis-based techniques for screening large gene libraries involve cloning the gene library in a reproducible expression vector, transforming suitable cells with the resulting vector database, and expressing the combinatorial gene under specific conditions, under which detection of the desired activity facilitates the isolation of the vector encoding the gene (whose product is detected). Recursive integration mutagenesis (REM) is a technique for increasing the frequency of functional mutants in a database and can be used in conjunction with screening tests to identify homologs.

[0236] The embodiments provided herein provide orthologs and paralogs of the polypeptides disclosed herein, as well as methods for identifying and separating such orthologs and paralogs.

[0237] c. The applicable coding nucleic acid sequence according to the present invention

[0238] In the context of this article, the following definitions apply:

[0239] The terms “nucleic acid sequence,” “nucleic acid,” “nucleic acid molecule,” and “polynucleotide” are used interchangeably and refer to a sequence of nucleotides. A nucleic acid sequence can be a single-stranded or double-stranded deoxyribonucleotide or ribonucleotide of any length and includes coding and non-coding sequences of genes, exons, introns, sense and antisense complementary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and / or RNA sequences, synthetic DNA and RNA sequences, fragments, primers, and nucleic acid probes. Those skilled in the art understand that the nucleic acid sequence of RNA is identical to that of DNA, except that thymine (T) is replaced by uracil (U). The term “nucleotide sequence” should also be understood to include polynucleotide or oligonucleotide molecules in the form of individual fragments or as components of larger nucleic acids.

[0240] "Isolated nucleic acid" or "isolated nucleic acid sequence" refers to a nucleic acid or nucleic acid sequence that exists in an environment different from that of naturally occurring nucleic acids or nucleic acid sequences, and may include those that are substantially free of contaminating endogenous substances. As used herein, the term "naturally occurring" for nucleic acids refers to a nucleic acid found in the cells of organisms in nature and that has not been intentionally modified by humans in a laboratory setting.

[0241] A “fragment” of a polynucleotide or nucleic acid sequence refers to a continuous sequence of nucleotides, particularly of a length of at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp, and / or at least 60 bp, according to one embodiment of this invention. Specifically, the polynucleotide fragment comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, and more particularly at least 1000 consecutive nucleotides of a polynucleotide sequence according to one embodiment of this invention. Without limitation, the polynucleotide fragments described herein can be used as PCR primers and / or probes, or for antisense gene silencing or RNAi.

[0242] As used herein, the term "hybridization" or hybridization under certain conditions is intended to describe the conditions under which hybridization and washing occur, under which significantly identical or homologous nucleotide sequences remain bound to each other. These conditions can result in sequences with at least about 70%, for example at least about 80%, and for example at least about 85%, 90%, or 95% identity remaining bound to each other. Definitions of low-tightness, medium-tightness, and high-tightness hybridization conditions are provided below. Those skilled in the art can select suitable hybridization conditions with minimal experimentation, for example, as illustrated by Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Additionally, tight conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0243] "Recombinant nucleic acid sequences" are nucleic acid sequences created by combining genetic material from more than one source using laboratory methods (such as molecular cloning), thereby creating or modifying nucleic acid sequences that are not naturally occurring and cannot be found in biological organisms in any other way.

[0244] “Recombinant DNA technology” refers to molecular biological methods used to prepare recombinant nucleic acid sequences, as described, for example, in Laboratory Manuals edited by Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook et al., 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0245] The term "gene" refers to a DNA sequence containing a region that is operatively linked to a suitable regulatory region (e.g., a promoter) and transcribed into an RNA molecule (e.g., mRNA in a cell). Therefore, a gene can contain several operatively linked sequences, such as a promoter, a 5' leader sequence (containing, for example, a sequence involved in translation initiation), a coding region of cDNA or genomic DNA, introns, exons, and / or a 3' untranslated sequence (containing, for example, a transcription termination site).

[0246] "Polycitronic" refers to nucleic acid molecules that can encode more than one polypeptide within the same nucleic acid molecule, particularly mRNA or the corresponding cDNA. Polycitronic genes allow translation initiation at two or more sites on a single sequence.

[0247] A "chimeric gene" is any gene that is not normally found in species in nature, particularly a gene in which one or more portions of the nucleic acid sequence are unrelated in nature. For example, a promoter that is unrelated in nature to part or all of the transcribed region or to another regulatory region. The term "chimeric gene" should be understood to include expression constructs in which a promoter or transcriptional regulatory sequence is operatively linked to one or more coding sequences or antisense (i.e., the inverse complementary strand of the sense strand) or inverted repeat sequences (sense and antisense, whereby the RNA transcript forms a double-stranded RNA post-transcriptionally). The term "chimeric gene" also includes genes obtained by combining portions of one or more coding sequences to produce new genes.

[0248] "3'URT" or "3' untranslated sequence" (also known as "3' untranslated region" or "3' end") refers to a nucleic acid sequence found downstream of the gene coding sequence that contains, for example, a transcription termination site and (in most, but not all, eukaryotic mRNAs) a polyadenylation signal, such as AAUAAA or its variants. After transcription termination, the mRNA transcript can be cleaved downstream of the polyadenylation signal and a poly(A) tail can be added, which is involved in the transport of mRNA to the translation site, such as the cytoplasm.

[0249] The term "primer" refers to a short nucleic acid sequence that is hybridized to a template nucleic acid sequence and used for the polymerization of nucleic acid sequences complementary to that template.

[0250] The term "selectable marker" refers to any gene that, after expression, can be used to select one or more cells containing that selectable marker. Examples of selectable markers are described below. Those skilled in the art will understand that different antibiotic, fungicide, auxotrophic, or herbicide selectable markers may be applicable to different target species.

[0251] This invention also relates to nucleic acid sequences encoding polypeptides as defined herein.

[0252] In particular, the present invention also relates to nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, such as cDNA, genomic DNA and mRNA) encoding one of the aforementioned polypeptides and their functional equivalents, which can be obtained, for example, by using artificial nucleotide analogs.

[0253] This invention relates to both isolated nucleic acid molecules encoding polypeptides or biologically active regions thereof according to the invention, and nucleic acid fragments that can be used, for example, as hybridization probes or primers for identifying or amplifying nucleic acids encoded according to the invention.

[0254] This invention also relates to nucleic acids that have a degree of "identity" with the sequences specifically disclosed herein. "Identity" between two nucleic acids refers to the identity of nucleotides along the entire length of the nucleic acid in each case.

[0255] The “identity” between two nucleotide sequences (and similarly, peptide or amino acid sequences) is a function of the number of nucleotide residues (or amino acid residues) when the two sequences are aligned, or the number of identical residues in both sequences. Identical residues are defined as the same residues at a given position in the alignment of the two sequences. The percentage of sequence identity used herein is calculated from the best alignment by dividing the number of identical residues between the two sequences by the total number of residues in the shortest sequence and multiplying by 100. The best alignment is the alignment with the highest probability of identity percentage. Vacancies can be introduced into one or more positions in the alignment of one or both sequences to obtain the best alignment. These vacancies are then considered as dissimilar residues used to calculate the percentage of sequence identity. Alignments used to determine the percentage of identity of amino acid or nucleic acid sequences can be performed in various ways using computer programs, such as those publicly available on the Internet.

[0256] Specifically, the BLAST program (Tatiana et al., FEMS Microbiol Lett., 1999, 174:247-250, 1999), which is available from the National Center for Biotechnology Information (NCBI) at http: / / www.ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi with default parameters, can be used to obtain the best alignment of protein or nucleic acid sequences and calculate the percentage of sequence identity.

[0257] In another example, identity can be calculated using the Clustal method (Higgins DG, Sharp PM. (1989)) through the Vector NTI Suite 7.1 program of Informax Corporation (USA) with the following settings:

[0258] Multiple alignment parameters:

[0259]

[0260] Comparison parameters:

[0261]

[0262] Alternatively, identity can be determined according to the method of Chenna et al. (2003), webpage: http: / / www.ebi.ac.uk / Tools / clustalw / index.html# and the following settings:

[0263]

[0264] All nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, such as cDNA and mRNA) mentioned in this article can be produced from nucleotide structural units by chemical synthesis in a known manner, for example, by fragment condensation of individual overlapping complementary nucleic acid structural units of a double helix. The chemical synthesis of oligonucleotides can be carried out, for example, by the phosphoramide process (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897) in a known manner. The accumulation of synthetic oligonucleotides, and the filling of vacancies in the ligation reaction using the Klenow fragment of DNA polymerase, as well as general cloning techniques, are described in Sambrook et al. (1989), see below.

[0265] In addition, the nucleic acid molecules according to the present invention may further include untranslated sequences from the 3' and / or 5' ends of the coding genetic region.

[0266] The present invention further relates to nucleic acid molecules that are complementary to the nucleotide sequences or segments thereof specifically described.

[0267] The nucleotide sequences according to the invention enable the generation of probes and primers that can be used to identify and / or clone homologous sequences in other cell types and organisms. Such probes or primers typically contain a nucleotide sequence region that hybridizes to at least about 12, preferably at least about 25, such as about 40, 50 or 75 consecutive nucleotides of the sense strand or corresponding antisense strand of the nucleic acid sequence according to the invention under “stringent” conditions (as defined elsewhere herein).

[0268] "Isolated" nucleic acid molecules are separated from other nucleic acid molecules present in natural sources of nucleic acids, and if produced by recombinant technology, they may be substantially free of other cell materials or culture media, or if produced by chemical synthesis, they may be free of chemical precursors or other chemicals.

[0269] Nucleic acid molecules according to the invention can be isolated using standard molecular biology techniques and the sequence information provided according to the invention. For example, cDNA can be isolated from a suitable cDNA library using one of the specifically disclosed complete sequences or fragments thereof as hybridization probes and standard hybridization techniques (e.g., described in Sambrook, (1989)).

[0270] Alternatively, nucleic acid molecules containing one or a fragment of the disclosed sequence can be isolated by polymerase chain reaction using oligonucleotide primers constructed based on that sequence. The nucleic acid amplified in this manner can be cloned into a suitable vector and characterized by DNA sequencing. The oligonucleotides according to the invention can also be prepared using standard synthetic methods, for example, using an automated DNA synthesizer.

[0271] According to the nucleic acid sequences or derivatives thereof of the present invention, homologs or portions of these sequences can be isolated from other bacteria, for example, by conventional hybridization techniques or PCR techniques, through genomic or cDNA libraries. These DNA sequences hybridize with the sequences according to the present invention under standard conditions.

[0272] "Hybridization" refers to the ability of polynucleotides or oligonucleotides to bind to nearly complementary sequences under standard conditions, while non-complementary pairs do not bind nonspecifically. For this purpose, the sequences can be 90–100% complementary. This property of complementary sequences being able to bind specifically to each other is used for primer binding, for example, in Northern or Southern blotting, or in PCR or RT-PCR.

[0273] Short oligonucleotides in conserved regions are advantageously used for hybridization. However, longer fragments or complete sequences of the nucleic acids of the present invention may also be used for hybridization. These “standard conditions” vary depending on the nucleic acid used (oligonucleotide, longer fragment, or complete sequence) or the type of nucleic acid used for hybridization (DNA or RNA). For example, the melting temperature of DNA:DNA hybrids is about 10°C lower than that of DNA:RNA hybrids of the same length.

[0274] For example, depending on the specific nucleic acid, standard conditions refer to a temperature of 42 to 58°C in a buffered aqueous solution with a concentration of 0.1 to 5x SSC (1x SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2), or additionally in the presence of 50% formamide (e.g., 42°C, 5x SSC, 50% formamide). Advantageously, hybridization conditions for DNA:DNA hybrids are 0.1× SSC and a temperature of about 20°C to 45°C, preferably about 30°C to 45°C. For DNA:RNA hybrids, hybridization conditions are advantageously 0.1× SSC and a temperature of about 30°C to 55°C, preferably about 45°C to 55°C. These hybridization temperatures are examples of calculated melting temperatures for nucleic acids of about 100 nucleotides in length and with a G+C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization have been described in relevant genetics textbooks (e.g., Sambrook et al., 1989) and can be calculated using molecular formulas known to those skilled in the art, depending on factors such as nucleic acid length, hybrid type, or G+C content. More information on hybridization can be obtained from textbooks such as Ausubel et al. (eds), (1985), and Brown (ed). (1991).

[0275] “Hybridization” can be carried out under particularly stringent conditions. Such hybridization conditions are described, for example, in Sambrook (1989), or in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6.

[0276] As used herein, the term "hybridization or hybridization under certain conditions" is intended to describe the conditions under which hybridization and washing occur, under which significantly identical or homologous nucleotide sequences remain bound to each other. These conditions are such that sequences with at least about 70%, for example, at least about 80%, and for example, at least about 85%, 90%, or 95% identity remain bound to each other. Definitions of low-tightness, medium-tightness, and high-tightness hybridization conditions are provided herein.

[0277] Those skilled in the art can select suitable hybridization conditions with minimal experiments, as illustrated, for example, by Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Furthermore, stringent conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).

[0278] As used herein, the low-tightness conditions are defined as follows. Filter membranes containing DNA were pretreated at 40°C for 6 hours in a solution containing 35% formamide, 5xSSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization was performed in the same solution, modified as follows: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and using 5-20x10 6 32P-labeled probe. The filter membrane was incubated in a hybridization mixture at 40°C for 18–20 h, followed by washing at 55°C for 1.5 h. In a solution containing 2x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS, the membrane was replaced with fresh solution and incubated again at 60°C for 1.5 h. The filter membrane was then blotted dry and subjected to autoradiography.

[0279] As used herein, the moderately stringent conditions are defined as follows. Filter membranes containing DNA were pretreated at 50°C for 7 hours in a solution containing 35% formamide, 5x SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg / ml denatured salmon sperm DNA. Hybridization was performed in the same solution, modified as follows: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (wt / vol) dextran sulfate, and using 5-20x10 632P-labeled probe. The filter membrane was incubated in a hybridization mixture at 50°C for 30 hours, followed by washing at 55°C for 1.5 hours. In a solution containing 2x SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS, the membrane was incubated with fresh solution instead of the washing solution at 60°C for another 1.5 hours. The filter membrane was then blotted dry and subjected to autoradiography.

[0280] As used herein, the stringent conditions are as follows: DNA-containing filter membranes were pre-hybridized at 65°C for 8 hours to overnight in a buffer consisting of 6x SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg / ml denatured salmon sperm DNA. The membranes were then pre-hybridized in a buffer containing 100 μg / ml denatured salmon sperm DNA and 5-20 x 10⁻⁶ ppm of DNA. 6 The filter membrane was hybridized in a prehybridization mixture of cpm 32P labeled probes at 65°C for 48 hours. The membrane was then washed at 37°C for 1 hour in a solution containing 2x SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. It was then washed in 0.1x SSC at 50°C for 45 minutes.

[0281] If the above conditions are not suitable (e.g., for interspecific hybridization), other low, medium and high stringency conditions well known in the art (e.g., for interspecific hybridization) may be used.

[0282] A detection kit for the nucleic acid sequence encoding the polypeptide of the present invention may include primers and / or probes specific to the nucleic acid sequence encoding the polypeptide, and a protocol for using the primers and / or probes to detect the nucleic acid sequence encoding the polypeptide in a sample. Such a detection kit can be used to determine whether a plant, organism, microorganism, or cell has been modified, i.e., whether it has been transformed with the sequence encoding the polypeptide.

[0283] To test the function of a variant DNA sequence according to one embodiment of this article, the target sequence is operatively linked to an optional or screenable marker gene, and the expression of the reporter gene is tested in a transient expression analysis using microorganisms or protoplasts or in stably transformed plants.

[0284] The present invention also relates to derivatives of specifically disclosed or derivable nucleic acid sequences.

[0285] Therefore, the additional nucleic acid sequences according to the invention may be derived from the sequences specifically disclosed herein and may be distinguished by one or more (e.g., 1 to 10) nucleotides, such as 1 to 20, particularly 1 to 15 or 5 to 10, additions, substitutions, insertions or deletions, and may also encode polypeptides having the desired properties.

[0286] The invention also includes nucleic acid sequences containing so-called silent mutations or altered sequences, depending on the codon usage of a particular original or host organism, compared to the specifically stated sequences.

[0287] According to specific embodiments of the invention, variant nucleic acids can be prepared to adapt their nucleotide sequences to a particular expression system. For example, bacterial expression systems are known to express polypeptides more efficiently if the amino acids are encoded by specific codons. Due to the degeneracy of the genetic code, more than one codon can encode the same amino acid sequence, and multiple nucleic acid sequences can encode the same protein or polypeptide; all such DNA sequences are covered in the embodiments herein. Where appropriate, the nucleic acid sequence encoding the polypeptide described herein can be optimized to increase expression in host cells. For example, nucleotides of the embodiments herein can be synthesized using host-specific codons to improve expression.

[0288] This invention also covers naturally occurring variants of the sequences described herein, such as splice variants or allele variants.

[0289] The allele variants have at least 60% homology at the derived amino acid level, preferably at least 80% homology, and very particularly preferably at least 90% homology across the entire amino acid range (for details on homology at the amino acid level, please refer to the information given above for peptides). Advantageously, homology may be even higher in certain regions of the sequence.

[0290] The present invention also relates to sequences that can be obtained by conserved nucleotide substitution (i.e., as a result, the amino acid in question is replaced by an amino acid having the same charge, size, polarity and / or solubility).

[0291] This invention also relates to molecules derived from specifically disclosed nucleic acids through sequence polymorphism. Such genetic polymorphism can exist in cells from different populations or from cells within a single population due to natural allelic variations. Allelic variants may also include functional equivalents. These natural variations typically produce changes of 1–5% in the nucleotide sequence of a gene. The polymorphism can lead to alterations in the amino acid sequence of the polypeptides disclosed herein. Allelic variants may also include functional equivalents.

[0292] Furthermore, derivatives should also be understood as homologs of the nucleic acid sequences according to the present invention, such as homologs of animals, plants, fungi, or bacteria, shortened sequences, single-stranded DNA or RNA encoding or non-coding DNA sequences. For example, at the DNA level, the homolog has at least 40%, preferably at least 60%, particularly preferably at least 70%, and very particularly preferably at least 80% homology in the entire DNA region given in the sequence specifically disclosed herein.

[0293] Furthermore, derivatives should be understood as, for example, fusions with promoters. Promoters added to the nucleotide sequence can be modified by at least one nucleotide exchange, at least one insertion, inversion, and / or deletion, without impairing the function or effectiveness of the promoter. Moreover, the effectiveness of promoters can be increased by altering their sequence, or by completely exchanging them with more effective promoters or even promoters from different genera of organisms.

[0294] d. Generation of functional polypeptide mutants

[0295] Those skilled in the art are familiar with methods for generating functional mutants, namely, a nucleotide sequence encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any amino acid-related SEQ ID NO disclosed herein; and / or encoded by a nucleic acid molecule containing a nucleotide sequence having at least 70% sequence identity with any nucleotide-related SEQ ID NO disclosed herein.

[0296] Depending on the techniques used, those skilled in the art can introduce completely random or more targeted mutations into gene or non-coding nucleic acid regions (e.g., those important for regulating expression) and subsequently generate a genetic library. The molecular biological methods required for this purpose are known to those skilled in the art, for example, as described in Sambrook and Russell, Molecular Cloning, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001.

[0297] Methods for modifying genes and thereby modifying the polypeptides encoded by them are known long to those skilled in the art, for example:

[0298] - Site-specific mutagenesis, in which single or multiple nucleotides of a gene are replaced in a directed manner (Trower MK (Ed.) 1996; In vitro mutagenesis protocols. Humana Press, New Jersey).

[0299] - Saturation mutagenesis, in which the codon of any amino acid can be exchanged or added at any site in the gene (Kegler-Ebo DM, Docktor CM, DiMaio D (1994) Nucleic Acids Res 22:1593; Barettino D, Feigenbutz M, Valcárel R, Stunnenberg HG (1994) Nucleic Acids Res 22:541; Barik S (1995) Mol Biotechnol 3:1),

[0300] - Error-prone polymerase chain reaction, in which the nucleotide sequence is mutated by error-prone DNA polymerase (Eckert KA, Kunkel TA (1990) Nucleic Acids Res 18:3739);

[0301] -SeSaM method (sequence saturation method), in which preferred exchanges are prevented by polymerase. Schenk et al., Biospektrum, Vol. 3, 2006, 277-279.

[0302] - Gene propagation in mutant strains, where, for example, due to defects in DNA repair mechanisms, the mutation rate of nucleotide sequences increases (Greener A, Callahan M, Jerpseth B (1996) An efficient random mutagenesis technique using an E. coli mutator strain. In: Trower MK (Ed.) In vitro mutagenesis protocols. Humana Press, New Jersey), or

[0303] -DNA shuffling, in which a set of closely related genes are formed and digested, and these fragments are used as templates for polymerase chain reactions, in which the full-length mosaic gene is eventually generated through repeated strand separation and recombination (Stemmer WPC (1994) Nature 370:389; Stemmer WPC (1994) Proc Natl Acad Sci USA 91:10747).

[0304] Using so-called directed evolution (particularly described in Reetz MT and Jaeger KE (1999), Topics Curr Chem 200:31; Zhao H, Moore JC, Volkov AA, Arnold FH (1999), Methods for optimizing industrial polypeptides by directed evolution, In: Demain AL, Davies JE (Ed.) Manual of industrial microbiology and biotechnology. American Society for Microbiology), skilled workers can mass-produce functional mutants in a directed manner. To this end, in the first step, gene libraries of the respective polypeptides are first generated, for example, using the methods given above. The gene libraries are expressed in a suitable manner, for example, through bacterial or phage display systems.

[0305] The relevant genes in the host organism expressing the functional mutant (whose function largely corresponds to the desired trait) can be submitted to another mutation cycle. The mutation and selection or screening steps can be repeated iteratively until the functional mutant of the present invention possesses a sufficient degree of the desired trait. Using this iterative process, a limited number of mutations, such as 1, 2, 3, 4, or 5 mutations, can be performed in stages, and their effects on the activity under study can be evaluated and selected. The selected mutants can then be subjected to further mutation steps in the same manner. This significantly reduces the number of individual mutants to be studied.

[0306] The results of this invention also provide important information regarding the structure and sequence of the relevant polypeptides, which is essential for the targeted generation of other polypeptides with desired modified properties. In particular, so-called "hot spots" can be defined as sequence segments potentially suitable for modification of properties by introducing targeted mutations.

[0307] Information about the location of amino acid sequences can also be derived, where mutations that may have little effect on activity can occur, and these can be designated as potential “silent mutations”.

[0308] e. Constructs expressing the polypeptides of the present invention

[0309] In the context of this article, the following definitions apply:

[0310] "Gene expression" encompasses both "heterologous expression" and "overexpression," and involves gene transcription and the translation of mRNA into proteins. Overexpression refers to the production of gene products, measured as mRNA, peptide, and / or enzyme activity levels, in transgenic cells or organisms exceeding the levels found in non-transformed cells or organisms with similar genetic backgrounds.

[0311] As used herein, an "expression vector" refers to a nucleic acid molecule engineered using molecular biology methods and recombinant DNA technology to deliver foreign or exogenous DNA into a host cell. Expression vectors typically include the sequences required for correct transcription of the nucleotide sequence. The coding region usually encodes the target protein, but it can also encode RNA, such as antisense RNA, siRNA, etc.

[0312] As used herein, “expression vector” includes any linear or circular recombinant vector, including but not limited to viral vectors, bacteriophages, and plasmids. Those skilled in the art can select a suitable vector based on the expression system. In one embodiment, the expression vector includes a nucleic acid of the embodiments described herein, operably linked to at least one “regulatory sequence” that controls transcription, translation, initiation, and termination, such as a transcription promoter, operon, or enhancer, or an mRNA ribosome binding site, and optionally includes at least one selection marker. When the regulatory sequence functionally relates to the nucleic acid of the embodiments described herein, the nucleotide sequence is “operably linked.”

[0313] As used herein, “expression system” encompasses any combination of nucleic acid molecules required to co-express two or more polypeptides in vivo or in vitro in a given expression host. The respective coding sequences may reside on a single nucleic acid molecule or vector, such as a vector containing multiple cloning sites, or on a polycistronic nucleic acid, or may be distributed across two or more physically distinct vectors.

[0314] As used herein, the terms “amplifying” and “amplification” refer to the use of any suitable amplification method to generate or detect recombinants of naturally expressed nucleic acids, as described in detail below. For example, the present invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo-dT primers) for amplifying (e.g., by polymerase chain reaction, PCR) naturally expressed (e.g., genomic DNA or mRNA) or recombinant nucleic acids (e.g., cDNA) of the present invention in vivo, in vitro, or in vitro.

[0315] A "regulatory sequence" refers to a nucleic acid sequence that determines the expression level of the nucleic acid sequence in the embodiment described herein and can regulate the transcription rate of a nucleic acid sequence operatively linked to that regulatory sequence. Regulatory sequences include promoters, enhancers, transcription factors, promoter elements, etc.

[0316] According to the present invention, "promoter," "nucleic acid with promoter activity," or "promoter sequence" should be understood as referring to a nucleic acid that, when functionally linked to a nucleic acid to be transcribed, regulates the transcription of said nucleic acid. "Promoter" specifically refers to a nucleic acid sequence that controls the expression of a coding sequence by providing a binding site for RNA polymerase and other factors suitable for transcription, including but not limited to transcription factor binding sites, repressor and activator protein binding sites. The term "promoter" also includes the term "promoter regulatory sequence." A promoter regulatory sequence may include upstream and downstream elements that may affect transcription, RNA processing, or the stability of the associated coding nucleic acid sequence. Promoters include naturally derived and synthetic sequences. The coding nucleic acid sequence is typically located downstream of the promoter relative to the transcription direction initiating from the transcription start site.

[0317] In this context, "functional" or "operationally" linked is understood, for example, to refer to the sequential arrangement of one of the nucleic acids having a regulatory sequence. For example, a sequence with promoter activity, and the nucleic acid sequence to be transcribed, along with optional other regulatory elements (e.g., nucleic acid sequences that ensure transcription) and, for example, a terminator, arranged such that each regulatory element can perform its function after transcription of the nucleic acid sequence. This does not necessarily require a direct chemical link. Genetic control sequences, such as enhancer sequences, can even act on the target sequence from more distant locations or even from other DNA molecules. A preferred arrangement is one where the nucleic acid sequence to be transcribed is located downstream (i.e., at the 3' end) of the promoter sequence, thereby covalently linking the two sequences together. The distance between the promoter sequence and the nucleic acid sequence to be recombined can be less than 200 base pairs, or less than 100 base pairs, or less than 50 base pairs.

[0318] Besides promoters and terminators, other examples of regulatory elements include: target sequences, enhancers, polyadenylation signals, selection markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0319] The term "constitutive promoter" refers to an unregulated promoter that allows for the continuous transcription of the nucleic acid sequence to which it is operatively linked.

[0320] As used herein, the term "operably linked" refers to the linking of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if a promoter or transcriptional regulatory sequence can influence the transcription of a coding sequence, then the promoter or transcriptional regulatory sequence is operably linked to the coding sequence. Operable linking means that the linked DNA sequences are typically adjacent. The nucleotide sequence associated with the promoter sequence can be homologous or heterologous in origin relative to the plant to be transformed. The sequence can also be wholly or partially synthetic. Regardless of origin, the nucleic acid sequence associated with the promoter sequence will be expressed or silenced depending on the nature of the promoter linked after binding to the polypeptide of the embodiments described herein. The associated nucleic acid can encode a protein that needs to be expressed or repressed throughout the organism or in a specific tissue, cell, or cell compartment at all times or alternatively at specific times. This nucleotide sequence specifically encodes a protein that confers the desired phenotypic trait to the host cell or organism altered or transformed by it. More specifically, the associated nucleotide sequence results in the production of vanillin in the cell or organism. Specifically, this nucleotide sequence encodes IEM.

[0321] The nucleotide sequence described above can be part of an "expression cassette". The terms "expression cassette" and "expression construct" are used synonymously. A (preferred recombinant) expression construct contains a nucleotide sequence that encodes a polypeptide according to the invention and is under the genetic control of a regulatory nucleic acid sequence.

[0322] In the method applied according to the present invention, the expression cassette may be an "expression vector", particularly a part of a recombinant expression vector.

[0323] According to the present invention, "expression unit" should be understood as a nucleic acid with expressive activity, which contains a promoter as defined herein, and regulates expression upon functional linkage with a nucleic acid or gene to be expressed, i.e., transcription and translation of said nucleic acid or gene. Therefore, it is also referred to in this respect as a "regulatory nucleic acid sequence". In addition to promoters, other regulatory elements, such as enhancers, may also be present.

[0324] According to the present invention, an "expression cassette" or "expression construct" should be understood as an expression unit functionally linked to a nucleic acid or gene to be expressed. Therefore, in contrast to an expression unit, an expression cassette contains not only nucleic acid sequences that regulate transcription and translation, but also nucleic acid sequences that are expressed as proteins due to transcription and translation.

[0325] In the context of this invention, the terms "expression" or "overexpression" describe the generation or increase of intracellular activity of one or more polypeptides encoded by corresponding DNA in a microorganism. For this purpose, for example, a gene may be introduced into the organism, an existing gene may be replaced with another gene, the copy number of a gene may be increased, a strong promoter may be used, or a gene encoding a corresponding polypeptide with high activity may be used. Optionally, these measures may be combined.

[0326] Preferably, such constructs according to the invention include a promoter upstream of the respective coding sequence 5' and a terminator sequence downstream of the respective coding sequence 3', as well as optionally other common regulatory elements, each operatively connected to the coding sequence.

[0327] The nucleic acid constructs according to the invention specifically comprise a sequence encoding a polypeptide, such as derived from the amino acid-related SEQ ID NO or its inverse complementary sequence as described herein, or derivatives and homologs thereof, and is operatively or functionally linked to one or more regulatory signals for advantageous control, for example, increasing gene expression.

[0328] In addition to these regulatory sequences, the natural regulation of these sequences may still exist before the actual structural genes, and optionally may have been genetically modified so that natural regulation has been turned off and gene expression is enhanced. However, nucleic acid constructs can also have simpler constructions, i.e., no additional regulatory signals are inserted before the coding sequence, and the natural promoter with regulatory function has not been removed. Instead, the natural regulatory sequences are mutated so that regulation no longer occurs and gene expression increases.

[0329] Preferred nucleic acid constructs advantageously also include one or more previously mentioned "enhancer" sequences functionally linked to a promoter, which enable enhanced expression of the nucleic acid sequence. Other advantageous sequences, such as other regulatory elements or terminators, may also be inserted at the 3' end of the DNA sequence. One or more copies of the nucleic acid according to the invention may be present in the construct. Optionally, other markers, such as genes complementary to auxotrophic or antibiotic resistance genes, may also be present in the construct for selection.

[0330] Examples of suitable regulatory sequences exist in promoters, such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, H9, H10, G6, C4, gal, trc, ara, rhaP(rhaP BAD SP6, lambda-P R Or lambda-P LIn promoters, they are advantageously used in Gram-negative bacteria. Other advantageous regulatory sequences are found, for example, in the Gram-positive promoters amy and SpO2, and in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, and ADH. Artificial promoters can also be used for regulation. See, for example, Jones et al. ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Sci. Rep. 2015, 5, 11301.

[0331] To facilitate expression in a host organism, nucleic acid constructs are advantageously inserted into vectors, such as plasmids or phages, enabling optimal gene expression in the host. Besides plasmids and phages, vectors should be understood to include all other vectors known to those skilled in the art, such as viruses like SV40, CMV, baculoviruses and adenoviruses, transposons, IS elements, phages, granules, and linear or circular DNA or artificial chromosomes. These vectors are capable of autonomous replication in the host organism or replication via chromosomes. These vectors represent a further development of the invention. Binary or CPO integration vectors are also suitable.

[0332] Suitable plasmids include, for example, those for *E. coli* pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, and pIN-III. 113 The plasmids listed above are a small selection of possible plasmids, found in *Streptomyces* pIJ101, pIJ364, pIJ702, or pIJ361; in *Bacillus* pUB110, pC194, or pBD214; in *Corynebacterium* pSA77 or pAJ667; in fungi pALS1, pIL2, or pBB116; in yeast 2alphaM, pAG-1, YEp6, YEp13, or pEMBLYe23; or in plants pLGV23, pGHlac+, pBIN19, pAK2004, or pDH51. Other plasmids are well known to technicians and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

[0333] In further development of the vector, vectors containing the nucleic acid constructs of the present invention or the nucleic acids of the present invention can also be advantageously introduced into microorganisms in the form of linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination. This linear DNA can consist of linearized vectors such as plasmids, or solely of the nucleic acid constructs or nucleic acids of the present invention.

[0334] For optimal expression of heterologous genes in an organism, it is advantageous to modify the nucleic acid sequence to match the specific “codon usage” used in the organism. “Codon usage” can be readily determined by computer evaluation of other known genes in the organism under discussion.

[0335] The expression cassette according to the invention is generated by fusing a suitable promoter to a suitable coding nucleotide sequence and a terminator or polyadenylation signal. Conventional recombination and cloning techniques are used for this purpose, as described in, for example, T. Maniatis, EFFritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); TJ Silhavy, MLBerman and LWEnquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984); and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

[0336] To facilitate expression in a suitable host organism, recombinant nucleic acid constructs or gene constructs are advantageously inserted into host-specific vectors, enabling optimal gene expression in the host. Vectors are well-known to those skilled in the art and can be found, for example, in "cloning vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

[0337] Alternative embodiments of the present invention provide a method for “altering gene expression in host cells.” For example, in certain contexts (e.g., exposure to certain temperatures or culture conditions), polynucleotides of the present invention can be enhanced, overexpressed, or induced in host cells or host organisms.

[0338] The altered expression of the polynucleotides described herein can also result in ectopic expression, which is a different expression pattern in altered and control or wild-type organisms. The alteration in expression occurs due to contact of the peptide of the embodiments described herein with an exogenous or endogenous modulator or is caused by chemical modification of the peptide. The term also refers to the altered expression pattern of the polynucleotides of the embodiments described herein, which is altered to below detectable levels or completely inhibited in activity.

[0339] In one embodiment, this document also provides isolated, recombinant, or synthetic polynucleotides encoding the polypeptide or variant polypeptide provided herein.

[0340] In one embodiment, multiple nucleic acid sequences encoding polypeptides are co-expressed in a single host, particularly under the control of different promoters. In another embodiment, multiple nucleic acid sequences encoding polypeptides may be present on a single transformation vector, or separate vectors may be used and transformants containing two chimeric genes may be selected for simultaneous co-transformation. Similarly, one or more polypeptide-encoding genes may be expressed together with other chimeric genes in a single plant, cell, microorganism, or organism.

[0341] f. Microorganisms applicable to this invention

[0342] Depending on the context, the term "microorganism" can refer to wild-type microorganisms or genetically modified recombinant microorganisms, or both.

[0343] In one embodiment, using the vector of the present invention, recombinant microorganisms can be produced, which, for example, can be transformed with at least one vector of the present invention, and can be used to produce the polypeptides of the present invention. Advantageously, the recombinant constructs according to the present invention, as described above, are introduced into and expressed in a suitable host system. Preferably, common cloning and transfection methods known to those skilled in the art, such as co-precipitation, protoplast fusion, electroporation, retroviral transfection, etc., are used to express the nucleic acids in their respective expression systems. Suitable systems are described in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0344] In principle, all prokaryotes or eukaryotes can be considered as recombinant host organisms for the nucleic acids or nucleic acid constructs according to the present invention. Advantageously, microorganisms such as bacteria, fungi / yeasts are used as host organisms. Advantageously, Gram-positive or Gram-negative bacteria are used, preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, or Nocardiaceae, particularly preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium, or Rhodococcus. The genus and species *Escherichia coli* are highly preferred. Furthermore, other advantageous bacteria have been found in the alpha-Proteobacteria, beta-Proteobacteria, or gamma-Proteobacteria groups.

[0345] Depending on the host organism, the organism used in the method according to the invention is grown or cultured in a manner known to those skilled in the art. Culture can be carried out in batches, semi-batch, or continuously. Nutrients can be provided at the start of fermentation or later, semi-continuously, or continuously. This is also described in more detail below.

[0346] g. Recombinant production of the polypeptide according to the present invention

[0347] The present invention further relates to a method for recombinantly producing polypeptides or functional biologically active fragments thereof according to the invention, wherein microorganisms that produce polypeptides are cultured, and optionally, expression of the polypeptides is induced by applying at least one inducer for gene expression, and the polypeptides are isolated from the culture. If desired, the polypeptides can also be produced on an industrial scale in this manner.

[0348] The microorganisms produced according to the present invention can be cultured continuously or discontinuously using batch culture, fed-batch culture, or repeated fed-batch culture. An overview of known culture methods can be found in Chmiel's textbook (Bioprozesstechnik 1.Einführungin die Bioverfahrenstechnik [Bioprocess technology 1.Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or Storhas's textbook (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheralequipment] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

[0349] The culture medium used must be appropriately suited to the requirements of each strain. Descriptions of culture media for various microorganisms are provided in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981).

[0350] These culture media, which can be used according to the present invention, typically contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0351] Preferred carbon sources are sugars, such as monosaccharides, disaccharides, or polysaccharides. Good carbon sources include, for example, glucose, fructose, mannose, galactose, ribose, sorbitol, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose. Sugars can also be added to the culture medium via malt extracts, complex compounds (e.g., molasses), or other byproducts of saccharification. Adding mixtures of different carbon sources is also advantageous. Other possible carbon sources are oils and fats, such as soybean oil, sunflower oil, peanut oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, or linoleic acid; alcohols such as glycerol, methanol, or ethanol; and organic acids such as acetic acid or lactic acid.

[0352] Nitrogen sources are typically organic or inorganic nitrogen compounds or materials containing these compounds. Examples of nitrogen sources include ammonia or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, or ammonium nitrate, nitrates, urea, amino acids, or complex nitrogen sources, such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. Nitrogen sources can be used alone or in combination.

[0353] Inorganic salt compounds that can be present in culture media include chlorides, phosphorus, or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, and iron.

[0354] Inorganic sulfur-containing compounds, such as sulfates, sulfites, dithionites, tetrasulfites, thiosulfates, sulfides, and organic sulfur compounds, such as thiols and thiols, can be used as sulfur sources.

[0355] Phosphoric acid, potassium dihydrogen phosphate, or dipotassium hydrogen phosphate, or their corresponding sodium-containing salts, can be used as phosphorus sources.

[0356] Chelating agents can be added to the culture medium to retain metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuic acid esters, or organic acids, such as citric acid.

[0357] The fermentation medium used according to the present invention typically also contains other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, niacin, pantothenic acid, and pyridoxine. The growth factors and salts are often derived from components of complex culture media, such as yeast extract, molasses, corn steep liquor, etc. Furthermore, suitable precursors may be added to the medium. The exact composition of the compounds in the medium depends largely on the specific experiment and is determined individually for each case. Information on medium optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Ed. PMRhodes, PFStanbury, IRL Press (1997), pp. 53-73, ISBN 0 19 963577 3). Growth media are also available from commercial suppliers such as Standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO).

[0358] All components of the culture medium are sterilized by heating (at 1.5 bar and 121°C for 20 minutes) or by aseptic filtration. These components can be sterilized together or individually as needed. All components of the culture medium can be given at the start of the culture or added continuously or in batches.

[0359] The culture temperature is typically between 15°C and 45°C, preferably between 25°C and 40°C, and can be varied or kept constant during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0. The pH during growth can be controlled by adding alkaline compounds (e.g., sodium hydroxide, potassium hydroxide, ammonia, or ammonia solution) or acidic compounds (e.g., phosphoric acid or sulfuric acid). Antifoaming agents such as fatty acid polyethylene glycol esters can be used to control foaming. To maintain plasmid stability, suitable selective substances such as antibiotics can be added to the culture medium. To maintain aerobic conditions, oxygen or a mixture of oxygen-containing gases (e.g., ambient air) is supplied to the culture. The culture temperature is typically in the range of 20°C to 45°C. Continue culturing until the maximum amount of the desired product is formed. This usually takes between 10 and 160 hours.

[0360] The fermentation broth is then further processed. Depending on the needs, the biomass can be completely or partially removed from the fermentation broth, or it can be left entirely in it, by separation techniques such as centrifugation, filtration, decantation, or a combination of these methods.

[0361] If the polypeptide is not secreted in the culture medium, the cells can also be lysed, and the product can be obtained from the lysate using known methods for protein separation. Cells can be disrupted optionally by high-frequency ultrasound, high pressure (e.g., in a French press), by osmosis, by the action of detergents, lysing enzymes, or organic solvents, by a homogenizer, or by a combination of these methods.

[0362] Peptides can be purified using known chromatographic techniques, such as molecular sieve chromatography (gel filtration), Q-agarose chromatography, ion exchange chromatography, and hydrophobic chromatography, as well as other conventional techniques such as ultrafiltration, crystallization, salting out, dialysis, and natural gel electrophoresis. Suitable methods are described, for example, in Cooper, TG, Biochemimsche Arbeitsmethoden [Biochemical Processes], Verlag Walter de Gruyter, Berlin, New York, or Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

[0363] For the isolation of recombinant proteins, the use of a carrier system or oligonucleotide may be advantageous, which extends cDNA by a defined nucleotide sequence and thus encodes an altered polypeptide or fusion protein, for example, for easier purification. Suitable modifications of this type are, for example, so-called “tags” that act as anchors, such as modifications known as hexahistine anchors or epitopes that can be recognized as antibody antigens (e.g., described in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press). These anchors can be used to attach proteins to solid supports, such as polymer matrices, which can be used, for example, as packing material in chromatographic columns, or on microtiter plates or other supports.

[0364] These anchors can also be used to identify proteins. To identify proteins, conventional markers, such as fluorescent dyes, enzyme markers (which react with a substrate to form a detectable reaction product), or radiolabels, can be used alone or in combination with anchors to derivatize proteins.

[0365] For the expression of mutants according to the present invention, reference can be made to the description of the expression of wild-type enzyme EbN1 and the expression systems that can be used in the expression system in WO2005 / 108590 and WO2006 / 094945, which are hereby explicitly referenced.

[0366] h. Immobilization of peptides

[0367] The enzymes or polypeptides according to the invention can be used in free form or immobilized in the methods described herein. Immobilized enzymes are enzymes immobilized on an inert support. Suitable support materials and enzymes immobilized thereon are known from EP-A-1149849, EP-A-1069183, and DE-OS 100193773 and the references cited therein. In this regard, reference is made to the full disclosure of these documents. Suitable support materials include, for example, clay, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, alumina, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger materials, synthetic polymers such as polystyrene, acrylic resins, phenolic resins, polyurethanes, and polyolefins such as polyethylene and polypropylene. For the preparation of loaded enzymes, the support material is generally used in the form of finely divided particles, preferably porous. The particle size of the support material is generally no greater than 5 mm, particularly no greater than 2 mm (particle size distribution profile). Similarly, when using dehydrogenases as whole-cell catalysts, either free or immobilized form can be selected. Carrier materials include, for example, calcium alginate and carrageenan. Enzymes and cells can also be directly cross-linked with glutaraldehyde (cross-linked with CLEAs). Corresponding and other immobilization techniques are described, for example, in J. Lalonde and A. Margolin, "Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol 3, Chapter 17, VCH, Weinheim provides further information on biotransformation and bioreactors for carrying out the methods according to the invention.

[0368] i. Reaction conditions of the biocatalytic production method of the present invention

[0369] At least one polypeptide / enzyme present in a single step of the method of the present invention or a multi-step method as defined above can be in the form of natural living cells or recombinant living cells that produce one or more enzymes, harvested cells, dead cells, permeabilized cells, crude cell extracts, purified extracts, or substantially pure or completely pure forms. The at least one enzyme can be present in solution or as an enzyme immobilized on a carrier. One or more enzymes can be present simultaneously in soluble and / or immobilized forms.

[0370] The method according to the invention can be carried out in common reactors known to those skilled in the art and can be carried out on various scales, from laboratory scale (a few milliliters to tens of liters of reaction volume) to industrial scale (a few liters to thousands of cubic meters of reaction volume). A chemical reactor can be used if the polypeptide is used in the form of encapsulation through non-living, optionally permeabilized cells, as a more or less purified cell extract, or in a purified form. Chemical reactors typically allow control of the amount of at least one enzyme, the amount of at least one substrate, pH, temperature, and the circulation of the reaction medium. When at least one polypeptide / enzyme is present in living cells, the process will be fermentation. In this case, biocatalytic production will be carried out in a bioreactor (fermenter) where parameters necessary for suitable survival conditions for living cells (e.g., nutrient-rich culture medium, temperature, pH, agitation, aeration, aerobic or anaerobic or other gases, antibiotics, etc.) can be controlled. Those skilled in the art are familiar with chemical or bioreactors, for example, procedures for scaling up chemical or biotechnological methods from laboratory to industrial scale or optimizing process parameters, which are also extensively described in the literature (for biotechnological methods, see, for example, Crueger und Crueger, Biotechnologie – Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, Munich, Wien, 1984).

[0371] Cells containing at least one enzyme can be permeated by physical or mechanical means, such as ultrasound or radio frequency pulses, high-pressure cell lysis (French press), or by chemical means, such as hypotonic media present in the culture medium, lysing enzymes, and detergents, or a combination of these methods. Examples of detergents are digitoxin, n-dodecyl maltodextrin, octyl glycoside, etc. X-100, 20, Deoxycholate, CHAPS (3-[(3-chloroamidopropyl)dimethylammonium]-1-propanesulfonate), P40 (ethylphenol poly(ethylene glycol ether)), etc.

[0372] Instead of living cells, non-living cell biomass containing the desired biocatalyst can also be used in the biotransformation reaction of this invention.

[0373] If at least one enzyme is immobilized, it is ligated to an inert carrier as described above.

[0374] The conversion reaction can be carried out in batches, semi-batches, or continuously. Reactants (and optional nutrients) can be provided at the start of the reaction, or they can be provided subsequently in a semi-continuous or continuous manner.

[0375] Depending on the specific reaction type, the reactions of this invention can be carried out in aqueous, aqueous-organic, or non-aqueous reaction media.

[0376] Aqueous or aqueous-organic media may contain suitable buffer solutions to adjust the pH to 5 to 11, such as 6 to 10.

[0377] In aqueous-organic media, organic solvents that are miscible, partially miscible, or immiscible with water can be used. Non-limiting examples of suitable organic solvents are listed below. Further examples are mono- or poly-aryl, aromatic or aliphatic alcohols, particularly poly-aliphatic alcohols such as glycerol.

[0378] Non-aqueous media may contain substantially no water, that is, will contain less than about 1% by weight or 0.5% by weight of water.

[0379] Biocatalytic methods can also be carried out in organic non-aqueous media. Suitable organic solvents include, for example, aliphatic hydrocarbons having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane, or cyclooctane; aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene, or dichlorobenzene; aliphatic acyclic and ethers, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether; or mixtures thereof.

[0380] The concentration of reactants / substrate can be adapted to optimal reaction conditions, which can depend on the specific enzyme being applied. For example, the initial substrate concentration can be 0.1 to 0.5 M, or, for example, 10 to 100 mM.

[0381] The reaction temperature can be adapted to optimal reaction conditions, which can depend on the specific enzyme used. For example, the reaction can be carried out at temperatures ranging from 0 to 70°C, such as 20 to 50 or 25 to 40°C. Examples of reaction temperatures are approximately 30°C, approximately 35°C, approximately 37°C, approximately 40°C, approximately 45°C, approximately 50°C, approximately 55°C, and approximately 60°C.

[0382] The process can continue until equilibrium is reached between the substrate and the subsequent product, but it can be stopped earlier. Typical process times range from 1 minute to 25 hours, particularly from 10 minutes to 6 hours, for example from 1 hour to 4 hours, and particularly from 1.5 hours to 3.5 hours. These parameters are non-limiting examples of suitable process conditions.

[0383] If the host is a genetically modified plant, it can provide optimal growth conditions, such as optimal light, water, and nutrients.

[0384] k. Product separation

[0385] The method of the present invention may further include the step of recovering a final product or intermediate product, which may optionally be a substantially pure form of a stereoisomer or enantiomer. The term "recovery" includes the extraction, harvesting, separation, or purification of a compound from a culture medium or reaction medium. The recovery of a compound can be carried out according to any conventional separation or purification method known in the art, including but not limited to treatment with conventional resins (e.g., anion or cation exchange resins, nonion adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silica, silica gel, cellulose, alumina, etc.), pH alteration, solvent extraction (e.g., using conventional solvents such as alcohols, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0386] The identity and purity of the separated products can be determined by known techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopy (e.g., IR, UV, NMR), staining methods, TLC, NIRS, enzyme or microbial assays (see, for example: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; und Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70; Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, pp. 89-90, 521-540, 540-547, 559-566, 575-581). S.581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.).

[0387] l. Fermentation production of vanillin

[0388] This invention also relates to a method for producing vanillin through fermentation.

[0389] The fermentation used according to the invention can be carried out, for example, in stirred fermenters, bubble columns, and loop reactors. For a comprehensive overview of possible method types, including stirrer types and geometries, see "Chmiel: Bioprozesstechnik: Einfuhrung in die Bioverfahrenstechnik, Band 1". Typical variations available in the method of the invention are those known to those skilled in the art or explained, for example, in "Chmiel, Hammes and Bailey: Biochemical Engineering", such as batch, fed-batch, repeatedly fed-batch, or continuous fermentation, with or without biomass recovery. Depending on the production strain, air, oxygen, carbon dioxide, hydrogen, nitrogen, or suitable gas mixtures can be injected to achieve good yields (YP / S).

[0390] The culture medium used must be appropriately suited to the requirements of the specific strain. Descriptions of various microbial culture media are provided in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981).

[0391] These culture media, which can be used according to the present invention, typically contain one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0392] Preferred carbon sources are sugars, such as monosaccharides, disaccharides, or polysaccharides. Very good carbon sources include, for example, glucose, fructose, mannose, galactose, ribose, sorbitol, ribulose, lactose, maltose, sucrose, raffinose, starch, or cellulose. Sugars can also be added to the culture medium via complex compounds (e.g., molasses) or other byproducts of saccharification. Adding mixtures of various carbon sources is also advantageous. Other possible sources of carbon are oils and fats, such as soybean oil, sunflower oil, peanut oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, or linoleic acid; alcohols such as glycerol, methanol, or ethanol; and organic acids such as acetic acid or lactic acid.

[0393] Nitrogen sources are typically organic or inorganic nitrogen compounds or materials containing these compounds. Examples of nitrogen sources include ammonia or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, or ammonium nitrate, nitrates, urea, amino acids, or complex nitrogen sources, such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. Nitrogen sources can be used alone or in combination.

[0394] Inorganic salt compounds that can be present in culture media include chlorides, phosphates, or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, and iron.

[0395] Inorganic sulfur-containing compounds, such as sulfates, sulfites, dithionites, tetrasulfites, thiosulfates, sulfides, and organic sulfur compounds, such as thiols and thiols, can be used as sulfur sources.

[0396] Phosphoric acid, potassium dihydrogen phosphate, or dipotassium hydrogen phosphate, or their corresponding sodium-containing salts, can be used as phosphorus sources.

[0397] Chelating agents can be added to the culture medium to retain metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuic acid esters, or organic acids, such as citric acid.

[0398] The fermentation medium used according to the present invention may also contain other growth factors, such as vitamins or growth promoters, including, for example, biotin, riboflavin, thiamine, folic acid, niacin, pantothenic acid, and pyridoxine. Growth factors and salts are typically derived from complex components of the medium, such as yeast extract, molasses, corn steep liquor, etc. Additionally, suitable precursors may be added to the medium. The precise composition of compounds in the medium depends heavily on the specific experiment and must be determined individually for each specific case. Information on medium optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (1997). Growth media are also available from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain Heart Infusion, DIFCO), etc.

[0399] All components of the culture medium are sterilized by heating (at 1.5 bar and 121°C for 20 minutes) or by aseptic filtration. These components can be sterilized together or individually as needed. All components of the culture medium can be given at the start of growth, or can be added continuously or in batches.

[0400] The culture temperature is typically between 15°C and 45°C, preferably between 25°C and 40°C, and can be kept constant or varied during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0. The pH during growth can be controlled by adding alkaline compounds (e.g., sodium hydroxide, potassium hydroxide, ammonia, or ammonia solution) or acidic compounds (e.g., phosphoric acid or sulfuric acid). Antifoaming agents such as fatty acid polyethylene glycol esters can be used to control foaming. To maintain plasmid stability, suitable substances with selective action, such as antibiotics, can be added to the culture medium. To maintain aerobic conditions, oxygen or an oxygen-containing gas mixture (e.g., ambient air) is supplied to the culture. The culture temperature is typically between 20°C and 45°C. Continue culturing until the maximum amount of the desired product is formed. This usually takes between 1 and 160 hours.

[0401] The method of the present invention may further include a step of recovering vanillin.

[0402] The term "recovery" includes the extraction, harvesting, separation, or purification of compounds from a culture medium. The recovery of compounds can be performed using any conventional separation or purification method known in the art, including but not limited to treatment with conventional resins (e.g., anion or cation exchange resins, nonion adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silica, silica gel, cellulose, alumina, etc.), pH alteration, solvent extraction (e.g., using conventional solvents such as alcohols, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0403] Before the intended separation, the biomass in the fermentation broth can be removed. Methods for removing biomass are known to those skilled in the art, such as filtration, sedimentation, and flotation. Therefore, biomass can be removed, for example, by centrifuges, separators, decanters, filters, or in flotation equipment. To maximize the recovery of valuable products, washing the biomass, for example by percolation, is generally recommended. The choice of method depends on the biomass content and properties in the fermentation broth, as well as the interaction between the biomass and the valuable products.

[0404] In one implementation, the fermentation broth can be sterilized or pasteurized. In another implementation, the fermentation broth is concentrated. This concentration can be carried out in batches or continuously, depending on the needs. Pressure and temperature ranges should be selected to ensure that product damage is not caused and to minimize equipment and energy consumption. Skillful selection of pressure and temperature levels for multi-stage evaporation can be particularly energy-efficient.

[0405] The following examples are illustrative only and are not intended to limit the scope of the invention as set forth in the abstract, specification or claims.

[0406] After considering the disclosure provided herein, a variety of possible variations that will immediately become apparent to those skilled in the art also fall within the scope of this invention.

[0407] Example

[0408] Material:

[0409] Unless otherwise stated, all chemical and biochemical materials, as well as microorganisms or cells used in this article, are commercially available products.

[0410] Unless otherwise stated, recombinant proteins are cloned and expressed using standard methods, such as those described, for example, in Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular cloning: A Laboratory Manual, 2008. nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0411] method:

[0412] In a typical isoeugenol monooxygenase assay, as described in the following examples, the reaction is centrifuged and extracted with MTBE, followed by analysis by gas chromatography.

[0413] Gas chromatography analysis was performed using an Agilent Technologies 6850 equipped with an FID detector and a DB-WAX column (30 μm, 250 microM, 0.25 microM) using the following program: 100 °C (1 min), 245 °C, 20 °C min. -1 245℃ (10 minutes). Carrier gas: hydrogen, 75 cm sec. -1 Split = 50, injection volume = 1 μL.

[0414] Example 1: Preparation and testing of the first-generation isoeugenol monooxygenase (IEM1)

[0415] The following first-generation biocatalysts were used:

[0416]

[0417] Freshly grown colonies of strain BL21(DE3):pIEM1 were selected from LB agar plates and transferred to 20 mL of LB medium containing 100 mg / L ampicillin. The culture was incubated at 37°C with shaking at 225 rpm for 20 hours. One mL of this culture was then inoculated into 200 mL of sterile LB medium using a 1 L Erlenmeyer flask containing 100 mg / L ampicillin. -1 Ampicillin. Growth was carried out at 37°C with shaking at 220 rpm until OD (dose expiratory volume) was reached. 600 The value was 0.4. The temperature was lowered to 20°C, while the culture continued to grow until the OD value was 0.4. 600 The OD value was 0.6. The culture was induced with 1 mM IPTG and then regrowth was performed for 18 hours. After overnight incubation, the OD value was typically reached. 600 It ranges from 5.5 to 7.

[0418] Cells were centrifuged at 9000g and 4°C for 20 minutes. The supernatant was discarded, and the cells were resuspended in 100mM glycine-NaOH buffer (pH 10.5) containing 10v / v % DMSO (for isoeugenol monooxygenase of Pseudomonas nitrate-reducingis Jin1, the optimal pH was subsequently found to be approximately 9). Preparations were then made corresponding to the OD... 600 Suspensions with cell densities ranging from 10 to 90 were prepared. To a 20 mL vial, add: 2 mL of cell suspension at the specified optical density, 75 mg of isoeugenol (Sigma Aldrich #17206). Seal the vial using a perforated aluminum foil seal and react at 500 rpm for 20 hours at room temperature (24°C). Acidify the reaction mixture with 6 drops of 15% HCl and add 10 mL of solution containing 1 g L... -1 MTBE extraction with tridecane as an internal standard was performed, followed by gas chromatography analysis. A negative control was established using empty cells in the same manner. The time-varying curve for vanillin formation is shown below. Figure 3 As shown.

[0419] Example 2: Production of vanillin via first-generation isoeugenol monooxygenase (IEM1)

[0420] Vanillin was prepared as follows: *Escherichia coli* cells expressing nitrifying Pseudomonas Jin1 isoeugenol monooxygenase (see Example 1) were transferred from an LB agar plate containing ampicillin to 50 mL of agar containing 15 g / L ampicillin. -1 The medium was grown in an inorganic salt medium with glucose as the carbon source, and under shaking conditions at 225 rpm and 37°C for 20 hours. The inorganic salt medium consisted of 15 g L... -1 Glucose, 5g / L -1 (NH4)2HPO4, 16g L -1 K2HPO4, 2g L -1 Citric acid, 1g / L -1There is still 30 mg L of MgSO4. -1 The composition of CaCl2, along with trace elements, vitamins and 100mg L -1 Ampicillin was administered. Then, 0.4 mL of this culture was inoculated into a 1 L flask with 200 mL of inorganic salt medium of the same composition. Several flasks were run in parallel. The culture was grown at 37°C with shaking at 180 rpm until OD (dose retardation) was achieved. 600 The value is 2.5. Lower the temperature to 20°C. Use OD... 600 The culture was induced with 1 mM IPTG at 3°C ​​and grown for another 17 hours at 20°C with shaking at 180 rpm. OD was observed at the end of the culture. 600 The culture was centrifuged at 4000g for 50 minutes at 4°C. Cells were resuspended in 100mM glycine-NaOH buffer at pH 9. The final OD value was 24. 600 It is 45.

[0421] Add 1.3L of OD to a 2L reaction flask. 600 The reaction mixture consisted of a 45 g cell suspension and 19.5 g isoeugenol. The reaction was stirred at room temperature for 20 hours. Gas chromatography analysis showed a yield of 7.3 g / L. -1 Vanillin, 1.2g / L -1 (Z)-isoeugenol and 4.1 g L -1The remaining (E)-isoeugenol was extracted. The temperature of the reaction broth was lowered to 12°C, the pH was adjusted to pH 12.5, and then extracted with distilled ethyl acetate (1 L). After phase separation and removal of the organic phase, the aqueous phase was again adjusted to 12°C and pH 12.5. A second extraction was performed with 700 mL of ethyl acetate, and the organic phase was discarded. The pH of the aqueous phase was then adjusted to pH 7 with 15% HCl. The aqueous phase was extracted twice with 1 L of distilled ethyl acetate. According to GC analysis, these combined extracts contained 7.7 g of vanillin. The combined organic extracts were then washed with 600 mL of water and then evaporated under vacuum. 7.7 g of yellow residue was obtained. An attempt to remove the yellow color by a second extraction with ethyl acetate at 10°C and pH 12.5 failed. Approximately 0.8 g of vanillin was lost in the process, leaving 6.9 g of pale yellow solid residue. The residue was dissolved in 153 mL of hot water at 55°C. Once all the crude vanillin had dissolved, the solution was left to stand at room temperature for 1 hour. The temperature was lowered to 4°C and maintained at that temperature for 3 hours. Finally, the solution was cooled in an ice-water bath for 18 hours. The solution with precipitated vanillin was passed through a pre-cooled paper filter. The crystals were washed with ice-cold water (30 mL). The crystals were then dried under vacuum in a desiccator at room temperature for 6 hours. A pale yellow solid residue containing 99.7% vanillin was recovered by GC according to an internal standard. Chemical identity was verified by GC-MS, 1H-NMR, and 13C-NMR. Some losses occurred during purification. Ethyl acetate is not well-suited for alkaline extraction and can be replaced with a more stable solvent (e.g., MTBE).

[0422] Example 3: Preparation of second-generation isoeugenol monooxygenase

[0423] The following second-generation biocatalysts (IEMs and helper peptides located on two different plasmids) were tested:

[0424]

[0425] Freshly grown cells were isolated from agar plates and grown in 2 mL LB medium containing ampicillin and chloramphenicol at 37°C with shaking until the optical density OD was reached. 600 =1. Take 1 mL of this culture and inoculate it into 200 mL of LB medium containing 100 mg L. -1 Ampicillin and 30mg L -1 Chloramphenicol was added, and 5 mg L was added to a 1000 mL flask. -1 FeCl3. According to the manufacturer's agreement (Takara Bio Inc.), 2 mg / mL was used at the start of the culture. -1 Arabinose and, if necessary, 5 ng / mL -1Tetracycline-induced molecular chaperone. Incubate at 37°C with shaking at 180 rpm until OD is reached. 600 It is 0.4. (Using OD) 600 Before induction with 1 mM IPTG (0.6 g / mL), the temperature was lowered to 20°C. After overnight culture, cells were harvested by centrifugation at 3600 g and resuspended in a cold buffer of 0.1 M glycine-NaOH (pH 9.5) to the desired optical density. Catalytic activity was tested as described in Example 1. Figure 4 The catalytic activities observed with different constructs are shown. Most second-generation catalysts yielded higher specific activities than the first-generation catalysts (control). Cells co-expressing isoeugenol monooxygenase from *Pseudomonas nitrate-reducing* Jin1 and the molecular chaperones GroES and GroEL were the most active. Cells carrying plasmid pKJE7 showed very weak growth and were not subjected to activity testing.

[0426] Example 4: Fed-batch culture of second-generation isoeugenol monooxygenase

[0427] The strain BL21(DE3):pIEM1_pGro7 was tested under fed-batch culture conditions. The fed-batch culture was conducted in a reactor with a working volume of 3.7 L (Bioengineering, Switzerland) using dissolved oxygen-based glucose feed. The feed contained 5 g L... -1 Yeast extract supplemented with carbenicillin and chloramphenicol at pH 7 was used as the growth medium, with glycerol used instead of glucose as the carbon source. Cultures were performed at 37°C. (OD...) 600 When the temperature is 48°C, the temperature is lowered to 25°C, and at OD... 600 Under conditions of 51, use 1.5g L -1 Arabinose-induced culture. At OD... 600 At a pH of 59, the culture was induced with 1 mM IPTG. The feed solution contained 70% sterile glycerol aqueous solution. The pH was controlled with NH3 aqueous solution. Fermentation lasted for 43 hours, and the final OD... 600 The value was 163. Plasmid stability was determined using agar plates with and without appropriate antibiotics. Bacterial cells were harvested by centrifugation as previously described, resuspended in 100 mM glycine-NaOH buffer at pH 9, and activity was tested. In the reaction described in Example 1, up to 3.7 g / L was observed by gas chromatography. -1 Vanillin, where the catalyst loading corresponds to OD 600 It is 5.

[0428] Example 5: Preparation of third-generation isoeugenol monooxygenase

[0429] The following third-generation biocatalysts (IEMs and accessory peptides located on a single polycistronic construct) were prepared and tested:

[0430]

[0431]

[0432] Schematic diagrams of different polycistronic constructs are also provided. Figure 2 Culture, induction, and activity testing were performed as described in Example 1. OD 600 Comparative activity tests with a catalyst loading of 5 showed that *E. coli* cells expressing one of the polycistronic constructs (the third-generation catalyst) exhibited higher specific catalytic activity compared to first- and second-generation catalysts. Figure 5 ).

[0433] Example 6: Generation of a mutant of isoeugenol monooxygenase from *Pseudomonas putida* IE27

[0434] An artificial mutant of isoeugenol monooxygenase from *Pseudomonas putida* IE27 (IEM2) was inserted into plasmid pJ431(T7, kan R Low copy number; provided by Atum, Newark, CA) to be expressed in Escherichia coli BL21(DE3)T1. Alternatively, E. coli cells producing one of the Pseudomonas putridae IE27 mutants were co-transformed with plasmid pGro7 to co-produce the molecular chaperones GroES and GroEL.

[0435] The following mutants were tested:

[0436]

[0437] Fresh colonies of *E. coli* carrying plasmid-coding mutants were selected from LB agar plates and then plated in a solution containing 30 mg L... -1 The culture was incubated overnight (16 h) at 2 mL LB liquid culture of kanamycin at 220 rpm and 37 °C. Then, 150 μL was transferred to a flask containing 50 mL LB medium with the desired antibiotic and incubated at 37 °C with shaking at 200 rpm for approximately 2 h, until OD (dose retardation) was achieved. 600 The value was 0.45. The temperature was lowered to 20°C, while the culture continued to grow to the OD value. 600 The OD value was 0.6. After induction with 0.5 mM IPTG, the culture was regrown for 16 hours under the same conditions. *E. coli* cells containing the *Pseudomonas putida* IE27 mutant isoeugenol monooxygenase were collected by centrifugation and resuspended in ice-cold 0.1 M glycine buffer (pH 9.5) containing 10 v / v % DMSO. 600The value was 45. For *E. coli* cells containing isoeugenol monooxygenase from *Pseudomonas nitroreductoids* Jin1 (used as a positive control), the same procedure was performed using 0.1 M glycine buffer (pH 9.0). Add the following to a 20 mL vial: 80 mg of isoeugenol, 2 mL of OD... 600 =45% cell suspension. Seal the vial with a perforated aluminum foil to allow air circulation. React at room temperature (23°C) with magnetic stirring at 450 rpm for 17 hours. Acidify the reaction with 3 drops of 15% HCl and use 10 mL of solution containing 1 g L -1 MTBE extraction with tridecane as an internal standard was used for analysis by GC. Vanillin concentrations obtained using single mutants and controls are shown below. Figure 6 As shown. Additional mutants of isoeugenol monooxygenase from *Pseudomonas putida* IE27 (IEM) were obtained through saturation mutagenesis experiments performed by a commercial manufacturer at amino acid positions T52, Q74, or D440, respectively. These additional mutants were tested similarly to the mutants identified above. The relative activities of each mutant are shown in the figure. Figure 10 As shown, and expressed as the fraction of the average vanillin titer obtained under standard assay conditions using mutant “C154” (i.e. mutant a154c; corresponding to PQD).

[0438] Example 7: Isoeugenol monooxygenase of *Pseudomonas putida* IE27 co-expressed with GroES and GroEL Three mutants

[0439] The plasmid pIEM2_c154_t222_a1318 (see [link]) was isolated from agar plates. Figure 8 The plasmid image and annotations of *Escherichia coli* BL21(DE3)T1 (SEQ ID NO: 16) were obtained, and pGro7 cells were isolated from agar plates and added to 2 mL of LB medium containing 30 mg / L kanamycin and 30 mg / L chloramphenicol. The culture was incubated at 37°C with shaking at 220 rpm for 16 hours. 150 μL was transferred to 50 mL of fresh LB medium containing the same antibiotics. The cells were incubated at 37°C with shaking at 220 rpm until OD (dose retardation). 600 The value was 0.45. The culture was induced with 1.5 g / L arabinose, and the temperature was lowered to 20°C to allow growth under shaking until OD reached 0.45. 600 The OD value was 0.6. The culture was induced with 0.5 mM IPTG and grown for another 16 hours at 20°C with shaking at 220 rpm. Cells were collected by centrifugation and resuspended in ice-cold 0.1 M glycine buffer, pH 9.5. Different cell dilutions were prepared, corresponding to OD values. 600The concentrations ranged from 2 to 45. In an open 20 mL reaction vial, 80 mg of isoeugenol and 2 mL of cell suspensions at different optical densities were added. The reaction mixture was stirred at 500 rpm with a magnetic rod at room temperature for 17 hours. The reaction solution was acidified with 3 drops of 15% HCl and extracted with 10 mL of MTBE containing 1 g / L tridecane as an internal standard for GC analysis. Figure 7 The vanillin concentrations observed in reactions with varying cell densities are shown.

[0440] Example 8: From the polycistronic construct PC1_triple(SEQ ID NO:25; Contains IEM IE27's three protrusions Different expression constructs of the variant co-expressed isoeugenol monooxygenase of *Pseudomonas putida* IE27 with GroES and GroEL. The three mutants.

[0441] Colonies of *E. coli* cells BL21(DE3)T1 were extracted from agar plates containing kanamycin, carrying different plasmid constructs (according to...). Figure 11 The general protocol was explained, and the nucleotide sequences were selected from SEQ ID NO:17 to 24, along with the corresponding constructs (without the transcription terminator encoding the IEM sequence) for expressing the triple mutant of isoeugenol monooxygenase IE27, along with the molecular chaperones GroES and GroEL, and inoculated into 2 mL of inorganic salt medium. The inorganic salt medium contained 30 g / L glycerol, 5 g L... -1 (NH4)2HPO4, 16g L -1 K2HPO4, 2g L -1 Citric acid, 1g / L -1 MgSO4, 30 mg / L -1 CaCl2, 5 g / L yeast extract, trace elements and 50 mg / L kanamycin.

[0442] The culture was incubated at 37°C with shaking at 230 rpm for 16 hours. 0.25 mL of the culture was then transferred to a flask containing 50 mL of the same culture medium and incubated at 37°C with shaking at 230 rpm until the optical density (OD) reached its maximum. 600 The value was 2.5. The temperature was lowered to 23°C, and incubation continued at 220 rpm until the OD value reached 2.5. 600 3. Induce the culture with 0.5 mM IPTG and incubate overnight under the same conditions.

[0443] At the end of the culture, samples were taken to measure the optical density at 600 nm. Cell dry weight was estimated using the standard gravimetric method, and cell viability was assessed. For the viability test, 15 mL of culture was centrifuged at 3500 g for 35 minutes at 4°C, and the supernatant was discarded. The cell pellet was frozen at -80°C for several hours. The frozen pellet was thawed on ice and resuspended in ice-cold 0.1 M glycine buffer (pH 9.5). The final OD... 600 It is 20.

[0444] Add 80 mg of isoeugenol and 1800 μL of ice-cold 0.1 M glycine buffer (pH 9.5) to a 20 mL flask. While magnetically stirring, add 200 μL of cell resuspension (OD200). 600 =20) to achieve the final OD 600 The reaction mixture was 2. The mixture was stirred at 450 rpm with a magnetic stirrer at 23°C for 16 hours.

[0445] Acidification was performed using 8 drops (80 μL) of 85% phosphoric acid, followed by 10 mL of solution containing 1 g L. -1 MTBE extraction was performed with tridecane as an internal standard. Vanillin concentration was determined by gas chromatography as described. Vanillin concentrations obtained with different plasmid constructs were normalized to the cell dry weight of the cell suspensions used in the reaction. Results are as follows. Figure 12 As shown.

[0446] The content of cross-referenced literature is incorporated here through citation.

[0447] The sequence referred to in this article is:

[0448] SEQ ID NO name source type 1 IMG1 codon modification Nitrification-reducing Pseudomonas Jin1 NA 2 IMG1 Nitrification-reducing Pseudomonas Jin1 AA 3 IMG2 Wild Type Pseudomonas putida IE27 NA 4 IMG2 Wild Type Pseudomonas putida IE27 AA 5 IMG2 codon modification Pseudomonas putida IE27 NA 6 IMG2 Pseudomonas putida IE27 AA 7 GroEL E. coli NA 8 GroEL E. coli AA 9 GroES E. coli NA 10 GroES E. coli AA 11 PC1 Expression builder NA 12 PC2 Expression builder NA 13 PC3 Expression builder NA 14 PC4 Expression builder NA 15 PC5 Expression builder NA 16 pIEM2_c154_t222_a1318 Expression plasmids NA 17 trc / +term Expression builder NA 18 tac / +term Expression builder NA 19 T7 / +term Expression builder NA 20 T5 / +term Expression builder NA 21 H10 / +term Expression builder NA 22 H9 / +term Expression builder NA 23 G6 / +term Expression builder NA 24 C4 / +term Expression builder NA 25 PC1_triple Expression builder NA

[0449] NA = Nucleic Acid

[0450] AA = amino acid.

[0451] sequence list

[0452] SEQ ID NO:1 - Isoeugenol monooxygenase of *Pseudomonas nitrification-reducing* Jin1 (codon-modified)

[0453]

[0454] SEQ ID NO:2 - Isoeugenol monooxygenase protein of *Pseudomonas nitrification-reducing* Jin1

[0455] MARLNRNDPQLVGTLLPTRIEADLFDLEVDGEIPKSINGTFYRNTPEPQVTPQKFHTFIDGDGMASAFHFEDGHVDFISRWVKTARFTAERLARKSLFGMYRNPYTDDTSVKGLDRTVA NTSIISHHGKVLAVKEDGLPYELDPRTLETRGRFDYDGQVTSQTHTAHPKYDPETGDLLFFGSAAKGEATPDMAYYIVDKHGKVTHETWFEQPYGAFMHDFAITRNWSIFPIMPATNSLS RLKAKQPIYMWEPELGSYIGVLPRRGQGSQIRWLKAPALWVFHVVNAWEVGTKIYIDLMESEILPFPFPNSQNQPFAPEKAVPRLTRWEIDLDSSSDEIKRTRLHDFFAEMPIMDFRFA LQCNRYGFMGVDDPRKPLAHQQAEKIFAYNSLGIWDNHRGDYDLWYSGEASAAQEPAFVPRSPTAAEGDGYLLTVVGRLDENRSDLVILDTQDIQSSGPVATIKLPFRLRAALHGCWVPRP

[0456] SEQ ID NO:3 - Isoeugenol monooxygenase of *Pseudomonas putida* IE27 (GenBank entry AB291707)

[0457]

[0458] SEQ ID NO:4 or 6 - Isoeugenol monooxygenase protein of *Pseudomonas putida* IE27

[0459] MATFDRNDPQLAGTMFPTRIEANVFDLEIEGEIPRAINGSFFRNTPEPQVTTQPFHTFIDGDGLASAFHFEDGQVDFVSRWVCTPRFEAERSARKSLFGMYRNPFTDDPSVEGIDRTVA NTSIITHHGKVLAAKEDGLPYELDPQTLETRGRYDYKGQVTSHTHTAHPKFDPQTGEMLLFGSAAKGERTLDMAYYIVDRYGKVTHETWFKQPYGAFMHDFAVTRNWSIFPIMPATNSLE RLKAKQPIYMWEPERGSYIGVLPRRGQGKDIRWFRAPALWVFHVVNAWEEGNRILIDLMESEILPFPFPNSQNLPFDPSKAVPRLTRWEIDLNSGNDEMKRTQLHEYFAEMPIMDFRFA LQDHRYAYMGVDDPRRPLAHQQAEKIFAYNSLGVWDNHRKDYELWFTGKMSAAQEPAFVPRSPDAPEGDGYLLSVVGRLDEDRSDLVILDTQCLAAGPVATVKLPFRLRAALHGCWQSKN

[0460] SEQ ID NO:5 - Isoeugenol monooxygenase (codon-modified) of *Pseudomonas putida* IE27

[0461]

[0462] SEQ ID NO:7-Molecular chaperone GroEL (GenBank ID CP009685.1, region 3964433..3966079)

[0463]

[0464] SEQ ID NO: 8 - Chaperonin GroEL protein

[0465] MAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTITKDGVSVAREIELEDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQAIITEGLKAVAAGMNPMDLKRGIDKAVTAAVEELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEAMDKVGKEGVITVEDGTGLQDELDVVEGMQFDRGYLSPYFINKPETGAVELESPFILLADKKISNIREMLPVLEAVAKAGKPLLIIAEDVEGEALATLVVNTMRGIVKVAAVKAPGFGDRRKAMLQDIATLTGGTVISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEAAIQGRVAQIRQQIEEATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGVVAGGGVALIRVASKLADLRGQNEDQNVGIKVALRAMEAPLRQIVLNCGEEPSVVANTVKGGDGNYGYNAATEEYGNMIDMGILDPTKVTRSALQYAASVAGLMITTECMVTDLPKNDAADLGAAGGMGGMGGMGGMM

[0466] SEQ ID NO: 9 - Chaperonin GroES (GenBank accession number CP009685.1, region 3966123..3966416)

[0467] ATGAATATTCGTCCATTGCATGATCGCGTGATCGTCAAGCGTAAAGAAGTTGAAACTAAATCTGCTGGCGGCATCGTTTCTGACCGGCTCTGCAGCGGCTAAATCCACCCGCGGCGAAGTGCTGGCTGTCGGCAATGGCCGTATCCTT GAAAATGGCGAAGTGAAGCCGCTGGATGTGAAAGTTGGCGACATCGTTATTTTCAACGATGGCTACGGTGTGAAATCTGAGAAGATCGACAATGAAGAAGTGTTGATCATGTCCGAAAGCGACATTCTGGCAATTGTTGAAGCGTAA

[0468] SEQ ID NO:10 - Molecular chaperone GroES protein

[0469] MNIRPLHDRVIVKRKEVETKSAGGIVLTGSAAAKSTRGEVLAVGNGRILENGEVKPLDVKVGDIVIFNDGYGVKSEKIDNEEVLIMSESDILAIVEA

[0470] SEQ ID NO:11-PC1 (a polycistronic construct containing codon-modified isoeugenol monooxygenase, groES, groEL from *Pseudomonas nitrificans* Jin1)

[0471]

[0472] SEQ ID NO:12-PC2 (a polycistronic construct containing codon-modified isoeugenol monooxygenase, groES, groEL from *Pseudomonas nitrificationis* Jin1)

[0473]

[0474] SEQ ID NO:13-PC3 (a polycistronic construct containing codon-modified isoeugenol monooxygenase, groES, groEL from *Pseudomonas nitrificationis* Jin1)

[0475]

[0476] SEQ ID NO:14-PC4 (a polycistronic construct containing codon-modified isoeugenol monooxygenase, groES, groEL from *Pseudomonas nitrificationis* Jin1)

[0477]

[0478] SEQ ID NO:15-PC5 (a polycistronic construct containing codon-modified isoeugenol monooxygenase, groES, groEL from Pseudomonas nitrificationis Jin1)

[0479]

[0480] SEQ ID NO:16 pIEM2_C154_T222_A1318

[0481]

[0482]

[0483]

[0484]

[0485] SEQ ID NO:17: Plasmid construct trc / +term

[0486]

[0487] SEQ ID NO:18: Plasmid construct tac / +term

[0488]

[0489] SEQ ID NO:19: Plasmid construct T7 / +term

[0490]

[0491] SEQ ID NO:20: Plasmid construct T5 / +term

[0492]

[0493] SEQ ID NO:21: Plasmid construct H10 / +term

[0494]

[0495] SEQ ID NO:22: Plasmid construct H9 / +term

[0496]

[0497] SEQ ID NO:23: Plasmid construct G6 / +term

[0498]

[0499] SEQ ID NO:24: Plasmid construct C4 / +term

[0500]

[0501] SEQ ID NO:25: Plasmid construct PC1_triple

[0502] sequence list <110> Firmenich SA <120> Methods for producing vanillin <130> 10790 / PCT <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 1437 <212> DNA <213> Isoeugenol monooxygenase of *Pseudomonas nitrification-reducing* Jin1 (codon-modified) <220> <221> CDS <222> (1)..(1437) <400> 1 atg gca cgt ctg aac cgt aat gat ccg caa ctc gtc ggc acc ctg tta 48 Met Ala Arg Leu Asn Arg Asn Asp Pro Gln Leu Val Gly Thr Leu Leu 1 5 10 15 cca acc cgt att gag gct gac ctg ttc gac ctg gaa gtg gac ggc gaa 96 Pro Thr Arg Ile Glu Ala Asp Leu Phe Asp Leu Glu Val Asp Gly Glu 20 25 30 att ccg aag tcc atc aac ggt acc ttc tac cgt aac acg ccg gag cct 144 Ile Pro Lys Ser Ile Asn Gly Thr Phe Tyr Arg Asn Thr Pro Glu Pro 35 40 45 cag gtg acg ccg cag aaa ttc cac acc ttc atc gat ggt gac ggc atg 192 Gln Val Thr Pro Gln Lys Phe His Thr Phe Ile Asp Gly Asp Gly Met 50 55 60 gcg tct gca ttt cat ttt gaa gat ggc cac gtg gac ttc atc agc cgc 240 Ala Ser Ala Phe His Phe Glu Asp Gly His Val Asp Phe Ile Ser Arg 65 70 75 80 tgg gtt aaa acc gcg cgt ttc acg gcg gag aga ctg gca cgt aaa agc 288 Trp Val Lys Thr Ala Arg Phe Thr Ala Glu Arg Leu Ala Arg Lys Ser 85 90 95 ctg ttc ggt atg tac cgt aat ccg tac acc gat gac acg tct gtg aag 336 Leu Phe Gly Met Tyr Arg Asn Pro Tyr Thr Asp Asp Thr Ser Val Lys 100 105 110 ggt ctg gat cgt acc gtt gcc aac acg agc atc atc agc cat cac ggt 384 Gly Leu Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Ser His His Gly 115 120 125 aag gtt ctg gcg gtg aaa gaa gat ggc ttg ccg tac gag ctg gac cca 432 Lys Val Leu Ala Val Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro 130 135 140 cgc act ctg gaa acc cgt ggt cgc ttt gac tat gat ggc caa gtc acc 480 Arg Thr Leu Glu Thr Arg Gly Arg Phe Asp Tyr Asp Gly Gln Val Thr 145 150 155 160 agc cag acg cac acc gcg cat ccg aag tat gac ccg gaa acc ggt gac 528 Ser Gln Thr His Thr Ala His Pro Lys Tyr Asp Pro Glu Thr Gly Asp 165 170 175 ctg ttg ttc ttt ggc tcc gca gcc aag ggt gag gca acg cct gat atg 576 Leu Leu Phe Phe Gly Ser Ala Ala Lys Gly Glu Ala Thr Pro Asp Met 180 185 190 gcc tat tac atc gtt gat aaa cat ggt aag gta acg cat gag act tgg 624 Ala Tyr Tyr Ile Val Asp Lys His Gly Lys Val Thr His Glu Thr Trp 195 200 205 ttc gag caa ccg tat ggc gcc ttt atg cat gat ttt gct att acc cgc 672 Phe Glu Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Ile Thr Arg 210 215 220 aat tgg agc atc ttc ccg atc atg ccg gct acc aat tcg ttg agc cgc 720 Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Ser Arg 225 230 235 240 ctg aaa gcg aag cag ccg att tac atg tgg gag ccg gaa ctg ggt tcc 768 Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Leu Gly Ser 245 250 255 tat att ggt gtt ctg ccg cgt cgc ggt caa ggt agc cag atc cgc tgg 816 Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Ser Gln Ile Arg Trp 260 265 270 ctg aaa gca cca gcg ctg tgg gtc ttt cac gtc gtc aac gcc tgg gaa 864 Leu Lys Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu 275 280 285 gtg ggc acc aaa atc ​​tac att gac ctt atg gag agc gaa att ctg cca 912 Val Gly Thr Lys Ile Tyr Ile Asp Leu Met Glu Ser Glu Ile Leu Pro 290 295 300 ttc ccg ttc ccg aat agc caa aat cag ccg ttc gct cct gag aaa gca 960 Phe Pro Phe Pro Asn Ser Gln Asn Gln Pro Phe Ala Pro Glu Lys Ala 305 310 315 320 gtg ccg cgt ctg acc cgt tgg gag att gat ctg gat agc agc agc gat 1008 Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asp Ser Ser Ser Asp 325 330 335 gag att aag cgt acg cgt ctg cac gac ttc ttt gca gag atg ccg atc 1056 Glu Ile Lys Arg Thr Arg Leu His Asp Phe Phe Ala Glu Met Pro Ile 340 345 350 atg gat ttt cgt ttt gcg ctg cag tgc aac cgc tac ggt ttt atg ggt 1104 Met Asp Phe Arg Phe Ala Leu Gln Cys Asn Arg Tyr Gly Phe Met Gly 355 360 365 gtc gat gac ccg cgc aag ccg ctg gcg cac caa gcg gag aag att 1152 Val Asp Asp Pro Arg Lys Pro Leu Ala His Gln Gln Ala Glu Lys Ile 370 375 380 ttt gcg tac aat agc ctg ggt atc tgg tgg gac aac cac cgt ggt gat tat 1200 Phe Ala Tyr Asn Is Leu Gly Ile Trp Asp Asn His Arg Gly Asp Tyr 385 390 395 400 gat ttg tgg tac agc ggc gaa gcc tca gcg gcg caa gaa ccg gct ttc 1248 Asp Leu Trp Tyr Ser Gly Glu Ala Ser Ala Ala Gln Glu Pro Ala Phe 405 410 415 gtt ccg cgt tct ccg act gca gcg gaa ggc gac ggt tat ctg ctg acc 1296 Val Pro Arg Ser Pro Thr Ala Ala Glu Gly Asp Gly Tyr Leu Leu Thr 420 425 430 gtt gtg ggc cgt ttg gac gag aac cgc agc gac ctg gtc att ctg gac 1344 Val Val Gly Arg Leu Asp Glu Asn Arg Ser Asp Leu Val Ile Leu Asp 435 440 445 acg cag gac atc cag agc ggt ccg gtt gcg acc att aaa ctg ccg ttt 1392 Thr Gln Asp Ile Gln Ser Gly Pro Val Ala Thr Ile Lys Leu Pro Phe 450 455 460 cgc ctg cgt gcg gcc ctg cac ggc tgt tgg gtt ccg cgt ccg taa 1437 Arg Leu Arg Ala Ala Leu His Gly Cys Trp Val Pro Arg Pro 465 470 475 <210> 2 <211> 478 <212> PRT <213> Isoeugenol monooxygenase of *Pseudomonas nitrification-reducing* Jin1 (codon-modified) <400> 2 Met Ala Arg Leu Asn Arg Asn Asp Pro Gln Leu Val Gly Thr Leu Leu 1 5 10 15 Pro Thr Arg Ile Glu Ala Asp Leu Phe Asp Leu Glu Val Asp Gly Glu 20 25 30 Ile Pro Lys Ser Ile Asn Gly Thr Phe Tyr Arg Asn Thr Pro Glu Pro 35 40 45 Gln Val Thr Pro Gln Lys Phe His Thr Phe Ile Asp Gly Asp Gly Met 50 55 60 Ala Ser Ala Phe His Phe Glu Asp Gly His Val Asp Phe Ile Ser Arg 65 70 75 80 Trp Val Lys Thr Ala Arg Phe Thr Ala Glu Arg Leu Ala Arg Lys Ser 85 90 95 Leu Phe Gly Met Tyr Arg Asn Pro Tyr Thr Asp Asp Thr Ser Val Lys 100 105 110 Gly Leu Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Ser His His Gly 115 120 125 Lys Val Leu Ala Val Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro 130 135 140 Arg Thr Leu Glu Thr Arg Gly Arg Phe Asp Tyr Asp Gly Gln Val Thr 145 150 155 160 Ser Gln Thr His Thr Ala His Pro Lys Tyr Asp Pro Glu Thr Gly Asp 165 170 175 Leu Leu Phe Phe Gly Ser Ala Ala Lys Gly Glu Ala Thr Pro Asp Met 180 185 190 Ala Tyr Tyr Ile Val Asp Lys His Gly Lys Val Thr His Glu Thr Trp 195 200 205 Phe Glu Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Ile Thr Arg 210 215 220 Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Ser Arg 225 230 235 240 Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Leu Gly Ser 245 250 255 Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Ser Gln Ile Arg Trp 260 265 270 Leu Lys Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu 275 280 285 Val Gly Thr Lys Ile Tyr Ile Asp Leu Met Glu Ser Glu Ile Leu Pro 290 295 300 Phe Pro Phe Pro Asn Ser Gln Asn Gln Pro Phe Ala Pro Glu Lys Ala 305 310 315 320 Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asp Ser Ser Ser Asp 325 330 335 Glu Ile Lys Arg Thr Arg Leu His Asp Phe Phe Ala Glu Met Pro Ile 340 345 350 Met Asp Phe Arg Phe Ala Leu Gln Cys Asn Arg Tyr Gly Phe Met Gly 355 360 365 Val Asp Asp Pro Arg Lys Pro Leu Ala His Gln Gln Ala Glu Lys Ile 370 375 380 Phe Ala Tyr Asn Ser Leu Gly Ile Trp Asp Asn His Arg Gly Asp Tyr 385 390 395 400 Asp Leu Trp Tyr Ser Gly Glu Ala Ser Ala Ala Gln Glu Pro Ala Phe 405 410 415 Val Pro Arg Ser Pro Thr Ala Ala Glu Gly Asp Gly Tyr Leu Leu Thr 420 425 430 Val Val Gly Arg Leu Asp Glu Asn Arg Ser Asp Leu Val Ile Leu Asp 435 440 445 Thr Gln Asp Ile Gln Ser Gly Pro Val Ala Thr Ile Lys Leu Pro Phe 450 455 460 Arg Leu Arg Ala Ala Leu His Gly Cys Trp Val Pro Arg Pro 465 470 475 <210> 3 <211> 1437 <212> DNA <213> Isoeugenol monooxygenase of Pseudomonas putida IE27 <220> <221> CDS <222> (1)..(1437) <400> 3 atg gca acg ttt gac cgc aat gat ccg cag ttg gca gga acg atg ttc 48 Met Ala Thr Phe Asp Arg Asn Asp Pro Gln Leu Ala Gly Thr Met Phe 1 5 10 15 ccc acc cga ata gag gcg aat gtc ttt gac ctt gaa att gag ggc gag 96 Pro Thr Arg Ile Glu Ala Asn Val Phe Asp Leu Glu Ile Glu Gly Glu 20 25 30 atc cca cgt gca atc ​​aac ggg agc ttc ttc cgc aac acc ccc gaa cct 144 Ile Pro Arg Ala Ile Asn Gly Ser Phe Phe Arg Asn Thr Pro Glu Pro 35 40 45 cag gtc acc acg caa cct ttc cac acc ttc atc gat ggg gat ggt ttg 192 Gln Val Thr Thr Gln Pro Phe His Thr Phe Ile Asp Gly Asp Gly Leu 50 55 60 gcg tct gct ttt cat ttc gaa gat ggc cag gtc gac ttt gtc agc cgt 240 Ala Ser Ala Phe His Phe Glu Asp Gly Gln Val Asp Phe Val Ser Arg 65 70 75 80 tgg gta tgt act cct cgc ttt gaa gct gag cgg tcg gct cgt aaa tca 288 Trp Val Cys Thr Pro Arg Phe Glu Ala Glu Arg Ser Ala Arg Lys Ser 85 90 95 ctc ttc ggt atg tac cgc aat ccg ttc act gat gat cca tcg gta gaa 336 Leu Phe Gly Met Tyr Arg Asn Pro Phe Thr Asp Asp Pro Ser Val Glu 100 105 110 ggt att gat cgt aca gtc gcc aac acc agt atc atc act cat cac ggg 384 Gly Ile Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Thr His His Gly 115 120 125 aaa gta ctg gcc gca aag gaa gat gga cta cct tat gag ctt gac ccc 432 Lys Val Leu Ala Ala Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro 130 135 140 caa acc ctg gaa acc cga ggt cgt tat gat tac aag ggg cag gta acc 480 Gln Thr Leu Glu Thr Arg Gly Arg Tyr Asp Tyr Lys Gly Gln Val Thr 145 150 155 160 agc cat aca cat aca gcg cac cct aag ttc gac ccc cag aca ggt gaa 528 Ser His Thr His Thr Ala His Pro Lys Phe Asp Pro Gln Thr Gly Glu 165 170 175 atg tta ctc ttc ggc tcc gct gct aaa ggc gaa cga acg ctt gat atg 576 Met Leu Leu Phe Gly Ser Ala Ala Lys Gly Glu Arg Thr Leu Asp Met 180 185 190 gcg tac tat att gtt gat cgc tac ggc aag gtg aca cat gag acc tgg 624 Ala Tyr Tyr Ile Val Asp Arg Tyr Gly Lys Val Thr His Glu Thr Trp 195 200 205 ttt aag cag cct tac ggt gca ttc atg cac gac ttt gct gtc acg cgc 672 Phe Lys Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Val Thr Arg 210 215 220 aac tgg tca atc ​​ttt ccg atc atg cct gcg aca aat agc ctt gag cgt 720 Asn Trp To Be There Pro Ile Met Pro Ala Thr Asn To Be Leu Glu Arg 225 230 235 240 ctt aaa gca aag cag ccc att tac atg tgg gag cct gag cga gga agc 768 Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Arg Gly Ser 245 250 255 tat ata gga gta ctt cct cgt cgt ggt cag ggc aag gac att cgt tgg 816 Tyr Ile Gly Val Leu Pro Arg Arg Gly Gly Gly Lys Asp Ile Arg Trp 260 265 270 ttc cgt gcc ccg gcg ttg tgg gtt ttc cat gtc gtg aat gct tgg gag 864 Phe Arg Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu 275 280 285 go ggg aat aga att ctg att gac ttg atg go agt gag att ttg ccg 912 Glu Gly Asn Arg Ile Leu Ile Asp Leu Met Glu Ser Glu Ile Leu Pro 290 295 300 ttc cca ttc ccg aac tcg cag aac ctt cca ttt gat ccc tcc aag gct 960 Phe Pro Phe Pro Asn Ser Gln Asn Leu Pro Phe Asp Pro Ser Lys Ala 305 310 315 320 gtt ccg cgt cta acc cgt tgg gaa att gat ctc aat agt ggt aac gat 1008 Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asn Ser Gly Asn Asp 325 330 335 gag atg aaa cgt acg cag cta cac gaa tat ttt gca gaa atg cct atc 1056 Glu Met Lys Arg Thr Gln Leu His Glu Tyr Phe Ala Glu Met Pro Ile 340 345 350 atg gat ttc cgt ttt gcg ctc cag gat cat cgc tac gcc tac atg ggg 1104 Met Asp Phe Arg Phe Ala Leu Gln Asp His Arg Tyr Ala Tyr Met Gly 355 360 365 gtt gac gat cct cgt cgc ccc tta gct cat cag caa gct gaaaaa atc ​​1152 Val Asp Asp Pro Arg Arg Pro Leu Ala His Gln Gln Ala Glu Lys Ile 370 375 380 ttt gcc tac aat tcg tta ggg gtt tgg gac aac cat cgt aaa gat tat 1200 Phe Ala Tyr Asn Serves Leu Gly Val Trp Asp Asn His Arg Lys Asp Tyr 385 390 395 400 gaa ctt tgg ttt acg gga aaa atg tct gca gcg cag gaa ccg gcg ttt 1248 Glu Leu Trp Phe Thr Gly Lys Met Ser Ala Ala Gln Glu Pro Ala Phe 405 410 415 gtt cct aga agc cca gat gcg cct gag ggc gat ggc tac cta ctc agt 1296 Val Pro Arg Ser Pro Asp Ala Pro Glu Gly Asp Gly Tyr Leu Leu Ser 420 425 430 gta gta ggg cgg ctc gat gaa gat cgt agc gat cta gtt atc ctt gat 1344 Val Val Gly Arg Leu Asp Glu Asp Arg Ser Asp Leu Val Ile Leu Asp 435 440 445 acg caa tgt ttg gca gct ggg cct gtg gcc act gtc aag ctt ccc ttc 1392 Thr Gln Cys Leu Ala Ala Gly Pro Val Ala Thr Val Lys Leu Pro Phe 450 455 460 cgt ctc cga gca gcg ttg cac ggt tgt tgg cag tct aag aac tga 1437 Arg Leu Arg Ala Ala Leu His Gly Cys Trp Gln Ser Lys Asn 465 470 475 <210> 4 <211> 478 <212> PRT <213> Isoeugenol monooxygenase of Pseudomonas putida IE27 <400> 4 Met Ala Thr Phe Asp Arg Asn Asp Pro Gln Leu Ala Gly Thr Met Phe 1 5 10 15 Pro Thr Arg Ile Glu Ala Asn Val Phe Asp Leu Glu Ile Glu Gly Glu 20 25 30 Ile Pro Arg Ala Ile Asn Gly Ser Phe Phe Arg Asn Thr Pro Glu Pro 35 40 45 Gln Val Thr Thr Gln Pro Phe His Thr Phe Ile Asp Gly Asp Gly Leu 50 55 60 Ala Ser Ala Phe His Phe Glu Asp Gly Gln Val Asp Phe Val Ser Arg 65 70 75 80 Trp Val Cys Thr Pro Arg Phe Glu Ala Glu Arg Ser Ala Arg Lys Ser 85 90 95 Leu Phe Gly Met Tyr Arg Asn Pro Phe Thr Asp Asp Pro Ser Val Glu 100 105 110 Gly Ile Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Thr His His Gly 115 120 125 Lys Val Leu Ala Ala Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro 130 135 140 Gln Thr Leu Glu Thr Arg Gly Arg Tyr Asp Tyr Lys Gly Gln Val Thr 145 150 155 160 Ser His Thr His Thr Ala His Pro Lys Phe Asp Pro Gln Thr Gly Glu 165 170 175 Met Leu Leu Phe Gly Ser Ala Ala Lys Gly Glu Arg Thr Leu Asp Met 180 185 190 Ala Tyr Tyr Ile Val Asp Arg Tyr Gly Lys Val Thr His Glu Thr Trp 195 200 205 Phe Lys Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Val Thr Arg 210 215 220 Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Glu Arg 225 230 235 240 Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Arg Gly Ser 245 250 255 Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Lys Asp Ile Arg Trp 260 265 270 Phe Arg Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu 275 280 285 Glu Gly Asn Arg Ile Leu Ile Asp Leu Met Glu Ser Glu Ile Leu Pro 290 295 300 Phe Pro Phe Pro Asn Ser Gln Asn Leu Pro Phe Asp Pro Ser Lys Ala 305 310 315 320 Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asn Ser Gly Asn Asp 325 330 335 Glu Met Lys Arg Thr Gln Leu His Glu Tyr Phe Ala Glu Met Pro Ile 340 345 350 Met Asp Phe Arg Phe Ala Leu Gln Asp His Arg Tyr Ala Tyr Met Gly 355 360 365 Val Asp Asp Pro Arg Arg Pro Leu Ala His Gln Gln Ala Glu Lys Ile 370 375 380 Phe Ala Tyr Asn Ser Leu Gly Val Trp Asp Asn His Arg Lys Asp Tyr 385 390 395 400 Glu Leu Trp Phe Thr Gly Lys Met Ser Ala Ala Gln Glu Pro Ala Phe 405 410 415 Val Pro Arg Ser Pro Asp Ala Pro Glu Gly Asp Gly Tyr Leu Leu Ser 420 425 430 Val Val Gly Arg Leu Asp Glu Asp Arg Ser Asp Leu Val Ile Leu Asp 435 440 445 Thr Gln Cys Leu Ala Ala Gly Pro Val Ala Thr Val Lys Leu Pro Phe 450 455 460 Arg Leu Arg Ala Ala Leu His Gly Cys Trp Gln Ser Lys Asn 465 470 475 <210> 5 <211> 1437 <212> DNA <213> Isoeugenol monooxygenase (codon-modified) of *Pseudomonas putida* IE27 <220> <221> CDS <222> (1)..(1437) <400> 5 atg gcc act ttt gac cgc aat gac ccg caa ctg gca ggc acc atg ttc 48 Met Ala Thr Phe Asp Arg Asn Asp Pro Gln Leu Ala Gly Thr Met Phe 1 5 10 15 ccg acg cgc atc gaa gcg aat gtt ttt gat ctg gag att gaa ggt gag 96 Pro Thr Arg Ile Glu Ala Asn Val Phe Asp Leu Glu Ile Glu Gly Glu 20 25 30 att ccg cgt gcg atc aac ggt agc ttt ttc cgc aac acg cca gag ccg 144 Ile Pro Arg Ala Ile Asn Gly Ser Phe Phe Arg Asn Thr Pro Glu Pro 35 40 45 caa gtc acc acg cag ccg ttt cat act ttc atc gac ggc gac ggc ctg 192 Gln Val Thr Thr Gln Pro Phe His Thr Phe Ile Asp Gly Asp Gly Leu 50 55 60 gcg tca gcg ttc cac ttc gaa gat ggc cag gtc gac ttt gtg agc cgc 240 Ala Ser Ala Phe His Phe Glu Asp Gly Gln Val Asp Phe Val Ser Arg 65 70 75 80 tgg gtc tgc acc ccg cgt ttc gag gca gag cgc agc gcg cgt aaa agc 288 Trp Val Cys Thr Pro Arg Phe Glu Ala Glu Arg Ser Ala Arg Lys Ser 85 90 95 ctg ttt ggt atg tat cgc aat ccg ttt acg gat gac ccg agc gtt gaa 336 Leu Phe Gly Met Tyr Arg Asn Pro Phe Thr Asp Asp Pro Ser Val Glu 100 105 110 ggc att gac cgt acc gtg gcg aat acc tcg atc att acc cac cac ggt 384 Gly Ile Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Thr His His Gly 115 120 125 aag gtc ctg gca gca aaa gaa gat ggc ttg ccg tac gag tta gat ccg 432 Lys Val Leu Ala Ala Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro 130 135 140 cag acc ctg gaa acg cgt ggt cgc tat gac tac aag ggc cag gtt acc 480 Gln Thr Leu Glu Thr Arg Gly Arg Tyr Asp Tyr Lys Gly Gln Val Thr 145 150 155 160 agc cat acc cac acg gct cac cct aag ttt gat ccg caa acg ggt gag 528 Ser His Thr His Thr Ala His Pro Lys Phe Asp Pro Gln Thr Gly Glu 165 170 175 atg ctg ctg ttc ggc agc gcg gca aag ggt gag cgt acc ctg gac atg 576 Met Leu Leu Phe Gly Ser Ala Ala Lys Gly Glu Arg Thr Leu Asp Met 180 185 190 gcg tac tat atc gtt gac cgt tac ggt aaa gtg acc cat gaa acc tgg 624 Ala Tyr Tyr Ile Val Asp Arg Tyr Gly Lys Val Thr His Glu Thr Trp 195 200 205 ttc aag caa ccg tac ggc gcc ttt atg cac gac ttc gca gtc acg cgc 672 Phe Lys Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Val Thr Arg 210 215 220 aac tgg tct atc ttt ccg att atg ccg gcc acc aat agc ctg gag cgt 720 Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Glu Arg 225 230 235 240 ctg aaa gct aag caa ccg att tac atg tgg gaa ccg gag cgt ggt tcc 768 Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Arg Gly Ser 245 250 255 tac atc ggc gtg ctg ccg cgt cgt ggt cag ggt aaa gat atc cgc tgg 816 Tyr Ile Gly Val Leu Pro Arg Arg Gly Gly Gly Lys Asp Ile Arg Trp 260 265 270 ttc cgt gcg cct gcc ctc tgg gtg ttc cac gtt gtg aac gca tgg gaa 864 Phe Arg Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu 275 280 285 gag ggc aat cgt att ctg atc gat ctg atg gag agc gaa atc ​​ctg cca 912 Glu Gly Asn Arg Ile Leu Ile Asp Leu Met Glu Ser Glu Ile Leu Pro 290 295 300 ttc ccg ttt ccg aac tct cag aat ctg ccg ttc gat ccg agc aaa gcc 960 Phe Pro Phe Pro Asn Ser Gln Asn Leu Pro Phe Asp Pro Ser Lys Ala 305 310 315 320 gta ccg cgc ttg acc cgt tgg gag att gat ttg aac agc ggt aat gac 1008 Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asn Ser Gly Asn Asp 325 330 335 gag atg aag cgt act cag ctg cac gaa tac ttc gct gag atg ccg att 1056 Glu Met Lys Arg Thr Gln Leu His Glu Tyr Phe Ala Glu Met Pro Ile 340 345 350 atg gac ttt cgt ttc gcg ctg caa gat cac cgt tac gcg tat atg ggt 1104 Met Asp Phe Arg Phe Ala Leu Gln Asp His Arg Tyr Ala Tyr Met Gly 355 360 365 gtt gat gat cca cgc cgt cca ttg gcg cat caa gcg gaa aag att 1152 Val Asp Asp Pro Arg Arg Pro Leu Ala His Gln Gln Ala Glu Lys Ile 370 375 380 ttt gcg tat aac agc ctg ggt gtt tgg gac aac cat cgt aaa gac tat 1200 Phe Ala Tyr Asn Serves Leu Gly Val Trp Asp Asn His Arg Lys Asp Tyr 385 390 395 400 gag ctg tgg ttt acg ggt aaa atg tcc gcg gct cag gaa ccg gcc ttc 1248 Glu Leu Trp Phe Thr Gly Lys Met Ser Ala Ala Gln Glu Pro Ala Phe 405 410 415 gtg ccg cgc agc ccg gac gcc cct gag ggt gat ggt tat ttg ctg tcc 1296 Val Pro Arg Ser Pro Asp Ala Pro Glu Gly Asp Gly Tyr Leu Leu Ser 420 425 430 gtc gtg ggt cgc ctg gat gaa gat cgt agc gac ctg gtt atc ctg gac 1344 Val Val Gly Arg Leu Asp Glu Asp Arg Ser Asp Leu Val Ile Leu Asp 435 440 445 acc cag tgc ctt gcg gca ggc ccg gtt gcg acc gtc aag ctg ccg ttc 1392 Thr Gln Cys Leu Ala Ala Gly Pro Val Ala Thr Val Lys Leu Pro Phe 450 455 460 cgt ctg cgt gca gct ctg cat ggt tgt tgg cag agc aaa aac taa 1437 Arg Leu Arg Ala Ala Leu His Gly Cys Trp Gln Ser Lys Asn 465 470 475 <210> 6 <211> 478 <212> PRT <213> Isoeugenol monooxygenase (codon-modified) of *Pseudomonas putida* IE27 <400> 6 Met Ala Thr Phe Asp Arg Asn Asp Pro Gln Leu Ala Gly Thr Met Phe 1 5 10 15 Pro Thr Arg Ile Glu Ala Asn Val Phe Asp Leu Glu Ile Glu Gly Glu 20 25 30 Ile Pro Arg Ala Ile Asn Gly Ser Phe Phe Arg Asn Thr Pro Glu Pro 35 40 45 Gln Val Thr Thr Gln Pro Phe His Thr Phe Ile Asp Gly Asp Gly Leu 50 55 60 Ala Ser Ala Phe His Phe Glu Asp Gly Gln Val Asp Phe Val Ser Arg 65 70 75 80 Trp Val Cys Thr Pro Arg Phe Glu Ala Glu Arg Ser Ala Arg Lys Ser 85 90 95 Leu Phe Gly Met Tyr Arg Asn Pro Phe Thr Asp Asp Pro Ser Val Glu 100 105 110 Gly Ile Asp Arg Thr Val Ala Asn Thr Ser Ile Ile Thr His His Gly 115 120 125 Lys Val Leu Ala Ala Lys Glu Asp Gly Leu Pro Tyr Glu Leu Asp Pro 130 135 140 Gln Thr Leu Glu Thr Arg Gly Arg Tyr Asp Tyr Lys Gly Gln Val Thr 145 150 155 160 Ser His Thr His Thr Ala His Pro Lys Phe Asp Pro Gln Thr Gly Glu 165 170 175 Met Leu Leu Phe Gly Ser Ala Ala Lys Gly Glu Arg Thr Leu Asp Met 180 185 190 Ala Tyr Tyr Ile Val Asp Arg Tyr Gly Lys Val Thr His Glu Thr Trp 195 200 205 Phe Lys Gln Pro Tyr Gly Ala Phe Met His Asp Phe Ala Val Thr Arg 210 215 220 Asn Trp Ser Ile Phe Pro Ile Met Pro Ala Thr Asn Ser Leu Glu Arg 225 230 235 240 Leu Lys Ala Lys Gln Pro Ile Tyr Met Trp Glu Pro Glu Arg Gly Ser 245 250 255 Tyr Ile Gly Val Leu Pro Arg Arg Gly Gln Gly Lys Asp Ile Arg Trp 260 265 270 Phe Arg Ala Pro Ala Leu Trp Val Phe His Val Val Asn Ala Trp Glu 275 280 285 Glu Gly Asn Arg Ile Leu Ile Asp Leu Met Glu Ser Glu Ile Leu Pro 290 295 300 Phe Pro Phe Pro Asn Ser Gln Asn Leu Pro Phe Asp Pro Ser Lys Ala 305 310 315 320 Val Pro Arg Leu Thr Arg Trp Glu Ile Asp Leu Asn Ser Gly Asn Asp 325 330 335 Glu Met Lys Arg Thr Gln Leu His Glu Tyr Phe Ala Glu Met Pro Ile 340 345 350 Met Asp Phe Arg Phe Ala Leu Gln Asp His Arg Tyr Ala Tyr Met Gly 355 360 365 Val Asp Asp Pro Arg Arg Pro Leu Ala His Gln Gln Ala Glu Lys Ile 370 375 380 Phe Ala Tyr Asn Ser Leu Gly Val Trp Asp Asn His Arg Lys Asp Tyr 385,390,395,400 Glu Leu Trp Phe Thr Gly Lys Met Ser Ala Ala Gln Glu Pro Ala Phe 405 410 415 Val Pro Arg Ser Pro Asp Ala Pro Glu Gly Asp Gly Tyr Leu Leu Ser 420 425 430 Val Val Gly Arg Leu Asp Glu Asp Arg Ser Asp Leu Val Ile Leu Asp 435 440 445 Thr Gln Cys Leu Ala Ala Gly Pro Val Ala Thr Val Lys Leu Pro Phe 450 455 460 Arg Leu Arg Ala Ala Leu His Gly Cys Trp Gln Ser Lys Asn 465 470 475 <210> 7 <211> 1647 <212> DNA <213> GroEL is the largest company in the world <220> <221> CDS <222> (1)..(1647) <400> 7 atg gca gct aaa gac gta aaa ttc ggt aac gac gct cgt gtg aaa atg 48 Met Ala Ala Lys Asp Val Lys Phe Gly Asn Asp Ala Arg Val Lys Met 1 5 10 15 ctg cgc ggc gta aac gta ctg gca gat gca gtg aaa gtt acc ctc ggt 96 Leu Arg Gly Val Asn Val Leu Ala Asp Ala Val Lys Val Thr Leu Gly 20 25 30 cca aaa ggc cgt aac gta gtt ctg gat aaa tct ttc ggt gca ccg acc 144 Pro Lys Gly Arg Asn Val Val Leu Asp Lys Ser Phe Gly Ala Pro Thr 35 40 45 atc acc aaa gat ggt gtt tcc gtt gct cgt gaa atc gaa ctg gaa gac 192 Ile Thr Lys Asp Gly Val Ser Val Ala Arg Glu Ile Glu Leu Glu Asp 50 55 60 aag ttc gaa aat atg ggt gcg cag atg gtg aaa gaa gtt gcc tct aaa 240 Lys Phe Glu Asn Met Gly Ala Gln Met Val Lys Glu Val Ala Ser Lys 65 70 75 80 gca aac gac gct gca ggc gac ggt acc acc act gca acc gta ctg gct 288 Ala Asn Asp Ala Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala 85 90 95 cag gct atc atc act gaa ggt ctg aaa gct gtt gct gcg ggc atg aac 336 Gln Ala Ile Ile Thr Glu Gly Leu Lys Ala Val Ala Gly Met Asn 100 105 110 ccg atg gac ctg aaa cgt ggt atc gac aaa gcg gtt acc gct gca gtt 384 Pro Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Val Thr Ala Ala Val 115 120 125 gaa gaa ctg aaa gcg ctg tcc gta cca tgc tct gac tct aaa gcg att 432 Glu Glu Leu Lys Wing Leu Ser Val Pro Cys Ser Asp Ser Lys Wing Ile 130 135 140 gct cag gtt gtt acc atc tcc gct aac tcc gac gaa acc gta gt aaa 480 Only Gln Val Gly Thr Ile Serves Only Asn Serves Asp Glu Thr Val Gly Lys 145 150 155 160 ctg atc gct gaa gcg atg gac aaa gtc ggt aaa gaa ggc gtt atc acc 528 Leu Ile Ala Glu Ala Met Asp Lys Val Gly Lys Glu Gly Val Ile Thr 165 170 175 gtt gaa gac ggt acc ggt ctg cag gac gaa ctg gac gtg gtt gaa ggt 576 Val Glu Asp Gly Thr Gly Leu Gln Asp Glu Leu Asp Val Val Glu Gly 180 185 190 atg cag ttc gac cgt ggc tac ctg tct cct tac ttc atc aac aag ccg 624 Met Gln Phe Asp Arg Gly Tyr Leu Ser Pro Tyr Phe Ile Asn Lys Pro 195 200 205 gaa act ggc gca gta gaa ctg gaa agc ccg ttc atc ctg ctg gct gac 672 Glu Thr Gly Ala Val Glu Leu Glu Ser Pro Phe Ile Leu Leu Ala Asp 210 215 220 aag aaa atc ​​tcc aac atc cgc gaa atg ctg ccg gtt ctg gaa gct gtt 720 Lys Lys Ile Ser Asn Ile Arg Glu Met Leu Pro Val Leu Glu Wing Val 225 230 235 240 gcc aaa gca ggc aaa ccg ctg ctg atc atc gct gagat gat gta gaa ggc 768 Ala Lys Ala Gly Lys Pro Leu Leu Ile Ala Glu Asp Val Glu Gly 245 250 255 gaa gcg ctg gca act ctg gtt gtt aac acc atg cgt ggc atc gtg aaa 816 Glu Ala Leu Ala Thr Leu Val Val Asn Thr Met Arg Gly Ile Val Lys 260 265 270 gtc gct gcg gtt aaa gca ccg ggc ttc gggc gat cgt cgt aaa gct atg 864 Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 275 280 285 ctg cag gat atc gca acc ctg act ggc ggt acc gtg atc tct gaa gag 912 Leu Gln Asp Ile Ala Thr Leu Thr Gly Gly Thr Val Ile Ser Glu Glu 290 295 300 atc ggt atg gag ctg gaa aaa gca acc ctg gaa gac ctg ggt cag gct 960 Ile Gly Met Glu Leu Glu Lys Ala Thr Leu Glu Asp Leu Gly Gln Ala 305 310 315 320 aaa cgt gtt gtg atc aac aaa gac acc acc act atc atc gat ggc gtg 1008 Lys Arg Val Val Ile Asn Lys Asp Thr Thr Thr Ile Ile Asp Gly Val 325 330 335 ggt gaa gaa gct gca atc cag ggc cgt gtt gct cag atc cgt cag cag 1056 Gly Glu Glu Ala Ala Ile Gln Gly Arg Val Ala Gln Ile Arg Gln Gln 340 345 350 att gaa gaa gca act tct gac tac gac cgt gaa aaa ctg cag gaa cgc 1104 Ile Glu Glu Ala Thr Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg 355 360 365 gta gcg aaa ctg gca ggc ggc gtt gca gtt atc aaa gtg ggt gct gct 1152 Val Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Val Gly Ala Ala 370 375 380 acc gaa gtt gaa atg aaa gag aaa aaa gca cgc gtt gaa gat gcc ctg 1200 Thr Glu Val Glu Met Lys Glu Lys Lys Ala Arg Val Glu Asp Ala Leu 385 390 395 400 cac gcg acc cgt gct gcg gta gaa gaa ggc gtg gtt gct ggt ggt ggt 1248 His Ala Thr Arg Ala Ala Val Glu Glu Gly Val Val Ala Gly Gly Gly 405 410 415 gtt gcg ctg atc cgc gta gcg tct aaa ctg gct gac ctg cgt ggt cag 1296 Val Ala Leu Ile Arg Val Ala Ser Lys Leu Ala Asp Leu Arg Gly Gln 420 425 430 aac gaa gac cag aac gtg ggt atc aaa gtt gca ctg cgt gca atg gaa 1344 Asn Glu Asp Gln Asn Val Gly Ile Lys Val Ala Leu Arg Ala Met Glu 435 440 445 gct ccg ctg cgt cag atc gta ttg aac tgc ggc gaa gaa ccg tct gtt 1392 Ala Pro Leu Arg Gln Ile Val Leu Asn Cys Gly Glu Glu Pro Ser Val 450 455 460 gtt gct aac acc gtt aaa ggc ggc gac ggc aac tac ggt tac aac gca 1440 Val Ala Asn Thr Val Lys Gly Gly Asp Gly Asn Tyr Gly Tyr Asn Ala 465 470 475 480 gca acc gaa gaa tac ggc aac atg atc gac atg ggt atc ctg gat cca 1488 Ala Thr Glu Glu Tyr Gly Asn Met Ile Asp Met Gly Ile Leu Asp Pro 485 490 495 acc aaa gta act cgt tct gct ctg cag tac gca gct tct gtg gct ggc 1536 Thr Lys Val Thr Arg Ser Ala Leu Gln Tyr Ala Ala Ser Val Ala Gly 500 505 510 ctg atg atc acc acc gaa tgc atg gtt acc gac ctg ccg aaa aac gat 1584 Leu Met Ile Thr Thr Glu Cys Met Val Thr Asp Leu Pro Lys Asn Asp 515 520 525 gca gct gac tta ggc gct gct ggc ggt atg ggc ggc atg ggt ggc atg 1632 Ala Ala Asp Leu Gly Ala Ala Gly Gly Met Gly Gly Met Gly Gly Met 530 535 540 ggc ggc atg atg taa 1647 Gly Gly Met Met 545 <210> 8 <211> 548 <212> PRT <213> GroEL <400> 8 Met Ala Ala Lys Asp Val Lys Phe Gly Asn Asp Ala Arg Val Lys Met 1 5 10 15 Leu Arg Gly Val Asn Val Leu Ala Asp Ala Val Lys Val Thr Leu Gly 20 25 30 Pro Lys Gly Arg Asn Val Val Leu Asp Lys Ser Phe Gly Ala Pro Thr 35 40 45 Ile Thr Lys Asp Gly Val Ser Val Ala Arg Glu Ile Glu Leu Glu Asp 50 55 60 Lys Phe Glu Asn Met Gly Ala Gln Met Val Lys Glu Val Ala Ser Lys 65 70 75 80 Ala Asn Asp Ala Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala 85 90 95 Gln Ala Ile Ile Thr Glu Gly Leu Lys Ala Val Ala Ala Gly Met Asn 100 105 110 Pro Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Val Thr Ala Ala Val 115 120 125 Glu Glu Leu Lys Ala Leu Ser Val Pro Cys Ser Asp Ser Lys Ala Ile 130 135 140 Ala Gln Val Gly Thr Ile Ser Ala Asn Ser Asp Glu Thr Val Gly Lys 145 150 155 160 Leu Ile Ala Glu Ala Met Asp Lys Val Gly Lys Glu Gly Val Ile Thr 165 170 175 Val Glu Asp Gly Thr Gly Leu Gln Asp Glu Leu Asp Val Val Glu Gly 180 185 190 Met Gln Phe Asp Arg Gly Tyr Leu Ser Pro Tyr Phe Ile Asn Lys Pro 195 200 205 Glu Thr Gly Ala Val Glu Leu Glu Ser Pro Phe Ile Leu Leu Ala Asp 210 215 220 Lys Lys Ile Ser Asn Ile Arg Glu Met Leu Pro Val Leu Glu Ala Val 225 230 235 240 Ala Lys Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly 245 250 255 Glu Ala Leu Ala Thr Leu Val Val Asn Thr Met Arg Gly Ile Val Lys 260 265 270 Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met 275 280 285 Leu Gln Asp Ile Ala Thr Leu Thr Gly Gly Thr Val Ile Ser Glu Glu 290 295 300 Ile Gly Met Glu Leu Glu Lys Ala Thr Leu Glu Asp Leu Gly Gln Ala 305 310 315 320 Lys Arg Val Val Ile Asn Lys Asp Thr Thr Thr Ile Ile Asp Gly Val 325 330 335 Gly Glu Glu Ala Ala Ile Gln Gly Arg Val Ala Gln Ile Arg Gln Gln 340 345 350 Ile Glu Glu Ala Thr Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg 355 360 365 Val Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Val Gly Ala Ala 370 375 380 Thr Glu Val Glu Met Lys Glu Lys Lys Ala Arg Val Glu Asp Ala Leu 385 390 395 400 His Ala Thr Arg Ala Ala Val Glu Glu Gly Val Val Ala Gly Gly Gly 405 410 415 Val Ala Leu Ile Arg Val Ala Ser Lys Leu Ala Asp Leu Arg Gly Gln 420 425 430 Asn Glu Asp Gln Asn Val Gly Ile Lys Val Ala Leu Arg Ala Met Glu 435 440 445 Ala Pro Leu Arg Gln Ile Val Leu Asn Cys Gly Glu Glu Pro Ser Val 450 455 460 Val Ala Asn Thr Val Lys Gly Gly Asp Gly Asn Tyr Gly Tyr Asn Ala 465 470 475 480 Ala Thr Glu Glu Tyr Gly Asn Met Ile Asp Met Gly Ile Leu Asp Pro 485 490 495 Thr Lys Val Thr Arg Ser Ala Leu Gln Tyr Ala Ala Ser Val Ala Gly 500 505 510 Leu Met Ile Thr Thr Glu Cys Met Val Thr Asp Leu Pro Lys Asn Asp 515 520 525 Ala Ala Asp Leu Gly Ala Ala Gly Gly Met Gly Gly Met Gly Gly Met 530 535 540 Gly Gly Met Met 545 <210> 9 <211> 294 <212> DNA <213> Molecular chaperonin GroES of Escherichia coli <220> <221> CDS <222> (1)..(294) <400> 9 atg aat att cgt cca ttg cat gat cgc gtg atc gtc aag cgt aaa gaa 48 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 gtt gaa act aaa tct gct ggc ggc atc gtt ctg acc tct gca gcg 96 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 gct aaa tcc acc cgc ggc gaa gtg ctg gct gtc ggc aat ggc cgt atc 144 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 ctt gaa aat ggc gaa gtg aag ccg ctg gat gtg aaa gtt ggc gac atc 192 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 gtt att ttc aac gat ggc tac ggt gtg aaa tct gag aag atc gac aat 240 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 gaa gaa gtg ttg atc atg tcc gaa agc gac att ctg gca att gtt gaa 288 Glu Glu Lion's Valve Met Ser Glu Ser Asp Lion's Wing Ala Glu Valve 85 90 95 gccg 294 Only <210> 10 <211> 97 <212> PRT <213> GroES, a molecular chaperone of Escherichia coli <400> 10 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala <210> 11 <211> 3451 <212> DNA <213> PC1 (a polycistronic construct of isoeugenol monooxygenase containing codon-modified nitrifying Pseudomonas Jin1, groES, groEL) <400> 11 gaaggagata tacatatggc acgtctgaac cgtaatgatc cgcaactcgt cggcaccctg 60 ttaccaaccc gtattgaggc tgacctgttc gacctggaag tggacggcga aattccgaag 120 tccatcaacg gtaccttcta ccgtaacacg ccggagcctc aggtgacgcc gcagaaattc 180 cacaccttca tcgatggtga cggcatggcg tctgcatttc attttgaaga tggccacgtg 240 gacttcatca gccgctgggt taaaaccgcg cgtttcacgg cggagagact ggcacgtaaa 300 agcctgttcg gtatgtaccg taatccgtac accgatgaca cgtctgtgaa gggtctggat 360 cgtaccgttg ccaacacgag catcatcagc catcacggta aggttctggc ggtgaaagaa 420 gatggcttgc cgtacgagct ggacccacgc actctggaaa cccgtggtcg ctttgactat 480 gatggccaag tcaccagcca gacgcacacc gcgcatccga agtatgaccc ggaaaccggt 540 gacctgttgt tctttggctc cgcagccaag ggtgaggcaa cgcctgatat ggcctattac 600 atcgttgata aacatggtaa ggtaacgcat gagacttggt tcgagcaacc gtatggcgcc 660 tttatgcatg attttgctat tacccgcaat tggagcatct tcccgatcat gccggctacc 720 aattcgttga gccgcctgaa agcgaagcag ccgatttaca tgtgggagcc ggaactgggt 780 tcctatattg gtgttctgcc gcgtcgcggt caaggtagcc agatccgctg gctgaaagca 840 ccagcgctgt gggtctttca cgtcgtcaac gcctgggaag tgggcaccaa aatctacatt 900 gaccttatgg agagcgaaat tctgccattc ccgttcccga atagccaaaa tcagccgttc 960 gctcctgaga aagcagtgcc gcgtctgacc cgttgggaga ttgatctgga tagcagcagc 1020 gatgagatta agcgtacgcg tctgcacgac ttctttgcag agatgccgat catggatttt 1080 cgttttgcgc tgcagtgcaa ccgctacggt tttatgggtg tcgatgaccc gcgcaagccg 1140 ctggcgcacc aacaagcgga gaagattttt gcgtacaata gcctgggtat ctgggacaac 1200 caccgtggtg attatgattt gtggtacagc ggcgaagcct cagcggcgca agaaccggct 1260 ttcgttccgc gttctccgac tgcagcggaa ggcgacggtt atctgctgac cgttgtgggc 1320 cgtttggacg agaaccgcag cgacctggtc attctggaca cgcaggacat ccagagcggt 1380 ccggttgcga cattaaact gccgtttcgc ctgcgtgcgg ccctgcacgg ctgttgggtt 1440 ccgcgtccgt aagaaggaga tatacatatg aatattcgtc cattgcatga tcgcgtgatc 1500 gtcaagcgta aagaagttga aactaaatct gctggcggca tcgttctgac cggctctgca 1560 gcggctaaat ccacccgcgg cgaagtgctg gctgtcggca atggccgtat ccttgaaaat 1620 ggcgaagtga agccgctgga tgtgaaagtt ggcgacatcg ttattttcaa cgatggctac 1680 1740 ctggcaattg ttgaagcgta atccgcgcac gacactgaac atacgaattt aaagtaaa 1800 gataatggca gctaaagacg taaaattcgg taacgacgct cgtgtgaaaa tgctgcgcgg 1860 cgtaaacgta ctggcagatg cagtgaaagt taccctcggt ccaaaaggcc gtaacgtagt 1920 tctggataaa tctttcggtg caccgaccat caccaaagat ggtgtttccg ttgctcgtga 1980 aatcgaactg gaagacaagt tcgaaaatat gggtgcgcag atggtgaaag aagttgcctc 2040 taagcaac gacgctgcag gcgacggtac caccactgca accgtactgg ctcaggctat 2100 catcactgaa ggtctgaaag ctgttgctgc gggcatgaac ccgatggacc tgaaacgtgg 2160 tatcgaaa gcggttaccg ctgcagttga agaatgaaa gcgctgtccg taccatgctc 2220 tgactctaa gcgattgctc aggttggtac catctccgct aactccgacg aaaccgtagg 2280 taaactgatc gctgaagcga tggacaaagt cggtaaagaa ggcgttatca ccgttgaaga 2340 cggtaccggt ctgcaggacg aactggacgt ggttgaaggt atgcagttcg accgtggcta 2400 cctgtctcct tacttcatca acaagccgga aactggcgca gtagaactgg aaagcccgtt 2460 catcctgctg gctgacaaga aaatctccaa catccgcgaa atgctgccgg ttctggaagc 2520 tgttgccaaa gcaggcaaac cgctgctgat catcgctgaa gatgtagaag gcgaagcgct 2580 ggcaactctg gttgttaaca ccatgcgtgg catcgtgaaa gtcgctgcgg ttaaagcacc 2640 gggcttcggc gatcgtcgta aagctatgct gcaggatatc gcaaccctga ctggcggtac 2700 cgtgatctct gaagagatcg gtatggagct ggaaaaagca accctggaag acctgggtca 2760 ggctaaacgt gttgtgatca acaaagacac caccactatc atcgatggcg tgggtgaaga 2820 agctgcaatc cagggccgtg ttgctcagat ccgtcagcag attgaagaag caacttctga 2880 ctacgaccgt gaaaaactgc aggaacgcgt agcgaaactg gcaggcggcg ttgcagttat 2940 caaagtgggt gctgctaccg aagttgaaat gaaaagaaaa aaagcacgcg ttgaagatgc 3000 cctgcacgcg acccgtgctg cggtagaaga aggcgtggtt gctggtggtg gtgttgcgct 3060 gatccgcgta gcgtctaaac tggctgacct gcgtggtcag aacgaagacc agaacgtggg 3120 tatcaaagtt gcactgcgtg caatggaagc tccgctgcgt cagatcgtat tgaactgcgg 3180 cgaagaaccg tctgttgttg ctaacaccgt taaaggcggc gacggcaact acggttacaa 3240 cgcagcaacc gaagaatacg gcaacatgat cgacatgggt atcctggatc caaccaaagt 3300 aactcgttct gctctgcagt acgcagcttc tgtggctggc ctgatgatca ccaccgaatg 3360 catggttacc gacctgccga aaaacgatgc agctgactta ggcgctgctg gcggtatggg 3420 cggcatgggt ggcatgggcg gcatgatgta a 3451 <210> 12 <211> 3475 <212> DNA <213> PC2 (a polycistronic construct of isoeugenol monooxygenase containing codon-modified nitrifying Pseudomonas Jin1, groES, groEL) <400> 12 gaaggagata tacatatggc acgtctgaac cgtaatgatc cgcaactcgt cggcaccctg 60 ttaccaaccc gtattgaggc tgacctgttc gacctggaag tggacggcga aattccgaag 120 tccatcaacg gtaccttcta ccgtaacacg ccggagcctc aggtgacgcc gcagaaattc 180 cacaccttca tcgatggtga cggcatggcg tctgcatttc attttgaaga tggccacgtg 240 gacttcatca gccgctgggt taaaaccgcg cgtttcacgg cggagagact ggcacgtaaa 300 agcctgttcg gtatgtaccg taatccgtac accgatgaca cgtctgtgaa gggtctggat 360 cgtaccgttg ccaacacgag catcatcagc catcacggta aggttctggc ggtgaaagaa 420 gatggcttgc cgtacgagct ggacccacgc actctggaaa cccgtggtcg ctttgactat 480 gatggccaag tcaccagcca gacgcacacc gcgcatccga agtatgaccc ggaaaccggt 540 gacctgttgt tctttggctc cgcagccaag ggtgaggcaa cgcctgatat ggcctattac 600 atcgttgata aacatggtaa ggtaacgcat gagacttggt tcgagcaacc gtatggcgcc 660 tttatgcatg attttgctat tacccgcaat tggagcatct tcccgatcat gccggctacc 720 aattcgttga gccgcctgaa agcgaagcag ccgatttaca tgtgggagcc ggaactgggt 780 tcctatattg gtgttctgcc gcgtcgcggt caaggtagcc agatccgctg gctgaaagca 840 ccagcgctgt gggtctttca cgtcgtcaac gcctgggaag tgggcaccaa aatctacatt 900 gaccttatgg agagcgaaat tctgccattc ccgttcccga atagccaaaa tcagccgttc 960 gctcctgaga aagcagtgcc gcgtctgacc cgttgggaga ttgatctgga tagcagcagc 1020 gatgagatta agcgtacgcg tctgcacgac ttctttgcag agatgccgat catggatttt 1080 cgttttgcgc tgcagtgcaa ccgctacggt tttatgggtg tcgatgaccc gcgcaagccg 1140 ctggcgcacc aacaagcgga gaagattttt gcgtacaata gcctgggtat ctgggacaac 1200 caccgtggtg attatgattt gtggtacagc ggcgaagcct cagcggcgca agaaccggct 1260 ttcgttccgc gttctccgac tgcagcggaa ggcgacggtt atctgctgac cgttgtgggc 1320 cgtttggacg agaaccgcag cgacctggtc attctggaca cgcaggacat ccagagcggt 1380 ccggttgcga cattaaact gccgtttcgc ctgcgtgcgg ccctgcacgg ctgttgggtt 1440 ccgcgtccgt aatccgcgca cgacactgaa catacggaag gagatataca tatgaatatt 1500 cgtccattgc atgatcgcgt gatcgtcaag cgtaaagaag ttgaaactaa atctgctggc 1560 ggcatcgttc tgaccggctc tgcagcggct aaatccaccc gcggcgaagt gctggctgtc 1620 ggcaatggcc gtatccttga aaatggcgaa gtgaagccgc tggatgtgaa agttggcgac 1680 atcgttattt tcaacgatgg ctacggtgtg aaatctgaga agatcgacaa tgaagaagtg 1740 ttgatcatgt ccgaaagcga cattctggca attgttgaag cgtaatccgc gcacgacact 1800 gaacatacga atttaaggaa taaagataat ggcagctaaa gacgtaaaat tcggtaacga 1860 cgctcgtgtg aaaatgctgc gcggcgtaaa cgtactggca gatgcagtga aagttaccct 1920 cggtccaaaa ggccgtaacg tagttctgga taaatctttc ggtgcaccga ccatcaccaa 1980 agatggtgtt tccgttgctc gtgaaatcga actggaagac aagttcgaaa atatgggtgc 2040 gcagatggtg aaagaagttg cctctaaagc aaacgacgct gcaggcgacg gtaccaccac 2100 tgcaaccgta ctggctcagg ctatcatcac tgaaggtctg aaagctgttg ctgcgggcat 2160 gaacccgatg gacctgaaac gtggtatcga caaagcggtt accgctgcag ttgaagaact 2220 gaaagcgctg tccgtaccat gctctgactc taaagcgatt gctcaggttg gtaccatctc 2280 cgctaactcc gacgaaaccg taggtaaact gatcgctgaa gcgatggaca aagtcggtaa 2340 agaaggcgtt atcaccgttg aagacggtac cggtctgcag gacgaactgg acgtggttga 2400 aggtatgcag ttcgaccgtg gctacctgtc tccttacttc atcaacaagc cggaaactgg 2460 cgcagtagaa ctggaaagcc cgttcatcct gctggctgac aagaaaatct ccaacatccg 2520 cgaaatgctg ccggttctgg aagctgttgc caaagcaggc aaaccgctgc tgatcatcgc 2580 tgaagatgta gaaggcgaag cgctggcaac tctggttgtt aacaccatgc gtggcatcgt 2640 gaaagtcgct gcggttaaag caccgggctt cggcgatcgt cgtaaagcta tgctgcagga 2700 tatcgcaacc ctgactggcg gtaccgtgat ctctgaagag atcggtatgg agctggaaaa 2760 agcaaccctg gaagacctgg gtcaggctaa acgtgttgtg atcaacaaag acaccaccac 2820 tatcatcgat ggcgtgggtg aagaagctgc aatccagggc cgtgttgctc agatccgtca 2880 gcagattgaa gaagcaactt ctgactacga ccgtgaaaaa ctgcaggaac gcgtagcgaa 2940 actggcaggc ggcgttgcag ttatcaaagt gggtgctgct accgaagttg aaatgaaaga 3000 gaaaaaagca cgcgttgaag atgccctgca cgcgacccgt gctgcggtag aagaaggcgt 3060 ggttgctggt ggtggtgttg cgctgatccg cgtagcgtct aaactggctg acctgcgtgg 3120 tcagaacgaa gaccagaacg tgggtatcaa agttgcactg cgtgcaatgg aagctccgct 3180 gcgtcagatc gtattgaact gcggcgaaga accgtctgtt gttgctaaca ccgttaaagg 3240 cggcgacggc aactacggtt acaacgcagc aaccgaagaa tacggcaaca tgatcgacat 3300 gggtatcctg gatccaacca aagtaactcg ttctgctctg cagtacgcag cttctgtggc 3360 tggcctgatg atcaccaccg aatgcatggt taccgacctg ccgaaaaacg atgcagctga 3420 cttaggcgct gctggcggta tgggcggcat gggtggcatg ggcggcatga tgtaa 3475 <210> 13 <211> 3456 <212> DNA <213> PC3 (a polycistronic construct of isoeugenol monooxygenase containing codon-modified nitrifying Pseudomonas Jin1, groES, groEL) <400> 13 gaaggagata tacatatggc acgtctgaac cgtaatgatc cgcaactcgt cggcaccctg 60 ttaccaaccc gtattgaggc tgacctgttc gacctggaag tggacggcga aattccgaag 120 tccatcaacg gtaccttcta ccgtaacacg ccggagcctc aggtgacgcc gcagaaattc 180 cacaccttca tcgatggtga cggcatggcg tctgcatttc attttgaaga tggccacgtg 240 gacttcatca gccgctgggt taaaaccgcg cgtttcacgg cggagagact ggcacgtaaa 300 agcctgttcg gtatgtaccg taatccgtac accgatgaca cgtctgtgaa gggtctggat 360 cgtaccgttg ccaacacgag catcatcagc catcacggta aggttctggc ggtgaaagaa 420 gatggcttgc cgtacgagct ggacccacgc actctggaaa cccgtggtcg ctttgactat 480 gatggccaag tcaccagcca gacgcacacc gcgcatccga agtatgaccc ggaaaccggt 540 gacctgttgt tctttggctc cgcagccaag ggtgaggcaa cgcctgatat ggcctattac 600 atcgttgata aacatggtaa ggtaacgcat gagacttggt tcgagcaacc gtatggcgcc 660 tttatgcatg attttgctat tacccgcaat tggagcatct tcccgatcat gccggctacc 720 aattcgttga gccgcctgaa agcgaagcag ccgatttaca tgtgggagcc ggaactgggt 780 tcctatattg gtgttctgcc gcgtcgcggt caaggtagcc agatccgctg gctgaaagca 840 ccagcgctgt gggtctttca cgtcgtcaac gcctgggaag tgggcaccaa aatctacatt 900 gaccttatgg agagcgaaat tctgccattc ccgttcccga atagccaaaa tcagccgttc 960 gctcctgaga aagcagtgcc gcgtctgacc cgttgggaga ttgatctgga tagcagcagc 1020 gatgagatta agcgtacgcg tctgcacgac ttctttgcag agatgccgat catggatttt 1080 cgttttgcgc tgcagtgcaa ccgctacggt tttatgggtg tcgatgaccc gcgcaagccg 1140 ctggcgcacc aacaagcgga gaagattttt gcgtacaata gcctgggtat ctgggacaac 1200 caccgtggtg attatgattt gtggtacagc ggcgaagcct cagcggcgca agaaccggct 1260 ttcgttccgc gttctccgac tgcagcggaa ggcgacggtt atctgctgac cgttgtgggc 1320 cgtttggacg agaaccgcag cgacctggtc attctggaca cgcaggacat ccagagcggt 1380 ccggttgcga cattaaact gccgtttcgc ctgcgtgcgg ccctgcacgg ctgttgggtt 1440 ccgcgtccgt aaaattaggt aaaaaataaa aaatgaatat tcgtccattg catgatcgcg 1500 tgatcgtcaa gcgtaaagaa gttgaaacta aatctgctgg cggcatcgtt ctgaccggct 1560 ctgcagcggc taaatccacc cgcggcgaag tgctggctgt cggcaatggc cgtatccttg 1620 aaaatggcga agtgaagccg ctggatgtga aagttggcga catcgttatt ttcaacgatg 1680 gctacggtgt gaaatctgag aagatcgaca atgaagaagt gttgatcatg tccgaaagcg 1740 acattctggc aattgttgaa gcgtaatccg cgcacgacac tgaacatacg aatttaagga 1800 ataaagataa tggcagctaa agacgtaaaa ttcggtaacg acgctcgtgt gaaaatgctg 1860 cgcggcgtaa acgtactggc agatgcagtg aaagttaccc tcggtccaaa aggccgtaac 1920 gtagttctgg ataaatcttt cggtgcaccg accatcacca aagatggtgt ttccgttgct 1980 cgtgaaatcg aactggaaga caagttcgaa aatatgggtg cgcagatggt gaaagaagtt 2040 gcctctaaag caaacgacgc tgcaggcgac ggtaccacca ctgcaaccgt actggctcag 2100 gctatcatca ctgaaggtct gaaagctgtt gctgcgggca tgaacccgat ggacctgaaa 2160 cgtggtatcg acaaagcggt taccgctgca gttgaagaac tgaaagcgct gtccgtacca 2220 tgctctgact ctaaagcgat tgctcaggtt ggtaccatct ccgctaactc cgacgaaacc 2280 2340 gaagacggta ccggtctgca ggacgaactg gacgtggttg aaggtatgca gttcgaccgt 2400 ggctacctgt ctccttactt catcaaaag ccggaaactg gcgcagtaga actggaaagc 2460 ccgttcatcc tgctggctga caagaaaatc tccaacatcc gcgaaatgct gccggttctg 2520 gaagctggtg ccaaagcagg caaaccgctg ctgatcatcg ctgaagatgt agaaggcgaa 2580 gcgctggcaa ctctggttgt taacaccatg cgtggcatcg tgaaagtcgc tgcggttaaa 2640 gcaccgggct tcggcgatcg tcgtaaagct atgctgcagg atatcgcaac cctgactggc 2700 ggtaccgtga tctctgaaga gatcggtatg gagctgggaaa aagcaaccct ggaagacctg 2760 ggtcaggcta aacgtgttgt gatcaaa gacaccacca ctatcatcga tggcgtgggt 2820 gaaagctg caatccaggg ccgtgttgct cagatccgtc agcagattga agaagcaact 2880 tctgactacg accgtgaaaa actgcaggaa cgcgtagcga aactggcagg cggcgttgca 2940 gttatcaaag tgggtgctgc taccgaagtt gaatgaaag agaaaaaagc acgcgttgaa 3000 gatgccctgc acgcgacccg tgctgcggta gaagaaggcg tggttgctgg tggtggtgtt 3060 gcgctgatcc gcgtagcgtc taaactggct gacctgcgtg gtcagaacga agaccagaac 3120 gtgggtatca aagttgcact gcgtgcaatg gaagctccgc tgcgtcagat cgtattgaac 3180 tgcggcgaag aaccgtctgt tgttgctaac accgttaaag gcggcgacgg caactacggt 3240 tacaacgcag caaccgaaga atacggcaac atgatcgaca tgggtatcct ggatccaacc 3300 aaagtaactc gttctgctct gcagtacgca gcttctgtgg ctggcctgat gatcaccacc 3360 gaatgcatgg ttaccgacct gccgaaaaac gatgcagctg acttaggcgc tgctggcggt 3420 atgggcggca tgggtggcat gggcggcatg atgtaa 3456 <210> 14 <211> 3423 <212> DNA <213> PC4 (a polycistronic construct of isoeugenol monooxygenase containing codon-modified nitrifying Pseudomonas Jin1, groES, groEL) <400> 14 gaaggagata tacatatggc acgtctgaac cgtaatgatc cgcaactcgt cggcaccctg 60 ttaccaaccc gtattgaggc tgacctgttc gacctggaag tggacggcga aattccgaag 120 tccatcaacg gtaccttcta ccgtaacacg ccggagcctc aggtgacgcc gcagaaattc 180 cacaccttca tcgatggtga cggcatggcg tctgcatttc attttgaaga tggccacgtg 240 gacttcatca gccgctgggt taaaaccgcg cgtttcacgg cggagagact ggcacgtaaa 300 agcctgttcg gtatgtaccg taatccgtac accgatgaca cgtctgtgaa gggtctggat 360 cgtaccgttg ccaacacgag catcatcagc catcacggta aggttctggc ggtgaaagaa 420 gatggcttgc cgtacgagct ggacccacgc actctggaaa cccgtggtcg ctttgactat 480 gatggccaag tcaccagcca gacgcacacc gcgcatccga agtatgaccc ggaaaccggt 540 gacctgttgt tctttggctc cgcagccaag ggtgaggcaa cgcctgatat ggcctattac 600 atcgttgata aacatggtaa ggtaacgcat gagacttggt tcgagcaacc gtatggcgcc 660 tttatgcatg attttgctat tacccgcaat tggagcatct tcccgatcat gccggctacc 720 aattcgttga gccgcctgaa agcgaagcag ccgatttaca tgtgggagcc ggaactgggt 780 tcctatattg gtgttctgcc gcgtcgcggt caaggtagcc agatccgctg gctgaaagca 840 ccagcgctgt gggtctttca cgtcgtcaac gcctgggaag tgggcaccaa aatctacatt 900 gaccttatgg agagcgaaat tctgccattc ccgttcccga atagccaaaa tcagccgttc 960 gctcctgaga aagcagtgcc gcgtctgacc cgttgggaga ttgatctgga tagcagcagc 1020 gatgagatta agcgtacgcg tctgcacgac ttctttgcag agatgccgat catggatttt 1080 cgttttgcgc tgcagtgcaa ccgctacggt tttatgggtg tcgatgaccc gcgcaagccg 1140 ctggcgcacc aacaagcgga gaagattttt gcgtacaata gcctgggtat ctgggacaac 1200 caccgtggtg attatgattt gtggtacagc ggcgaagcct cagcggcgca agaaccggct 1260 ttcgttccgc gttctccgac tgcagcggaa ggcgacggtt atctgctgac cgttgtgggc 1320 cgtttggacg agaaccgcag cgacctggtc attctggaca cgcaggacat ccagagcggt 1380 ccggttgcga cattaaact gccgtttcgc ctgcgtgcgg ccctgcacgg ctgttgggtt 1440 ccgcgtccgt aagaaggaga tatacatatg aatattcgtc cattgcatga tcgcgtgatc 1500 gtcaagcgta aagaagttga aactaaatct gctggcggca tcgttctgac cggctctgca 1560 gcggctaaat ccacccgcgg cgaagtgctg gctgtcggca atggccgtat ccttgaaaat 1620 ggcgaagtga agccgctgga tgtgaaagtt ggcgacatcg ttattttcaa cgatggctac 1680 ggtgtgaaat ctgagaagat cgacaatgaa gaagtgttga tcatgtccga aagcgacatt 1740 ctggcaattg ttgaagcgta agaaggagat atacatatgg cagctaaaga cgtaaaattc 1800 ggtaacgacg ctcgtgtgaa aatgctgcgc ggcgtaaacg tactggcaga tgcagtgaaa 1860 gttaccctcg gtccaaaagg ccgtaacgta gttctggata aatctttcgg tgcaccgacc 1920 atcaccaaag atggtgtttc cgttgctcgt gaaatcgaac tggaagacaa gttcgaaaat 1980 atgggtgcgc agatggtgaa agaagttgcc tctaaagcaa acgacgctgc aggcgacggt 2040 accaccactg caaccgtact ggctcaggct atcatcactg aaggtctgaa agctgttgct 2100 gcgggcatga acccgatgga cctgaaacgt ggtatcgaca aagcggttac cgctgcagtt 2160 gaagaactga aagcgctgtc cgtaccatgc tctgactcta aagcgattgc tcaggttggt 2220 accatctccg ctaactccga cgaaaccgta ggtaaactga tcgctgaagc gatggacaaa 2280 gtcggtaaag aaggcgttat caccgttgaa gacggtaccg gtctgcagga cgaactggac 2340 gtggttgaag gtatgcagtt cgaccgtggc tacctgtctc cttacttcat caacaagccg 2400 gaaactggcg cagtagaact ggaaagcccg ttcatcctgc tggctgacaa gaaaatctcc 2460 aacatccgcg aaatgctgcc ggttctggaa gctgttgcca aagcaggcaa accgctgctg 2520 atcatcgctg aagatgtaga aggcgaagcg ctggcaactc tggttgttaa caccatgcgt 2580 ggcatcgtga aagtcgctgc ggttaaagca ccgggcttcg gcgatcgtcg taaagctatg 2640 ctgcaggata tcgcaaccct gactggcggt accgtgatct ctgaagagat cggtatggag 2700 ctggaaaaag caaccctgga agacctgggt caggctaaac gtgttgtgat caacaaagac 2760 accaccacta tcatcgatgg cgtgggtgaa gaagctgcaa tccagggccg tgttgctcag 2820 atccgtcagc agattgaaga agcaacttct gactacgacc gtgaaaaact gcaggaacgc 2880 gtagcgaaac tggcaggcgg cgttgcagtt atcaaagtgg gtgctgctac cgaagttgaa 2940 atgaaagaga aaaaagcacg cgttgaagat gccctgcacg cgacccgtgc tgcggtagaa 3000 gaaggcgtgg ttgctggtgg tggtgttgcg ctgatccgcg tagcgtctaa actggctgac 3060 ctgcgtggtc agaacgaaga ccagaacgtg ggtatcaaag ttgcactgcg tgcaatggaa 3120 gctccgctgc gtcagatcgt attgaactgc ggcgaagaac cgtctgttgt tgctaacacc 3180 gttaaaggcg gcgacggcaa ctacggttac aacgcagcaa ccgaagaata cggcaacatg 3240 atcgacatgg gtatcctgga tccaaccaaa gtaactcgtt ctgctctgca gtacgcagct 3300 tctgtggctg gcctgatgat caccaccgaa tgcatggtta ccgacctgcc gaaaaacgat 3360 gcagctgact taggcgctgc tggcggtatg ggcggcatgg gtggcatggg cggcatgatg 3420 taa 3423 <210> 15 <211> 3456 <212> DNA <213> PC5 (a polycistronic construct of isoeugenol monooxygenase, groES, groEL, codon-modified from Pseudomonas nitrificationis Jin1) <400> 15 gaaggagata tacatatggc acgtctgaac cgtaatgatc cgcaactcgt cggcaccctg 60 ttaccaaccc gtattgaggc tgacctgttc gacctggaag tggacggcga aattccgaag 120 tccatcaacg gtaccttcta ccgtaacacg ccggagcctc aggtgacgcc gcagaaattc 180 cacaccttca tcgatggtga cggcatggcg tctgcatttc attttgaaga tggccacgtg 240 gacttcatca gccgctgggt taaaaccgcg cgtttcacgg cggagagact ggcacgtaaa 300 agcctgttcg gtatgtaccg taatccgtac accgatgaca cgtctgtgaa gggtctggat 360 cgtaccgttg ccaacacgag catcatcagc catcacggta aggttctggc ggtgaaagaa 420 gatggcttgc cgtacgagct ggacccacgc actctggaaa cccgtggtcg ctttgactat 480 gatggccaag tcaccagcca gacgcacacc gcgcatccga agtatgaccc ggaaaccggt 540 gacctgttgt tctttggctc cgcagccaag ggtgaggcaa cgcctgatat ggcctattac 600 atcgttgata aacatggtaa ggtaacgcat gagacttggt tcgagcaacc gtatggcgcc 660 tttatgcatg attttgctat tacccgcaat tggagcatct tcccgatcat gccggctacc 720 aattcgttga gccgcctgaa agcgaagcag ccgatttaca tgtgggagcc ggaactgggt 780 tcctatattg gtgttctgcc gcgtcgcggt caaggtagcc agatccgctg gctgaaagca 840 ccagcgctgt gggtctttca cgtcgtcaac gcctgggaag tgggcaccaa aatctacatt 900 gaccttatgg agagcgaaat tctgccattc ccgttcccga atagccaaaa tcagccgttc 960 gctcctgaga aagcagtgcc gcgtctgacc cgttgggaga ttgatctgga tagcagcagc 1020 gatgagatta agcgtacgcg tctgcacgac ttctttgcag agatgccgat catggatttt 1080 cgttttgcgc tgcagtgcaa ccgctacggt tttatgggtg tcgatgaccc gcgcaagccg 1140 ctggcgcacc aacaagcgga gaagattttt gcgtacaata gcctgggtat ctgggacaac 1200 caccgtggtg attatgattt gtggtacagc ggcgaagcct cagcggcgca agaaccggct 1260 ttcgttccgc gttctccgac tgcagcggaa ggcgacggtt atctgctgac cgttgtgggc 1320 cgtttggacg agaaccgcag cgacctggtc attctggaca cgcaggacat ccagagcggt 1380 ccggttgcga cattaaact gccgtttcgc ctgcgtgcgg ccctgcacgg ctgttgggtt 1440 ccgcgtccgt aaaagatagc aaaaaataaa aaatgaatat tcgtccattg catgatcgcg 1500 tgatcgtcaa gcgtaaagaa gttgaaacta aatctgctgg cggcatcgtt ctgaccggct 1560 ctgcagcggc taaatccacc cgcggcgaag tgctggctgt cggcaatggc cgtatccttg 1620 aaaatggcga agtgaagccg ctggatgtga aagttggcga catcgttatt ttcaacgatg 1680 gctacggtgt gaaatctgag aagatcgaca atgaagaagt gttgatcatg tccgaaagcg 1740 acattctggc aattgttgaa gcgtaatccg cgcacgacac tgaacatacg aatttaagga 1800 ataaagataa tggcagctaa agacgtaaaa ttcggtaacg acgctcgtgt gaaaatgctg 1860 cgcggcgtaa acgtactggc agatgcagtg aaagttaccc tcggtccaaa aggccgtaac 1920 gtagttctgg ataaatcttt cggtgcaccg accatcacca aagatggtgt ttccgttgct 1980 cgtgaaatcg aactggaaga caagttcgaa aatatgggtg cgcagatggt gaaagaagtt 2040 gcctctaaag caaacgacgc tgcaggcgac ggtaccacca ctgcaaccgt actggctcag 2100 gctatcatca ctgaaggtct gaaagctgtt gctgcgggca tgaacccgat ggacctgaaa 2160 cgtggtatcg acaaagcggt taccgctgca gttgaagaac tgaaagcgct gtccgtacca 2220 tgctctgact ctaaagcgat tgctcaggtt ggtaccatct ccgctaactc cgacgaaacc 2280 gtaggtaaac tgatcgctga agcgatggac aaagtcggta aagaaggcgt tatcaccgtt 2340 gaagacggta ccggtctgca ggacgaactg gacgtggttg aaggtatgca gttcgaccgt 2400 ggctacctgt ctccttactt catcaaaag ccggaaactg gcgcagtaga actggaaagc 2460 ccgttcatcc tgctggctga caagaaaatc tccaacatcc gcgaaatgct gccggttctg 2520 gaagctggtg ccaaagcagg caaaccgctg ctgatcatcg ctgaagatgt agaaggcgaa 2580 gcgctggcaa ctctggttgt taacaccatg cgtggcatcg tgaaagtcgc tgcggttaaa 2640 gcaccgggct tcggcgatcg tcgtaaagct atgctgcagg atatcgcaac cctgactggc 2700 ggtaccgtga tctctgaaga gatcggtatg gagctgggaaa aagcaaccct ggaagacctg 2760 ggtcaggcta aacgtgttgt gatcaaa gacaccacca ctatcatcga tggcgtgggt 2820 gaaagctg caatccaggg ccgtgttgct cagatccgtc agcagattga agaagcaact 2880 tctgactacg accgtgaaaa actgcaggaa cgcgtagcga aactggcagg cggcgttgca 2940 gttatcaaag tgggtgctgc taccgaagtt gaatgaaag agaaaaaagc acgcgttgaa 3000 gatgccctgc acgcgacccg tgctgcggta gaaaggcg tggttgctgg tggtggtgtt 3060 gcgctgatcc gcgtagcgtc taaactggct gacctgcgtg gtcagaacga agaccagaac 3120 gtgggtatca aagttgcact gcgtgcaatg gaagctccgc tgcgtcagat cgtattgaac 3180 tgcggcgaag aaccgtctgt tgttgctaac accgttaaag gcggcgacgg caactacggt 3240 tacaacgcag caaccgaaga atacggcaac atgatcgaca tgggtatcct ggatccaacc 3300 aaagtaactc gttctgctct gcagtacgca gcttctgtgg ctggcctgat gatcaccacc 3360 gaatgcatgg ttaccgacct gccgaaaaac gatgcagctg acttaggcgc tgctggcggt 3420 atgggcggca tgggtggcat gggcggcatg atgtaa 3456 <210> 16 <211> 5424 <212> DNA <213> pIEM2_C154_T222_A1318? <220> <221> Ori_p15a? <222> (1)..(827) <220> <221> Term_rpoC? <222> (1009?..(1128? <220> <221> Term_bla? <222> (1129)..(1429) <220> <221> P_T7_Inducible? <222> (1456?..(1497? <220> <221> LacO1? <222> (1477?..(1497? <220> <221> IEM_C154_T222_A1318? <222> (1528)..(2997) <220> <221> Term_T7? <222> (3002?..(3049? <220> <221> P_Amp? <222> (3322?..(3439? <220> <221> Kanamycin-r? <222> (3450)..(4259? <220> <221> P_widthI? <222> (4260?..(4341? <220> <221> wide? <222> (4342?..(5424? <400> 16 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtgga tgagacaaac gcggccata cagcggatg acaccggta accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg tagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgagga gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840. cagccgaacg accgagcgca gcgagtcagt gagcgagga gcggaaggcg aggtaggga actgccaggc atcaaacta gcagaaggcc cctgacggat ggccttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaaataa cgtaaaaacc cgcttcggcg ggttttttta 1080 tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct 1140 tgatatatga gaattattta accttataaaa tgaaaaaa gcaacgcact ttaaataaga 1200 tacgttgctt ttcgattga tgaacaccta taattaaact attcatctat tattatgat 1260 tttttgtata tacaatattt ctagttgtt aaagagaatt aagaaaataa atctcgaaaa 1320 tataaaggg aaaatcagtt tttgatatca aaattataca tgtcaacgat aatacaaaat 1380 ataatacaaa ctataagatg ttatcagtat ttattatgca tttagaataa atttgtgtc 1440 gcccttccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc 1500 ctctagaaat aatttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc 1560 aatgatccgc agttggcagg aacgatgttc cccacccgaa tagaggcgaa tgtctttgac 1620 cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680 gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740 gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800. tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat cacgggaag tactggccgc aaaggagat ggactacctt atgagcttga cccccaaacc ctggaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg caccctaagt tcgacccccca gacaggtgaa atgttactct tcggctccgc tgctaaaggc gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacacatgag acctggttta agcagcctta cggtgcattc atgcacgact ttgctgtcac gcgcaactgg 2220 tcaatctttc cgatcatgcc tgcgacaaat agccttgagc gtcttaagc aaagcagccc 2280. atttacatgt gggagcctga gcgaggaagc tatataggag tacttcctcg tcgtggtcag 2340. ggcaaggaca ttcgttggtt ccgtgccccg gcgttgtggg ttttccatgt cgtgaatgct tgggaggag ggatagaat tctgattgac ttgatggaa gtgagatttt gccgttccca ttcccgaact cgcagaacct tccatttgat ccctccaagg ctgttccgcg tctaacccgt 2520 tgggaaattg atctcaatag tggtaacgat gagatgaaac gtacgcagct acacgaatat 2580 tttgcagaaa tgcctatcat ggatttccgt tttgcgctcc aggatcatcg ctacgcctac 2640 atggggttg acgatcctcg tcgcccctta gctcatcagc aagctgaaaa aatctttgcc 2700 tacaattcgt taggggtttg ggacaccat cgtaaagatt atgaactttg gtttacggga 2760 aaaatgtctg cagcgcagga accggcgttt gttcctagaa gcccagatgc gcctgagggc 2820 gatggctacc tactcagtgt agtagggcgg ctcgatgaaa atcgtagcga tctagttatc 2880 cttgatacgc aatgtttggc agctgggcct gtggccactg tcaagcttcc cttccgtctc 2940 cgagcagcgt tgcacggttg ttggcagtct aagaactgag gatccgaatt cgagctcccc 3000 cctagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc ccctgagacg 3060 cgtcaatcga gttcgtacct aagggcgaca cccctaatt agcccgggcg aaaggcccag 3120 tctttcgact gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcatgggg 3180 agtccccaca ctaccatcgg cgctacggcg tttcacttct gagttcggca tggggtcagg 3240 tgggaccacc gcgctactgc cgccaggcaa acaaggggtg ttatgagcca tattcaggta 3300 taaatgggct cgcgataatg ttcagaattg gttaattggt tgtaacactg acccctattt 3360 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 3420 tgcttcaata atattgaaaa aggaagaata tgagccatat tcaacgggaa acgtcgaggc 3480 cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg 3540 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt 3600 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa 3660 actggctgac ggaatttatg ccacttccga ccatcaagca ttttatccgt actcctgatg 3720 atgcatggtt actcaccact gcgatccccg gaaaaacagc gttccaggta ttagaagaat 3780 atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcact 3840 cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgcctc gctcaggcgc 3900 aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct 3960 ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag 4020 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag 4080 gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat 4140 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta 4200 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag 4260 cggcgcgcca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag 4320 agtcaattca gggtggtgaa tatgaaacca gtaacgttat acgatgtcgc agagtatgcc 4380 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa 4440 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca 4500 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg 4560 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc 4620 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat 4680 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc 4740 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag 4800 acacccatca acagtattat tttctcccat gaggacggta cgcgactggg cgtggagcat 4860 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg 4920 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata 4980 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg 5040 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca 5100 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac 5160 gacgataccg aagatagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt 5220 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg 5280 aagggcaatc agctgttgcc agtctcactg gtgaaaagaa aaaccaccct ggcgcccaat 5340 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt 5400 tcccgactgg aaagcgggca gtga 5424 <210> 17 <211> 7557 <212> DNA <213> Construct trc / +term <400> 17 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa 420 accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg tagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgagga gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840. cagccgaacg accgagcgca gcgagtcagt gagcgagga gcggaaggcg aggtaggga actgccaggc atcaaacta gcagaaggcc cctgacggat ggccttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaata cgtaaaaacc cgcttcggcg ggtttttta tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct tgatatatga gattatta accttataaa tgagaaaaaa gcaacgcact ttaataaga tacgttgctt tttcgattga tgaacaccta taattaaact attcatctat tatttatgat tttttgtata tacaatattt ctagtttgtt aaagagaatt aagaaaata atctcgaaaa fatheraggg aaaatcagtt tttgatatca aaattataca tgtcaacgat fathercaaaat father ctataagatg ttatcagtat ttattatgca tttagaata attttgtgtc gcccttccgc gathering cgactcacta tagged gtgagcggat aacaattccc ctctagaaat aattttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc aatgatccgc agttggcagg aacgatgttc cccacccga tagaggcga tgtctttgac cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680. gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740. gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800. tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat cacgggaag tactggccgc aaaggagat ggactacctt atgagcttga cccccaaacc ctggaaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg 2040 caccctaagt tcgaccccca gacaggtgaa atgttactct tcggctccgc tgctaaaggc 2100 gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacacatgag 2160 acctggttta agcagcctta cggtgcattc atgcacgact ttgctgtcac gcgcaactgg 2220 tcaatctttc cgatcatgcc tgcgacaaat agccttgagc gtcttaaagc aaagcagccc 2280 atttacatgt gggagcctga gcgaggaagc tatataggag tacttcctcg tcgtggtcag 2340 ggcaaggaca ttcgttggtt ccgtgccccg gcgttgtggg ttttccatgt cgtgaatgct 2400 tgggaggaag ggaatagaat tctgattgac ttgatggaaa gtgagattttt gccgttccca 2460 ttcccgaact cgcagaacct tccatttgat ccctccaagg ctgttccgcg tctaacccgt 2520 tgggaaattg atctcaatag tggtaacgat gagatgaaac gtacgcagct acacgaatat 2580 tttgcagaaa tgcctatcat ggatttccgt tttgcgctcc aggatcatcg ctacgcctac 2640 atgggggtg acgatcctcg tcgcccctta gctcatcagc aagctgaaaa aatctttgcc 2700 tacaattcgt taggggtttg ggacaaccat cgtaaagatt atgaactttg gtttacggga 2760 aaaatgtctg cagcgcagga accggcgttt gttcctagaa gcccagatgc gcctgagggc 2820 gatggctacc tactcagtgt agtagggcgg ctcgatgaaa atcgtagcga tctagttatc 2880 cttgatacgc aatgtttggc agctgggcct gtggccactg tcaagcttcc cttccgtctc 2940 cgagcagcgt tgcacggttg ttggcagtct aagaactgac tagcataacc ccttggggcc 3000 tctaaacggg tcttgagggg ttttttggca tcctgtccat gactcggtga caattaatta 3060 tccggctcgt ataatgtgtg gaattgtgag cggataacaa ttccctctag aaataatttt 3120 gtttaacttt taaaggagag ttatcaatga atattcgtcc attgcatgat cgcgtgatcg 3180 tcaagcgtaa agaagttgaa actaaatctg ctggcggcat cgttctgacc ggctctgcag 3240 cggctaaatc cacccgcggc gaagtgctgg ctgtcggcaa tggccgtatc cttgaaaatg 3300 gcgaagtgaa gccgctggat gtgaaagttg gcgacatcgt tattttcaac gatggctacg 3360 gtgtgaaatc tgagaagatc gacaatgaag aagtgttgat catgtccgaa agcgacattc 3420 tggcaattgt tgaagcgtaa tccgcgcacg acactgaaca tacgaattta aggaataaag 3480 ataatggcag ctaaagacgt aaaattcggt aacgacgctc gtgtgaaaat gctgcgcggc 3540 gtaaacgtac tggcagatgc agtgaaagtt accctcggtc caaaaggccg taacgtagtt 3600 ctggataaat ctttcggtgc accgaccatc accaaagatg gtgtttccgt tgctcgtgaa 3660 atcgaactgg aagacaagtt cgaaaatatg ggtgcgcaga tggtgaaaga agttgcctct 3720 aaagcaaacg acgctgcagg cgacggtacc accactgcaa ccgtactggc tcaggctatc 3780 atcactgaag gtctgaaagc tgttgctgcg ggcatgaacc cgatggacct gaaacgtggt 3840 atcgacaaag cggttaccgc tgcagttgaa gaactgaaag cgctgtccgt accatgctct 3900 gactctaaag cgattgctca ggttggtacc atctccgcta actccgacga aaccgtaggt 3960 aaactgatcg ctgaagcgat ggacaaagtc ggtaaagaag gcgttatcac cgttgaagac 4020 ggtaccggtc tgcaggacga actggacgtg gttgaaggta tgcagttcga ccgtggctac 4080 ctgtctcctt acttcatcaa caagccggaa actggcgcag tagaactgga aagcccgttc 4140 atcctgctgg ctgaagaa aatctccaac atccgcgaaa tgctgccggt tctggaagct 4200 gttgccaaag caggcaaacc gctgctgatc atcgctgaag atgtagaagg cgaagcgctg 4260 gcaactctgg ttgttaacac catgcgtggc atcgtgaaag tcgctgcggt taaagcaccg 4320 ggcttcggcg atcgtcgtaa agctatgctg caggatatcg caaccctgac tggcggtacc 4380 gtgatctctg aagagatcgg tatggagctg gaaaaagcaa ccctgggaaga cctgggtcag 4440 gctaaacgtg ttgtgatcaa caaagacacc accactatca tcgatggcgt gggtgaagaa 4500 gctgcaatcc agggccgtgt tgctcagatc cgtcagcaga ttgaagaagc aacttctgac 4560 tacgaccgtg aaaaactgca ggaacgcgta gcgaaactgg caggcggcgt tgcagttatc 4620 aaagtgggtg ctgctaccga agttgaaatg aaagagaaaa aagcacgcgt tgaagatgcc 4680 ctgcacgga cccgtgctgc ggtagaagaa ggcgtggttg ctggtggtgg tgttgcgctg 4740 atccgcgtag cgtctaaact ggctgacctg cgtggtcaga acgaagacca gaacgtgggt 4800 atcaaagttg cactgcgtgc aatggaagct ccgctgcgtc agatcgtatt gaactgcggc 4860 gaagaaccgt ctgttgttgc taacaccgtt aaaggcggcg acggcaacta cggttacaac 4920 gcagcaaccg aagaatacgg caacatgatc gacatgggta tcctggatcc aaccaaagta 4980 actcgttctg ctctgcagta cgcagcttct gtggctggcc tgatgatcac caccgaatgc 5040 atggttaccg acctgccgaa aaacgatgca gctgacttag gcgctgctgg cggtatgggc 5100 ggcatgggtg gcatgggcgg catgatgtaa ccccctagca taaccccttg gggcccttaa 5160 acgggtcttg aggggttttt tgcccctgag acgcgtcaat cgagttcgta cctaagggcg 5220 acacccccta attagcccgg gcgaaaggcc cagtctttcg actgagcctt tcgttttatt 5280 tgatgcctgg cagttcccta ctctcgcatg gggagtcccc acactaccat cggcgctacg 5340 gcgtttcact tctgagttcg gcatggggtc aggtgggacc accgcgctac tgccgccagg 5400 caaacaaggg gtgttatgag ccatattcag gtataaatgg gctcgcgata atgttcagaa 5460 ttggttaatt ggttgtaaca ctgaccccta tttgtttatt tttctaaata cattcaaata 5520 tgtatccgct catgagacaa taaccctgat aaatgcttca samaattga aaaaggaaga 5580 atatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg 5640 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc 5700 gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg 5760 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgccacttc 5820 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc 5880 ccggaaaaac agcgttccag gtattagaag aatatcctga ttcaggtgaa aatattgttg 5940 atgcgctggc agtgttcctg cgccggttgc actcgattcc tgtttgtaat tgtcctttta 6000 acagcgatcg cgtatttcgc ctcgctcagg cgcaatcacg aatgaataac ggtttggttg 6060 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa 6120 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg 6180 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa 6240 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt 6300 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc 6360 agtttcattt gatgctcgat gagttttct aagcggcgcg ccatcgaatg gcgcaaaacc 6420 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatatgaaa 6480 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc 6540 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg 6600 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg 6660 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg 6720 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc 6780 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg 6840 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat 6900 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc 6960 catgaggacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc 7020 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat 7080 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc 7140 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg 7200 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg 7260 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagatag ctcatgttat 7320 atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac 7380 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gccagtctca 7440 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 7500 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtga 7557 <210> 18 <211> 7556 <212> DNA <213> Construct tac / +term <400> 18 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa 420 accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg taagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgaggaa gcggaatata tcctgtatca 720 catattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaaggcg agagtaggga 900 actgccaggc atcaaactaa gcagaaggcc cctgacggat ggcctttttg cgtttctaca 960 aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct 1020 gataacgagt aatcgttaat ccgcaaataa cgtaaaaacc cgcttcggcg ggttttttta 1080 tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct 1140 tgatatatga gaattattta accttataaa tgaaaaaa gcaacgcact ttaaataaga 1200 tacgttgctt ttcgattga tgaacaccta taattaaact attcatctat tattatgat 1260 tttttgtata tacaatattt ctagttgtt aaagagaatt aagaaaataa atctcgaaaa 1320 tataaaggg aaaatcagtt tttgatatca aaattataca tgtcaacgat aatacaaaat 1380 ataatacaaa ctataagatg ttatcagtat ttattatgca tttagaataa atttgtgtc 1440 gcccttccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc 1500 ctctagaaat aattttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc aatgatccgc agttggcagg aacgatgttc cccacccga tagaggcga tgtctttgac cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680. gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740. gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800. tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat cacgggaag tactggccgc aaaggagat ggactacctt atgagcttga cccccaaacc ctggaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg caccctaagt tcgacccccca gacaggtgaa atgttactct tcggctccgc tgctaaaggc gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacacatgag acctggttta agcagcctta cggtgcattc atgcacgact ttgctgtcac gcgcaactgg 2220 tcaatctttc cgatcatgcc tgcgacaaat agccttgagc gtcttaaagc aaagcagccc 2280 atttacatgt gggagcctga gcgaggaagc tatataggag tacttctcg tcgtggtcag 2340 ggcaaggaca ttcgttggtt ccgtgccccg gcgttgtggg ttttccatgt cgtgaatgct 2400 tgggaggaag ggaatagaat tctgattgac ttgatggaaa gtgagatttt gccgttccca 2460 ttcccgaact cgcagaacct tccatttgat ccctccaagg ctgttccgcg tctaacccgt 2520 tgggaaattg atctcaatag tggtaacgat gagatgaaac gtacgcagct acacgaatat 2580 tttgcagaaa tgcctatcat ggatttccgt tttgcgctcc aggatcatcg ctacgcctac 2640 atggggttg acgatcctcg tcgcccctta gctcatcagc aagctgaaaa aatctttgcc 2700 tacaattcgt taggggtttg ggacaccat cgtaaagatt atgaactttg gtttacggga 2760 aaaatgtctg cagcgcagga accggcgttt gttcctagaa gcccagatgc gcctgagggc 2820 gatggctacc tactcagtgt agtagggcgg ctcgatgaaa atcgtagcga tctagttatc 2880 cttgatacgc aatgtttggc agctgggcct gtggccactg tcaagcttcc cttccgtctc 2940 cgagcagcgt tgcacggttg ttggcagtct aagaactgac tagcataacc ccttggggcc 3000 tctaaacggg tcttgagggg ttttttggca tcctgtccat gactcggtga caattaatca 3060 tcggctcgta taatgtgtgg aattgtgagc ggataacaat tccctctaga aataatttg 3120 tttaactttt aaaggagagt tatcaatgaa tattcgtcca ttgcatgatc gcgtgatcgt 3180 caagcgtaaa gaagttgaaa ctaaatctgc tggcggcatc gttctgaccg gctctgcagc 3240 ggctaaatcc acccgcggcg aagtgctggc tgtcggcaat ggccgtatcc ttgaaaatgg 3300 cgaagtgaag ccgctggatg tgaaagttgg cgacatcgtt atttcaacg atggctacgg 3360 tgtgaaatct gagaagatcg acaatgaaga agtgttgatc atgtccgaaa gcgacattct 3420 ggcaattgtt gaagcgtaat ccgcgcacga cactgaacat acgaatttaa ggaataaaga 3480 taatggcagc taaagacgta aaattcggta acgacgctcg tgtgaaaatg ctgcgcggcg 3540 taaacgtact ggcagatgca gtgaaagtta ccctcggtcc aaaaggccgt aacgtagttc 3600 tggataaatc ttcggtgca ccgaccatca ccaaagatgg tgtttccgtt gctcgtgaaa 3660 tcgaactgga agacaagttc gaaaatatgg gtgcgcagat ggtgaaaga gttgcctcta aagcaaacga cgctgcaggc gacggtacca ccactgcaac cgtactggct caggctatca tcactgaagg tctgaaagct gttgctgcgg gcatgaaccc gatggacctg aaacgtggta 3840 3900. tcgacaaagc ggttaccgct gcagttgaag aactgaaagc gctgtccgta ccatgctctg actctaaagc gattgctcag gttggtacca tctccgctaa ctccgacgaa accgtaggta aactgatcgc tgaagcgatg gacaaagtcg gtaaagaagg cgttatcacc gttgaagacg gtaccggtct gcaggacgaa ctggacgtgg ttgaaggtat gcagttcgac cgtggctacc 4080 tgtctcctta cttcatcaac aagccggaa ctggcgcagt agaactgga agcccgttca tcctgctggc tgacaagaaa atctccaaca tccgcgaat gctgccggtt ctggaagctg ttgccaaagc aggcaaaccg ctgctgatca tcgctgaaga tgtagaaggc gaagcgctgg caactctggt tgttaacacc atgcgtggca tcgtgaaagt cgctgcggtt aaagcaccgg 4320. gcttcggcga tcgtcgtaaa gctatgctgc aggatatcgc aaccctgact ggcggtaccg 4380 tgatctctga agagatcggt atggagctgg aaaaagcaac cctggaagac ctgggtcagg 4440 ctaaacgtgt tgtgatcaac aaagacacca ccactatcat cgatggcgtg ggtgaagaag 4500 ctgcaatcca gggccgtgtt gctcagatcc gtcagcagat tgaagaagca acttctgact 4560 acgaccgtga aaaactgcag gaacgcgtag cgaaactggc aggcggcgtt gcagttatca 4620 aagtgggtgc tgctaccgaa gttgaaatga aagagaaaaa agcacgcgtt gaagatgccc 4680 tgcacgcgac ccgtgctgcg gtagaaag gcgtggttgc tggtggtgt gttgcgctga 4740 tccgcgtagc gtctaaactg gctgacctgc gtggtcagaa cgaagaccag aacgtgggta 4800 tcaaagttgc actgcgtgca atggaagctc cgctgcgtca gatcgtattg aactgcggcg 4860 aagaaccgtc tgttgttgct aacaccgtta aaggcggcga cggcaactac ggttacaacg 4920 cagcaaccga agaatacggc aacatgatcg acatgggtat cctggatcca accaaagtaa 4980 ctcgttctgc tctgcagtac gcagcttctg tggctggcct gatgatcacc accgaatgca 5040 tggttaccga cctgccgaaa aacgatgcag ctgacttagg cgctgctggc ggtatgggcg 5100 gcatgggtgg catgggcggc atgatgtaac cccctagcat aaccccttgg ggcctctaaa 5160 cgggtcttga ggggtttttt gcccctgaga cgcgtcaatc gagttcgtac ctaagggcga 5220 caccccctaa ttagcccggg cgaaaggccc agtctttcga ctgagccttt cgttttattt 5280 gatgcctggc agttccctac tctcgcatgg ggagtcccca cactaccatc ggcgctacgg 5340 cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact gccgccaggc 5400 aaaacaagggg tgttatgagc catattcagg tataaatggg ctcgcgataa tgttcagaat 5460 tggttaattg gttgtaacac tgacccctat ttgtttattt ttctaaatac attcaaatat 5520 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagaa 5580 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga 5640 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg 5700 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc 5760 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgccacttcc 5820 gaccatcaag catttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc 5880 cggaaaaaca gcgttccagg tattagaaga atatcctgat tcaggtgaaa atattgttga 5940 tgcgctggca gtgttcctgc gccggttgca ctcgattcct gtttgtaatt gtccttttaa 6000 cagcgatcgc gtatttcgcc tcgctcaggc gcaatcacga atgaataacg gtttggttga 6060 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat 6120 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga 6180 taaccttatt ttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat 6240 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc 6300 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca 6360 gtttcatttg atgctcgatg agttttcta agcggcgcgc catcgaatgg cgcaaaacct 6420 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatatgaaac 6480 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc tttcagacc gtttcccgcg 6540 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg 6600 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc 6660 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga 6720 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg 6780 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga 6840 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg 6900 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc 6960 atgaggacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg 7020 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata 7080 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca 7140 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc 7200 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc 7260 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagatagc tcatgttata 7320 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc 7380 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg ccagtctcac 7440 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg 7500 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtga 7556 <210> 19 <211> 7544 <212> DNA <213> Construct T7 / +term <400> 19 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtgga tgagacaaac gcggccata cagcggatg acaccggta accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg tagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgagga gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840. cagccgaacg accgagcgca gcgagtcagt gagcgagga gcggaaggcg aggtaggga actgccaggc atcaaacta gcagaaggcc cctgacggat ggccttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaaataa cgtaaaaacc cgcttcggcg ggttttttta 1080 tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct 1140 tgatatatga gaattattta accttataaaa tgaaaaaa gcaacgcact ttaaataaga 1200 tacgttgctt ttcgattga tgaacaccta taattaaact attcatctat tattatgat 1260 tttttgtata tacaatattt ctagttgtt aaagagaatt aagaaaataa atctcgaaaa 1320 tataaaggg aaaatcagtt tttgatatca aaattataca tgtcaacgat aatacaaaat 1380 ataatacaaa ctataagatg ttatcagtat ttattatgca tttagaataa atttgtgtc 1440 gcccttccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc 1500 ctctagaaat aatttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc 1560 aatgatccgc agttggcagg aacgatgttc cccacccgaa tagaggcgaa tgtctttgac 1620 cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680 gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740 gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800. tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat cacgggaag tactggccgc aaaggagat ggactacctt atgagcttga cccccaaacc ctggaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg caccctaagt tcgacccccca gacaggtgaa atgttactct tcggctccgc tgctaaaggc gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacacatgag acctggttta agcagcctta cggtgcattc atgcacgact ttgctgtcac gcgcaactgg 2220 tcaatctttc cgatcatgcc tgcgacaaat agccttgagc gtcttaagc aaagcagccc 2280. atttacatgt gggagcctga gcgaggaagc tatataggag tacttcctcg tcgtggtcag 2340. ggcaaggaca ttcgttggtt ccgtgccccg gcgttgtggg ttttccatgt cgtgaatgct tgggaggag ggatagaat tctgattgac ttgatggaa gtgagatttt gccgttccca ttcccgaact cgcagaacct tccatttgat ccctccaagg ctgttccgcg tctaacccgt 2520 tgggaaattg atctcaatag tggtaacgat gagatgaaac gtacgcagct acacgaatat 2580 tttgcagaaa tgcctatcat ggatttccgt tttgcgctcc aggatcatcg ctacgcctac 2640 atgggggttg acgatcctcg tcgcccctta gctcatcagc aagctgaaaa aatctttgcc 2700 tacaattcgt taggggtttg ggacaaccat cgtaaagatt atgaactttg gtttacggga 2760 aaaatgtctg cagcgcagga accggcgttt gttcctagaa gcccagatgc gcctgagggc 2820 gatggctacc tactcagtgt agtagggcgg ctcgatgaaa atcgtagcga tctagttatc 2880 cttgatacgc aatgtttggc agctgggcct gtggccactg tcaagcttcc cttccgtctc 2940 cgagcagcgt tgcacggttg ttggcagtct aagaactgac tagcataacc ccttggggcc 3000 tctaaacggg tcttgagggg ttttttggca tcctgtccat gactcggtaa tacgactcac 3060 tataggggaa ttgtgagcgg ataacaattc cctctagaaa taattttgtt taacttttaa 3120 aggagagtta tcaatgaata ttcgtccatt gcatgatcgc gtgatcgtca agcgtaaaga 3180 agttgaaact aaatctgctg gcggcatcgt tctgaccggc tctgcagcgg ctaaatccac 3240 ccgcggcgaa gtgctggctg tcggcaatgg ccgtatcctt gaaaatggcg aagtgaagcc 3300 gctggatgtg aaagttggcg acatcgttat tttcaacgat ggctacggtg tgaaatctga 3360 gaagatcgac aatgaagaag tgttgatcat gtccgaaagc gacattctgg caattgttga 3420 agcgtaatcc gcgcacgaca ctgaacatac gaatttaagg aataaagata atggcagcta 3480 aagacgtaaa attcggtaac gacgctcgtg tgaaaatgct gcgcggcgta aacgtactgg 3540 cagatgcagt gaaagttacc ctcggtccaa aaggccgtaa cgtagttctg gataaatctt 3600 tcggtgcacc gaccatcacc aaagatggtg tttccgttgc tcgtgaaatc gaactggaag 3660 acaagttcga aaatatgggt gcgcagatgg tgaaagaagt tgcctctaaa gcaaacgacg 3720 ctgcaggcga cggtaccacc actgcaaccg tactggctca ggctatcatc actgaaggtc 3780 tgaaagctgt tgctgcgggc atgaacccga tggacctgaa acgtggtatc gacaaagcgg 3840 ttaccgctgc agttgaagaa ctgaaagcgc tgtccgtacc atgctctgac tctaaagcga 3900 ttgctcaggt tggtaccatc tccgctaact ccgacgaaac cgtaggtaaa ctgatcgctg 3960 aagcgatgga caaagtcggt aaagaaggcg ttatcaccgt tgaagacggt accggtctgc 4020 aggacgaact ggacgtggtt gaaggtatgc agttcgaccg tggctacctg tctcctact 4080 tcatcaacaa gccggaaact ggcgcagtag aactggaaag cccgttcatc ctgctggctg 4140 acaagaaaat ctccaacatc cgcgaaatgc tgccggttct ggaagctgtt gccaaagcag 4200 gcaaaccgct gctgatcatc gctgaagatg tagaaggcga agcgctggca actctggttg 4260 ttaacaccat gcgtggcatc gtgaaagtcg ctgcggttaa agcaccgggc ttcggcgatc 4320 gtcgtaaagc tatgctgcag gatatcgcaa ccctgactgg cggtaccgtg atctctgaag 4380 agatcggtat ggagctggaa aaagcaaccc tggaagacct gggtcaggct aaacgtgttg 4440 tgatcaacaa agacaccacc actatcatcg atggcgtggg tgaagaagct gcaatccagg 4500 gccgtgttgc tcagatccgt cagcagattg aagaagcaac ttctgactac gaccgtgaaa 4560 aactgcagga acgcgtagcg aaactggcag gcggcgttgc agttatcaaa gtgggtgctg 4620 ctaccgaagt tgaaatgaaa gagaaaaaag cacgcgttga agatgccctg cacgcgaccc 4680 gtgctgcggt agaagaaggc gtggttgctg gtggtggtgt tgcgctgatc cgcgtagcgt 4740 ctaaactggc tgacctgcgt ggtcagaacg aagaccagaa cgtgggtatc aaagttgcac 4800 tgcgtgcaat ggaagctccg ctgcgtcaga tcgtattgaa ctgcggcgaa gaaccgtctg 4860 ttgttgctaa caccgttaaa ggcggcgacg gcaactacgg ttacaacgca gcaaccgaag 4920 aatacggcaa catgatcgac atgggtatcc tggatccaac caaagtaact cgttctgctc 4980 tgcagtacgc agcttctgtg gctggcctga tgatcaccac cgaatgcatg gttaccgacc 5040 tgccgaaaaa cgatgcagct gacttaggcg ctgctggcgg tatgggcggc atgggtggca 5100 tgggcggcat gatgtaaccc cctagcataa ccccttgggg cctctaaacg ggtcttgagg 5160 ggttttttgc ccctgagacg cgtcaatcga gttcgtacct aagggcgaca ccccctaatt 5220 agcccgggcg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctggcag 5280 ttccctactc tcgcatgggg agtccccaca ctaccatcgg cgctacggcg tttcacttct 5340 gagttcggca tggggtcagg tgggaccacc gcgctactgc cgccaggcaa acaaggggtg 5400 ttatgagcca tattcaggta taaatgggct cgcgataatg ttcagaattg gttaattggt 5460 tgtaacactg acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 5520 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagaata tgagccatat 5580 tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta 5640 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa 5700 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac 5760 agatgagatg gtcagactaa actggctgac ggaatttatg ccacttccga ccatcaagca 5820 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc 5880 gttccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt 5940 gttcctgcgc cggttgcact cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt 6000 atttcgcctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt 6060 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt 6120 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt 6180 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata 6240 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg 6300 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat 6360 gctcgatgag tttttctaag cggcgcgcca tcgaatggcg caaaaccttt cgcggtatgg 6420 catgatagcg cccggaagag agtcaattca gggtggtgaa tatgaaacca gtaacgttat 6480 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg 6540 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt 6600 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg 6660 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg 6720 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct 6780 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc 6840 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat 6900 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaggacggta 6960 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg 7020 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc 7080 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc 7140 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg 7200 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg 7260 atatctcggt agtgggatac gacgataccg aagatagctc atgttatatc ccgccgttaa 7320 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac 7380 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc agtctcactg gtgaaaagaa 7440 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 7500 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtga 7544 <210> 20 <211> 7550 <212> DNA <213> Construct T5 / +term <400> 20 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa 420 accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg tagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgagga gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840. cagccgaacg accgagcgca gcgagtcagt gagcgagga gcggaaggcg aggtaggga actgccaggc atcaaacta gcagaaggcc cctgacggat ggccttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaata cgtaaaaacc cgcttcggcg ggtttttta tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct tgatatatga gattatta accttataaa tgagaaaaaa gcaacgcact ttaataaga tacgttgctt tttcgattga tgaacaccta taattaaact attcatctat tatttatgat tttttgtata tacaatattt ctagtttgtt aaagagaatt aagaaaata atctcgaaaa fatheraggg aaaatcagtt tttgatatca aaattataca tgtcaacgat fathercaaaat father ctataagatg ttatcagtat ttattatgca tttagaata attttgtgtc gcccttccgc gather cgactcacta tag gtgagcggat aacaattccc ctctagaaat aattttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc aatgatccgc agttggcagg aacgatgttc cccacccga tagaggcga tgtctttgac cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680. gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740. gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800. tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat cacgggaag tactggccgc aaaggagat ggactacctt atgagcttga cccccaaacc ctggaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg caccctaagt tcgaccccca gacaggtgaa atgttactct tcggctccgc tgctaaaggc 2100 gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacacatgag 2160 acctggttta agcagcctta cggtgcattc atgcacgact ttgctgtcac gcgcaactgg 2220 tcaatctttc cgatcatgcc tgcgacaaat agccttgagc gtcttaaagc aaagcagccc 2280 atttacatgt gggagcctga gcgaggaagc tatataggag tacttcctcg tcgtggtcag 2340 ggcaaggaca ttcgttggtt ccgtgccccg gcgttgtggg ttttccatgt cgtgaatgct 2400 tgggaggaag ggaatagaat tctgattgac ttgatggaaa gtgagattttt gccgttccca 2460 ttcccgaact cgcagaacct tccatttgat ccctccaagg ctgttccgcg tctaacccgt 2520 tgggaaattg atctcaatag tggtaacgat gagatgaaac gtacgcagct acacgaatat 2580 tttgcagaaa tgcctatcat ggatttccgt tttgcgctcc aggatcatcg ctacgcctac 2640 atgggggtg acgatcctcg tcgcccctta gctcatcagc aagctgaaaa aatctttgcc 2700 tacaattcgt taggggtttg ggacaaccat cgtaaagatt atgaactttg gtttacggga 2760 aaaatgtctg cagcgcagga accggcgttt gttcctagaa gcccagatgc gcctgagggc 2820 gatggctacc tactcagtgt agtagggcgg ctcgatgaaa atcgtagcga tctagttatc 2880 cttgatacgc aatgtttggc agctggggcct gtggccactg tcaagcttcc cttccgtctc 2940 cgagcagcgt tgcacggttg ttggcagtct aagaactgac tagcataacc ccttggggcc 3000 tctaaacggg tcttgagggg ttttttggca tcctgtccat gactcggaaa tcataaaaaa 3060 tttatttgct ttgtgagcgg ataacaatta taataccctc tagaataat tttgtttaac 3120 ttttaaagga gagttatcaa tgaatattcg tccattgcat gatcgcgtga tcgtcaagcg 3180 taaagaagtt gaaactaaat ctgctggcgg catcgttctg accggctctg cagcggctaa 3240 atccacccgc ggcgaagtgc tggctgtcgg caatggccgt atccttgaaa atggcgaagt 3300 gaagccgctg gatgtgaaag ttggcgacat cgttattttc aacgatggct acggtgtgaa 3360 atctgagaag atcgacaatg aagaagtgtt gatcatgtcc gaaagcgaca ttctggcaat 3420 tgttgaagcg taatccgcgc acgacactga acatacgaat ttaaggaata aagataatgg 3480 cagctaaa cgtaaaaattc ggtaacgacg ctcgtgtgaa aatgctgcgc ggcgtaaacg 3540 3600 aatctttcgg tgcaccgacc atcaccaaag atggtgtttc cgttgctcgt gaaatcgaac 3660 tggagacaa gttcgaaaat atgggtgcgc agatggtgaa agaagttgcc tctaaagcaa 3720 acgacgctgc aggcgacggt accaccactg caaccgtact ggctcaggct atcatcactg 3780 aaggtctgaa agctgttgct gcgggcatga acccgatgga cctgaaacgt ggtatcgaca 3840 aagcggttac cgctgcagtt gaaactga aagcgctgtc cgtaccatgc tctgactcta 3900 aagcgattgc tcaggttggt accatctccg ctaactccga cgaaaccgta ggtaaactga 3960 tcgctgaagc gatggaaa gtcggtaaag aaggcgttat caccgttgaa gacggtaccg 4020 gtctgcagga cgaactggac gtggttgaag gtatgcagtt cgaccgtggc tacctgtctc 4080 cttacttcat caacaagccg gaaactggcg footgaact ggaaagcccg ttcatcctgc 4140 tggctgacaa gaaaatctcc aacatccgcg aaatgctgcc ggttctggaa gctgttgcca 4200 aagcaggcaa accgctgctg atcatcgctg aagatgtaga aggcgaagcg ctggcaactc 4260 tggttgttaa caccatgcgt ggcatcgtga aagtcgctgc ggttaaagca ccgggcttcg 4320 gcgatcgtcg taaagctatg ctgcaggata tcgcaaccct gactggcggt accgtgatct 4380 ctgaagagat cggtatggag ctggaaaaag caaccctgga agacctgggt caggctaaac 4440 gtgttgtgat caacaaagac accaccacta tcatcgatgg cgtgggtgaa gaagctgcaa 4500 tccagggccg tgttgctcag atccgtcagc agattgaaga agcaacttct gactacgacc 4560 gtgaaaaact gcaggaacgc gtagcgaaac tggcaggcgg cgttgcagtt atcaaagtgg 4620 gtgctgctac cgaagttgaa atgaaagaga aaaaagcacg cgttgaagat gccctgcacg 4680 cgacccgtgc tgcggtagaa gaaggcgtgg ttgctggtgg tggtgttgcg ctgatccgcg 4740 tagcgtctaa actggctgac ctgcgtggtc agaacgaaga ccagaacgtg ggtatcaaag 4800 ttgcactgcg tgcaatggaa gctccgctgc gtcagatcgt attgaactgc ggcgaagaac 4860 cgtctgttgt tgctaacacc gttaaaggcg gcgacggcaa ctacggttac aacgcagcaa 4920 ccgaagaata cggcaacatg atcgacatgg gtatcctgga tccaaccaaa gtaactcgtt 4980 ctgctctgca gtacgcagct tctgtggctg gcctgatgat caccaccgaa tgcatggtta 5040 ccgacctgcc gaaaaacgat gcagctgact taggcgctgc tggcggtatg ggcggcatgg 5100 gtggcatggg cggcatgatg taaccccta gcataacccc ttggggcctc taaacgggtc 5160 ttgaggggtt ttttgcccct gagacgcgtc aatcgagttc gtacctaagg gcgacacccc 5220 ctaattagcc cgggcgaaag gcccagtctt tcgactgagc cttcgtttt atttgatgcc 5280 tggcagttcc ctactctcgc atggggagtc cccacactac catcggcgct acggcgtttc 5340 acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc aggcaaacaa 5400 ggggtgttat gagccatatt caggtataaa tgggctcgcg ataatgttca gaattggtta 5460 attggttgta acactgaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 5520 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agaatatgag 5580 ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata 5640 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta 5700 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga 5760 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgccac ttccgaccat 5820 caagcatttt atccgtactc ctgatgatgc atggtactc accactgcga tccccggaaa 5880 aacagcgttc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct 5940 ggcagtgttc ctgcgccggt tgcactcgat tcctgtttgt aattgtcctt ttaacagcga 6000 tcgcgtattt cgcctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag 6060 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa 6120 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct 6180 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga 6240 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca 6300 gaaacggctt tttcaaaat atggtattga taatcctgat atgaataaat tgcagtttca 6360 tttgatgctc gatgagtttt tctaagcggc gcgccatcga atggcgcaaa acctttcgcg 6420 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatatg aaaccagtaa 6480 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga 6540 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc 6600 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg 6660 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat 6720 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg 6780 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta 6840 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg 6900 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgagg 6960 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt 7020 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc 7080 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg 7140 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg 7200 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg 7260 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga tagctcatgt tatatcccgc 7320 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc 7380 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgccagtc tcactggtga 7440 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 7500 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga 7550 <210> 21 <211> 7544 <212> DNA [[ID=​​​​​​​cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa ccggcgcatg acttcaagac taactcctct aaatcaatta ccgtggctg ctgccagtgg tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtgga tgagacaaac gcggccata cagcggatg acaccggta accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg tagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgagga gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840. cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaaggcg agagtaggga 900 actgccaggc atcaaactaa gcagaaggcc cctgacggat ggcctttttg cgtttctaca 960 aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct 1020 gataacgagt aatcgttaat ccgcaaataa cgtaaaaacc cgcttcggcg ggttttttta 1080 tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct 1140 tgatatatga gaattattta accttataaaa tgaaaaaa gcaacgcact ttaaataaga 1200 tacgttgctt ttcgattga tgaacaccta taattaaact attcatctat tattatgat 1260 tttttgtata tacaatattt ctagttgtt aaagagaatt aagaaaataa atctcgaaaa 1320 tataaaggg aaaatcagtt tttgatatca aaattataca tgtcaacgat aatacaaaat 1380 ataatacaaa ctataagatg ttatcagtat ttattatgca tttagaataa atttgtgtc 1440 gcccttccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc 1500 ctctagaaat aatttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc 1560 aatgatccgc agttggcagg aacgatgttc cccacccga tagaggcga tgtctttgac cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680. gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740. gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800. tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat cacgggaag tactggccgc aaaggagat ggactacctt atgagcttga cccccaaacc ctggaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg caccctaagt tcgaccccca gacaggtgaa atgttactct tcggctccgc tgctaaaggc gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacacatgag acctggttta agcagcctta cggtgcattc atgcacgact ttgctgtcac gcgcaactgg 2220 tcaatctttc cgatcatgcc tgcgacaaat agccttgagc gtcttaagc aaagcagccc atttacatgt gggagcctga gcgaggaagc tatataggag tacttcctcg tcgtggtcag 2340 ggcaaggaca ttcgttggtt ccgtgccccg gcgttgtggg ttttccatgt cgtgaatgct 2400 tgggaggaag ggaatagaat tctgattgac ttgatggaaa gtgagatttt gccgttccca 2460 ttcccgaact cgcagaacct tccatttgat ccctccaagg ctgttccgcg tctaacccgt 2520 tgggaaattg atctcaatag tggtaacgat gagatgaaac gtacgcagct acacgaatat 2580 tttgcagaaa tgcctatcat ggatttccgt tttgcgctcc aggatcatcg ctacgcctac 2640 atggggttg acgatcctcg tcgcccctta gctcatcagc aagctgaaaa aatctttgcc 2700 tacaattcgt taggggtttg ggacaccat cgtaaagatt atgaactttg gtttacggga 2760 aaaatgtctg cagcgcagga accggcgttt gttcctagaa gcccagatgc gcctgagggc 2820 gatggctacc tactcagtgt agtagggcgg ctcgatgaaa atcgtagcga tctagttatc 2880 cttgatacgc aatgtttggc agctgggcct gtggccactg tcaagcttcc cttccgtctc 2940 cgagcagcgt tgcacggttg ttggcagtct aagaactgac tagcataacc ccttggggcc 3000 tctaaacggg tcttgagggg ttttttggca tcctgtccat gactcggtaa tacgactcac 3060 tacggaaga ttgtgagcgg ataacaattc cctcgaaaa taattttgtt taacttttaa aggagagtta tcaatgaata ttcgtccatt gcatgatcgc gtgatcgtca agcgtaaaga agttgaaact aaatctgctg gcggcatcgt tctgaccggc tctgcagcgg ctaaatccac 3240 ccgcggcga gtgctggctg tcggcaatgg ccgtatcctt gaaaatggcg aagtgaagcc gctggatgtg aaagttggcg acatcgttat tttcaacgat ggctacggtg tgaaatctga gaagatcgac aatgaagaag tgttgatcat gtccgaaagc gacattctgg caattgttga agcgtaatcc gcgcacgaca ctgaacatac gaatttaagg aataagata atggcagcta aagacgtaaa attcggtaac gacgctcgtg tgaaaatgct gcgcggcgta aacgtactgg cagatgcagt gaaagttacc ctcggtccaa aaggccgta cgtagttctg gataaatctt tcggtgcacc gaccatcacc aaagatggtg tttccgttgc tcgtgaaatc gaactggaag acaagttcga aaatatgggt gcgcagatgg tgaaagaagt tgcctctaaa gcaaacgacg ctgcaggcga cggtaccacc actgcaaccg tactggctca ggctatcatc actgaaggtc 3780 tgaaagctgt tgctgcgggc atgaacccga tggacctgaa acgtggtatc gacaaagcgg 3840 ttaccgctgc agttgaagaa ctgaaagcgc tgtccgtacc atgctctgac tctaaagcga 3900 ttgctcaggt tggtaccatc tccgctaact ccgacgaaac cgtaggtaaa ctgatcgctg 3960 aagcgatgga caaagtcggt aaagaaggcg ttatcaccgt tgaagacggt accggtctgc 4020 aggacgaact ggacgtggtt gaaggtatgc agttcgaccg tggctacctg tctcctact 4080 tcatcaacaa gccggaaact ggcgcagtag aactggaaag cccgttcatc ctgctggctg 4140 acaagaaaat ctccaacatc cgcgaaatgc tgccggttct ggaagctgtt gccaaagcag 4200 gcaaaccgct gctgatcatc gctgaagatg tagaaggcga agcgctggca actctggttg 4260 ttaacaccat gcgtggcatc gtgaaagtcg ctgcggttaa agcaccgggc ttcggcgatc 4320 gtcgtaaagc tatgctgcag gatatcgcaa ccctgactgg cggtaccgtg atctctgaag 4380 agatcggtat ggagctggaa aaagcaaccc tggaagacct gggtcaggct aaacgtgttg 4440 tgatcaacaa agacaccacc actatcatcg atggcgtggg tgaagaagct gcaatccagg 4500 gccgtgttgc tcagatccgt cagcagattg aagaagcaac ttctgactac gaccgtgaaa 4560 aactgcagga acgcgtagcg aaactggcag gcggcgttgc agttatcaaa gtgggtgctg 4620 ctaccgaagt tgaaatgaaa gagaaaaaag cacgcgttga agatgccctg cacgcgaccc 4680 gtgctgcggt agaagaaggc gtggttgctg gtggtggtgt tgcgctgatc cgcgtagcgt 4740 ctaaactggc tgacctgcgt ggtcagaacg aagaccagaa cgtgggtatc aaagttgcac 4800 tgcgtgcaat ggaagctccg ctgcgtcaga tcgtattgaa ctgcggcgaa gaaccgtctg 4860 ttgttgctaa caccgttaaa ggcggcgacg gcaactacgg ttacaacgca gcaaccgaag 4920 aatacggcaa catgatcgac atgggtatcc tggatccaac caaagtaact cgttctgctc 4980 tgcagtacgc agcttctgtg gctggcctga tgatcaccac cgaatgcatg gttaccgacc 5040 tgccgaaaaa cgatgcagct gacttaggcg ctgctggcgg tatgggcggc atgggtggca 5100 tgggcggcat gatgtaaccc cctagcataa ccccttgggg cctctaaacg ggtcttgagg 5160 ggttttttgc ccctgagacg cgtcaatcga gttcgtacct aagggcgaca ccccctaatt 5220 agcccgggcg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctggcag 5280 ttccctactc tcgcatgggg agtccccaca ctaccatcgg cgctacggcg tttcacttct 5340 gagttcggca tggggtcagg tgggaccacc gcgctactgc cgccaggcaa acaaggggtg 5400 ttatgagcca tattcaggta taaatgggct cgcgataatg ttcagaattg gttaattggt 5460 tgtaacactg acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 5520 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagaata tgagccatat 5580 tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta 5640 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa 5700 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac 5760 agatgagatg gtcagactaa actggctgac ggaatttatg ccacttccga ccatcaagca 5820 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc 5880 gttccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt 5940 gttcctgcgc cggttgcact cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt 6000 atttcgcctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt 6060 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt 6120 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt 6180 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata 6240 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg 6300 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat 6360 gctcgatgag tttttctaag cggcgcgcca tcgaatggcg caaaaccttt cgcggtatgg 6420 catgatagcg cccggaagag agtcaattca gggtggtgaa tatgaaacca gtaacgttat 6480 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg 6540 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt 6600 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg 6660 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg 6720 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct 6780 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc 6840 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat 6900 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gagacggta 6960 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg 7020 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc 7080 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc 7140 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg 7200 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg 7260 atatctcggt agtgggatac gacgataccg aagatagctc atgttatatc ccgccgttaa 7320 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac 7380 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc agtctcactg gtgaaaagaa 7440 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 7500 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtga 7544 <210> 22 <211> 7544 <212> DNA <213> Construct H9 / +term <400> 22 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa 420 accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg tagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgagga gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840. cagccgaacg accgagcgca gcgagtcagt gagcgagga gcggaaggcg aggtaggga actgccaggc atcaaacta gcagaaggcc cctgacggat ggccttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaata cgtaaaaacc cgcttcggcg ggtttttta tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct tgatatatga gaattattta accttataaaa tgaaaaaa gcaacgcact ttaaataaga 1200 tacgttgctt ttcgattga tgaacaccta taattaaact attcatctat tattatgat 1260 tttttgtata tacaatattt ctagttgtt aaagagaatt aagaaaataa atctcgaaaa 1320 tataaaggg aaaatcagtt tttgatatca aaattataca tgtcaacgat aatacaaaat 1380 ataatacaaa ctataagatg ttatcagtat ttattatgca tttagaataa atttgtgtc 1440 gcccttccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc 1500 ctctagaaat aatttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc 1560 aatgatccgc agttggcagg aacgatgttc cccacccgaa tagaggcgaa tgtctttgac 1620 cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680 gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740 gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800 tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact 1860 gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat 1920 cacgggaaag tactggccgc aaaggagat ggactacctt atgagcttga ccccaaacc 1980 ctggaaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg 2040 caccctaagt tcgaccccca gandaggtgaa atgttactct tcggctccgc tgctaaaggc 2100 gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacatgag 2160 acctggttta agcagcctta cggtgcattc atgcacgact tgctgtcac gcgcaactgg 2220 tcaatctttc cgatcatgcc tgcgacaaat agccttgagc gtctttaagc aaagcagccc 2280 atttacatgt gggagcctga gcgaggaagc tatataggag tacttccctcg tcgtggtcag 2340 ggcaaggaca tcgttggtt ccgtgccccg gcgttgtggg tttccatgt cgtgaatgct 2400 tgggaggaag ggaatagaat tctgattgac ttgatggaa gtgagatttt gccgttccca 2460 ttcccgaact cgcagaacct tccattgat ccctccaagg ctgttccgcg tctaacccgt 2520 tgggaaattg atctcaatag tggtaacgat gagatgaac gtacgcagct acacgaat 2580 tttgcagaaa tgcctatcat ggatttccgt tttgcgctcc aggatcatcg ctacgcctac 2640 atggggggttg acgatcctcg tcgcccctta gctcatcagc aagctgaaaa aatctttgcc 2700. 2760. tacaattcgt tagggtttg ggacaaccat cgtaaagatt atgaactttg gtttacggga aaaatgtctg cagcgcagga accggcgttt gttcctagaa gcccagatgc gcctgagggc 2820. gatggctacc tactcagtgt agtagggcgg ctcgatgaaa atcgtagcga tctagttatc 2880. cttgatacgc aatgtttggc agctgggcct gtggccactg tcaagcttcc cttccgtctc 2940. cgagcagcgt tgcacggttg ttggcagtct aagaactgac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttggca tcctgtccat gactcggtaa tacgactcac 3060 father ttgtgagcgg father cctctgaaa tattttgtt tacttttaa aggagagtta tcaatgaata ttcgtccatt gcatgatcgc gtgatcgtca agcgtaaaga agttgaaact aaatctgctg gcggcatcgt tctgaccggc tctgcagcgg ctaaatccac 3240 ccgcggcga gtgctggctg tcggcaatgg ccgtatcctt gaaaatggcg aagtgaagcc gctggatgtg aaagttggcg acatcgttat tttcaacgat ggctacggtg tgaaatctga 3360 gaagatcgac aatgaagaag tgttgatcat gtccgaaagc gacattctgg caattgttga 3420 agcgtaatcc gcgcacgaca ctgaacatac gaatttaagg aataaagata atggcagcta 3480 aagacgtaaa attcggtaac gacgctcgtg tgaaaatgct gcgcggcgta aacgtactgg 3540 cagatgcagt gaaagttacc ctcggtccaa aaggccgtaa cgtagttctg gataaatctt 3600 tcggtgcacc gaccatcacc aaagatggtg tttccgttgc tcgtgaaatc gaactggaag 3660 acaagttcga aaatatgggt gcgcagatgg tgaaagaagt tgcctctaaa gcaaacgacg 3720 ctgcaggcga cggtaccacc actgcaaccg tactggctca ggctatcatc actgaaggtc 3780 tgaaagctgt tgctgcgggc atgaacccga tggacctgaa acgtggtatc gacaaagcgg 3840 ttaccgctgc agttgaagaa ctgaaagcgc tgtccgtacc atgctctgac tctaaagcga 3900 ttgctcaggt tggtaccatc tccgctaact ccgacgaaac cgtaggtaaa ctgatcgctg 3960 aagcgatgga caaagtcggt aaagaaggcg ttatcaccgt tgaagacggt accggtctgc 4020 aggacgaact ggacgtggtt gaaggtatgc agttcgaccg tggctacctg tctcctact 4080 tcatcaacaa gccggaaact ggcgcagtag aactggaaag cccgttcatc ctgctggctg 4140 acaagaaaat ctccaacatc cgcgaaatgc tgccggttct ggaagctgtt gccaaagcag 4200 gcaaaccgct gctgatcatc gctgaagatg tagaaggcga agcgctggca actctggttg 4260 ttaacaccat gcgtggcatc gtgaaagtcg ctgcggttaa agcaccgggc ttcggcgatc 4320 gtcgtaaagc tatgctgcag gatatcgcaa ccctgactgg cggtaccgtg atctctgaag 4380 agatcggtat ggagctggaa aaagcaaccc tggaagacct gggtcaggct aaacgtgttg 4440 tgatcaacaa agacaccacc actatcatcg atggcgtggg tgaagaagct gcaatccagg 4500 gccgtgttgc tcagatccgt cagcagattg aagaagcaac ttctgactac gaccgtgaaa 4560 aactgcagga acgcgtagcg aaactggcag gcggcgttgc agttatcaaa gtgggtgctg 4620 ctaccgaagt tgaaatgaaa gagaaaaaag cacgcgttga agatgccctg cacgcgaccc 4680 gtgctgcggt agaagaaggc gtggttgctg gtggtggtgt tgcgctgatc cgcgtagcgt 4740 ctaaactggc tgacctgcgt ggtcagaacg aagaccagaa cgtgggtatc aaagttgcac 4800 tgcgtgcaat ggaagctccg ctgcgtcaga tcgtattgaa ctgcggcgaa gaaccgtctg 4860 ttgttgctaa caccgttaaa ggcggcgacg gcaactacgg ttacaacgca gcaaccgaag 4920 aatacggcaa catgatcgac atgggtatcc tggatccaac caaagtaact cgttctgctc 4980 tgcagtacgc agcttctgtg gctggcctga tgatcaccac cgaatgcatg gttaccgacc 5040 tgccgaaaaa cgatgcagct gacttaggcg ctgctggcgg tatgggcggc atgggtggca 5100 tgggcggcat gatgtaaccc cctagcataa ccccttgggg cctctaaacg ggtcttgagg 5160 ggttttttgc ccctgagacg cgtcaatcga gttcgtacct aagggcgaca ccccctaatt 5220 agcccgggcg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctggcag 5280 ttccctactc tcgcatgggg agtccccaca ctaccatcgg cgctacggcg tttcacttct 5340 gagttcggca tggggtcagg tgggaccacc gcgctactgc cgccaggcaa acaaggggtg 5400 ttatgagcca tattcaggta taaatgggct cgcgataatg ttcagaattg gttaattggt 5460 tgtaacactg acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 5520 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagaata tgagccatat 5580 tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta 5640 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa 5700 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac 5760 agatgagatg gtcagactaa actggctgac ggaatttatg ccacttccga ccatcaagca 5820 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc 5880 gttccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt 5940 gttcctgcgc cggttgcact cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt 6000 atttcgcctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt 6060 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt 6120 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt 6180 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata 6240 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg 6300 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat 6360 gctcgatgag tttttctaag cggcgcgcca tcgaatggcg caaaaccttt cgcggtatgg 6420 catgatagcg cccggaagag agtcaattca gggtggtgaa tatgaaacca gtaacgttat 6480 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg 6540 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt 6600 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg 6660 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg 6720 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct 6780 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc 6840 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat 6900 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaggacggta 6960 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg 7020 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc 7080 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc 7140 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg 7200 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg 7260 atatctcggt agtgggatac gacgataccg aagatagctc atgttatatc ccgccgttaa 7320 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac 7380 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc agtctcactg gtgaaaagaa 7440 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 7500 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtga 7544 <210> 23 <211> 7545 <212> DNA <213> Construct G6 / +term <400> 23 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa 420 accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg taagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgaggaa gcggaatata tcctgtatca 720 catattctgc tgacgcaccg gtgcagcctt tttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaaggcg agagtaggga 900 actgccaggc atcaaactaa gcagaaggcc cctgacggat ggcctttttg cgtttctaca 960 aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct 1020 gataacgagt aatcgttaat ccgcaaataa cgtaaaaacc cgcttcggcg ggttttttta 1080 tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct 1140 tgatatatga gaattattta accttataaa tgaaaaaa gcaacgcact ttaaataaga 1200 tacgttgctt ttcgattga tgaacaccta taattaaact attcatctat tattatgat 1260 tttttgtata tacaatattt ctagttgtt aaagagaatt aagaaaataa atctcgaaaa 1320 tataaaggg aaaatcagtt tttgatatca aaattataca tgtcaacgat aatacaaaat 1380 ataatacaaa ctataagatg ttatcagtat ttattatgca tttagaataa atttgtgtc 1440 gcccttccgc gather cgactcacta tag gtgagcggat aacaattccc ctctagaaat aattttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc aatgatccgc agttggcagg aacgatgttc cccacccga tagaggcga tgtctttgac cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680. gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740. gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800. tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat cacgggaag tactggccgc aaaggagat ggactacctt atgagcttga cccccaaacc ctggaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg caccctaagt tcgaccccca gacaggtgaa atgttactct tcggctccgc tgctaaaggc gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacacatgag acctggttta agcagcctta cggtgcattc atgcacgact ttgctgtcac gcgcaactgg 2220 tcaatctttc cgatcatgcc tgcgacaaat agccttgagc gtcttaaagc aaagcagccc 2280 atttacatgt gggagcctga gcgaggaagc tatataggag tacttcctcg tcgtggtcag 2340 ggcaaggaca ttcgttggtt ccgtgccccg gcgttgtggg ttttccatgt cgtgaatgct 2400 tgggaggaag ggaatagaat tctgattgac ttgatggaaa gtgagatttt gccgttccca 2460 ttcccgaact cgcagaacct tccatttgat ccctccaagg ctgttccgcg tctaacccgt 2520 tgggaaattg atctcaatag tggtaacgat gagatgaaac gtacgcagct acacgaatat 2580 tttgcagaaa tgcctatcat ggatttccgt tttgcgctcc aggatcatcg ctacgcctac 2640 atggggttg acgatcctcg tcgcccctta gctcatcagc aagctgaaaa aatctttgcc 2700 tacaattcgt taggggtttg ggacaccat cgtaaagatt atgaactttg gtttacggga 2760 aaaatgtctg cagcgcagga accggcgttt gttcctagaa gcccagatgc gcctgagggc 2820 gatggctacc tactcagtgt agtagggcgg ctcgatgaaa atcgtagcga tctagttatc 2880 cttgatacgc aatgtttggc agctggggcct gtggccactg tcaagcttcc cttccgtctc 2940 cgagcagcgt tgcacggttg ttggcagtct aagaactgac tagcataacc ccttggggcc 3000 tctaaacggg tcttgagggg ttttttggca tccctgtcca tgactcggta atacgactca 3060 ctatttcgga attgtgagcg gataacaatt ccctctagaa ataattttgt ttaactttta 3120 aaggagtt atcaatgaat attcgtccat tgcatgatcg cgtgatcgtc aagcgtaaag 3180 aagttgaaac taaatctgct ggcggcatcg ttctgaccgg ctctgcagcg gctaaatcca 3240 cccgcggcga agtgctggct gtcggcaatg gccgtatcct tgaaaatggc gaagtgaagc 3300 cgctggatgt gaaagttggc gacatcgtta ttttcaacga tggctacggt gtgaaatctg 3360 3420 aagcgtaatc cgcgcacgac actgaacata cgaatttaag gaataaagat aatggcagct 3480 aaagacgtaa aattcggtaa cgacgctcgt gtgaaaatgc tgcgcggcgt aaacgtactg 3540 gcagatgcag tgaaagttac cctcggtcca aaaggccgta acgtagttct ggataaatct 3600 ttcggtgcac cgaccatcac caaagatggt gtttccgttg ctcgtgaaat cgaactggaa 3660 gacaagttcg aaaatatggg tgcgcagatg gtgaaagaag ttgcctctaa agcaaacgac 3720 gctgcaggcg acggtaccac cactgcaacc gtactggctc aggctatcat cactgaaggt 3780 ctgaaagctg ttgctgcggg catgaacccg atggacctga aacgtggtat cgacaaagcg 3840 gttaccgctg cagttgaaga actgaaagcg ctgtccgtac catgctctga ctctaaagcg 3900 attgctcagg ttggtaccat ctccgctaac tccgacgaaa ccgtaggtaa actgatcgct 3960 gaagcgatgg acaaagtcgg taaagaaggc gttatcaccg ttgaagacgg taccggtctg 4020 caggacgaac tggacgtggt tgaaggtatg cagttcgacc gtggctacct gtctccttac 4080 ttcatcaaca agccggaaac tggcgcagta gaactggaaa gcccgttcat cctgctggct 4140 gacaagaaaa tctccaacat ccgcgaaatg ctgccggttc tggaagctgt tgccaaagca 4200 ggcaaaccgc tgctgatcat cgctgaagat gtagaaggcg aagcgctggc aactctggtt 4260 gttaacacca tgcgtggcat cgtgaaagtc gctgcggtta aagcaccggg cttcggcgat 4320 cgtcgtaaag ctatgctgca ggatatcgca accctgactg gcggtaccgt gatctctgaa 4380 gagatcggta tggagctggga aaaagcaacc ctggaagacc tgggtcaggc taaacgtgtt 4440 gtgatcaaca aagacaccac cactatcatc gatggcgtgg gtgaagaagc tgcaatccag 4500 ggccgtgttg ctcagatccg tcagcagatt gaagaagcaa cttctgacta cgaccgtgaa 4560 aaactgcagg aacgcgtagc gaaactggca ggcggcgttg cagttatcaa agtgggtgct 4620 gctaccgaag ttgaaatgaa agaaaaaa gcacgcgttg aagatgccct gcacgcgacc 4680 cgtgctgcgg tagagaagg cgtggttgct ggtggtggtg ttgcgctgat ccgcgtagcg 4740 tctaaactgg ctgacctgcg tggtcagaac gaagaccaga acgtgggtat caaagttgca 4800 ctgcgtgcaa tggaagctcc gctgcgtcag atcgtattga actgcggcga agaaccgtct 4860 gttgttgcta acaccgttaa aggcggcgac ggcaactacg gttacaacgc agcaaccgaa 4920 gaatacggca acatgatcga catgggtatc ctggatccaa ccaaagtaac tcgttctgct 4980 ctgcagtacg cagcttctgt ggctggcctg atgatcacca ccgaatgcat ggttaccgac 5040 ctgccgaaaa acgatgcagc tgacttaggc gctgctggcg gtatgggcgg catgggtggc 5100 atgggcggca tgatgtaacc ccctagcata accccttggg gcccttaaac gggtcttgag 5160 gggttttttg cccctgagac gcgtcaatcg agttcgtacc taagggcgac acccccctaat 5220 tagcccgggc gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggca 5280 gttccctact ctcgcatggg gagtccccac actaccatcg gcgctacggc gtttcacttc 5340 tgagttcggc atggggtcag gtgggaccac cgcgctactg ccgccaggca aacaaggggt 5400 gttatgagcc atattcaggt ataaatgggc tcgcgataat gttcagaatt ggttaattgg 5460 ttgtaacact gacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 5520 tgagacaata accctgataa atgcttcaat aatattgaaa aaagaagaat atgagccata 5580 ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt 5640 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga 5700 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta 5760 cagatgagat ggtcagacta aactggctga cggaatttat gccacttccg accatcaagc 5820 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag 5880 cgttccaggt attagaaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag 5940 tgttcctgcg ccggttgcac tcgattcctg tttgtaattg tccttttaac agcgatcgcg 6000 tatttcgcct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt 6060 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt 6120 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt 6180 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat 6240 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac 6300 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga 6360 tgctcgatga gttttctaa gcggcgcgcc atcgaatggc gcaaaacctt tcgcggtatg 6420 gcatgatagc gcccggaaga gagtcaattc agggtggtga atatgaaacc agtaacgtta 6480 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag 6540 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat 6600 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt 6660 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc 6720 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc 6780 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat 6840 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta 6900 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaggacggt 6960 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg 7020 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact 7080 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt 7140 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac 7200 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg 7260 gatatctcgg tagtgggata cgacgatacc gaagatagct catgttatat cccgccgtta 7320 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 7380 ctctctcagg gccaggcggt gaagggcaat cagctgttgc cagtctcact ggtgaaaaga 7440 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 7500 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtga 7545 <210> 24 <211> 7544 <212> DNA <213> Construct C4 / +term <400> 24 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtgga tgagacaaac gcggccata cagcggatg acaccggta accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg tagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgagga gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840. cagccgaacg accgagcgca gcgagtcagt gagcgagga gcggaaggcg aggtaggga actgccaggc atcaaacta gcagaaggcc cctgacggat ggccttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaaataa cgtaaaaacc cgcttcggcg ggttttttta 1080 tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct 1140 tgatatatga gaattattta accttataaaa tgaaaaaa gcaacgcact ttaaataaga 1200 tacgttgctt ttcgattga tgaacaccta taattaaact attcatctat tattatgat 1260 tttttgtata tacaatattt ctagttgtt aaagagaatt aagaaaataa atctcgaaaa 1320 tataaaggg aaaatcagtt tttgatatca aaattataca tgtcaacgat aatacaaaat 1380 ataatacaaa ctataagatg ttatcagtat ttattatgca tttagaataa atttgtgtc 1440 gcccttccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc 1500 ctctagaaat aatttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc 1560 aatgatccgc agttggcagg aacgatgttc cccacccgaa tagaggcgaa tgtctttgac 1620 cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680 gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740 gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800. tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat cacgggaag tactggccgc aaaggagat ggactacctt atgagcttga cccccaaacc ctggaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg caccctaagt tcgaccccca gacaggtgaa atgttactct tcggctccgc tgctaaaggc gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacacatgag acctggttta agcagcctta cggtgcattc atgcacgact ttgctgtcac gcgcaactgg 2220 tcaatctttc cgatcatgcc tgcgacaaat agccttgagc gtcttaagc aaagcagccc atttacatgt gggagcctga gcgaggaagc tatataggag tacttcctcg tcgtggtcag 2340. ggcaaggaca ttcgttggtt ccgtgccccg gcgttgtggg ttttccatgt cgtgaatgct tgggaggag ggatagaat tctgattgac ttgatggaa gtgagatttt gccgttccca ttcccgaact cgcagaacct tccatttgat ccctccaagg ctgttccgcg tctaacccgt 2520 tgggaaattg atctcaatag tggtaacgat gagatgaaac gtacgcagct acacgaatat 2580 tttgcagaaa tgcctatcat ggatttccgt tttgcgctcc aggatcatcg ctacgcctac 2640 atgggggttg acgatcctcg tcgcccctta gctcatcagc aagctgaaaa aatctttgcc 2700 tacaattcgt taggggtttg ggacaaccat cgtaaagatt atgaactttg gtttacggga 2760 aaaatgtctg cagcgcagga accggcgttt gttcctagaa gcccagatgc gcctgagggc 2820 gatggctacc tactcagtgt agtagggcgg ctcgatgaaa atcgtagcga tctagttatc 2880 cttgatacgc aatgtttggc agctgggcct gtggccactg tcaagcttcc cttccgtctc 2940 cgagcagcgt tgcacggttg ttggcagtct aagaactgac tagcataacc ccttggggcc 3000 tctaaacggg tcttgagggg ttttttggca tcctgtccat gactcggtaa tacgactcac 3060 tatcaaggaa ttgtgagcgg ataacaattc cctctagaaa taattttgtt taacttttaa 3120 aggagagtta tcaatgaata ttcgtccatt gcatgatcgc gtgatcgtca agcgtaaaga 3180 agttgaaact aaatctgctg gcggcatcgt tctgaccggc tctgcagcgg ctaaatccac 3240 ccgcggcgaa gtgctggctg tcggcaatgg ccgtatcctt gaaaatggcg aagtgaagcc 3300 gctggatgtg aaagttggcg acatcgttat tttcaacgat ggctacggtg tgaaatctga 3360 gaagatcgac aatgaagaag tgttgatcat gtccgaaagc gacattctgg caattgttga 3420 agcgtaatcc gcgcacgaca ctgaacatac gaatttaagg aataaagata atggcagcta 3480 aagacgtaaa attcggtaac gacgctcgtg tgaaaatgct gcgcggcgta aacgtactgg 3540 cagatgcagt gaaagttacc ctcggtccaa aaggccgtaa cgtagttctg gataaatctt 3600 tcggtgcacc gaccatcacc aaagatggtg tttccgttgc tcgtgaaatc gaactggaag 3660 acaagttcga aaatatgggt gcgcagatgg tgaaagaagt tgcctctaaa gcaaacgacg 3720 ctgcaggcga cggtaccacc actgcaaccg tactggctca ggctatcatc actgaaggtc 3780 tgaaagctgt tgctgcgggc atgaacccga tggacctgaa acgtggtatc gacaaagcgg 3840 ttaccgctgc agttgaagaa ctgaaagcgc tgtccgtacc atgctctgac tctaaagcga 3900 ttgctcaggt tggtaccatc tccgctaact ccgacgaaac cgtaggtaaa ctgatcgctg 3960 aagcgatgga caaagtcggt aaagaaggcg ttatcaccgt tgaagacggt accggtctgc 4020 aggacgaact ggacgtggtt gaaggtatgc agttcgaccg tggctacctg tctcctact 4080 tcatcaacaa gccggaaact ggcgcagtag aactggaaag cccgttcatc ctgctggctg 4140 acaagaaaat ctccaacatc cgcgaaatgc tgccggttct ggaagctgtt gccaaagcag 4200 gcaaaccgct gctgatcatc gctgaagatg tagaaggcga agcgctggca actctggttg 4260 ttaacaccat gcgtggcatc gtgaaagtcg ctgcggttaa agcaccgggc ttcggcgatc 4320 gtcgtaaagc tatgctgcag gatatcgcaa ccctgactgg cggtaccgtg atctctgaag 4380 agatcggtat ggagctggaa aaagcaaccc tggaagacct gggtcaggct aaacgtgttg 4440 tgatcaacaa agacaccacc actatcatcg atggcgtggg tgaagaagct gcaatccagg 4500 gccgtgttgc tcagatccgt cagcagattg aagaagcaac ttctgactac gaccgtgaaa 4560 aactgcagga acgcgtagcg aaactggcag gcggcgttgc agttatcaaa gtgggtgctg 4620 ctaccgaagt tgaaatgaaa gagaaaaaag cacgcgttga agatgccctg cacgcgaccc 4680 gtgctgcggt agaagaaggc gtggttgctg gtggtggtgt tgcgctgatc cgcgtagcgt 4740 ctaaactggc tgacctgcgt ggtcagaacg aagaccagaa cgtgggtatc aaagttgcac 4800 tgcgtgcaat ggaagctccg ctgcgtcaga tcgtattgaa ctgcggcgaa gaaccgtctg 4860 ttgttgctaa caccgttaaa ggcggcgacg gcaactacgg ttacaacgca gcaaccgaag 4920 aatacggcaa catgatcgac atgggtatcc tggatccaac caaagtaact cgttctgctc 4980 tgcagtacgc agcttctgtg gctggcctga tgatcaccac cgaatgcatg gttaccgacc 5040 tgccgaaaaa cgatgcagct gacttaggcg ctgctggcgg tatgggcggc atgggtggca 5100 tgggcggcat gatgtaaccc cctagcataa ccccttgggg cctctaaacg ggtcttgagg 5160 ggttttttgc ccctgagacg cgtcaatcga gttcgtacct aagggcgaca ccccctaatt 5220 agcccgggcg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctggcag 5280 ttccctactc tcgcatgggg agtccccaca ctaccatcgg cgctacggcg tttcacttct 5340 gagttcggca tggggtcagg tgggaccacc gcgctactgc cgccaggcaa acaaggggtg 5400 ttatgagcca tattcaggta taaatgggct cgcgataatg ttcagaattg gttaattggt 5460 tgtaacactg acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 5520 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagaata tgagccatat 5580 tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta 5640 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa 5700 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac 5760 agatgagatg gtcagactaa actggctgac ggaatttatg ccacttccga ccatcaagca 5820 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc 5880 gttccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt 5940 gttcctgcgc cggttgcact cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt 6000 atttcgcctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt 6060 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt 6120 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt 6180 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata 6240 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg 6300 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat 6360 gctcgatgag tttttctaag cggcgcgcca tcgaatggcg caaaaccttt cgcggtatgg 6420 catgatagcg cccggaagag agtcaattca gggtggtgaa tatgaaacca gtaacgttat 6480 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg 6540 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt 6600 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg 6660 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg 6720 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct 6780 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc 6840 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat 6900 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaggacggta 6960 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg 7020 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc 7080 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc 7140 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg 7200 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg 7260 atatctcggt agtgggatac gacgataccg aagatagctc atgttatatc ccgccgttaa 7320 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac 7380 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc agtctcactg gtgaaaagaa 7440 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 7500 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtga 7544 <210> 25 <211> 7405 <212> DNA <213> Construct PC1_triple <400> 25 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa 420 accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg tagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgagga gcggaatata tcctgtatca catattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtcccg 840. cagccgaacg accgagcgca gcgagtcagt gagcgagga gcggaaggcg aggtaggga actgccaggc atcaaacta gcagaaggcc cctgacggat ggccttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaata cgtaaaaacc cgcttcggcg ggtttttta tggggggagt ttagggaaag agcatttgtc agaatattta agggcgcctg tcactttgct tgatatatga gattatta accttataaa tgagaaaaaa gcaacgcact ttaataaga tacgttgctt tttcgattga tgaacaccta taattaaact attcatctat tatttatgat tttttgtata tacaatattt ctagtttgtt aaagagaatt aagaaaata atctcgaaaa fatheraggg aaaatcagtt tttgatatca aaattataca tgtcaacgat fathercaaaat father ctataagatg ttatcagtat ttattatgca tttagaata attttgtgtc gcccttccgc gather cgactcacta tag gtgagcggat aacaattccc ctctagaaat aattttgttt aacttttgaa ggagatatac atatggcaac gtttgaccgc aatgatccgc agttggcagg aacgatgttc cccacccga tagaggcga tgtctttgac cttgaaattg agggcgagat cccacgtgca atcaacggga gcttcttccg caacaccccc 1680. gaacctcagg tcaccccgca acctttccac accttcatcg atggggatgg tttggcgtct 1740. gcttttcatt tcgaagatgg ccatgtcgac tttgtcagcc gttgggtatg tactcctcgc 1800. tttgaagctg agcggtcggc tcgtaaatca ctcttcggta tgtaccgcaa tccgttcact gatgatccat cggtagaagg tattgatcgt acagtcgcca acaccagtat catcactcat cacgggaag tactggccgc aaaggagat ggactacctt atgagcttga cccccaaacc ctggaaccc gaggtcgtta tgattacaag gggcaggtaa ccagccatac acatacagcg caccctaagt tcgaccccca gacaggtgaa atgttactct tcggctccgc tgctaaaggc 2100 gaacgaacgc ttgatatggc gtactatatt gttgatcgct acggcaaggt gacacatgag 2160 acctggttta agcagcctta cggtgcattc atgcacgact ttgctgtcac gcgcaactgg 2220 tcaatc...

Claims

1. An expression system for recombinantly expressing a polypeptide having isoeugenol oxidation activity, the expression system comprising a single nucleic acid construct with the following sequence: a. a nucleotide sequence A encoding a polypeptide having enzymatic isoeugenol oxidation activity, and b. at least one nucleotide sequence B encoding at least one auxiliary polypeptide that assists the functional expression of the polypeptide encoded by the nucleotide sequence A alone or synergistically; wherein the expression system provides for the co-expression of the nucleotide sequences A and B, The amino acid sequence of the polypeptide having isoeugenol oxidation activity is SEQ ID NO: 4, and the mutation in the amino acid sequence of the polypeptide is: a. Single mutant T52X1; single mutant Q74X2; single mutant D440X3; b. Double mutant T52X1, Q74X2; Double mutant T52X1, D440X3; Double mutant Q74X2, D440X3; or c. Triple mutant T52X1, Q74X2, D440X3, in X1 is P; X2 is H; and X3 is N, A, C, G, H, Q, or S.

2. The expression system of claim 1 which is a single polycistronic nucleic acid construct.

3. The expression system of any of the preceding claims, wherein the at least one nucleotide sequence B comprises nucleotide sequences B1 and B2, wherein a nucleotide sequence B1 encoding a polypeptide having chaperone activity, the amino acid sequence of the polypeptide is SEQ IDNO: 8; and b. The nucleotide sequence B2 encodes a polypeptide having molecular chaperone activity, the amino acid sequence of which is SEQ ID NO:

10.

4. A polypeptide having isoeugenol oxidation activity, wherein the amino acid sequence is SEQ ID NO: 4, and the mutation in the amino acid sequence of the polypeptide is: a. Single mutant T52X1; single mutant Q74X2; single mutant D440X3; b. Double mutant T52X1, Q74X2; Double mutant T52X1, D440X3; Double mutant Q74X2, D440X3; or c. Triple mutant T52X1, Q74X2, D440X3, in X1 is P; X2 is H; and X3 is N, A, C, G, H, Q, or S.

5. A recombinant nucleic acid, the nucleotide sequence of which encodes the polypeptide of claim 4. A recombinant nucleic acid construct comprising the recombinant nucleic acid of claim 5 or its reverse complementary sequence.

7. An expression vector comprising: a. a single nucleic acid construct comprised by the recombinant expression system of any one of claims 1 to 3, or its reverse complement; or b. the recombinant nucleic acid of claim 5, or its reverse complementary sequence; or c. The recombinant nucleic acid construct of claim 6, or its reverse complementary sequence.

8. A host cell selected from a prokaryotic or eukaryotic microorganism and comprising the following substances, which are optionally stably integrated into its genome, a. a single nucleic acid construct comprised by the recombinant expression system of any one of claims 1 to 3; or b. the recombinant nucleic acid of claim 5; or c. the recombinant nucleic acid construct of claim 6; or d. The expression vector of claim 7.

9. A method for producing an isolated catalytically active polypeptide having isoeugenol oxidation activity, the method comprising co-expressing in a host cell system said polypeptide having isoeugenol oxidation activity and at least one auxiliary polypeptide, each of which is encoded by an expression system as defined in any one of claims 1 to 3; and optionally, isolating said polypeptide having isoeugenol oxidation activity.

10. A method for producing vanillin, comprising: a. in the presence of oxygen, contacting isoeugenol with a polypeptide having isoeugenol oxidation activity as defined in claim 4 or a polypeptide having isoeugenol oxidation activity prepared by the method of claim 9 to produce vanillin; and b. Optionally, isolating the vanillin produced in step a.

11. The process of claim 10, further comprising the step of chemically or biochemically isomerizing eugenol to isoeugenol and, optionally, separating the isoeugenol from the reaction medium.

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