CN111100886B

CN111100886B - Biosynthesis method of N-methyl pyrroline

Info

Publication number: CN111100886B
Application number: CN201811265624.0A
Authority: CN
Inventors: 肖友利; 平羽; 李晓东; 周志华
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2018-10-29
Filing date: 2018-10-29
Publication date: 2021-07-20
Anticipated expiration: 2038-10-29
Also published as: CN111100886A

Abstract

The invention provides a method for preparing N-methylpyrrolidine by three-enzyme combined catalysis, which comprises the following steps: the L-ornithine is taken as a substrate, and is obtained by combined catalysis of ornithine decarboxylase EcODC, putrescine-N-methyltransferase AtPMT and amine oxidase AaDAO 3. The above method can also be realized by fermentation of genetically engineered bacteria expressing these three enzymes. The method of the invention is environment-friendly, has mild reaction conditions and has industrial development prospect.

Description

Biosynthesis method of N-methyl pyrroline

Technical Field

The invention belongs to the technical field of biocatalysis and metabolic engineering, and particularly relates to a biosynthesis method of N-methylpyrrolidine.

Background

The compound N-methylpyrrolidinium (shown as formula I) mainly exists in the form of salt, and the molecular formula is C₅H₁₀N⁺In equilibrium with N-methylaminobutyraldehyde under certain solution environments) are common key intermediates in the biosynthetic pathway of plant-derived nicotine and plant natural product drug, tropine alkaloids. The N-methylpyrrolidine biosynthetic route starting from L-ornithine is reported ((Chase et al 2003;

2004；

et al.2005; ruetsch et al.2001.), see fig. 1, comprising three steps: l-ornithine is catalyzed by ornithine decarboxylase to generate putrescine, the putrescine is catalyzed by N-methyltransferase to generate N-methylated putrescine, the N-methylated putrescine is further catalyzed by amine oxidase to generate N-methylamino butyraldehyde, and the N-methylamino butyraldehyde spontaneously forms ring through the spontaneous reaction of Schiff base to form N-methylpyrrolidine.

At present, nicotine and tropine alkaloids taking N-methylpyrrolidine as a biosynthesis intermediate are mainly extracted from plants. In the synthetic biology technology developed in recent years, microbial strains are used as chassis cells, genes related to biosynthesis pathways of target compounds are introduced, heterologous high-efficiency synthesis (Kotopka et al.2018; Li et al.2018.) of some important medicinal compounds from plant sources is realized, and the establishment of the biosynthesis method of N-methylpyrrolidine can not only make up for the defects of the chemical synthesis method, but also provides a basis for pathway analysis and heterologous synthesis of nicotine and tropine alkaloid taking N-methylpyrrolidine as an intermediate.

In the biosynthetic pathway of N-methylpyrrolidine, only the metabolism of putrescine is engineered in the microbial host (Nguyen et al 2015; Qian et al 2010). Related enzymes in the biosynthetic pathway of N-methylpyrrolidine are also identified in many species at present (Docimo et al 2012; Heim et al 2007; Liu et al 2005), and since the solubility of tobacco-derived amine oxidases in the large intestine is low, amine oxidases in other species were found and identified as key steps in achieving the biosynthesis of N-methylpyrrolidine. The thirteen-thirds plant of solanaceae is a unique resource plant for producing toline alkaloid in China, also called wild , the content of total alkaloids in a dry product is up to 1.2 percent, the total content of the toline alkaloid is higher than that of common solanaceae plants such as belladonna, hyoscyamine, stramonium and the like (Kai et al.2012), and the transcriptome data of the solanaceae plants is published (Cui et al.2015.), so that the function of amine oxidase (AaDAOs) of the thirteen-thirds plant is researched.

As is well known, the chemical synthesis of N-methyl pyrroline has the problem of great environmental pollution, so that the development of an environment-friendly green preparation process is necessary. Therefore, biological preparation methods such as bio-enzyme catalysis methods, which have the advantages of mild reaction conditions, high catalytic efficiency, few byproducts, and the like, are attractive and promising methods.

Disclosure of Invention

In order to realize the biosynthesis of N-methylpyrrolidine, cocaine-derived ornithine decarboxylase (EcODC) with known function and high activity, putrescine-N-methyltransferase (AtPMT) from Shan downy and amine oxidase (AaDAOs) from Satsubishi are selected to carry out in-vitro co-catalysis reaction of a substrate L-ornithine, and the feasibility of the selected enzyme combination is verified. Because the genetic background of the large intestine and yeast is clear and the genetic manipulation is easy, we select the recombinant DNA as a chassis cell to explore the in vivo de novo synthesis of N-methylpyrrolidine, and hopefully provide a foundation for the industrial production of N-methylpyrrolidine and the heterologous synthesis of important alkaloids from N-methylpyrrolidine sources.

Specifically, the invention provides a method for preparing N-methylpyrrolidine, which comprises the following steps: the L-ornithine is taken as a substrate, and is obtained by carrying out combined catalysis on ornithine decarboxylase EcODC with an amino acid sequence of SEQ ID NO. 2, putrescine-N-methyltransferase AtPMT with an amino acid sequence of SEQ ID NO. 6 and amine oxidase from acutangular anisodus sources.

Preferably, pyridoxal phosphate aldehyde PLP, S-adenosylmethionine SAM and CuSO are also added into the reaction system₄。

The amine oxidase derived from anisodus acutangula is preferably AaDAO1 having an amino acid sequence of SEQ ID NO. 10, AaDAO2 having an amino acid sequence of SEQ ID NO. 14, or AaDAO3 having an amino acid sequence of SEQ ID NO. 18. More preferably, the amine oxidase is AaDAO3 having the amino acid sequence of SEQ ID NO 18.

According to a second aspect of the present invention, there is provided a microorganism expressing the ornithine decarboxylase EcODC, putrescine-N-methyltransferase attpmt and amine oxidase described above.

Preferably, the microorganism is selected from the group consisting of Escherichia coli and Saccharomyces cerevisiae.

When the microorganism is Escherichia coli, it is preferable that the EcODC gene and the AtPMT gene are constructed on one plasmid and the amine oxidase gene is constructed on the other plasmid, and the two plasmids are transformed into Escherichia coli to form a genetically engineered bacterium for producing N-methylpyrrolidine.

The above-mentioned Escherichia coli is preferably BL21(DE 3).

Preferably, the plasmid for cloning the EcODC gene and AtPMT gene is pET24a or pACYCDuet-1, and the plasmid for cloning the amine oxidase AaDAO1, AaDAO2 or AaDAO3 gene is pET30 a. Preferably the amine oxidase is AaDAO 3.

When the microorganism is saccharomyces cerevisiae, the EcODC gene and the AtPMT gene are preferably constructed on one plasmid and transformed into saccharomyces cerevisiae to obtain saccharomyces cerevisiae containing the EcODC gene and the AtPMT gene; the amine oxidase gene AaDAO1, AaDAO2 or AaDAO3 gene is integrated into the genome of Saccharomyces cerevisiae containing EcODC and AtPMT genes to form a genetically engineered bacterium for producing N-methylpyrrolidine.

The Saccharomyces cerevisiae is preferably BY 4742.

Preferably, the plasmid used to clone the EcODC gene and the AtPMT gene is the plasmid p2M (constructed in the Zhou Shiwa laboratory, the Shanghai Life sciences research institute of Chinese academy of sciences) having the sequence of SEQ ID NO: 35.

In a preferred embodiment, the microorganism is Saccharomyces cerevisiae and the genes ALD4, ALD5 and HFD1 metabolizing N-methylaminobutyraldehyde in the genome are knocked out.

Preferably, the saccharomyces cerevisiae overexpresses the cofactor synthesis gene SAM 2.

The preferred sequence of the cofactor synthesizing gene SAM2 is SEQ ID NO: 84.

According to a second aspect of the present invention, there is provided the use of the above-mentioned microorganism for the preparation of N-methylpyrrolidine.

Preferably, N-methylpyrrolidine is produced by fermentation of the above-mentioned microorganisms.

When the microorganism is Escherichia coli, preferably the culture medium is LB medium, or any medium suitable for Escherichia coli fermentation; when the microorganism is Saccharomyces cerevisiae, preferably the medium is YPD medium, or any medium suitable for fermentation by Saccharomyces cerevisiae.

The ornithine decarboxylase EcODC, putrescine-N-methyltransferase AtPMT, and amine oxidases AaDAO1, AaDAO2, and AaDAO3 may be in the form of an enzyme or in the form of a cell containing an enzyme.

Accordingly, the combination of the above three enzymes may be in the form of enzyme + enzyme, enzyme + cell, or cell + cell in the catalytic system.

The three-enzyme combined catalytic system can catalyze L-ornithine reaction to obtain N-methylpyrrolidine by a one-pot method, and escherichia coli expressing ornithine decarboxylase EcODC, putrescine-N-methyltransferase AtPMT and amine oxidase can produce 4.33mg/L of N-methylpyrrolidine through fermentation; the saccharomyces cerevisiae expressing the three enzymes can produce 3.22mg/L of N-methyl pyrroline through fermentation; the saccharomyces cerevisiae which expresses the three enzymes and has genes ALD4, ALD5 and HFD1 knocked out from the genome can generate 9.88mg/L of N-methylpyrrolidine through fermentation; the saccharomyces cerevisiae expressing the three enzymes, knocking out genes ALD4, ALD5 and HFD1 in a genome and over-expressing SAM2 gene can generate 21.92mg/L of N-methylpyrrolidine through fermentation, and has industrial development and application prospects.

Drawings

FIG. 1 shows the biosynthetic pathway from the reaction starting with L-ornithine up to the product N-methylpyrrolidine. The detection reaction of N-methylpyrrolidine to N-methylpyrrolidine is shown in the box.

FIG. 2A shows the mass spectra of the results of AaDAO2 and AaDAO3 reactions catalyzing the conversion of N-methylated putrescine to N-methylpyrrolidine. Wherein (i) is a deuterated N-methylpyrrolidine standard; (ii) AaDAO2 boil control; (iii) is AaDAO2 reaction; (iv) AaDAO3 boil control; (v) is the reaction product of AaDAO 3.

FIG. 2B shows the mass spectrum of the results of the reaction in which EcODC, AtPMT and AaDAO3 mix enzymes jointly catalyze the conversion of L-ornithine to N-methylpyrrolidine. Wherein (i) is a deuterated N-methylpyrrolidine standard; (ii) AtPMT and AaDAO3 blanks; (iii) AaDAO3 blank; (iv) mixing the enzyme reaction products.

FIG. 3 shows SDS-PAGE gel photographs of purified proteins of various enzymes involved in the N-methylpyrrolidine synthesis pathway.

FIG. 4 shows a schematic diagram of construction of an engineered Escherichia coli bacterium producing N-methylpyrrolidine.

FIG. 5 shows the fermentation process and N-methylpyrrolidine yield of Escherichia coli BL 21-OP-A3.

FIG. 6 shows a schematic diagram of construction of engineered N-methylpyrrolidine-producing Saccharomyces cerevisiae.

FIG. 7A shows the fermentation process and N-methylpyrrolidine yield of Saccharomyces cerevisiae engineering bacteria BY 4742-OP-A3.

FIG. 7B shows a graph of fermentation history and N-methylpyrrolidine yield of Saccharomyces cerevisiae engineering bacteria Δ BY 4742-OP-A3.

FIG. 7C shows a graph of fermentation history and N-methylpyrrolidine yield of Saccharomyces cerevisiae engineering bacteria Δ BY4742-OP-A3-SAM 2.

FIG. 8A is the NMR spectrum of N-methylated putrescine (N-methylated putrescine¹H NMR)。

FIG. 8B is the NMR spectrum of N-methylated putrescine (C:)¹³C NMR)。

FIG. 9A is a NMR spectrum of N-methylpyrrolidine (N-methylpyrrolidine¹H NMR)。

FIG. 9B is the NMR spectrum of N-methylpyrrolidine (NMR)¹³C NMR)。

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.

The N-methylpyrrolidine is synthesized by catalyzing L-ornithine for a plurality of reactions by a combined catalytic system consisting of three enzymes in a one-pot method. Wherein the term "combined catalytic system" refers to a combination of ornithine decarboxylase EcODC, putrescine-N-methyltransferase AtPMT and amine oxidase (AaDAO1, AaDAO2 or AaDAO3), including but not limited to a combination of enzyme expression strains.

When used as a biocatalyst for the production of compound I, the enzymes EcODC, attpmt, AaDAO1, AaDAO2 and AaDAO3 of the present invention may take the form of an enzyme or the form of a bacterial cell. The enzyme forms comprise free enzyme and immobilized enzyme, including purified enzyme, crude enzyme, fermentation liquor, carrier-immobilized enzyme and the like. Moreover, techniques for the isolation and purification of these enzymes, including the preparation of immobilized enzymes, are well known to those skilled in the art. The thallus form comprises a viable thallus and a dead thallus, and comprises a freeze-thaw thallus and an immobilized thallus.

When the microorganism fermentation is adopted to produce the N-methylpyrrolidine, the L-ornithine is not required to be added as a substrate, because the L-ornithine is generated in cells in the growth process of escherichia coli and saccharomyces cerevisiae, and the L-ornithine can be used as a carbon source and a nitrogen source in a culture solution to be converted into the N-methylpyrrolidine through metabolism only by fermentation.

In this context, sometimes for the sake of simplicity of description, the names of enzymes such as ornithine decarboxylase EcODC protein will be mixed with the names of the genes (DNA) encoding them, and the skilled person will understand that they represent different substances in different description situations. For example, for EcODC (Gene), when used to describe an ornithine decarboxylase function or class, a protein is referred to; when described as a gene, refers to the gene encoding the ornithine decarboxylase, and so on, as will be readily understood by those skilled in the art.

The enzymes EcODC, attpmt, AaDAO1, AaDAO2, and AaDAO3 used in the present invention are well defined in sequence, and thus those skilled in the art can easily obtain the genes encoding them, expression cassettes and plasmids containing the genes, and transformants containing the plasmids. These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction means well known to those skilled in the art.

The invention also provides a method for detecting the content of the N-methylpyrrolidine by using the NaBD₄And (3) reducing N-methylpyrrolidine by using isotope (preferably 1M dissolved in 0.1M sodium borate buffer solution, and having the pH value of 10.0) to generate deuterated N-methylpyrrolidine, and determining the content of the N-methylpyrrolidine in the liquid by combining liquid phase-mass spectrometry detection. By the detection method, the activity of amine oxidase (namely AaDAO1, AaDAO2 and AaDAO3) for catalyzing the conversion of N-methylated putrescine into N-methylpyrrolidine can be determined; and the activity of the EcODC, AtPMT and amine oxidase (i.e., AaDAO1, AaDAO2 or AaDAO3) in combination catalyzing the conversion of L-ornithine to N-methylpyrrolidine can be determined. The boxes in FIG. 1 show N-methylpyrrolidine and NaBD₄Fig. 2A and 2B show the results of mass spectrometric detection of deuterated N-methylpyrrolidine.

Examples

Materials and methods

The whole gene synthesis, primer synthesis and sequencing in the examples were performed by Jinzhi Biotechnology, Inc., Suzhou.

The molecular biological experiments in the examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, culture medium preparation, and the like, and are mainly performed with reference to "molecular cloning experimental manual" (third edition), sambrook, d.w. rasel (american), translation of huang peitang et al, scientific press, beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.

PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.

LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH 7.0.

YPD medium: 1 wt% yeast extract, 2 wt% peptone, 2 wt% glucose.

YPAD medium: 10.0g/L yeast extract, 20.0g/L peptone, 20.0g/L D-glucose, 0.4g/L adenine sulfate.

HPLC determination conditions: the liquid phase-mass spectrometer is an Agilent 1290UHPLC-Agilent 6545Q-TOF ESI mass spectrum; the column was Agilent C18 (4.6X 150mm,3.5 μm), mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% acetonitrile at a flow rate of 0.35 mL/min. The separation procedure was: 0-1min, 2% B; 1-2min, 2-3% of B; 2-10min, 3-5% B; 10-12min, 5-95% B; 12-15min, 95% B; 15-16min, 95-2% B. Mass spectrometry Using Positive mode, N-Methylpyrroline by NaBD₄After reduction, N-methyl pyrrolidine is generated, and the molecular weight of the target compound is 87.1027.

Example 1 preparation of enzymes related to the biosynthetic pathway of N-Methylpyrroline

1. Preparation of Ornithine decarboxylase EcODC

1.1 Using the EcODC coding gene (SEQ ID NO:1) synthesized by Jinzhi Biotechnology Ltd, Suzhou as a template, PCR amplification was performed with KOD DNA polymerase (94 ℃ C. for 2 min; 98 ℃ C. for 10s, 55 ℃ C. for 30s, 68 ℃ C. for 1 min; 30 cycles; 68 ℃ C. for 7min) using a primer pair (SEQ ID NO:3, 4), and a target DNA fragment was recovered using Axygen gel recovery kit (AxyPrep DNASELL extraction kit).

1.2 digestion of the fragment with NdeI and NotI double-enzyme Gel-cutting from Thermofish for 2h at 37 ℃ and clean recovery of the digested product with AxyPrep PCR clean Kit from AXYGEN, and recovery of the desired fragment with Axygen Gel recovery Kit (AxyPrep DNA Gel Extraction Kit). Connecting with T4DNA ligase from NEB at 20 deg.C for 2h, adding Top10 competent cells into the connection system, ice-cooling for 10min, heat-shocking at 42 deg.C for 1min30s, placing on ice for 5min, adding 1mL LB, shake culturing at 37 deg.C, 200rpm, and incubating for 45 min. Centrifugation was carried out at 12000rpm for 1min, 800. mu.l of the supernatant was discarded, and 200. mu.l of the supernatant was spread on a solid LB plate containing 50. mu.g/mL of kanamycin antibiotic and incubated overnight in an incubator at 37 ℃.

1.3 Single-clone transformants were picked, sequenced, and the correctly sequenced plasmid pET24a-EcODC was transformed into E.coli BL21(DE3) and spread on LB plates containing kanamycin. A single colony was cultured overnight at 37 ℃ and 200rpm in 10mL of LB containing kanamycin antibiotic (50. mu.g/mL). Transferring 10mL of the bacterial liquid into a 1L shake flask, culturing at 37 ℃ and 200rpm until the bacterial liquid is OD₆₀₀About 0.6. mu.L of 1M IPTG (final concentration of 0.5mM) was added and the induction was carried out at 16 ℃ for 20 hours.

1.4 the cells were collected from the shake flask, and 5 times the weight volume of lysis buffer (50mM potassium phosphate, pH 8.0, 150mM NaCl) was added thereto, and crushed by squeezing at 800bar for 3 times. Centrifuge at 18000rpm for 45min at 4 ℃. Mixing the supernatant with a Ni column, incubating for 40min, collecting the effluent supernatant Flow-through (FT), eluting 20 column volumes of the hybrid protein by using a lysis buffer containing 25mM imidazole, eluting the target protein by 250mM, and detecting by SDS-PAGE to obtain the target protein EcODC with high purity, wherein the amino acid sequence of the EcODC is SEQ ID NO: 2.

2. Preparation of putrescine N-methyltransferase AtPMT, amine oxidases AaDAO1, AaDAO2 and AaDAO3

Plasmids pET24a-AtPMT, pET30a-AaDAO1, pET30a-AaDAO2 and pET30a-AaDAO3 were obtained, respectively, according to the same method as the above-mentioned step, and the objective protein was purified.

SDS-PAGE gel electrophoresis of the purified proteins of these enzymes is shown in FIG. 3, and soluble and high-purity proteins EcODC, AaDAO2 and AaDAO3 are obtained, which can be used for the next enzyme activity experiment. Wherein

The gene sequence of AtPMT is SEQ ID NO. 5, obtained by amplification of primer pair (SEQ ID NO:7, 8), and the amino acid sequence of coded enzyme AtPMT is SEQ ID NO. 6.

AaDAO1 gene (SEQ ID NO:9) is obtained by using plant acutangular anisodus cDNA as a template and amplifying the cDNA by using a primer pair (SEQ ID NO:11, 12), and the amino acid sequence of the coded enzyme AaDAO1 is SEQ ID NO: 10.

The AaDAO2 gene (SEQ ID NO:13) is obtained by amplifying the three-thirds cDNA as a template by using a primer pair (SEQ ID NO:15, 16), and the amino acid sequence of the coded enzyme AaDAO2 is SEQ ID NO: 14.

AaDAO3 gene (SEQ ID NO:17) is obtained by using the acutangular anisodus cDNA as a template and amplifying the primer pair (SEQ ID NO:19, 20), and the amino acid sequence of the coded enzyme AaDAO3 is SEQ ID NO: 18.

Example 2AaDAO2, AaDAO3 Activity test and Synthesis of N-Methylpyrroline by in vitro Mixed enzyme reaction

2.1 amine oxidases AaDAO2 and AaDAO3 Activity experiments (100 microliters):

2mM substrate N-methylated putrescine, 20. mu.M CuSO₄50mM potassium phosphate (pH 8.0) and 10. mu.M of an enzyme protein were added to the mixture, and the mixture was made into a 100. mu.l system and reacted at 30 ℃ for 30 min. Add 10. mu.l NaBD₄(mother liquor 1M in 0.1M sodium borate buffer, pH 10.0). The liquid phase-mass spectrum detects that the N-methyl pyrroline can be reduced to generate N-methyl pyrrolidine, and the molecular weight of the target compound is 87.1027. As shown in FIG. 2A, the experimental results show that AaDAO2 and AaDAO3 can both catalyze N-methylated putrescine to generate N-methylpyrrolidine.

2.2EcODC, AtPMT and AaDAO3 mix enzyme reactions (100. mu.l):

5mM substrate L-ornithine, 5mM PLP (pyridoxal phosphate aldehyde), 5mM SAM (S-adenosylmethionine), 20. mu.M CuSO ₄10 μ M EcODC,10 μ M AtPMT, 10 μ M AaDAO 3. The reaction solution was incubated at 30 ℃ for 2h, and the product detection procedure was the same as in the above step 1 experiment. As shown in FIG. 2B, the results of the experiments show that the combination of EcODC, AtPMT and AaDAO3 can catalyze the conversion of L-ornithine to N-methylpyrrolidine in vitro.

Example 3 construction and fermentation of E.coli engineering bacteria producing N-methylpyrrolidine

3.1 PCR amplification (94 ℃ for 2 min; 98 ℃ for 10s, 55 ℃ for 30s, 68 ℃ for 1 min; 30 cycles; 68 ℃ for 7min) with KOD DNA polymerase using the EcODC coding gene (SEQ ID NO:1) as a template and a primer pair (SEQ ID NO:21, 22). And (5) performing agarose gel electrophoresis and recovering the target fragment.

3.2 using BamHI and HindIII to double enzyme digestion to recover fragment and pACYCDuet-1 plasmid, carrying out enzyme digestion for 2h at 37 ℃, cleanly recovering the enzyme digestion product, carrying out gel recovery on the pACYCDuet-1 plasmid after enzyme digestion, and connecting for 2h at 20 ℃ by using T4DNA ligase. E.coli TOP10 competent was added to the ligation system, ice-washed for 10min, heat-shocked at 42 ℃ for 1min30s, placed on ice for 5min, added with 1mL LB, incubated at 37 ℃ for 45min with 200rpm shaking. Centrifugation was carried out at 12000rpm for 1min, 800. mu.l of the supernatant was discarded, and 200. mu.l of the supernatant was spread on a solid LB plate containing 34. mu.g/mL of chloramphenicol, followed by overnight incubation in an incubator at 37 ℃. And (4) selecting a monoclonal transformant for sequencing to obtain a correct plasmid pACYCDuet-EcODC.

3.3 PCR amplification (94 ℃ for 2 min; 98 ℃ for 10s, 55 ℃ for 30s, 68 ℃ for 1 min; 30 cycles; 68 ℃ for 7min) with KOD DNA polymerase using the coding gene (SEQ ID NO:5) of AtPMT as a template and a primer pair (SEQ ID NO:23, 24), agarose gel electrophoresis, and recovering the target fragment.

3.4 the fragments and pACYCDuet-EcODC plasmid were digested at 37 ℃ for 2h with NdeI and KpnI double-enzyme digestion, the desired fragment was recovered from the gel and ligated with T4DNA ligase at 20 ℃ for 2 h. E.coli TOP10 competent was added to the ligation system, ice-washed for 10min, heat-shocked at 42 ℃ for 1min30s, placed on ice for 5min, added with 1mL LB, incubated at 37 ℃ for 45min with 200rpm shaking. Centrifugation was carried out at 12000rpm for 1min, 800. mu.l of the supernatant was discarded, and 200. mu.l of the supernatant was spread on a solid LB plate containing 34. mu.g/mL of chloramphenicol, followed by overnight incubation in an incubator at 37 ℃. And (4) selecting a monoclonal transformant for sequencing to obtain a correct plasmid pACYCDuet-EcODC-AtPMT. The plasmid contains two genes, EcODC and AtPMT.

3.5 mu.l of pACYCDuet-EcODC-AtPMT plasmid was transformed into E.coli BL21(DE3) to obtain strain BL 21-OP. BL21-OP strain was made competent, and plasmid pET30a-AaDAO3 prepared in example 1 was transformed into competent cells to obtain strain BL21-OP-A3, and a schematic diagram of the strain construction is shown in FIG. 4.

3.6 Single colonies of the strain were picked from the plate and shake-cultured overnight in a test tube containing LB liquid medium (containing 34. mu.g/mL chloramphenicol and 50. mu.g/mL kanamycin). Transfer into 50ml LB-containing Erlenmeyer flask to OD₆₀₀To 0.6-0.8, 0.15mM IPTG, and 50. mu.M CuSO were added₄Placing the bacterial liquid in a shaking table at 25 ℃, fermenting at 120rpm, sampling at different time points and detecting OD₆₀₀And N-methylpyrrolidine yield. As shown in FIG. 5, the experimental result shows that the strain can synthesize N-methylpyrrolidine, and the yield reaches 4.33mg/L in 48 h.

Example 4 construction of engineered Saccharomyces cerevisiae producing N-methylpyrrolidine

4.1 construction of BY4742-OP Yeast strains:

4.1.1 the primers and templates shown in Table 1 are used for amplifying EcODC, AtPMT gene, yeast endogenous promoter PGK1(SEQ ID NO:33) and terminator FBA1(SEQ ID NO:34) by a PCR method, and 15-21 bp homologous sequences are introduced between adjacent fragments through the primers. The DNA polymerase is high-fidelity DNA polymerase I-5 from TSINGKE^TM2X HighFidelity Master Mix, the PCR program was set up with reference to the description: 2min at 98 ℃; 35 cycles of 98 ℃ for 15s, 58 ℃ for 10s and 72 ℃ for 1 min; 5min at 72 ℃; keeping the temperature at 10 ℃.

4.1.2 detecting the PCR product by agarose gel electrophoresis, and cutting off a band with the same size with the target DNA under ultraviolet light. Then, the DNA fragment was recovered from the agarose gel using AxyPrep DNASELExtractionkit from AXYGEN. The yeast high copy plasmid p2M (supplied by Zhoujihua laboratory of Shanghai Life sciences research institute of China academy of sciences, full length sequence is SEQ ID NO:35) was digested with both the restriction enzymes BamHI and EcoRI from Takara for 30min, and the digested product was collected by cleaning with AxyPrep PCR clean Kit from AXYGEN. By using the Hieff Clone of Yeasen^TMThe recombinant enzyme of Plus Multi One Step Cloning Kit recombines the recovered gel product and the plasmid of p2M double digestion (the reaction conditions refer to the Kit instructions).

4.1.3 ligation products E.coli TOP10 competent cells were transformed and plated on LB plates supplemented with 100. mu.g/mL ampicillin. Positive transformants were verified by colony PCR and further verified by sequencing, which indicated that the yeast expression plasmid p2M-EcODC-AtPMT was successfully constructed. The plasmid contains two genes, EcODC and AtPMT.

4.1.4 transfer the yeast expression plasmid p2M-EcODC-AtPMT into Saccharomyces cerevisiae BY4742 competence (reference for competence preparation and conversion method (Gietz and Schiestl 2007.), pick out BY4742 single clone to 5mLYPAD liquid culture medium with sterile inoculating loop, culture at 200rpm and 30 ℃, transfer to 50mL YPAD culture medium after 12h-16h, culture at 200rpm and 30 ℃ to OD₆₀₀When the concentration is 2, centrifuging for 5min at 3000g, collecting thalli, and suspending in 25mL sterile water; 3000g, centrifuging for 5min to collect thalli, suspending the thalli in 1mL sterile water, sucking 100 microliters of bacteria liquid into a 1.5mL centrifuge tube, 13000g, centrifuging for 30s, and discarding the supernatant. Preparing a transformation system: mu.L of PEG 3350 (50% w/v), 36. mu.L of 1.0M LiAc, 50. mu.L of Single-stranded carrier DNA (Invitrogen) (2.0mg/mL), 34. mu.L of plasmid p2M-EcODC-AtPMT, in a total volume of 360. mu.L. The transformation system is added into BY4742 competence, the thalli is resuspended, the thalli is thermally shocked at 42 ℃ for 40min, 13000g is centrifuged for 30s, the supernatant is discarded, 1mL of YPD culture medium is added, the temperature is 30 ℃, the rpm is 200, and the mixture is incubated for 2 h. 200. mu.l of the suspension was spread on YPAD plates supplemented with 300. mu.g/mL of hygromycin B (hygromycin B), and cultured at 30 ℃ for 48 hours. And (4) obtaining a positive transformant through yeast colony PCR verification, namely obtaining the BY4742-OP strain.

TABLE 1

Segment names	Form panel	Forward primer	Reverse primer
				EcODC gene	pACYCDuet-EcODC-AtPMT plasmid	SEQ ID NO:25	SEQ ID NO:26
FBA1 terminator	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:27	SEQ ID NO:28
				PGK1 promoter	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:29	SEQ ID NO:30
AtPMT gene	pACYCDuet-EcODC-AtPMT plasmid	SEQ ID NO:31	SEQ ID NO:32

4.2 construction of BY4742-OP-A3 Yeast Strain:

4.2.1 amplification of the transformed fragment by PCR using the primers and templates of Table 2: an upstream homologous sequence (SEQ ID NO:48) of an X-4 site, a TEF1 promoter (SEQ ID NO:49), an AaDAO3 gene, a PRM9 terminator (SEQ ID NO:50), a KanMX expression cassette (SEQ ID NO:51) and a downstream homologous sequence (SEQ ID NO:52) of the X-4 site, wherein 60-80 bp homologous sequences are introduced between adjacent fragments through primers. The DNA polymerase is high-fidelity DNA polymerase I-5 from TSINGKE^TM2X High Fidelity Master Mix, the PCR program was set up with reference to the description: 2min at 98 ℃; 35 cycles of 98 ℃ for 15s, 58 ℃ for 10s and 72 ℃ for 1 min; 5min at 72 ℃; keeping the temperature at 10 ℃.

4.2.2 agarose gel electrophoresis detection is carried out on the PCR product, and a band with the same size with the target DNA is cut off under ultraviolet light. Then, the DNA fragment was recovered from the agarose Gel using AxyPrep DNA Gel Extraction Kit from AXYGEN.

4.2.3 the 6 fragments are transformed into BY4742-OP competence (reference for competence making and transformation method (Gietz and Schiestl 2007)), and spread on YPAD plates added with 300. mu.g/mL hygromycin B (hygromycin B) and 200mg/L G418, and cultured at 30 ℃ for 48h, and positive transformants are obtained through yeast colony PCR verification, namely the BY4742-OP-A3 strain, and genes of three enzymes of EcODC, AtPMT and AaDAO3 are introduced into the saccharomyces cerevisiae.

TABLE 2

Segment names	Form panel	Forward primer	Reverse primer
				Homologous sequence at the upstream of X-4 site	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:36	SEQ ID NO:37
TEF1 promoter	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:38	SEQ ID NO:39
				AaDAO3 gene	pET30a-AaDAO3 plasmid	SEQ ID NO:40	SEQ ID NO:41
PRM9 terminator	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:42	SEQ ID NO:43
				KanMX expression cassette	pMEL13 plasmid	SEQ ID NO:44	SEQ ID NO:45
Downstream homologous sequence of X-4 site	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:46	SEQ ID NO:47

pMEL13 plasmid is a commercial plasmid that can be used to transcribe sgrnas, purchased from euroscaf.

4.3 construction of dehydrogenase knockout strain Δ BY 4742:

4.3.1 design sgRNA sequences (PAM sequences in bold) knocking out Saccharomyces cerevisiae endogenous genes ALD4(SEQ ID NO:53), ALD5(SEQ ID NO:54), HFD1(SEQ ID NO:55) on the Yeast website (https:// yeastraction. tnw. tudelft. nl /): ALD 4: 5'-CTTGTCCTGTTCAATTAATTCGG-3', respectively; ALD 5: 5'-TGGCAAATGATTCTCAATATGGG-3', respectively; HFD 1: 5'-AGGGTAAAATCATTCCAATATGG-3' are provided.

Using the primers and templates shown in Table 3, a fragment of CAS9 gene (SEQ ID NO:56) was amplified by PCR, and 21bp homologous sequences were introduced at both ends via the primers. The DNA polymerase is high-fidelity DNA polymerase I-5 from TSINGKE^TM2X High Fidelity Master Mix, the PCR program was set up with reference to the description: 2min at 98 ℃; 15s at 98 ℃,10 s at 58 ℃ and 1.5mi at 72 DEG Cn, 35 cycles; 5min at 72 ℃; keeping the temperature at 10 ℃.

4.3.2 agarose gel electrophoresis detection is carried out on the PCR product, and a band with the same size with the target DNA is cut off under ultraviolet light. Then, the DNA fragment was recovered from the agarose Gel using AxyPrep DNA Gel Extraction Kit from AXYGEN. The yeast high copy plasmid p2M (provided by Zhou Shiwa laboratory of Shanghai Life sciences institute of Chinese academy of sciences, full length sequence shown in SEQ ID NO:43) was double digested with the restriction enzymes BamHI and XhoI from Takara for 30min, and the digested product was recovered by cleaning with AxyPrep PCR clean Kit from AXYGEN. By using the Hieff Clone of Yeasen^TMThe recombinant enzyme of Plus Mlti One Step Cloning Kit recombines and ligates the recovered gel product with the p2M double-digested plasmid.

4.3.3 recombinant products were transformed into E.coli TOP10 competent cells and plated on LB plates supplemented with 100. mu.g/mL ampicillin. Positive transformants were verified by colony PCR and further verified by sequencing, indicating that the construction of the CAS9 expression plasmid p2M-CAS9 was successful.

TABLE 3

Segment names	Form panel	Forward primer	Reverse primer
				CAS9 fragment	IMX673 Strain genome	SEQ ID NO:63	SEQ ID NO:64
Linearized pMEL13-ALD4	pMEL13 plasmid	SEQ ID NO:65	SEQ ID NO:66
				ALD4-repair-UP	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:67	SEQ ID NO:68
ALD4-repair-DOWN	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:69	SEQ ID NO:70
				Linearized pMEL13-ALD5	pMEL13 plasmid	SEQ ID NO:71	SEQ ID NO:72
ALD5-repair-UP	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:73	SEQ ID NO:74
				ALD5-repair-DOWN	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:75	SEQ ID NO:76
Linearized pMEL13-HFD1	pMEL13 plasmid	SEQ ID NO:77	SEQ ID NO:78
				HFD1-repair-UP	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:79	SEQ ID NO:80
HFD1-repair-DOWN	Saccharomyces cerevisiae BY4742 genome	SEQ ID NO:81	SEQ ID NO:82

IMX673 strain was a commercial saccharomyces cerevisiae strain purchased from euroscaf.

4.3.4 knock-out ALD4 gene:

the primers and templates in Table 3 are used for amplification by a PCR method to obtain a linearized pMEL13-ALD4 plasmid, and a sgRNA homologous sequence of 20bp is introduced at two ends through the primers. The DNA polymerase is high-fidelity DNA polymerase I-5 from TSINGKE^TM2X High Fidelity Master Mix, the PCR program was set up with reference to the description: 2min at 98 ℃; 15s at 98 ℃,10 s at 58 ℃ and 1.5min at 72 ℃ for 35 cycles; 5min at 72 ℃; keeping the temperature at 10 ℃.

And (3) carrying out agarose gel electrophoresis detection on the PCR product, and cutting off a band with the size consistent with that of the target DNA under ultraviolet light. Then, the DNA fragment was recovered from the agarose Gel using AxyPrep DNA Gel Extraction Kit from AXYGEN. By using the Hieff Clone of Yeasen^TMThe recombinant enzyme of Plus Multi One Step Cloning Kit recombines the recovered gel products for cyclization (reaction conditions refer to Kit instructions).

Coli TOP10 competent cells were transformed with the cyclization product and plated on LB plates supplemented with 100. mu.g/mL ampicillin. Positive transformants were verified by colony PCR and further verified by sequencing, which indicated successful construction of sgRNA expression plasmid pMEL13-ALD 4.

Using the primers and templates in Table 3, ALD4-repair-UP (SEQ ID NO:57) and ALD4-repair-DOWN (SEQ ID NO:58) were amplified by PCR, and 53bp homologous sequences were introduced into the junction by the primers. The DNA polymerase is high-fidelity DNA polymerase I-5 from TSINGKE^TM2X High Fidelity Master Mix, the PCR program was set up with reference to the description: 2min at 98 ℃; 15s at 98 ℃,10 s at 58 ℃ and 0.5min at 72 ℃ for 35 cycles; 5min at 72 ℃; keeping the temperature at 10 ℃.

And (3) carrying out agarose gel electrophoresis detection on the PCR product, and cutting off a band with the size consistent with that of the target DNA under ultraviolet light. Then, the DNA fragment was recovered from the agarose Gel using AxyPrep DNA Gel Extraction Kit from AXYGEN.

The two fragments obtained were transformed into Saccharomyces cerevisiae BY4742 competence together with the two plasmids constructed (p2M-CAS9 and pMEL13-ALD4), spread on YPAD plates supplemented with 300. mu.g/mL hygromycin B (hygromycin B) and 200mg/LG418, and cultured at 30 ℃ for 48 h. ALD4 knockout transformants were verified by yeast colony PCR. The Δ BY4742-1 strain was obtained BY subculturing missing the p2M-CAS9 and pMEL13-ALD4 plasmids.

4.3.5 knock-out of ALD5 and HFD1 genes:

with reference to the method for knocking out the ALD4 gene in the step 4.3.4, a pMEL13-ALD5 plasmid is constructed, ALD5-repair-UP (SEQ ID NO:59) and ALD5-repair-DOWN (SEQ ID NO:60) fragments are amplified, ALD5 is knocked out on the basis of delta BY4742-1, and a delta BY4742-2 strain is obtained through plasmid loss.

With reference to the method for knocking out the ALD4 gene in the step 4.3.4, a pMEL13-HFD1 plasmid is constructed, HFD1-repair-UP (SEQ ID NO:61) and HFD1-repair-DOWN (SEQ ID NO:62) fragments are amplified, gene HFD1 is continuously knocked out on the basis of delta BY4742-2, and an ALD4-ALD5-HFD1 knock-out strain, namely the delta BY4742 strain, is finally obtained through plasmid loss.

4.3.6 construction of the Δ BY4742-OP-A3 Strain:

referring to the method for constructing the BY4742-OP-A3 strain in the step 4.2, genes EcODC, AtPMT and AaDAO3 are transferred into the delta BY4742 strain to obtain the delta BY4742-OP-A3 strain. The saccharomyces cerevisiae is introduced with genes of EcODC, AtPMT and AaDAO3, and is knocked out with genes of ALD4, ALD5 and HFD 1.

4.4 construction of a.DELTA.BY 4742-OP-A3-SAM2 yeast strain overexpressing Gene SAM 2:

4.4.1 amplification of the transformed fragment by PCR using the primers and templates of Table 4: upstream homologous sequence of X-4 site, TEF1 promoter, AaDAO3 gene, PRM9 terminator, PGI terminator (SEQ ID NO:83), SAM2 gene (SEQ ID NO:84), TCCTDH promoter (enhanced TDH3 promoter, sequence SEQ ID NO:85), KanMX expression cassette, downstream homologous sequence of X-4 site, and 60-80 bp homologous sequence is introduced between adjacent fragments through primers. The DNA polymerase is high-fidelity DNA polymerase I-5 from TSINGKE^TM2X High Fidelity Master Mix, the PCR program was set up with reference to the description: 2min at 98 ℃; 35 cycles of 98 ℃ for 15s, 58 ℃ for 10s and 72 ℃ for 1 min; 5min at 72 ℃; keeping the temperature at 10 ℃.

4.4.2 detecting the PCR product by agarose gel electrophoresis, and cutting off a band with the same size with the target DNA under ultraviolet light. Then, the DNA fragment was recovered from the agarose Gel using AxyPrep DNA Gel extraction kit from AXYGEN.

4.4.3 transformation of 9 fragments above with p2M-EcODC-AtPMT plasmid into Δ BY4742 competence (competence creation and transformation method reference (Gietz and Schiestl 2007), spread on YPAD plates supplemented with 300. mu.g/mL hygromycin B (hygromycin B) and 200mg/L G418, and culture at 30 ℃ for 48 h. Positive results were confirmed BY yeast colony PCR, i.e., Δ BY4742-OP-A3-SAM2 strain, in which genes for EcODC, AtPMT and AaDAO3 were introduced, and ALD4, ALD5 and HFD1 genes were knocked out, and cofactor synthesis gene SAM2 was introduced. the yeast strain was constructed as shown in FIG. 6.

TABLE 4

TCCTDH sequence was ligated into pUC57 plasmid.

EXAMPLE 5 fermentation of engineered Saccharomyces cerevisiae producing N-methylpyrrolidine

Wild-type Saccharomyces cerevisiae BY4742, engineered Saccharomyces cerevisiae BY4742-OP, BY4742-OP-A3, Δ BY4742-OP-A3, Δ BY4742-OP-A3-SAM2 constructed in examples 3 and 4 above were picked from the plate and shake-cultured overnight in a test tube containing 5mL of YPD medium. The flask was transferred to a 50ml YPD flask and 50. mu.M CuSO was added₄Placing the bacterial liquid in a 30 ℃ shaking table, performing fermentation culture at 200rpm, sampling at different time points and detecting OD₆₀₀And N-methylpyrrolidine yield. As shown in FIG. 7A, N-methylpyrrolidine was accumulated in yeast strain BY4742-OP-A3 containing EcODC, AtPMT and AaDAO3 genes, and the yield of N-methylpyrrolidine was 3.22mg/L when the fermentation time reached 120 hours. To further increase the target product yield, we knocked out the dehydrogenase genes ALD4, ALD5 and HFD1 for converting N-methylaminobutyraldehyde to N-methylaminobutyric acid, and the fermentation results indicated that the N-methylpyrrolidine yield of yeast strain Δ BY4742-OP-A3 increased to 9.88mg/L (fig. 7B). In order to further improve the yield of the N-methylpyrrolidine, SAM2 is overexpressed to improve the supply amount of a cofactor SAM required BY AtPMT catalysis, and the optimized strain delta BY4742-OP-A3-SAM2 finally generates 21.92mg/L of the N-methylpyrrolidine (figure 7C), and compared with the yeast strain BY4742-OP-A3, the yield is improved BY about 7 times.

Example 7 chemical Synthesis of N-methylated putrescine and N-methylpyrroline

Since N-methylated putrescine and N-methylpyrroline are difficult to obtain by commercial purchase, the N-methylated putrescine and N-methylpyrroline are synthesized by a chemical method to serve as reaction substrates and product standards. The specific method comprises the following steps:

7.1 Synthesis of N-methylated putrescine

Adding Et₃N (1mmol) and (Boc)₂O (262mg, 1.2mmol) was added to 10mL of methanol containing 1.0mol putrescine, and the mixture was stirred at room temperatureFor 2h, 10mL of 0.1N aqueous NaOH solution was added and extracted with 15mL of ethyl acetate. The organic phase was then back extracted with 20mL of 0.1N HCl, the resulting aqueous phase adjusted to pH10, extracted three times with 20mL of ethyl acetate, the organic phases combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin dried to give Boc-putrescine. Mixing LiAlH₄(6.33mmol) was added to 10mL of anhydrous THF, ice-cooled, the flask was purged with nitrogen to maintain an oxygen-free environment, Boc-putrescine (1mmol) was dissolved in 5mL of anhydrous THF, LiAlH was added dropwise₄In THF solution, the temperature of the reaction solution is gradually increased to 85 ℃, reflux is carried out for 18h, the reaction solution is placed in an ice bath after being cooled to room temperature, sodium sulfate is slowly added to stop the reaction, the filtration is carried out, 20mL of 0.1N NaOH aqueous solution is added into the filtrate, 25mL of ethyl acetate is added to extract, then 25mL of 0.1N HCl is added to back extract the organic phase, and the obtained aqueous solution is dried in a rotating mode to obtain the pure N-methylated putrescine.

FIG. 8A and FIG. 8B show the NMR spectra of N-methylated putrescine (R) ((R))¹H NMR), and nuclear magnetic resonance carbon spectrum (C¹³C NMR)。

¹H NMR(400MHz,D₂O)δ3.09(dd,J＝15.2,7.4Hz,4H),2.74(s,3H),1.94-1.64(m,4H)。

¹³C NMR(101MHz,D₂O)δ48.26,38.79,32.68,23.83,22.56。

7.2 Synthesis of N-methylpyrrolidine

1mL Et₃N and 0.1mmol DMAP were added dropwise to a solution of 1mmol N-methylaminobutanol in 5mL THF, 1.1mmol (Boc) were added slowly₂O, the solution was stirred at room temperature for 1h, then 10mL of 0.1N aqueous NaOH solution was added and extracted with 15mL of ethyl acetate. 15mL of 0.1N HCl was added to the organic phase, the resulting aqueous solution was adjusted to pH10, and then extracted 3 times with 15mL of ethyl acetate, the organic phases were combined, dried with saturated brine, filtered, and spin-dried to give Boc-N-methylaminobutanol. 0.5mmol Boc-N-methylaminobutanol, 1mL DMSO and 0.5mL Et₃N was dissolved in 4mL of anhydrous dichloromethane,adding 1.5mmol of SO₃And maintaining the temperature of the/Pyr compound at 0 ℃, stirring for 30min, purifying by a silica gel column to obtain Boc-N-methylamino butyraldehyde, dissolving 0.4mmol of Boc-N-methylamino butyraldehyde in 10mL of water, adding 2N HCl, stirring for 1h at room temperature, and carrying out rotary drying to obtain the pure N-methylpyrrolidine.

FIG. 9A and FIG. 9B show the NMR spectra of N-methylpyrrolidine ((H))¹H NMR), and nuclear magnetic resonance carbon spectrum (C¹³C NMR)。

¹H NMR(500MHz,D₂O)δ8.63(s,1H),4.16(t,J＝7.4Hz,2H),3.63(s,3H),3.16(d,J＝29.4Hz,2H),2.44–2.27(m,2H)。

¹³C NMR(101MHz,D₂O)δ181.56,60.59,40.39,35.74,19.48。

In a word, the EcODC, AtPMT and AaDAO2-3 three-enzyme combined catalytic system can catalyze L-ornithine reaction to obtain N-methylpyrrolidine by a one-pot method, and the constructed escherichia coli engineering bacteria and saccharomyces cerevisiae engineering bacteria can directly produce the N-methylpyrrolidine through fermentation. It is obvious to those skilled in the art that the biosynthesis method of the present invention has natural advantages of environmental protection compared with the chemical synthesis method, and thus has industrial development and application prospects.

It should also be noted that the listing or discussion of a prior-published document in this specification should not be taken as an admission that the document is prior art or common general knowledge.

Reference to the literature

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Kai G,Zhang A,Guo Y,Li L,Cui L,Luo X,Liu C,Xiao J.2012.Enhancing the production of tropane alkaloids in transgenic Anisodus acutangulus hairy root cultures by over-expressing tropinone reductase I and hyoscyamine-6β-hydroxylase.Molecular BioSystems 8(11):2883-2890.

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Sequence listing

<110> Shanghai Life science research institute of Chinese academy of sciences

Biosynthesis method of <120> N-methyl pyrroline

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Met Ile Val His Leu Pro Leu Gly Ser Ile Pro Ser Pro Lys Lys Val

115 120 125

Leu Ile Ile Gly Gly Gly Ile Gly Phe Thr Leu Phe Glu Val Ser Arg

130 135 140

Tyr Pro Thr Ile Glu Thr Ile Asp Ile Val Glu Ile Asp Asp Val Val

145 150 155 160

Val Asp Val Ser Arg Lys Phe Phe Pro Tyr Leu Ala Ala Gly Phe Asp

165 170 175

Asp Pro Arg Val Thr Leu Ile Ile Gly Asp Gly Ala Ala Phe Val Lys

180 185 190

Ala Ala Gln Pro Gly Tyr Tyr Asp Ala Ile Ile Val Asp Ser Ser Asp

195 200 205

Pro Ile Gly Pro Ala Lys Asp Leu Phe Glu Arg Pro Phe Phe Glu Ala

210 215 220

Leu Ala Lys Ala Leu Arg Pro Gly Gly Val Val Cys Thr Gln Ala Glu

225 230 235 240

Ser Ile Trp Leu His Met His Leu Ile Lys Gln Ile Ile Ala Asn Cys

245 250 255

Arg Gln Val Phe Lys Gly Ser Val Asn Tyr Ala Trp Thr Thr Val Pro

260 265 270

Thr Tyr Pro Thr Gly Val Ile Gly Tyr Met Leu Cys Ser Thr Glu Gly

275 280 285

Pro Glu Val Asn Phe Lys Asn Pro Val Asn Ser Ile Asp Lys Asp Thr

290 295 300

Ser His Val Lys Ser Lys Gly Pro Leu Lys Phe Tyr Asn Ser Asp Ile

305 310 315 320

His Lys Ala Ala Phe Ile Leu Pro Ser Phe Ala Arg Asp Leu Val Glu

325 330 335

Phe

<210> 7

<211> 33

<212> DNA

<213> Artificial sequence ()

<400> 7

ggaattccat atgatggaag ttatttctaa tca 33

<210> 8

<211> 36

<212> DNA

<213> Artificial sequence ()

<400> 8

ataagaatgc ggccgcaaat tcaacgagat ccctag 36

<210> 9

<211> 2127

<212> DNA

<213> three thirds ()

<400> 9

atgagatata tcttctttct cattagcata attttgcttt taattttcac ttttttaaac 60

ttaccatctc ctctttctca cacaactgag ttattagact gcaccactta ctctccatgg 120

tgcactaagc ccttttccca acacaaaaat cactatgcca atttcccaaa atacccttta 180

gatccattaa caataaaaga aattcaaaaa gtgaaaaaaa tcattaattc aattgatgag 240

tttagtaaaa aaggttatgt tctccactct gtggtacttg aagagccggc aaaggaggtg 300

gtggtgaact ggcggaaagg tcgccgcctt ccgccacgga aggcggcggt ggttgtacgt 360

gcggttggtg ttgtgcacgt gcttactgtg gatattgaaa cgggtcgggt gacccgacgt 420

gaaacgggtg attattcggg ttacccgatc atgacgatcg aggatatgat tacggcgact 480

actgcaccac ttgcgaacgc tgattttaac cgttcgattc tcgagcgtgg cgttgatttg 540

gctgaccttg catgtttgcc tattgccact ggttggtacg ggaaagctga ggagaataga 600

agagtcatca aagtacagtg ctacacaatg aaggacacaa ttaatttcta catgagaccc 660

attgaaggac taacagtact tcttgattta gacacccaac aagtcataga tattctagat 720

gaaggaaaga acataccaat accaaaggcc gccaacacag actatcgctt ctctactaaa 780

aaacacacac aaaaaataaa cttactcaaa ccaatatcta tcgaacaacc aaatggtcca 840

agtttcacta tagagaacaa ccatatagtg aaatgggcaa attgggaatt tcacctcaag 900

cccgacccga gagccggggt gattatatcc caggtcatga tccaagatcc ggatacgggg 960

aagattagaa atgtcatgta taaagggttt acgtcggagt tgtttgtgcc ttacatggat 1020

ccatcagatg catggtattt taagacgtat atggatgcgg gtgaatacgg gttcgggtta 1080

caagcaatgc cgcttgaccc gttaaatgat tgtccacgta atgcgtatta tatggatggt 1140

gtgtttgtgg cagctgatgg aacaccatat gttcgtgaaa atatgatttg tgtgtttgag 1200

agatatgctg gtgatattgg atggcgacat gctgaatctc ccattactgg tttgccgatt 1260

agagaagtga ggccgaaggt gacgttagta gttagaatgg cagcgtcagt ggctaactat 1320

gattacattg tggattggga gttccagact gatggactta tccgagctaa ggttggacta 1380

agtggaatat tgatggtgaa aggctctcca tatgtgaaca tgaaccaagt gaaccaaaat 1440

gagtatctct acggcaccct cttatctgaa aacataatag gagtcatcca tgatcactac 1500

ataactttcc atctcgacat ggacatcgat ggtccatcga acaattcttt cgtgaaggtg 1560

aatctccaga aggagatgac ttctcctgga gaatccccgc gaaggagcta cttaaaagct 1620

gttcgacatg tggctaaaac tgaaaaggat gcacagatta agctcaagtt atatgatcct 1680

tcagaattcc atgtgattaa tcctaacaag aagtcaagag ttgggaaccc tgttggttac 1740

aaagtggttc ctggtggcac tgcagctagt ttactggacc atgatgatcc tccacagaag 1800

agaggtgctt ttactaataa ccaaatttgg gtcacacctt ataatgaatc tgagcaatgg 1860

gctgctggct tgtttgttta ccaaagtcaa ggtgatgata ctcttgctgt ttggtctgac 1920

agagacaggc caattgagaa taaagacata gtggtgtggt atacattagg gttccatcat 1980

attccatgcc aagaggattt cccaatcatg ccaacagtat catcaagctt tgagataaag 2040

ccagtgaatt tctttgagag caatccaatt atgaggattc aacctaattc tccaaaggac 2100

ctgccaattt gcaaagcttc tgcttga 2127

<210> 10

<211> 707

<212> PRT

<213> three thirds ()

<400> 10

Met Arg Tyr Ile Phe Phe Leu Ile Ser Ile Ile Leu Leu Leu Ile Phe

1 5 10 15

Thr Phe Leu Asn Leu Pro Ser Pro Leu Ser His Thr Thr Glu Leu Leu

20 25 30

Asp Cys Thr Thr Tyr Ser Pro Trp Cys Thr Lys Pro Phe Ser Gln His

35 40 45

Lys Asn His Tyr Ala Asn Phe Pro Lys Tyr Pro Leu Asp Pro Leu Thr

50 55 60

Ile Lys Glu Ile Gln Lys Val Lys Lys Ile Ile Asn Ser Ile Asp Glu

65 70 75 80

Phe Ser Lys Lys Gly Tyr Val Leu His Ser Val Val Leu Glu Glu Pro

85 90 95

Ala Lys Glu Val Val Val Asn Trp Arg Lys Gly Arg Arg Leu Pro Pro

100 105 110

Arg Lys Ala Ala Val Val Val Arg Ala Val Gly Val Val His Val Leu

115 120 125

Thr Val Asp Ile Glu Thr Gly Arg Val Thr Arg Arg Glu Thr Gly Asp

130 135 140

Tyr Ser Gly Tyr Pro Ile Met Thr Ile Glu Asp Met Ile Thr Ala Thr

145 150 155 160

Thr Ala Pro Leu Ala Asn Ala Asp Phe Asn Arg Ser Ile Leu Glu Arg

165 170 175

Gly Val Asp Leu Ala Asp Leu Ala Cys Leu Pro Ile Ala Thr Gly Trp

180 185 190

Tyr Gly Lys Ala Glu Glu Asn Arg Arg Val Ile Lys Val Gln Cys Tyr

195 200 205

Thr Met Lys Asp Thr Ile Asn Phe Tyr Met Arg Pro Ile Glu Gly Leu

210 215 220

Thr Val Leu Leu Asp Leu Asp Thr Gln Gln Val Ile Asp Ile Leu Asp

225 230 235 240

Glu Gly Lys Asn Ile Pro Ile Pro Lys Ala Ala Asn Thr Asp Tyr Arg

245 250 255

Phe Ser Thr Lys Lys His Thr Gln Lys Ile Asn Leu Leu Lys Pro Ile

260 265 270

Ser Ile Glu Gln Pro Asn Gly Pro Ser Phe Thr Ile Glu Asn Asn His

275 280 285

Ile Val Lys Trp Ala Asn Trp Glu Phe His Leu Lys Pro Asp Pro Arg

290 295 300

Ala Gly Val Ile Ile Ser Gln Val Met Ile Gln Asp Pro Asp Thr Gly

305 310 315 320

Lys Ile Arg Asn Val Met Tyr Lys Gly Phe Thr Ser Glu Leu Phe Val

325 330 335

Pro Tyr Met Asp Pro Ser Asp Ala Trp Tyr Phe Lys Thr Tyr Met Asp

340 345 350

Ala Gly Glu Tyr Gly Phe Gly Leu Gln Ala Met Pro Leu Asp Pro Leu

355 360 365

Asn Asp Cys Pro Arg Asn Ala Tyr Met Asp Gly Val Phe Val Ala Ala

370 375 380

Asp Gly Thr Pro Tyr Val Arg Glu Asn Met Ile Cys Val Phe Glu Arg

385 390 395 400

Tyr Ala Gly Asp Ile Gly Trp Arg His Ala Glu Ser Pro Ile Thr Gly

405 410 415

Leu Pro Ile Arg Glu Val Arg Pro Lys Val Thr Leu Val Val Arg Met

420 425 430

Ala Ala Ser Val Ala Asn Tyr Asp Tyr Ile Val Asp Trp Glu Phe Gln

435 440 445

Thr Asp Gly Leu Ile Arg Ala Lys Val Gly Leu Ser Gly Ile Leu Met

450 455 460

Val Lys Gly Ser Pro Tyr Val Asn Met Asn Gln Val Asn Gln Asn Glu

465 470 475 480

Tyr Leu Tyr Gly Thr Leu Leu Ser Glu Asn Ile Ile Gly Val Ile His

485 490 495

Asp His Tyr Ile Thr Phe His Leu Asp Met Asp Ile Asp Gly Pro Ser

500 505 510

Asn Asn Ser Phe Val Lys Val Asn Leu Gln Lys Glu Met Thr Ser Pro

515 520 525

Gly Glu Ser Pro Arg Arg Ser Tyr Leu Lys Ala Val Arg His Val Ala

530 535 540

Lys Thr Glu Lys Asp Ala Gln Ile Lys Leu Lys Leu Tyr Asp Pro Ser

545 550 555 560

Glu Phe His Val Ile Asn Pro Asn Lys Lys Ser Arg Val Gly Asn Pro

565 570 575

Val Gly Tyr Lys Val Val Pro Gly Gly Thr Ala Ala Ser Leu Leu Asp

580 585 590

His Asp Asp Pro Pro Gln Lys Arg Gly Ala Phe Thr Asn Asn Gln Ile

595 600 605

Trp Val Thr Pro Tyr Asn Glu Ser Glu Gln Trp Ala Ala Gly Leu Phe

610 615 620

Val Tyr Gln Ser Gln Gly Asp Asp Thr Leu Ala Val Trp Ser Asp Arg

625 630 635 640

Asp Arg Pro Ile Glu Asn Lys Asp Ile Val Val Trp Tyr Thr Leu Gly

645 650 655

Phe His His Ile Pro Cys Gln Glu Asp Phe Pro Ile Met Pro Thr Val

660 665 670

Ser Ser Ser Phe Glu Ile Lys Pro Val Asn Phe Phe Glu Ser Asn Pro

675 680 685

Ile Met Arg Ile Gln Pro Asn Ser Pro Lys Asp Leu Pro Ile Cys Lys

690 695 700

Ala Ser Ala

705

<210> 11

<211> 43

<212> DNA

<213> Artificial sequence ()

<400> 11

gatatcatga gatatatctt ctttctcatt agcataattt tgc 43

<210> 12

<211> 46

<212> DNA

<213> Artificial sequence ()

<400> 12

ataagaatgc ggccgcctag tatatattca cgaatcataa ttacac 46

<210> 13

<211> 2322

<212> DNA

<213> three thirds ()

<400> 13

atggcctcaa ctcaggaaaa ggcgacgtca cgaaatgttt ccgccgcttc tgcaacggaa 60

tggactgccg gaagcgtagg tcaagtttcc gatctttcct catcggcgaa cgtcaaaagt 120

actgctgctg ctgctccgtc tcctttgacg gctgtgatcg actctattga tcgtccttct 180

ccaccaaatc cgcctgccgt taaaggactg cctaccctgt tgagggctca aactcaccat 240

cccttggacc ccttaactgc tgctgaaatt tccgtggccg tggcgactgt cagggcagct 300

ggaagcactc ctgaggtgag agatagcatg cgctttgtcg aggctgtttt ggtggaacca 360

gataagagtg tggttgcttt agctgatgca tacttcttcc ctcctttcca accatcttta 420

ttgccgagaa caaaaggtgg gcctgtaatt cctagtaaac ttcctccaag gaaggctaaa 480

ctgattgttt ataacaagaa atcaaatgag accagcatat ggattgttga gctctcagag 540

gttcatgctg ttactcgagg aggccaccac cgtggcaaag ttatttcatc taaggttgta 600

cccgatatac agcctccaat ggacgctgct gaatatgctg aatgtgaatc tgtggtgaag 660

gatttccctc cttttcgtga tgcaatgaag aagagaggaa tcgatgatat ggatcttgtg 720

atggtcgatg cctggtgtgc aggttatttc agtgaggctg atgcacctaa ccgccgtcta 780

ggaaaacctc ttatcttttg cagaactgag agcgactgtc caatggaaaa tggctatgct 840

cggccagttg aagggattca tatactggtt gatatgcaga atatggttgt gatagagttt 900

gaagatcgta aactggttcc attaccacca gctgatccat tgagaaatta cacttccggt 960

gaaacgagag gaggggttga ccgaagtgat gttaaacctc ttcttattag tcagcctgaa 1020

ggtcctagct tccgtgtcag tgggcacttt gttcaatggc agaagtggaa ctttcgtatt 1080

ggctttaccc ccaaagaggg tctggttatc tattctgttg catatattga cggtagcaga 1140

ggccgtagac cagttgcgca tagactgagc ttcattgaaa tggtagtccc ctatggggat 1200

ccaaatgagc cacattacag gaaaaatgct tttgatgcag gggaagatgg gcttggcaaa 1260

aatgcgcatt ccttaaagaa gggttgtgat tgcttaggct atattaaata tttcgatgca 1320

cattttacaa acttcacagg cggtgttgag acaatagaaa attgtgtctg cttacatgag 1380

gaggatcatg gaatcttatg gaagcatcaa gattggagaa caggtttagc tgaagtacga 1440

cgttcccgac ggctaacagt gtctttcatc tgtactgtgg caaactatga atatggattt 1500

tactggcatt tttatcagga tgggaaaatc gaggctgagg ttaagctgac cggaattctc 1560

agcttaggtg ctcttctacc aggggaagtt cgaaaatatg ggacaactat tgcacctggc 1620

ttgtatgcac ctgtacatca gcacttcttt gtggctcgca tggatatggc tgttgattgc 1680

aaagcagggg agtcacataa tcaggtggtt gaagtaaatg ctagaattga accaccagga 1740

gaaaataatg ttcataataa tgcattttat gctgaagaga gattgcttaa aactgaactg 1800

gaagcaatgc gtgattgtaa tcctctaact gctcggcatt ggattatccg gaacacaaga 1860

acagtcaatc gcactgggca gttaactggt tacaagctag taccaggtac aaactgtctg 1920

ccactagctg gctcagaggc aaaattcttg aggagagctg ctttcttaaa gcataatctt 1980

tgggtcaccc cttactctct tgatgagatg tttcctggag gagaatttcc aaatcaaaat 2040

cctcgtgttg gtgagggatt ggctacttgg gttcagcaga atcgatccct ggaagaaacg 2100

caaatagtac tttggtatgt ttttggactt atacatgttc cacggctaga agactggccg 2160

gtcatgccag tagagcacat tggttttatg ctacagcctc atgggttctt caattgctcc 2220

ccagctgttg atgttcctcc cagcacggct gacttggata ttaaggaaaa tggggtagtg 2280

gcaaagtctt gccacaatga tggtataatg gctaagcttt ga 2322

<210> 14

<211> 773

<212> PRT

<213> three thirds ()

<400> 14

Met Ala Ser Thr Gln Glu Lys Ala Thr Ser Arg Asn Val Ser Ala Ala

1 5 10 15

Ser Ala Thr Glu Trp Thr Ala Gly Ser Val Gly Gln Val Ser Asp Leu

20 25 30

Ser Ser Ser Ala Asn Val Lys Ser Thr Ala Ala Ala Ala Pro Ser Pro

35 40 45

Leu Thr Ala Val Ile Asp Ser Ile Asp Arg Pro Ser Pro Pro Asn Pro

50 55 60

Pro Ala Val Lys Gly Leu Pro Thr Leu Leu Arg Ala Gln Thr His His

65 70 75 80

Pro Leu Asp Pro Leu Thr Ala Ala Glu Ile Ser Val Ala Val Ala Thr

85 90 95

Val Arg Ala Ala Gly Ser Thr Pro Glu Val Arg Asp Ser Met Arg Phe

100 105 110

Val Glu Ala Val Leu Val Glu Pro Asp Lys Ser Val Val Ala Leu Ala

115 120 125

Asp Ala Tyr Phe Phe Pro Pro Phe Gln Pro Ser Leu Leu Pro Arg Thr

130 135 140

Lys Gly Gly Pro Val Ile Pro Ser Lys Leu Pro Pro Arg Lys Ala Lys

145 150 155 160

Leu Ile Val Tyr Asn Lys Lys Ser Asn Glu Thr Ser Ile Trp Ile Val

165 170 175

Glu Leu Ser Glu Val His Ala Val Thr Arg Gly Gly His His Arg Gly

180 185 190

Lys Val Ile Ser Ser Lys Val Val Pro Asp Ile Gln Pro Pro Met Asp

195 200 205

Ala Ala Glu Tyr Ala Glu Cys Glu Ser Val Val Lys Asp Phe Pro Pro

210 215 220

Phe Arg Asp Ala Met Lys Lys Arg Gly Ile Asp Asp Met Asp Leu Val

225 230 235 240

Met Val Asp Ala Trp Cys Ala Gly Tyr Phe Ser Glu Ala Asp Ala Pro

245 250 255

Asn Arg Arg Leu Gly Lys Pro Leu Ile Phe Cys Arg Thr Glu Ser Asp

260 265 270

Cys Pro Met Glu Asn Gly Tyr Ala Arg Pro Val Glu Gly Ile His Ile

275 280 285

Leu Val Asp Met Gln Asn Met Val Val Ile Glu Phe Glu Asp Arg Lys

290 295 300

Leu Val Pro Leu Pro Pro Ala Asp Pro Leu Arg Asn Tyr Thr Ser Gly

305 310 315 320

Glu Thr Arg Gly Gly Val Asp Arg Ser Asp Val Lys Pro Leu Leu Ile

325 330 335

Ser Gln Pro Glu Gly Pro Ser Phe Arg Val Ser Gly His Phe Val Gln

340 345 350

Trp Gln Lys Trp Asn Phe Arg Ile Gly Phe Thr Pro Lys Glu Gly Leu

355 360 365

Val Ile Tyr Ser Val Ala Tyr Ile Asp Gly Ser Arg Gly Arg Arg Pro

370 375 380

Val Ala His Arg Leu Ser Phe Ile Glu Met Val Val Pro Tyr Gly Asp

385 390 395 400

Pro Asn Glu Pro His Tyr Arg Lys Asn Ala Phe Asp Ala Gly Glu Asp

405 410 415

Gly Leu Gly Lys Asn Ala His Ser Leu Lys Lys Gly Cys Asp Cys Leu

420 425 430

Gly Tyr Ile Lys Tyr Phe Asp Ala His Phe Thr Asn Phe Thr Gly Gly

435 440 445

Val Glu Thr Ile Glu Asn Cys Val Cys Leu His Glu Glu Asp His Gly

450 455 460

Ile Leu Trp Lys His Gln Asp Trp Arg Thr Gly Leu Ala Glu Val Arg

465 470 475 480

Arg Ser Arg Arg Leu Thr Val Ser Phe Ile Cys Thr Val Ala Asn Tyr

485 490 495

Glu Tyr Gly Phe Tyr Trp His Phe Tyr Gln Asp Gly Lys Ile Glu Ala

500 505 510

Glu Val Lys Leu Thr Gly Ile Leu Ser Leu Gly Ala Leu Leu Pro Gly

515 520 525

Glu Val Arg Lys Tyr Gly Thr Thr Ile Ala Pro Gly Leu Tyr Ala Pro

530 535 540

Val His Gln His Phe Phe Val Ala Arg Met Asp Met Ala Val Asp Cys

545 550 555 560

Lys Ala Gly Glu Ser His Asn Gln Val Val Glu Val Asn Ala Arg Ile

565 570 575

Glu Pro Pro Gly Glu Asn Asn Val His Asn Asn Ala Phe Tyr Ala Glu

580 585 590

Glu Arg Leu Leu Lys Thr Glu Leu Glu Ala Met Arg Asp Cys Asn Pro

595 600 605

Leu Thr Ala Arg His Trp Ile Ile Arg Asn Thr Arg Thr Val Asn Arg

610 615 620

Thr Gly Gln Leu Thr Gly Tyr Lys Leu Val Pro Gly Thr Asn Cys Leu

625 630 635 640

Pro Leu Ala Gly Ser Glu Ala Lys Phe Leu Arg Arg Ala Ala Phe Leu

645 650 655

Lys His Asn Leu Trp Val Thr Pro Tyr Ser Leu Asp Glu Met Phe Pro

660 665 670

Gly Gly Glu Phe Pro Asn Gln Asn Pro Arg Val Gly Glu Gly Leu Ala

675 680 685

Thr Trp Val Gln Gln Asn Arg Ser Leu Glu Glu Thr Gln Ile Val Leu

690 695 700

Trp Tyr Val Phe Gly Leu Ile His Val Pro Arg Leu Glu Asp Trp Pro

705 710 715 720

Val Met Pro Val Glu His Ile Gly Phe Met Leu Gln Pro His Gly Phe

725 730 735

Phe Asn Cys Ser Pro Ala Val Asp Val Pro Pro Ser Thr Ala Asp Leu

740 745 750

Asp Ile Lys Glu Asn Gly Val Val Ala Lys Ser Cys His Asn Asp Gly

755 760 765

Ile Met Ala Lys Leu

770

<210> 15

<211> 31

<212> DNA

<213> Artificial sequence ()

<400> 15

gatatcatgg cctcaactca ggaaaaggcg a 31

<210> 16

<211> 46

<212> DNA

<213> Artificial sequence ()

<400> 16

ataagaatgc ggccgctcaa agcttagcca ttataccatc attgtg 46

<210> 17

<211> 2310

<212> DNA

<213> three thirds ()

<400> 17

atggccacaa cctcgcataa gccttctttc tgtcatccac cttctgcttc tgtcgtccgt 60

cgtgaaacgg cctccgcctc cgccgtggct ccgtccgtgg acgatcagca gaaacaaacg 120

ccgccgctag cgtcaatatt ggttgactct caaatacctt cctccaactc gtctaccaaa 180

gggatccaaa tcatgctaag agctcaaact tgtcatcctt tggacccttt atctgctgct 240

gagatctctg tggctgtggc aaccgttaga gctgctggtg acacacctga ggtcagagat 300

ggcatgcggt ttgttgaggt ggttcttttg gaaccagata aaactttcat tgcactcgca 360

gacgcctatt tcttcccacc ttttcaatca tcattgatgc ccagaaccaa aggagggctt 420

cttgttcctt ctaaacttcc cccaaggcaa gctagactta ttgtttacaa taagaaaaca 480

aatgagacaa gcgtatggat tgttcagcta actgaagtac atgctgctgc tcgaggtgga 540

caacacaggg gaaaagtgat ttcatccaaa gttgttccag atgttcagcc acctatagat 600

gcacaagagt atgctgactg tgaatccgtg gttaaaaatt atccttcttt tatagaagca 660

atgaagagaa ggggtattga tgacatggat cttgtgatgg ttgacccctg gtgtgttggt 720

tatcacagtg aagctgatgc tcctagccgc aggcttgcca aaccactggt attctgcaga 780

acagagagcg actgtccaat ggaaaatgga tatgcaagac cagttgaagg aatacatgtg 840

cttgttgatg tgcaaaacat gcaggtgata gagttcgaag accgcaaagt tgtaccttta 900

ccccctgctg atccactgag gaattacact gctggtgaga caagaggagg ggttgatcga 960

agtgatgtga aacccctaca aattattcag ccagatggtc caagctttcg cgtcgatggg 1020

aactatgtac aatggcaaaa gtggaacttc cgagtaggtt tcacccctag ggagggtttg 1080

gttatacact ctgtggcata tcttgacggt agcaggggtc gaagatccat agcccatagg 1140

ttgagttttg tggagatggt tgtcccctat ggagatccaa atgacccaca ttacagaaag 1200

aacgcatttg atgcaggaga agatggcctt ggaaagaatg ctcattcact taagagggga 1260

tgcgattgtc taggatacat aaaatacttc gatgccaatt ttacaaattt cacgggagga 1320

gtagaaacca ttgaaaattg tgtatgtttg catgaagaag atcacgggat gctttggaag 1380

catcaagatt ggagaactgg ccttgctgaa gttagacggt ctagacgact gacagtttct 1440

tttatttgca ctgtggccaa ttatgaatat ggattctact ggcactttta ccaggatggg 1500

aaaattgaag cagaagtcaa actcacagga attctcagtt tgggagcatt gcaacctgga 1560

gagtctcgta aatatggcac cacaataaca ccaggattgt atgcacctgt tcatcagcac 1620

ttctttgttg ctcgtatgaa tatggcagtt gattgtaagc caggagaagc acacaatcag 1680

attgttgaag taaatgtcaa agttgaagaa cctgggaagg aaaatgttca caataatgca 1740

ttctatgctg aagaaacact gcttaggtct gaattgcaag caatgcgtga ctgtgatcct 1800

ttatctgctc ggcattgggt tgttaggaac acaagaacgg ccaacagaac aggaaagcta 1860

acaggttaca agctggtacc tggtccgaac tgtttgccat tggctggtcc tgaggctaag 1920

ttcttgagaa gagcggcatt tttgaagcac aatttatggg ttacacaata tgcacctgga 1980

gaagattttc ccgggggaga gttccctaat caaaatccac gtgttggtga aggattagct 2040

tcttgggtta agcaagatcg ttctctggaa gaaagtgata ttgttctctg gtatgttttt 2100

ggaatcaccc atgtccctcg gttggaggac tggcctgtta tgcctgttga acatatcggc 2160

tttatgcttc agccacacgg attctttaac tgctctcctg ctgtagatgt acctccatct 2220

cggggatgtg actcggaaag caaagacagt gatgtttcag aaaatggtgt agcaaagccc 2280

actaccactg gtttgatggc caagctttga 2310

<210> 18

<211> 769

<212> PRT

<213> three thirds ()

<400> 18

Met Ala Thr Thr Ser His Lys Pro Ser Phe Cys His Pro Pro Ser Ala

1 5 10 15

Ser Val Val Arg Arg Glu Thr Ala Ser Ala Ser Ala Val Ala Pro Ser

20 25 30

Val Asp Asp Gln Gln Lys Gln Thr Pro Pro Leu Ala Ser Ile Leu Val

35 40 45

Asp Ser Gln Ile Pro Ser Ser Asn Ser Ser Thr Lys Gly Ile Gln Ile

50 55 60

Met Leu Arg Ala Gln Thr Cys His Pro Leu Asp Pro Leu Ser Ala Ala

65 70 75 80

Glu Ile Ser Val Ala Val Ala Thr Val Arg Ala Ala Gly Asp Thr Pro

85 90 95

Glu Val Arg Asp Gly Met Arg Phe Val Glu Val Val Leu Leu Glu Pro

100 105 110

Asp Lys Thr Phe Ile Ala Leu Ala Asp Ala Tyr Phe Phe Pro Pro Phe

115 120 125

Gln Ser Ser Leu Met Pro Arg Thr Lys Gly Gly Leu Leu Val Pro Ser

130 135 140

Lys Leu Pro Pro Arg Gln Ala Arg Leu Ile Val Tyr Asn Lys Lys Thr

145 150 155 160

Asn Glu Thr Ser Val Trp Ile Val Gln Leu Thr Glu Val His Ala Ala

165 170 175

Ala Arg Gly Gly Gln His Arg Gly Lys Val Ile Ser Ser Lys Val Val

180 185 190

Pro Asp Val Gln Pro Pro Ile Asp Ala Gln Glu Tyr Ala Asp Cys Glu

195 200 205

Ser Val Val Lys Asn Tyr Pro Ser Phe Ile Glu Ala Met Lys Arg Arg

210 215 220

Gly Ile Asp Asp Met Asp Leu Val Met Val Asp Pro Trp Cys Val Gly

225 230 235 240

Tyr His Ser Glu Ala Asp Ala Pro Ser Arg Arg Leu Ala Lys Pro Leu

245 250 255

Val Phe Cys Arg Thr Glu Ser Asp Cys Pro Met Glu Asn Gly Tyr Ala

260 265 270

Arg Pro Val Glu Gly Ile His Val Leu Val Asp Val Gln Asn Met Gln

275 280 285

Val Ile Glu Phe Glu Asp Arg Lys Val Val Pro Leu Pro Pro Ala Asp

290 295 300

Pro Leu Arg Asn Tyr Thr Ala Gly Glu Thr Arg Gly Gly Val Asp Arg

305 310 315 320

Ser Asp Val Lys Pro Leu Gln Ile Ile Gln Pro Asp Gly Pro Ser Phe

325 330 335

Arg Val Asp Gly Asn Tyr Val Gln Trp Gln Lys Trp Asn Phe Arg Val

340 345 350

Gly Phe Thr Pro Arg Glu Gly Leu Val Ile His Ser Val Ala Tyr Leu

355 360 365

Asp Gly Ser Arg Gly Arg Arg Ser Ile Ala His Arg Leu Ser Phe Val

370 375 380

Glu Met Val Val Pro Tyr Gly Asp Pro Asn Asp Pro His Tyr Arg Lys

385 390 395 400

Asn Ala Phe Asp Ala Gly Glu Asp Gly Leu Gly Lys Asn Ala His Ser

405 410 415

Leu Lys Arg Gly Cys Asp Cys Leu Gly Tyr Ile Lys Tyr Phe Asp Ala

420 425 430

Asn Phe Thr Asn Phe Thr Gly Gly Val Glu Thr Ile Glu Asn Cys Val

435 440 445

Cys Leu His Glu Glu Asp His Gly Met Leu Trp Lys His Gln Asp Trp

450 455 460

Arg Thr Gly Leu Ala Glu Val Arg Arg Ser Arg Arg Leu Thr Val Ser

465 470 475 480

Phe Ile Cys Thr Val Ala Asn Tyr Glu Tyr Gly Phe Tyr Trp His Phe

485 490 495

Tyr Gln Asp Gly Lys Ile Glu Ala Glu Val Lys Leu Thr Gly Ile Leu

500 505 510

Ser Leu Gly Ala Leu Gln Pro Gly Glu Ser Arg Lys Tyr Gly Thr Thr

515 520 525

Ile Thr Pro Gly Leu Tyr Ala Pro Val His Gln His Phe Phe Val Ala

530 535 540

Arg Met Asn Met Ala Val Asp Cys Lys Pro Gly Glu Ala His Asn Gln

545 550 555 560

Ile Val Glu Val Asn Val Lys Val Glu Glu Pro Gly Lys Glu Asn Val

565 570 575

His Asn Asn Ala Phe Tyr Ala Glu Glu Thr Leu Leu Arg Ser Glu Leu

580 585 590

Gln Ala Met Arg Asp Cys Asp Pro Leu Ser Ala Arg His Trp Val Val

595 600 605

Arg Asn Thr Arg Thr Ala Asn Arg Thr Gly Lys Leu Thr Gly Tyr Lys

610 615 620

Leu Val Pro Gly Pro Asn Cys Leu Pro Leu Ala Gly Pro Glu Ala Lys

625 630 635 640

Phe Leu Arg Arg Ala Ala Phe Leu Lys His Asn Leu Trp Val Thr Gln

645 650 655

Tyr Ala Pro Gly Glu Asp Phe Pro Gly Gly Glu Phe Pro Asn Gln Asn

660 665 670

Pro Arg Val Gly Glu Gly Leu Ala Ser Trp Val Lys Gln Asp Arg Ser

675 680 685

Leu Glu Glu Ser Asp Ile Val Leu Trp Tyr Val Phe Gly Ile Thr His

690 695 700

Val Pro Arg Leu Glu Asp Trp Pro Val Met Pro Val Glu His Ile Gly

705 710 715 720

Phe Met Leu Gln Pro His Gly Phe Phe Asn Cys Ser Pro Ala Val Asp

725 730 735

Val Pro Pro Ser Arg Gly Cys Asp Ser Glu Ser Lys Asp Ser Asp Val

740 745 750

Ser Glu Asn Gly Val Ala Lys Pro Thr Thr Thr Gly Leu Met Ala Lys

755 760 765

Leu

<210> 19

<211> 32

<212> DNA

<213> Artificial sequence ()

<400> 19

gatatcatgg ccacaacctc gcataagcct tc 32

<210> 20

<211> 44

<212> DNA

<213> Artificial sequence ()

<400> 20

ataagaatgc ggccgctcaa agcttggcca tcaaaccagt ggca 44

<210> 21

<211> 28

<212> DNA

<213> Artificial sequence ()

<400> 21

ggatcccatg ggttctaatg ctagaaac 28

<210> 22

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 22

cccaagcttt taagggtttg aatatgcta 29

<210> 23

<211> 31

<212> DNA

<213> Artificial sequence ()

<400> 23

ggaattccat atgatggaag ttatttctaa t 31

<210> 24

<211> 32

<212> DNA

<213> Artificial sequence ()

<400> 24

cggggtacct taaaattcaa cgagatccct ag 32

<210> 25

<211> 46

<212> DNA

<213> Artificial sequence ()

<400> 25

ctaagtttta attacaaagg atccatgggt tcgaacgcca ggaacc 46

<210> 26

<211> 30

<212> DNA

<213> Artificial sequence ()

<400> 26

tgaattaacc tacggattgg aataggcaag 30

<210> 27

<211> 33

<212> DNA

<213> Artificial sequence ()

<400> 27

aatccgtagg ttaattcaaa ttaattgata tag 33

<210> 28

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 28

tctgtgcgtt aaaagatgat gttgaactt 29

<210> 29

<211> 29

<212> DNA

<213> Artificial sequence ()

<400> 29

catcttttaa cgcacagata ttataacat 29

<210> 30

<211> 26

<212> DNA

<213> Artificial sequence ()

<400> 30

cattgtttta tatttgttgt aaaaag 26

<210> 31

<211> 42

<212> DNA

<213> Artificial sequence ()

<400> 31

aacaaatata aaacaatgat ggaagttatt tctaatcata ac 42

<210> 32

<211> 47

<212> DNA

<213> Artificial sequence ()

<400> 32

gtgtgatgga tatctgcaga attcttaaaa ttcaacgaga tccctag 47

<210> 33

<211> 750

<212> DNA

<213> Artificial sequence ()

<400> 33

acgcacagat attataacat ctgcataata ggcatttgca agaattactc gtgagtaagg 60

aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt 120

tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt 180

cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc 240

gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct 300

gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca 360

caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat 420

gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc 480

tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt 540

tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat 600

atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt 660

agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa 720

ttatctactt tttacaacaa atataaaaca 750

<210> 34

<211> 480

<212> DNA

<213> Artificial sequence ()

<400> 34

gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60

gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120

tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180

caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 240

gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 300

ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 360

actcttcgag ttctttgtaa agtctttcat agtagcttac tttatcctcc aacatattta 420

acttcatgtc aatttcggct cttaaatttt ccacatcatc aagttcaaca tcatctttta 480

<210> 35

<211> 6312

<212> DNA

<213> Artificial sequence ()

<400> 35

atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca 60

tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc 120

tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt 180

tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat 240

tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg 300

tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc 360

ttgctcatta gaaagaaagc atagcaatct aatctaagtt ttaattacaa aggatccact 420

agtaacggcc gccagtgtgc tggaattctg cagatatcca tcacactggc ggccgctcga 480

gcatgcatct agagggccgc atcatgtaat tagttatgtc acgcttacat tcacgccctc 540

cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta 600

tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 660

tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 720

ttttgggacg ctcgaaggct ttaatttgca agctgcggcc ctgcattaat gaatcggcca 780

acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 840

gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 900

gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 960

gcccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 1020

cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 1080

ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 1140

taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 1200

ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 1260

ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 1320

aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 1380

tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac 1440

agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 1500

ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 1560

tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 1620

tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 1680

cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 1740

aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 1800

atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggagcg 1860

cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 1920

tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 1980

atccgcctcc attcagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 2040

taatagtttg cgcaacgttg ttggcattgc tacaggcatc gtggtgtcac tctcgtcgtt 2100

tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 2160

gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 2220

cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 2280

cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 2340

gcggcgaccg agttgctctt gcccggcgtc aatacgggat aatagtgtat cacatagcag 2400

aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 2460

accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 2520

ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 2580

gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatgagctcg 2640

ttttcgacac tggatggcgg cgttagtatc gaatcgacag cagtatagcg accagcattc 2700

acatacgatt gacgcatgat attactttct gcgcacttaa cttcgcatct gggcagatga 2760

tgtcgaggcg aaaaaaaata taaatcacgc taacatttga ttaaaataga acaactacaa 2820

tataaaaaaa ctatacaaat gacaagttct tgaaaacaag aatcttttta ttgtcagtac 2880

tgattattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac 2940

ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac 3000

agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc 3060

gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc 3120

ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg 3180

ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc 3240

cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac 3300

attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca 3360

aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt 3420

ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta 3480

ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc 3540

agcgatcgca tccatggcct ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg 3600

caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct 3660

gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata 3720

aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg 3780

ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc 3840

ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg 3900

ctttttaccc atggttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt 3960

ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaat cctaaccttt tatatttctc 4020

tacaggggcg cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg 4080

caggtccgca gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg 4140

gatgcaaatg attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat 4200

gcccctgagc tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtcgctg 4260

gccgggtgac ccggcgggga cgaggcaagc taaacagagc ttatcgatga taagctgtca 4320

aagatgagaa ttaattccac ggactataga ctatactaga tactccgtct actgtacgat 4380

acacttccgc tcaggtcctt gtcctttaac gaggccttac cactcttttg ttactctatt 4440

gatccagctc agcaaaggca gtgtgatcta agattctatc ttcgcgatgt agtaaaacta 4500

gctagaccga gaaagagact agaaatgcaa aaggcacttc tacaatggct gccatcatta 4560

ttatccgatg tgacgctgca gcttctcaat gatattcgaa tacgctttga ggagatacag 4620

cctaatatcc gacaaactgt tttacagatt tacgatcgta cttgttaccc atcattgaat 4680

tttgaacatc cgaacctggg agttttccct gaaacagata gtatatttga acctgtataa 4740

taatatatag tctagcgctt tacggaagac aatgtatgta tttcggttcc tggagaaact 4800

attgcatcta ttgcataggt aatcttgcac gtcgcatccc cggttcattt tctgcgtttc 4860

catcttgcac ttcaatagca tatctttgtt aacgaagcat ctgtgcttca ttttgtagaa 4920

caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca 4980

gaacagaaat gcaacgcgaa agcgctattt taccaacgaa gaatctgtgc ttcatttttg 5040

taaaacaaaa atgcaacgcg acgagagcgc taatttttca aacaaagaat ctgagctgca 5100

tttttacaga acagaaatgc aacgcgagag cgctatttta ccaacaaaga atctatactt 5160

cttttttgtt ctacaaaaat gcatcccgag agcgctattt ttctaacaaa gcatcttaga 5220

ttactttttt tctcctttgt gcgctctata atgcagtctc ttgataactt tttgcactgt 5280

aggtccgtta aggttagaag aaggctactt tggtgtctat tttctcttcc ataaaaaaag 5340

cctgactcca cttcccgcgt ttactgatta ctagcgaagc tgcgggtgca ttttttcaag 5400

ataaaggcat ccccgattat attctatacc gatgtggatt gcgcatactt tgtgaacaga 5460

aagtgatagc gttgatgatt cttcattggt cagaaaatta tgaacggttt cttctatttt 5520

gtctctatat actacgtata ggaaatgttt acattttcgt attgttttcg attcactcta 5580

tgaatagttc ttactacaat ttttttgtct aaagagtaat actagagata aacataaaaa 5640

atgtagaggt cgagtttaga tgcaagttca aggagcgaaa ggtggatggg taggttatat 5700

agggatatag cacagagata tatagcaaag agatactttt gagcaatgtt tgtggaagcg 5760

gtattcgcaa tgggaagctc caccccggtt gataatcaga aaagccccaa aaacaggaag 5820

attgtataag caaatattta aattgtaaac gttaatattt tgttaaaatt cgcgttaaat 5880

ttttgttaaa tcagctcatt ttttaacgaa tagcccgaaa tcggcaaaat cccttataaa 5940

tcaaaagaat agaccgagat agggttgagt gttgttccag tttccaacaa gagtccacta 6000

ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaggg tctatcaggg cgatggccca 6060

ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcagtaaat 6120

cggaagggta aacggatgcc cccatttaga gcttgacggg gaaagccggc gaacgtggcg 6180

agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc 6240

acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtgggga 6300

tgatccacta gt 6312

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 36

cccaaagcta agagtcccat 20

<210> 37

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 37

tggaagagta aaaaaggagt agaaacattt tgaagctatc tgctcttgaa tggcgacag 59

<210> 38

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 38

ggaacactgg ggcaataggc tgtcgccatt caagagcaga tagcttcaaa atgtttcta 59

<210> 39

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 39

accagaccag aagaatgatg atgatgatgg tgcattttgt aattaaaact tagattaga 59

<210> 40

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 40

aaagcatagc aatctaatct aagttttaat tacaaaatgc accatcatca tcatcattc 59

<210> 41

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 41

cctggtaaag ttgtgtgcta gtgtctcccg tcttctgttc aaagcttggc catcaaacc 59

<210> 42

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 42

agcaaagccc actaccactg gtttgatggc caagctttga acagaagacg ggagacact 59

<210> 43

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 43

ctgtcgattc gatactaacg ccgccatcca gtgtcgaatt ttcaacatcg tattttccg 59

<210> 44

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 44

cattatgcaa cgcttcggaa aatacgatgt tgaaaattcg acactggatg gcggcgtta 59

<210> 45

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 45

aattcaaaaa aaaaaagcga atcttcccat gcctgttcag cgacatggag gcccagaat 59

<210> 46

<211> 59

<212> DNA

<213> Artificial sequence ()

<400> 46

agactgtcaa ggagggtatt ctgggcctcc atgtcgctga acaggcatgg gaagattcg 59

<210> 47

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 47

tctggtgagg atttacggta tg 22

<210> 48

<211> 522

<212> DNA

<213> Artificial sequence ()

<400> 48

cccaaagcta agagtcccat tttattcttc tatatgtata ttttcgatac tctaaaccac 60

cctacaatgt agccctatac taaatctgct caattttcag cttctacaag tgactcgaga 120

ccacgtggaa agatccaact actccagcac aacgattcaa tataatcgat tgctccactc 180

ataagaggca agaacaagct tcaacttttg gtaagccgcc gtttataaac agggaagatg 240

tcctttgtca agggaggcac agagcatggc caatttggca aattgcaggt ttttctgagt 300

gaaaaatgaa aaagcattgt agtagagtcg gctcactgaa aaaccgggga ggacgaaaag 360

gtttccagcc acagttgtag tcacgtgcgc gccatgctga ctaatggcag ccgtcgttgg 420

gcagaagaga attagtatgg tacaggatac gctaattgcg ctccaactac caaggttgtt 480

gagggaacac tggggcaata ggctgtcgcc attcaagagc ag 522

<210> 49

<211> 430

<212> DNA

<213> Artificial sequence ()

<400> 49

agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 60

tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 120

ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 180

aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 240

tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 300

tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 360

ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 420

taattacaaa 430

<210> 50

<211> 250

<212> DNA

<213> Artificial sequence ()

<400> 50

acagaagacg ggagacacta gcacacaact ttaccaggca aggtatttga cgctagcatg 60

tgtccaattc agtgtcattt atgatttttt gtagtaggat ataaatatat acagcgctcc 120

aaatagtgcg gttgccccaa aaacaccacg gaacctcatc tgttctcgta ctttgttgtg 180

acaaagtagc tcactgcctt attatcacat tttcattatg caacgcttcg gaaaatacga 240

tgttgaaaat 250

<210> 51

<211> 1398

<212> DNA

<213> Artificial sequence ()

<400> 51

cagcgacatg gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc 60

atgatgtgac tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc 120

catacatttt gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg 180

cgagcaggga aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta 240

gagaaatata aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa 300

aatcttgcta ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaggaa 360

aagactcacg tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat 420

aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag 480

cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca 540

gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat 600

tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca 660

ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg 720

ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta 780

tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt 840

gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg 900

ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt 960

gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac 1020

caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg 1080

ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg 1140

ctcgatgagt ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt 1200

catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt 1260

atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa 1320

tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac 1380

gccgccatcc agtgtcga 1398

<210> 52

<211> 452

<212> DNA

<213> Artificial sequence ()

<400> 52

aacaggcatg ggaagattcg cttttttttt ttgaattaca atagtatgtc tgatgtctgc 60

aagaagtaac aggcgtgtgc acaagaatac gtgtgtgtgc gtaagcgtat gcactggtgg 120

cataacttat ctaagaagta tatatcactg acatagaaat gtagatatac aggtattttt 180

ctcgataatc gataaaaatc tcgtcgcgct gaaccaaact tggtggttac ggagagtttt 240

tctctcatca ttactgtctt tcgcattgat ttcccctttg accgataaaa tcccttggat 300

tcataagatt aaacaaagag gtgatcaaag agaaccctgt gaaagtttat gtttataacc 360

gggcataaag tgaactagac actttcaaga agccaaccaa agcatgagta acgaagctta 420

ccagcatgat cataccgtaa atcctcacca ga 452

<210> 53

<211> 1560

<212> DNA

<213> Saccharomyces cerevisiae BY4742()

<400> 53

atgttcagta gatctacgct ctgcttaaag acgtctgcat cctccattgg gagacttcaa 60

ttgagatatt tctcacacct tcctatgaca gtgcctatca agctgcccaa tgggttggaa 120

tatgagcaac caacggggtt gttcatcaac aacaagtttg ttccttctaa acagaacaag 180

accttcgaag tcattaaccc ttccacggaa gaagaaatat gtcatattta tgaaggtaga 240

gaggacgatg tggaagaggc cgtgcaggcc gccgaccgtg ccttctctaa tgggtcttgg 300

aacggtatcg accctattga caggggtaag gctttgtaca ggttagccga attaattgaa 360

caggacaagg atgtcattgc ttccatcgag actttggata acggtaaagc tatctcttcc 420

tcgagaggag atgttgattt agtcatcaac tatttgaaat cttctgctgg ctttgctgat 480

aaaattgatg gtagaatgat tgatactggt agaacccatt tttcttacac taagagacag 540

cctttgggtg tttgtgggca gattattcct tggaatttcc cactgttgat gtgggcctgg 600

aagattgccc ctgctttggt caccggtaac accgtcgtgt tgaagactgc cgaatccacc 660

ccattgtccg ctttgtatgt gtctaaatac atcccacagg cgggtattcc acctggtgtg 720

atcaacattg tatccgggtt tggtaagatt gtgggtgagg ccattacaaa ccatccaaaa 780

atcaaaaagg ttgccttcac agggtccacg gctacgggta gacacattta ccagtccgca 840

gccgcaggct tgaaaaaagt gactttggag ctgggtggta aatcaccaaa cattgtcttc 900

gcggacgccg agttgaaaaa agccgtgcaa aacattatcc ttggtatcta ctacaattct 960

ggtgaggtct gttgtgcggg ttcaagggtg tatgttgaag aatctattta cgacaaattc 1020

attgaagagt tcaaagccgc ttctgaatcc atcaaggtgg gcgacccatt cgatgaatct 1080

actttccaag gtgcacaaac ctctcaaatg caactaaaca aaatcttgaa atacgttgac 1140

attggtaaga atgaaggtgc tactttgatt accggtggtg aaagattagg tagcaagggt 1200

tacttcatta agccaactgt ctttggtgac gttaaggaag acatgagaat tgtcaaagag 1260

gaaatctttg gccctgttgt cactgtaacc aaattcaaat ctgccgacga agtcattaac 1320

atggcgaacg attctgaata cgggttggct gctggtattc acacctctaa tattaatacc 1380

gccttaaaag tggctgatag agttaatgcg ggtacggtct ggataaacac ttataacgat 1440

ttccaccacg cagttccttt cggtgggttc aatgcatctg gtttgggcag ggaaatgtct 1500

gttgatgctt tacaaaacta cttgcaagtt aaagcggtcc gtgccaaatt ggacgagtaa 1560

<210> 54

<211> 1563

<212> DNA

<213> Saccharomyces cerevisiae BY4742()

<400> 54

atgctttctc gcacaagagc tgcagctccg aattccagaa tattcactag aagcttgtta 60

cgtctttatt ctcaagcacc attacgcgtt ccaattactc ttccaaatgg tttcacctac 120

gaacagccaa cagggttatt catcaatggt gaatttgttg cctcgaagca aaagaaaacg 180

tttgacgtga tcaatccatc taacgaagaa aagataacaa ctgtatacaa ggctatggaa 240

gatgatgttg atgaagccgt tgcagcggct aaaaaagctt ttgaaacgaa gtggtctatt 300

gtagagccgg aggttcgcgc taaagcttta ttcaatctcg ctgacttggt tgagaaacac 360

caagaaacac tggctgccat tgagtcaatg gataatggta agtcattgtt ttgtgcgcgc 420

ggtgacgtcg ctttagtatc taaatacttg cgttcttgcg gtggttgggc agataaaatc 480

tacggtaacg ttattgacac aggtaaaaac cattttacct actcaattaa ggaaccatta 540

ggcgtttgcg gccaaataat cccttggaac ttccctttat tgatgtggtc atggaaaatt 600

gggcctgctc tggctacagg taacaccgtc gtattgaaac ccgctgaaac aacaccttta 660

tctgcccttt tcgcttccca gttgtgtcag gaagcaggca tacccgctgg tgtagtcaat 720

atccttccgg gttccggtag agttgttgga gaaagattga gtgcacaccc agacgtgaag 780

aagattgctt ttacaggctc tactgccacc ggccgccata ttatgaaggt cgctgccgat 840

actgtcaaga aagtcacttt ggagctggga ggtaaatcac caaatattgt gtttgctgac 900

gctgatctag ataaagccgt caagaacatt gccttcggta ttttttacaa ctctggtgaa 960

gtttgctgcg ctggttccag aatatacatt caagatacag tatacgagga ggtgttgcaa 1020

aaactaaagg attacaccga gtcactaaag gtcggtgacc catttgatga ggaagttttc 1080

caaggtgctc aaacatctga caaacagctg cataaaattt tagactatgt cgatgtagca 1140

aaatcagagg gggctcgtct tgtgactgga ggggccagac atggcagtaa aggttatttt 1200

gtcaagccaa cagtgtttgc tgatgtcaaa gaagatatga gaattgttaa ggaggaagtg 1260

tttggtccca ttgtaactgt atccaagttt tctactgttg atgaagtgat tgctatggca 1320

aatgattctc aatatgggtt agccgcaggt attcacacta acgatattaa caaggctgtt 1380

gatgtgtcca aaagagtgaa agctggtact gtttggataa atacctataa caacttccac 1440

caaaatgttc ctttcggtgg cttcggccag tcaggtattg gccgtgaaat gggtgaggct 1500

gctttaagta actacactca aacaaaatct gtcagaattg ccattgacaa gccaattcgt 1560

tga 1563

<210> 55

<211> 1599

<212> DNA

<213> Saccharomyces cerevisiae BY4742()

<400> 55

atgtcaaacg acggctcaaa aatattgaat tataccccag tgtctaaaat agatgaaata 60

gttgaaatct caagaaattt cttctttgag aaacaattga aattgtccca cgaaaataac 120

ccaaggaaaa aagatctaga attcaggcag ttgcagttga aaaaactcta ttatgccgtc 180

aaagatcatg aggaagaact gatcgatgct atgtacaagg actttcatcg gaacaaaatt 240

gaatcggttc tgaatgaaac gaccaaactt atgaacgata tacttcacct aattgagatt 300

ttaccaaaat tgatcaaacc tcggagagta tctgattctt ctcctccatt tatgtttggt 360

aaaacaatcg tggagaaaat atcaaggggc agtgtcttga ttattgctcc tttcaatttt 420

cccctacttt tagcatttgc cccattggca gcagctcttg ctgcaggtaa caccattgtt 480

ctgaagccaa gtgaactaac accacacact gctgtagtta tggaaaattt gttaaccaca 540

gctggtttcc ctgatggatt gattcaagta gttcagggag ctatagatga aactacaaga 600

ctactagatt gtggaaaatt tgacctaata ttctacacag gttctccccg tgtcggatca 660

atagttgctg agaaagcagc aaaaagtcta acaccttgtg tacttgaact tggtggtaaa 720

tcacctacct ttattacaga aaatttcaaa gcaagtaaca taaaaattgc tttgaaaagg 780

attttttttg gtgctttcgg aaattctggc cagatttgtg tttcaccaga ttatttgtta 840

gtacataaat ctatctatcc aaaagtcatt aaagagtgtg aatcagtact aaatgaattt 900

tatccaagct ttgatgaaca aacagatttc actcgtatga ttcatgagcc tgcttacaaa 960

aaggccgttg caagtataaa ctcaactaac ggctccaaga ttgtgccttc aaaaatttct 1020

atcaattcag atactgagga tctatgcctt gtaccaccaa ccatagttta taacattggt 1080

tgggatgatc ctttgatgaa acaggaaaac tttgctcctg tattgcccat cattgagtac 1140

gaggatcttg atgagaccat taacaagata atagaagaac atgacactcc attggtgcaa 1200

tacatattct ctgatagcca aactgaaata aatcgtatct tgacgcgctt aagatctggt 1260

gactgtgttg tcggtgatac agtgattcat gtaggaatta ccgacgctcc atttggaggg 1320

atcggtactt caggttatgg taactatggt ggatattatg gattcaatac ctttagtcat 1380

gaaagaacaa tttttaaaca accatattgg aatgatttta ccctttttat gagataccct 1440

ccaaatagcg cacaaaagga aaagctcgtc cgttttgcga tggaaagaaa accttggttt 1500

gacagaaatg gcaataacaa gtgggggtta cgccaatatt tttcattatc tgccgccgtt 1560

attttaatta gtaccattta cgctcattgt tcttcctga 1599

<210> 56

<211> 4140

<212> DNA

<213> Artificial sequence ()

<400> 56

atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540

gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600

atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660

cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720

cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780

gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840

cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960

atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200

aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620

tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920

catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040

gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280

atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580

gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700

actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420

tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960

cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080

gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtga 4140

<210> 57

<211> 591

<212> DNA

<213> Artificial sequence ()

<400> 57

cacgagcttg cattaccggc agttgctcca gcttgctggc ctctgccagc agcaatgtcc 60

cgccctggcg accctctggt tagatgacac tcctgcccca actgccacga atctgtaacc 120

ccataactat acccgtacgc agtactaaaa atgtatgtaa ttagtaaatg tatgtaacaa 180

tttcaccgtt ttgtgtaaca attcattcat tcattctttt gatcctttag taccgtccgc 240

acatgatgtc atttccccct catttttgtt tgctggtatg attccccgcc cgggcgacgg 300

tacggctgtt atccagcgat gcgggacttc cgtccacagg tatctttttc tccaactcca 360

acagagatgg aaaatgaggg gcgggtgtag gtaagcagaa tgaggagaaa tttgtaatga 420

aaatggaagt tcggcggtta tataaatggg gggggtttgt cggtgacaat tgacttcact 480

ctcctttcct caaaaattct tgggtgttag gattagaagt atctggaaaa ccaaccaaga 540

aaactacaat aacaaaaata aataaagcgg tcatcaataa gcctggtgtc c 591

<210> 58

<211> 588

<212> DNA

<213> Artificial sequence ()

<400> 58

gaaaactaca ataacaaaaa taaataaagc ggtcatcaat aagcctggtg tccaatcgat 60

gcttacatac ataaaattaa atattctgtc tctgttatat ttccacatgt catcatttca 120

aatatatgta ctttaaagaa aataaaataa aaaataaaat ttttttctcc cgataatcaa 180

ttttcttaat taattaattg cgttacgatt ccgttttttt acttctttta tctcattatc 240

tatctaagtt atttaaaaaa aagaaagaac tttttatgaa ctttcctctt ttctttcttt 300

tagactattt aaaatacatc accttggtca aacatagcat cagagacctt gatgaaactt 360

gcgatattag cacctttgac caaagatggc aagaccttac cgtccttagt gtacttcttg 420

gcatagtcga tacattcatt gaaacagttg atcataattc tcttcaactc ttggtcaact 480

ctttcgctag tccatgtgat tctttgagag ttttgtgcca tttctaaacc agaaacagca 540

acaccaccca agttagcagc ctttggtgga ccgtaccaaa cagcttcg 588

<210> 59

<211> 576

<212> DNA

<213> Artificial sequence ()

<400> 59

gccgtttaca catcaatgat aaataagtat acaaaaaggg ttccattttt ttttttggcc 60

gctaccggac tagcaagggc ctaatggtac gctgagcgta gtacaaccaa gcgcttgtta 120

gccgtagagt taagctcttg actactatta cggtaaaagc cgggaacgtg cgtaacaatt 180

tttttttgca ttaggttaaa gaggctcgct cgcggagcct ttagaatacc gcttaaggcg 240

ccaaaagatg gaagctattc tctttctttt ttttttcaca aactgagagg ggttgtgtat 300

cgttaaaaat gttggaagac ttctgactca tcactacgca gattgttaga gtttttcgat 360

gagaatggct tcaaagaaca gaacaaaaca cgattatata agccccatgt aaaaagaacg 420

tcttaattta ttttgaattt aggacttctt gacattttta gcatatataa caatacgata 480

agtgtctcat caaacgtggt taagacagaa aacttcttca caacattaac aaaaagccaa 540

agaagaagaa agtaactcag gcccgagttg actgct 576

<210> 60

<211> 556

<212> DNA

<213> Artificial sequence ()

<400> 60

cattaacaaa aagccaaaga agaagaaagt aactcaggcc cgagttgact gctcattgaa 60

ggtatgtatg ataaacatta tagaataatg aaaaatgcct cgtgacatac agataaaatc 120

taacaaggat atattcaaaa aaaaaaaaac aaaacaaaaa aataataacg tgataaacat 180

taatgaacaa tgtatttaca ttcttaagca taggtgagaa attaccttct ttactttttt 240

tttttttttt ggtgatattg tatattgaaa tatatagtaa tcaaattcgt ttcattgatc 300

aaattgctca ctagttctgt ttttcaaaat ttcatcttta taggtagata caagtgccag 360

agagatatat aaacagaaaa ctctatcgat gtgataatgt atgccaatat cgggactgta 420

cacccacaca tttacaagcc cacacatcct acaactttct tttcatttct tgcgcttctt 480

ccactgtcat aaaatatgat tgtccgatgc cgcagcctac gcctcggcga gtattcatca 540

cctaagcgtc ctgtag 556

<210> 61

<211> 473

<212> DNA

<213> Artificial sequence ()

<400> 61

tggtctctcg ggaaacttct ggatggctct aaggaaggtt gggtgccaac agcttacatg 60

aaaccacatt ccggaaataa taatatccct actcctcctc aaaacaggga tgttcctaaa 120

cccgttctga attctgtaca acatgataac acctctgcaa acgttattcc agctgcagcc 180

caagcaagtt tgggtgatgg tttggcgaat gcgcttgctg ctagggccaa taaaatgaga 240

ttagagagtg atgacgagga ggctaacgaa gatgaagagg aagatgattg gtaatcaaaa 300

tgtatttagc gagtatatac tctatacgag caaatagagt aaattgtaat taataaatat 360

tgatatatta tcgcccatat gaaattttta aacgaaaggt tacttataca tcaaataatt 420

aattaacctt aaacattacg tgttggtgat aaattactat ggctatggtt tta 473

<210> 62

<211> 468

<212> DNA

<213> Artificial sequence ()

<400> 62

aataattaat taaccttaaa cattacgtgt tggtgataaa ttactatggc tatggtttta 60

gaatattcct tttagataat caatatgcct gaagttttgt tttttatata tggagtatcc 120

gtttatccct aaaatccgaa gaatctcctc ctaaaacgac aaaaaaaagg gtaagcaatc 180

caccatctaa ttaaatcact aatattagat tgaattttcg aacctatcta aaagtgggat 240

cttgatgtaa aattgactga tagataaaag gcttggagtc gatagttgat atatcagtac 300

accggtaatt gtctattata taccctgttg gcatgaaatc catataggac ggtatatatt 360

tatatataaa atgcatgtaa aaatctttca tcatgaacta ctcggaattt ttggctggaa 420

attcgttgtt ccatttcctc taagtgtctt gatcatgttc tggtgtgc 468

<210> 63

<211> 40

<212> DNA

<213> Artificial sequence ()

<400> 63

agttttaatt acaaaggatc catggacaag aagtactcca 40

<210> 64

<211> 40

<212> DNA

<213> Artificial sequence ()

<400> 64

ccctctagat gcatgctcga gtcacacctt cctcttcttc 40

<210> 65

<211> 44

<212> DNA

<213> Artificial sequence ()

<400> 65

cttgtcctgt tcaattaatt gttttagagc tagaaatagc aagt 44

<210> 66

<211> 47

<212> DNA

<213> Artificial sequence ()

<400> 66

aattaattga acaggacaag gatcatttat ctttcactgc ggagaag 47

<210> 67

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 67

cacgagcttg cattaccg 18

<210> 68

<211> 48

<212> DNA

<213> Artificial sequence ()

<400> 68

ggacaccagg cttattgatg accgctttat ttatttttgt tattgtag 48

<210> 69

<211> 48

<212> DNA

<213> Artificial sequence ()

<400> 69

gaaaactaca ataacaaaaa taaataaagc ggtcatcaat aagcctgg 48

<210> 70

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 70

cgaagctgtt tggtacgg 18

<210> 71

<211> 44

<212> DNA

<213> Artificial sequence ()

<400> 71

tggcaaatga ttctcaatat gttttagagc tagaaatagc aagt 44

<210> 72

<211> 47

<212> DNA

<213> Artificial sequence ()

<400> 72

atattgagaa tcatttgcca gatcatttat ctttcactgc ggagaag 47

<210> 73

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 73

gccgtttaca catcaatg 18

<210> 74

<211> 46

<212> DNA

<213> Artificial sequence ()

<400> 74

agcagtcaac tcgggcctga gttactttct tcttctttgg cttttt 46

<210> 75

<211> 47

<212> DNA

<213> Artificial sequence ()

<400> 75

cattaacaaa aagccaaaga agaagaaagt aactcaggcc cgagttg 47

<210> 76

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 76

ctacaggacg cttaggtg 18

<210> 77

<211> 44

<212> DNA

<213> Artificial sequence ()

<400> 77

agggtaaaat cattccaata gttttagagc tagaaatagc aagt 44

<210> 78

<211> 47

<212> DNA

<213> Artificial sequence ()

<400> 78

cttctccgca gtgaaagata aatgatcagg gtaaaatcat tccaata 47

<210> 79

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 79

tggtctctcg ggaaactt 18

<210> 80

<211> 50

<212> DNA

<213> Artificial sequence ()

<400> 80

taaaaccata gccatagtaa tttatcacca acacgtaatg tttaaggtta 50

<210> 81

<211> 50

<212> DNA

<213> Artificial sequence ()

<400> 81

aataattaat taaccttaaa cattacgtgt tggtgataaa ttactatggc 50

<210> 82

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 82

gcacaccaga acatgatc 18

<210> 83

<211> 400

<212> DNA

<213> Artificial sequence ()

<400> 83

acaaatcgct cttaaatata tacctaaaga acattaaagc tatattataa gcaaagatac 60

gtaaattttg cttatattat tatacacata tcatatttct atatttttaa gatttggtta 120

tataatgtac gtaatgcaaa ggaaataaat tttatacatt attgaacagc gtccaagtaa 180

ctacattatg tgcactaata gtttagcgtc gtgaagactt tattgtgtcg cgaaaagtaa 240

aaattttaaa aattagagca ccttgaactt gcgaaaaagg ttctcatcaa ctgtttaaaa 300

ggaggatatc aggtcctatt tctgacaaac aatatacaaa tttagtttca aagatgaatc 360

agtgcgcgaa ggacataact catgaagcct ccagtatacc 400

<210> 84

<211> 1152

<212> DNA

<213> Saccharomyces cerevisiae BY4742()

<400> 84

atgtccaaga gcaaaacttt cttatttacc tctgaatccg tcggtgaagg tcacccagac 60

aagatttgtg accaagtttc tgatgctatt ttggacgctt gtttagaaca agatccattc 120

tccaaggttg cctgtgaaac agctgccaaa actggtatga ttatggtttt cggtgaaatt 180

accaccaaag ctagacttga ctaccaacaa atagtaagag ataccatcaa gaagattggt 240

tatgacgatt ctgccaaggg tttcgactac aagacatgta atgttttagt agctatcgaa 300

caacaatctc cagatatcgc tcaaggtctg cactatgaaa agagcttaga agacttaggt 360

gctggtgacc aaggtataat gtttggttac gctacagacg aaactccaga agggttacca 420

ttgaccattc ttttggctca caaattgaac atggctatgg cagatgctag aagagatggt 480

tctctcccat ggttgagacc agacacaaag actcaagtca ctgtcgaata cgaagacgac 540

aatggtagat gggttccaaa gaggatagat accgttgtta tttctgctca acatgctgat 600

gaaatttcca ccgctgactt gagaactcaa cttcaaaaag atattgttga aaaggtcata 660

ccaaaggata tgttagacga aaataccaaa tatttcatcc aaccatccgg tagattcgtc 720

atcggtggtc ctcaaggtga cgctggtttg accggtagaa agattattgt cgacgcttac 780

ggtggtgcct catccgtcgg tggtggtgcc ttctccggta aggactattc caaggtcgat 840

cgttccgctg cttacgctgc tagatgggtt gccaagtctc tagttgccgc tggtttgtgt 900

aagagagtcc aagtccaatt ttcatatgct attggtattg ctgaaccatt gtctttacat 960

gtggacacct atggtacagc tacaaaatca gatgacgaaa tcattgaaat tattaagaag 1020

aacttcgact tgagaccagg tgtgttagta aaggaattag atttggctag accaatttac 1080

ttaccaaccg cttcttatgg tcacttcact aatcaagagt actcatggga aaaaccaaag 1140

aaattggaat tt 1152

<210> 85

<211> 1556

<212> DNA

<213> Artificial sequence ()

<400> 85

cgcgccatag cttcaaaatg tttctactcc ttttttactc ttccagattt tctcggactc 60

cgcgcatcgc cgtaccactt caaaacaccc aagcacagca tactaaattt cccctctttc 120

ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc 180

ctcgtttctt tttcttcgtc gaaaaaggct taattaatag agattactac atattccaac 240

aagaccttcg caggaaagta tacctaaact aattaaagaa atctccgaag ttcgcatttc 300

attgaacggc tcaattaatc tttgtaaata tgagcgtttt tacgttcaca ttgccttttt 360

ttttatgtat ttaccttgca tttttgtgct aaaaggcgtc acgttttttt ccgccgcagc 420

cgcccggaaa tgaaaagtat gacccccgct agaccaaaaa tacttttgtg ttattggagg 480

atcgcaatcc ctaagcttag tggaattatt agaatgacca ctactccttc taatcaaaca 540

cgcggaaata gccgccaaaa gacagatttt attccaaatg cgggtaacta tttgtataat 600

atgtttacat attgagcccg tttaggaaag tgcaagttca aggcactaat caaaaaagga 660

gatttgtaaa tatagcgacc gaatcaggaa aaggtcaaca acgaagttcg cgatatggat 720

gaacttcggt gcctgtccgt ttaaacatcg gatcccatac tagcgttgaa tgttagcgtc 780

aacaacaaga agtttaatga cgcggaggcc aaggcaaaaa gattccttga ttacgtaagg 840

gagttagaat cattttgaat aaaaaacacg ctttttcagt tcgagtttat cattatcaat 900

actgccattt caaagaatac gtaaataatt aatagtagtg attttcctaa ctttatttag 960

tcaaaaaatt agccttttaa ttctgctgta acccgtacat gcccaaaata gggggcgggt 1020

tacacagaat atataacatc gtaggtgtct gggtgaacag tttattcctg gcatccacta 1080

aatataatgg agcccgcttt ttaagctggc atccagaaaa aaaaagaatc ccagcaccaa 1140

aatattgttt tcttcaccaa ccatcagttc ataggtccat tctcttagcg caactacaga 1200

gaacaggggc acaaacaggc aaaaaacggg cacaacctca atggagtgat gcaacctgcc 1260

tggagtaaat gatgacacaa ggcaattgac ccacgcatgt atctatctca ttttcttaca 1320

ccttctatta ccttctgctc tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca 1380

gttccctgaa attattcccc tacttgacta ataagtatat aaagacggta ggtattgatt 1440

gtaattctgt aaatctattt cttaaacttc ttaaattcta cttttatagt tagtcttttt 1500

tttagtttta aaacaccaag aacttagttt cgaataaaca cacataaaca aacaaa 1556

<210> 86

<211> 58

<212> DNA

<213> Artificial sequence ()

<400> 86

gcgcgaagga cataactcat gaagcctcca gtataccatt ttcaacatcg tattttcc 58

<210> 87

<211> 53

<212> DNA

<213> Artificial sequence ()

<400> 87

atgcaacgct tcggaaaata cgatgttgaa aatggtatac tggaggcttc atg 53

<210> 88

<211> 57

<212> DNA

<213> Artificial sequence ()

<400> 88

agtactcatg ggaaaaacca aagaaattgg aatttacaaa tcgctcttaa atatata 57

<210> 89

<211> 56

<212> DNA

<213> Artificial sequence ()

<400> 89

aatgttcttt aggtatatat ttaagagcga tttgtaaatt ccaatttctt tggttt 56

<210> 90

<211> 56

<212> DNA

<213> Artificial sequence ()

<400> 90

aacttagttt cgaataaaca cacataaaca aacaaaatgt ccaagagcaa aacttt 56

<210> 91

<211> 55

<212> DNA

<213> Artificial sequence ()

<400> 91

tcagaggtaa ataagaaagt tttgctcttg gacattttgt ttgtttatgt gtgtt 55

<210> 92

<211> 53

<212> DNA

<213> Artificial sequence ()

<400> 92

gtcgattcga tactaacgcc gccatccagt gtcgacgcgc catagcttca aaa 53

<210> 93

<211> 53

<212> DNA

<213> Artificial sequence ()

<400> 93

aaaaaggagt agaaacattt tgaagctatg gcgcgtcgac actggatggc ggc 53

Claims

1. A method for preparing N-methylpyrrolidine, comprising the steps of: is prepared by using L-ornithine as a substrate and performing combined catalysis on ornithine decarboxylase EcODC with an amino acid sequence of SEQ ID NO. 2, putrescine-N-methyltransferase AtPMT with an amino acid sequence of SEQ ID NO. 6 and amine oxidase from acutangular anisotrophin, wherein the L-ornithine is used as a substrate, and the amino acid is obtained by using the L-ornithine decarboxylase EcODC, the putrescine-N-methyltransferase AtPMT with an amino acid sequence of SEQ ID NO. 6 and the amine oxidase from acutangular anisotrophin

The amine oxidase is AaDAO2 with the amino acid sequence of SEQ ID NO. 14 or AaDAO3 with the amino acid sequence of SEQ ID NO. 18.

2. The method of claim 1, wherein the amine oxidase is AaDAO3 having the amino acid sequence of SEQ ID NO. 18.

3. A microorganism expressing the ornithine decarboxylase EcODC, putrescine-N-methyltransferase AtPMT of claim 1 or 2, and an amine oxidase.

4. The microorganism of claim 3, wherein the microorganism is selected from the group consisting of E.coli and Saccharomyces cerevisiae.

5. The microorganism according to claim 3, wherein the microorganism is Saccharomyces cerevisiae and the genes ALD4, ALD5 and HFD1 metabolizing N-methylaminobutyraldehyde in the genome are knocked out.

6. The microorganism of claim 5, wherein the Saccharomyces cerevisiae overexpresses the cofactor synthesis gene SAM2 having the sequence of SEQ ID NO 84.

7. Use of a microorganism as claimed in any one of claims 3 to 6 for the preparation of N-methylpyrrolidine.

8. Use according to claim 7, for the production of N-methylpyrrolidine by fermentation of a microorganism according to any of claims 3 to 6.

9. The use according to claim 8, wherein when the microorganism is Escherichia coli, the medium is LB medium; when the microorganism is Saccharomyces cerevisiae, the medium is YPD medium.