CN109266596B - Escherichia coli Recombinant Bacteria Efficiently Utilizing Fatty Acids to Synthesize Glycine and Its Construction Method and Application - Google Patents

Abstract

The invention discloses an Escherichia coli recombinant bacteria that efficiently utilizes fatty acids to synthesize glycine, and a construction method and application thereof. The Escherichia coli recombinant bacteria contain one or more of the following genetic modifications: (1) enhancement of fatty acid intake pathway and fatty acid beta oxidation pathway; (2) modification of glyoxylate cycle-related gene targets to enhance glyoxylate (3) Introduce/enhance the expression of glutamate glyoxylate transaminase gene. The engineering strain of the present invention has the ability to convert whole cells of fatty acid substrates of different lengths into glycine, and the fatty acids can be stearic acid (C18), palmitic acid (C16), myristic acid (C14), lauric acid (C12) , capric acid (C10), caprylic acid (C8) or caproic acid (C6). The ability of the engineered strain to utilize fatty acid to synthesize glycine makes it suitable for industrial production of glycine products.

Description

Escherichia coli recombinant bacterium for efficiently synthesizing glycine by using fatty acid and construction method and application thereof

Technical Field

The invention relates to the field of biotechnology and biochemical industry of industrial microorganisms, in particular to an escherichia coli recombinant bacterium for synthesizing glycine by efficiently utilizing fatty acid and a construction method and application thereof.

Background

With the development of society, people face a series of serious challenges such as energy shortage, resource shortage, environmental deterioration, economic decline, climate change and the like, and novel industries such as biological manufacturing, biological energy and the like are emerging. Compared with the traditional chemical production, the biotechnology has the advantages of energy conservation, emission reduction and consumption reduction. By carrying out genetic modification on industrial microbial strains, the utilization of microbial cells on raw materials can be enhanced, the product conversion rate is improved, and the production cost is reduced.

Glycine (also known as Glycine in english) is also known as Glycine, and is the α -amino acid with the simplest structure. It is an important fine chemical synthesis intermediate, and is widely applied in the fields of medicines, foods, pesticides, feed additives and the like. (1) Glycine is a synthetic precursor of some important small molecule metabolites in organisms, and the addition of a proper amount of glycine into the diet can effectively treat cardiovascular diseases, inflammation, obesity, tumors and metabolic disorders of patients with diabetes. (2) The glycine is an intermediate for synthesizing various medicaments such as delapril hydrochloride, oxalacetic acid aspirin calcium, paracetamol glycinate, and a limemine injection. (3) Glycine is widely used in the food industry as an amino acid enhancer, a flavoring agent, a sweetening agent and an antioxidant. (4) Glycine is the precursor for the synthetic herbicide glyphosate, which has been evaluated by the united states government as the most elegant pesticide.

The existing methods for synthesizing glycine mainly comprise chloroacetic acid ammonolysis, Strecker method, catalytic dehydrogenation oxidation method, radiation synthesis method, biosynthesis method and the like. Among them, the biosynthesis method has been conventionally obtained by transformation with microorganisms such as aerobic Agrobacterium, Brevibacterium, Corynebacterium, etc., using ethanolamine as a substrate, and also has been used for producing glycine by hydrolyzing glycinamide with the use of genera such as Pseudomonas, Casein, Alcaligenes, etc. Although the above method can realize the production of glycine, the method has the problems of high raw material cost, low enzyme activity and large quantity of strains, so that the biotransformation still needs to establish a cheaper raw material route. Fatty acids are highly reduced substances, a large amount of two-carbon substance acetyl coenzyme A is generated through beta oxidation, then glyoxylic acid is obtained through glyoxylic acid circulation to finally generate glycine, the glycine is generated by taking fatty acid (palmitic acid) as a substrate, the theoretical conversion rate can reach 234.21 percent and is obviously higher than that of glucose, and fatty acid raw materials for bioconversion can be obtained from oil crude processing products, illegal cooking oil and the like at low cost, so that the glycine production by taking the fatty acid as the substrate has great potential.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the synthesis efficiency of glycine.

In order to solve the technical problems, the invention improves the utilization capacity of fatty acid substrates by enhancing a fatty acid intake pathway and a fatty acid beta oxidation pathway, improves the synthesis capacity of glycine by modifying primary metabolism to strengthen the accumulation of intermediate product glyoxylate and introducing/enhancing the expression of a glutamate glyoxylate aminotransferase II gene required by a glycine synthesis pathway, and simultaneously improves the supply of auxiliary factors such as NADPH, glutamic acid and the like to further improve the synthesis efficiency of the glycine. Provides a recombinant Escherichia coli for synthesizing glycine by efficiently using fatty acid.

In the first aspect, the invention provides a construction method of recombinant escherichia coli for efficiently synthesizing glycine by using fatty acid.

The construction method of the recombinant escherichia coli for efficiently synthesizing the glycine by using the fatty acid comprises the steps of carrying out (1) - (13) modification on the recipient bacterium or carrying out (1) - (7) modification on the recipient bacterium to obtain the recombinant escherichia coli;

(1) inhibiting the expression of the fadR gene in the receptor bacterium or/and inhibiting the activity of a protein coded by the fadR gene in the receptor bacterium, namely a fatty acid degradation transcription factor;

(2) enhancing the expression of sthA gene in the recipient bacterium or/and enhancing the activity of pyridine nucleotide transhydrogenase which is protein coded by the sthA gene in the recipient bacterium;

(3) inhibiting the expression of the pntA gene in the recipient bacterium or/and inhibiting the activity of a protein coded by the pntA gene in the recipient bacterium, namely pyridine nucleotide transhydrogenase alpha subunit;

(4) enhancing the expression of fadL gene in the recipient bacterium or/and enhancing the activity of the protein coded by the fadL gene in the recipient bacterium, namely long-chain fatty acid, in the outer membrane protein;

(5) enhancing the expression of a gene involved in the fatty acid beta-oxidation pathway in the recipient bacterium or/and enhancing the activity of a protein encoded by a gene involved in the fatty acid beta-oxidation pathway in the recipient bacterium;

(6) enhancing the expression of genes involved in short-chain fatty acid degradation pathways in the recipient bacterium or/and enhancing the activity of proteins encoded by genes involved in short-chain fatty acid degradation pathways in the recipient bacterium;

(7) enhancing the expression of the exogenous alkL gene in the recipient bacterium or/and enhancing the activity of the protein coded by the exogenous alkL gene in the recipient bacterium, namely the exogenous alkane uptake outer membrane protein;

(8) inhibiting the expression of the iclR gene in the recipient bacterium or/and inhibiting the activity of a protein coded by the iclR gene in the recipient bacterium, namely a glyoxylate pathway transcription inhibitor;

(9) inhibiting the expression of the aceB gene in the recipient bacterium or/and inhibiting the activity of a protein coded by the aceB gene in the recipient bacterium, namely malate synthase A;

(10) inhibiting the expression of the glcB gene in the recipient bacterium or/and inhibiting the activity of the protein coded by the glcB gene, namely malate synthase G, in the recipient bacterium;

(11) enhancing the expression of the glyoxylate pathway aceA gene in the recipient bacterium or/and enhancing the activity of the protein coded by the glyoxylate pathway aceA gene in the recipient bacterium, namely isocitrate lyase;

(12) enhancing the expression of the exogenous cgaT gene in the recipient bacterium or/and enhancing the activity of a protein coded by the exogenous cgaT gene in the recipient bacterium, namely glutamate glyoxylate aminotransferase II;

(13) enhancing the expression of the gdhA gene in the recipient bacterium or/and enhancing the activity of the protein coded by the gdhA gene, namely glutamate dehydrogenase, in the recipient bacterium.

In the above method, GenBank of amino acid sequence of fatty acid degradation transcription factor, which is protein encoded by fadR gene: NP _415705.1 (date of filing 2016, 8 months, 8 days). GenBank of amino acid sequence of pyridine nucleotide transhydrogenase, protein encoded by sthA gene: NP _418396.1 (date of filing 2016, 8 months, 8 days). GenBank of amino acid sequence of protein encoded by pntA gene, i.e. pyridine nucleotide transhydrogenase alpha subunit: NP _416120.1 (date of filing 2016, 8 months, 8 days). GenBank of amino acid sequence of protein encoded by fadL gene, i.e. long chain fatty acid uptake outer membrane protein: NP _416846.2 (date of filing 2016, 8 months, 8 days). GenBank of amino acid sequence of protein encoded by foreign alkL gene, i.e. foreign alkane uptake outer membrane protein: CCG96306.1 (date of filing 2015, 2 months and 27 days). GenBank of amino acid sequence of protein encoded by iclR gene, glyoxylate pathway transcription repressing factor: NP _418442.2 (date of filing 2016, 8 months, 8 days). GenBank of amino acid sequence of malic acid synthase a, protein encoded by aceB gene: NP _418438.1 (date of filing 2016, 8 months, 8 days). GenBank of amino acid sequence of malate synthase G, protein encoded by glcB gene: NP _417450.1 (date of filing 2016, 8 months, 8 days). GenBank of amino acid sequence of protein encoded by aceA gene, i.e., isocitrate lyase: NP _418439.1 (date of filing 2016, 8 months, 8 days). GenBank of amino acid sequence of glutamate dehydrogenase, protein encoded by gdhA gene: NP _416275.1 (date of filing 2016, 8 months, 8 days).

In the above method, in the (5), the gene involved in the fatty acid β oxidation pathway is selected from one or more of the following genes: a gene fadD encoding a fatty acyl-CoA synthase, a gene fadE encoding a fatty acyl-CoA dehydrogenase, a gene fadB encoding a 3-hydroxyacyl-CoA dehydrogenase, a gene fadA encoding a 3-ketoacyl-CoA thiolase, a gene fadI encoding a 3-ketoacyl-CoA thiolase, a gene fadJ encoding a 3-hydroxyacyl-CoA dehydrogenase, and a gene fadK encoding a short-chain fatty acyl-CoA synthase. In one embodiment of the invention, the gene involved in the fatty acid beta oxidation pathway is specifically the gene fadD encoding fatty acyl-coa synthase.

In the above method, in the above (6), the gene involved in the short-chain fatty acid degradation pathway is selected from the group consisting of genes included in the following gene clusters: short chain fatty acid degradation regulation gene cluster atoSC and short chain fatty acid degradation gene cluster atoDAEB. Wherein the short-chain fatty acid degradation regulatory gene cluster atoSC comprises the following genes: a gene atoC encoding an atoC transcriptional activator, a gene atoS encoding an atoS-sensitive histidine kinase; the short-chain fatty acid degradation gene cluster atoDAEB comprises the following genes: gene atoA coding for the alpha subunit of acetoacetyl-CoA transferase, gene atoD coding for the beta subunit of acetoacetyl-CoA transferase, gene atoE coding for the acetoacetate transporter, gene atoB coding for acetyl-CoA acetyltransferase. In one embodiment of the invention, the genes involved in the short chain fatty acid degradation pathway are specifically the gene atoC encoding an atoC transcriptional activator and the gene atoS encoding an atoS-sensitive histidine kinase. GenBank of amino acid sequence of atoC transcriptional activator encoded by atoC gene: NC _416724.1 (date of submission 2016, 8 months and 8 days); GenBank of amino acid sequence of the atoS-sensitive histidine kinase encoded by the tos gene: NC _416723.1 (date of submission 2016, 8 months and 8 days).

In the above method, in the step (7), the enhancing of the expression of the exogenous alkL gene in the recipient bacterium or/and the enhancing of the activity of the protein encoded by the exogenous alkL gene in the recipient bacterium, that is, the exogenous alkane uptake outer membrane protein gene alkL, is overexpression in the recipient bacterium. The over-expression method is realized by introducing exogenous alkane into an outer membrane protein gene alkL into a recipient bacterium.

The foreign alkane uptake outer membrane protein gene alkL can be derived from Haemophilus hydrocarbonoclasus or Pseudomonas aeruginosa. In one embodiment of the present invention, the foreign alkane uptake outer membrane protein gene alkL is specifically derived from Haemophilus hydrocarbonoclasus (Marinobacter hydrocarbonoclasus), and the sequence thereof is shown in SEQ ID No.4, positions 15-686.

In the above method, the enhancing of the expression of the exogenous cgaT gene in the recipient bacterium or/and the enhancing of the activity of glutamate glyoxylate aminotransferase II, which is a protein encoded by the exogenous cgaT gene in the recipient bacterium in (12) above means that the exogenous glutamate glyoxylate aminotransferase II gene cgaT is overexpressed in the recipient bacterium. The overexpression method is realized by introducing exogenous glutamate glyoxylate aminotransferase II gene cgAT into a recipient bacterium.

The exogenous glutamate glyoxylate aminotransferase II gene cgAT has the following 7 sources: beet (Beta vulgaris), soybean (Glycine max), spinach (Spinacia oleracea), sweet potato (Ipomoea nil), quinoa (Chenopodium quinoa), tobacco (Nicotiana attenuata) and corn (Zea mays). In one embodiment of the invention, the exogenous glutamate glyoxylate aminotransferase II gene cgAT is respectively an engineered glutamate glyoxylate aminotransferase II gene cgAT from beet (Beta vulgaris) (SEQ ID No.2), an engineered glutamate glyoxylate aminotransferase II gene cgAT from soybean (Glycine max) (SEQ ID No.5), an engineered glutamate glyoxylate aminotransferase II gene cgAT from spinach (Spinacia oleracea) (SEQ ID No.6), an engineered glutamate glyoxylate aminotransferase II gene cgAT from sweet potato (ipoea nil) (SEQ ID No.7), an engineered glutamate glyoxylate aminotransferase II gene cgAT from quinoa (Chenopodium quinoa) (SEQ ID No.8), an engineered glutamate glyoxylate aminotransferase II gene cgAT from tobacco (Nicotiana attentuata) (SEQ ID No.9) or an engineered glutamate aminotransferase II gene cgAT from maize (Zeysaa) respectively Bits 1-1399).

In the above method, in the step (13), the enhancing of the expression of the gdhA gene in the recipient bacterium or/and the enhancing of the activity of the glutamate dehydrogenase, which is a protein encoded by the gdhA gene in the recipient bacterium, may be the enhancing of the expression of an endogenous glutamate dehydrogenase gene gdhA in the recipient bacterium, or the overexpression of an exogenous glutamate dehydrogenase gene gdhA in the recipient bacterium. The exogenous glutamate dehydrogenase gene gdhA can be derived from Bacillus subtilis or Escherichia coli. In one embodiment of the invention, the exogenous glutamate dehydrogenase gdhA gene is specifically derived from Escherichia coli (Escherichia coli).

Inhibition and enhancement in the above methods can be achieved by those skilled in the art using means well known in the art, and also by the cost of creative work.

Further, in the above (1), (3), (8), (9) and (10), the method for suppressing gene expression is to knock out the gene in the recipient bacterium. The genes are particularly fadR gene, pntA gene, iclR gene, aceB gene and glcB gene.

In the above (2), (4), (5), (6), (11) and (13), the method for enhancing gene expression is to replace the promoter of the gene in the recipient bacterium with a CPA1 promoter. The gene is sthA gene, fadL gene, fadD gene, atoC gene, atoS gene, aceA gene or gdhA gene.

Both the knockout and the substitution can be achieved by homologous recombination.

In the above method, the recipient bacterium may be Escherichia coli (e.g., K-series, B-series, and W-series Escherichia coli) commonly used in the prior art. In one embodiment of the invention, the escherichia coli is specifically escherichia coli BW 25113.

The modification methods of the steps (1) to (13) involved in the above method have no sequence requirement, and the recombinant Escherichia coli efficiently utilizing fatty acid to synthesize glycine of the present invention can be obtained as long as the modifications of the steps (1) to (13) or the modifications of the steps (1) to (7) are completed on the recipient bacterium.

The recombinant Escherichia coli constructed by the method also belongs to the protection scope of the invention.

In a second aspect, the invention protects a new use of the recombinant escherichia coli.

The invention provides application of the recombinant escherichia coli in glycine production.

In a third aspect, the present invention protects a process for producing glycine.

The method for producing the glycine provided by the invention takes the fatty acid as a substrate, and adopts the recombinant escherichia coli for biotransformation to synthesize the glycine.

In the method for producing glycine, the biotransformation method comprises the steps of carrying out arabinose-induced culture on the recombinant escherichia coli to obtain induced recombinant bacteria, and converting the fatty acid substrate into glycine by using the induced recombinant bacteria.

The arabinose induction culture is carried out in a culture medium with the arabinose concentration of 0.2%, the temperature of the induction culture can be 30-37 ℃, and the time of the induction culture can be 20-25 hours.

Further, the temperature of the induction culture may be 37 ℃, and the induction culture time may be 24 hours.

In the above method for producing glycine, the fatty acid is stearic acid (C18), palmitic acid (C16), myristic acid (C14), lauric acid (C12), capric acid (C10), caprylic acid (C8), or caproic acid (C6). In one embodiment of the invention, the fatty acid is in particular palmitic acid (C16).

The invention provides a recombinant escherichia coli engineering strain and a method for synthesizing glycine by using fatty acid as a raw material. The recombinant escherichia coli engineering strain contains one or more of the following genetic modifications: (1) enhancement of fatty acid uptake pathway and fatty acid beta oxidation pathway; (2) the gene target point related to the glyoxylate cycle is modified, and the synthesis capacity of the glyoxylate is enhanced; (3) introducing/improving the expression of glutamate glyoxylate aminotransferase; the engineered strain has the capacity of whole-cell conversion of fatty acid substrates with different lengths into glycine, wherein the fatty acids comprise stearic acid (C18), palmitic acid (C16), myristic acid (C14), lauric acid (C12), capric acid (C10), caprylic acid (C8) and caproic acid (C6). The ability of the engineering strain to synthesize glycine by using fatty acid enables the engineering strain to have the application of industrial production of glycine products. The method for synthesizing glycine by utilizing the recombinant escherichia coli engineering strain can ferment and convert various cheap fatty acids serving as raw materials in industrial production; meanwhile, the conversion rate of the glycine is higher than that of glucose by taking fatty acid as a substrate, so that the production cost of the glycine is further reduced.

Drawings

FIG. 1 shows the glycine yield of recombinant engineered Escherichia coli strains using fatty acids as raw materials.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Coli BW25113 in the following examples is described in the literature: datsenko KA, Wanner bl.one-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.proc.natl.acad.sci.u.s.a.2000; 97(12) 6640 and 6645, wherein the strain is a non-pathogenic bacterium, has clear genetic background, short generation time, easy culture and low cost of culture medium raw materials. The biological material is only used for repeating the relevant experiments of the present invention and is not used for other purposes.

The invention specifically comprises the following embodiments:

example 1 construction of recombinant E.coli engineered strains FG11, FG12, FG13, FG14, FG15, FG16, FG17

The construction of 7 recombinant E.coli engineered strains FG11, FG12, FG13, FG14, FG15, FG16, FG17, comprising the following twenty-six steps:

(1) fatty acid degradation transcription factor fadR knockout.

(1-a) first, a P1 phage containing an E.coli gene fragment having a fadR knock-out property was prepared. An E.coli gene fragment containing a fade-out trait is derived from E.coli strain JW1176, which is a W3110 series strain containing a fade-out trait and purchased from national institute of genetics (NIG, Japan), in which the gene fade encoding a fatty acid degradation transcription factor is replaced with a kanamycin-resistant gene (about 1300bp) having FRT sites at both ends to thereby knock out the fade-R gene (Baba T, Ara T, et al. The P1 phage was prepared as follows: the JW1176 strain is inoculated to the CaCl containing 5mmol/L after being cultured at 37 ℃ overnight₂And 0.1% glucose in LB medium, cultured at 37 ℃ for 1h, and then added with wild type P1 phage to continue culturing for 1-3 h. Adding a few drops of chloroform, culturing for a few minutes, centrifuging and taking the supernatant to obtain the phage P1vir delta fadR containing the escherichia coli gene fragment with the fadR knockout character.

(1-b) constructing an escherichia coli strain FG01-Kan by utilizing a P1 phage transduction technology, and specifically comprising the following steps: culturing BW25113 (recipient bacterium) overnight, centrifuging 1.5mL bacterial solution at 10000g for 2min, and adding 0.75mL P1 salt solution (solvent is water, and solute is 10mM CaCl)₂And 5mM MgSO₄) Resuspending BW25113 somatic cells100 μ L of phage P1vir Δ fadR was mixed with 100 μ L of BW25113 cell suspension, incubated at 37 ℃ for 30min, then 1mL of LB medium and 200 μ L of 1mol/L sodium citrate were added, further culture was continued at 37 ℃ for 1h, the cells were collected by centrifugation, resuspended in 100 μ L of LB medium, plated on a kanamycin-containing LB plate (kanamycin concentration was 50 μ g/mL), cultured overnight at 37 ℃ and then colonies were selected, and PCR-amplified with fadR-IF/fadR-IR primers to identify them (the band of 1700bp amplified was positive), and the selected positive colonies were named as 01-Kan.

(1-c) Elimination of resistance: the pCP20 plasmid (purchased from Clontech) was transformed into FG01-Kan by calcium chloride transformation, and after overnight culture at 30 ℃ on LB plates containing ampicillin, clones were selected to obtain recombinant E.coli FG01-Kan/pCP20 containing the plasmid pCP 20. After culturing in LB culture medium containing ampicillin resistance at 30 ℃, spreading on an LB plate without resistance for culturing overnight at 42 ℃, selecting clones, amplifying and identifying by using fade-IF/fade-IR primer PCR (amplified 400bp target band is positive), selecting positive clones and naming the positive clones as recombinant escherichia coli FG 01.

(2) Expression of the fadL gene is enhanced by promoter replacement.

(2-a) preparation of host bacteria containing pKD46 plasmid: the plasmid pKD46 (from Clontech) was transformed into the FG01 strain obtained in the previous step by calcium chloride transformation, and after overnight culture at 30 ℃ on LB plates containing ampicillin, clones were selected to obtain recombinant E.coli FG01/pKD46 containing the plasmid pKD 46. After the recombinant Escherichia coli FG01/pKD46 expresses 3 recombinant proteins of lambda phage after arabinose induction, the host bacteria have the capacity of homologous recombination. FG01/pKD46 competent cells were then prepared by 10% glycerol wash.

(2-b) preparation of plasmid for amplifying replacement promoter targeting gene fragment: the nucleotide sequence of the CPA1-Lox66-Kan-Lox71 fragment is shown as SEQ ID No.1, wherein, the constitutive promoter P_CPA1The nucleotide sequence of (a) is 1443-1622 of SEQ ID No.1, and the nucleotide sequence containing the kanamycin resistance gene (LOXP-kan-LOXP) flanking the LOXP is 21-1433 of SEQ ID No. 1. The sequence was ligated to pUC57 vector by whole-gene synthesis (Nanjing Kinshiri Biotech Co., Ltd.)The vector pUC57-9K was obtained.

(2-c) preparation of targeting fragment fadLup-kan-CPA 1-fadLdown: a fadLup-kan-CPA1-fadLdown fragment is amplified by using pUC57-9K as a template and adopting a primer fadL-PF/fadLFR, and a target fragment is recovered by agarose gel electrophoresis (Tiangen Biochemical technology Co., Ltd., product catalog is DP 209).

(2-d) homologous recombination: the fadLup-kan-CPA1-fadLdown fragment was electroporated into FG01/pKD46 competent cells prepared in (2-a), and then overnight at 37 ℃ on an LB plate containing kanamycin (50. mu.g/mL), colonies were selected, and PCR-amplified and identified using fadL-PIF/fadL-PIR primers (2000 bp bands were amplified as positive), and the positive colonies were selected and named FG 02-kan.

(2-e) Elimination of resistance: the pCP20 plasmid (purchased from Clontech) was transformed into FG01-Kan by calcium chloride transformation, and after overnight culture at 30 ℃ on LB plates containing ampicillin, clones were selected to obtain recombinant E.coli FG01-Kan/pCP20 containing the plasmid pCP 20. After being cultured in LB culture medium containing ampicillin resistance at 30 ℃, the mixed solution is spread on an LB plate without resistance and cultured at 43 ℃ overnight, clones are selected, fadL-PIF/fadL-PIR primers are used for PCR amplification and identification (the amplified 600bp target band is positive), meanwhile, corresponding primers are used for determining whether the strain keeps the character obtained by the previous modification, and the selected positive clone is named as recombinant escherichia coli FG 02.

(3) Expression of the fadD gene is enhanced by promoter replacement.

Starting from recombinant strain FG02, the promoter of the gene fadD encoding fatty acyl-CoA synthase was replaced with CPA1 promoter in the same manner as in section (2) above. Recombinant E.coli FG03 was obtained. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that fadL is changed to fadD.

(4) The expression of sthA gene was enhanced by promoter substitution.

Starting from recombinant strain FG03, the promoter of sthA, a gene encoding fatty acyl-CoA synthase, was replaced with CPA1 promoter in the same manner as in section (2) above. Recombinant E.coli FG04 was obtained. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that fadL is changed to sthA.

(5) Knock-out of pyridine nucleotide transhydrogenase alpha subunit pntA

Starting from the recombinant bacterium FG04, the pyridine nucleotide transhydrogenase alpha subunit pntA was knocked out in the same manner as in section (1) above. Recombinant E.coli FG05 was obtained. Wherein the difference of the primer names from the strains used in the section (1) is that: the E.coli strain containing the pntA knockout trait was changed to JW1595 (this strain was purchased from the national institute of genetics of Japan); the name of the corresponding primer was changed from fadR to pntA.

(6) Expression of the atoS and atoC genes is enhanced by promoter replacement.

Starting from the recombinant strain FG05, the promoter of the short-chain fatty acid degradation regulatory gene cluster atoSC was replaced with the CPA1 promoter using the same method as in section (2) above. Recombinant E.coli FG06 was obtained. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that fadL is changed to atoSC.

(7) Expression of the glutamate dehydrogenase gdhA gene is enhanced by promoter replacement.

Starting from recombinant strain FG06, the promoter of glutamate dehydrogenase was replaced with CPA1 promoter by the same method as in section (2) above. Recombinant E.coli FG07 was obtained. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that fadL is changed to gdhA.

(8) Knock-out of the glyoxylate pathway transcription repressor gene iclR.

Starting from a recombinant bacterium FG07, the glyoxylate pathway transcription repressor gene iclR was knocked out in the same manner as in section (1) above. Recombinant E.coli FG08 was obtained. Wherein the difference of the primer names from the strains used in the section (1) is that: the E.coli strain containing the iclR knockout trait was changed to JW3978 (this strain was purchased from the national institute of genetics); the corresponding primer is named after changing from fadR to iclR.

(9) The knockout of the malate synthase A gene aceB and the enhancement of the expression of the aceA gene through the replacement of a promoter.

Starting from recombinant bacterium FG08, recombinant Escherichia coli FG09 was obtained by knocking out malate synthase A gene aceB while replacing the promoter of gene aceA encoding isocitrate lyase with CPA1, using the same method as in section (2) above. Wherein the differences from the primer names used in section (2) are: the name of the corresponding primer was changed from fadL to aceA.

(10) Knock-out of the G gene glcB of malate synthase.

From the recombinant strain FG09, E.coli FG10 was obtained by knocking out the G gene glcB malate synthase in the same manner as in section (1) above. Wherein the difference of the primer names from the strains used in the section (1) is that: the E.coli strain containing the glcB knockout trait was changed to JW3179 (this strain was purchased from the national institute of genetics); the corresponding primer is named after the change from fadR to glcB.

(11) Construction of a plasmid overexpressing exogenous glutamic acid glyoxylate aminotransferase II gene cgAT of beet (Beta vulgaris).

(11-a) PCR amplification of LOC104897276 gene. The nucleotide sequence of the modified beet (Beta vulgaris) exogenous glutamic acid glyoxylate aminotransferase II gene cgAT is shown in SEQ ID No. 2. The sequence was ligated to pUC57 vector by whole gene synthesis to obtain vector pUC57-cgAT (Bvu). The cgAT (Bvu) gene fragment was PCR-amplified using cgAT (Bvu) -F and cgAT (Bvu) -R as primers and pUC57-cgAT (Bvu) plasmid as a template with high fidelity TransStart Fastpfu DNA polymerase (AP 221, Kyoto Total gold Biotech, Inc., Beijing).

(11-b) construction of a recombinant expression vector containing the cgAT (Bvu) gene. Subjecting the PCR amplified fragment obtained in (11-a) to agarose gel electrophoresis, and recovering a target fragment; the vector pSB1s (the nucleotide sequence of the vector pSB1s is shown in SEQ ID No.3) was digested simultaneously with NcoI and XhoI, and the large fragment SB1s-NX of the vector was recovered. The cgAT (Bvu) fragment was ligated to the SB1s-NX fragment using the Gibson assembly method (Gibson DG, Young L, et al. enzymatic assembly of DNA molecules up to recombinant human clones. Nat. methods. 2009; 6(5): 343-345). With CaCl₂Escherichia coli DH5 alpha competent cells (purchased from Beijing Quanjin Biotechnology Ltd., catalog No. CD201) were transformed by the method. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. Selecting clones, identifying clones capable of amplifying target fragments by using primers F105-F/cgAT (Bvu) -R, sequencing, selecting positive clonesThe plasmid was taken and the positive plasmid obtained was designated pSB1s-cgAT (Bvu).

(12) Construction of plasmid for over-expressing the foreign alkane uptake outer membrane protein gene MhalklL of marinobacter hydrocarbonoclases.

Using the same method as in section (11) above, DNA was extracted from Marinobacter hydrocarbonoclasus (Marinobacter hydrocarbonoclasus), and the MhalkL gene fragment was amplified using the primers MhalkL-F/MhalkL-R, while the RBS sequence was introduced into the primers. The vector pSB1s-cgAT (Bvu) was digested with XhoI and SpeI to obtain large fragment SB1s-cgAT (Bvu) -HS. The MhalkL fragment was ligated with the SB1s-cgAT (Bvu) -HS fragment. Coli DH 5. alpha. was transformed, clones capable of amplifying the desired fragment were identified with primer MhalkL-F/T58 and sequenced, positive clones were screened, and the obtained positive plasmid was named pSB1s-cgAT (Bvu) -MhalkL. The nucleotide sequence of MhalkL containing RBS is shown as SEQ ID No.4, wherein the sequence of RBS is shown as 2 nd to 7 th positions of SEQ ID No.4, and the nucleotide sequence of MhalkL is shown as 15 th to 686 th positions of SEQ ID No. 4.

(13) Construction of recombinant E.coli FG 11.

Competent cells were prepared from strain FG10, and plasmid pSB1s-cgAT (Bvu) -MhalklL with CaCl₂FG10 was transformed. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. The positive clone was selected and named recombinant E.coli FG 11.

(14) Construction of plasmid for overexpression of exogenous glutamic acid glyoxylate transaminase II gene cgAT of soybean (Glycine max) and exogenous alkane uptake outer membrane protein gene MhalklL.

Using the same method as in section (11) above, the nucleotide sequence of the modified soybean (Beta vulgaris) exogenous glutamic acid glyoxylate transaminase II gene cgaT (Gma) is shown in SEQ ID No.5, and plasmid pSB1s-cgaT (Gma) -MhalkL is constructed. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that cgAT (Bvu) is changed to cgAT (Gma).

(15) Construction of recombinant E.coli FG 12.

Strain FG10 was used to prepare competent cells, plasmid pSB1s-cgAT (Gma) -MhalklL with CaCl₂FG10 was transformed. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. Selecting positive clone and naming it as recombination large intestine rodBacteria FG 12.

(16) Construction of a plasmid overexpressing spinach (Spinacia oleracea) foreign glutamic acid glyoxylate aminotransferase II gene cgAT and foreign alkane uptake outer membrane protein gene MhalkL.

Using the same method as in section (11) above, the nucleotide sequence of the modified spinach (Spinacia oleracea) foreign glutamic acid glyoxylate transaminase II gene cgAT (Sol) is shown in SEQ ID No.6, and plasmid pSB1s-cgAT (Sol) -MhalkL was constructed. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that cgAT (Bvu) is changed to cgAT (Sol).

(17) Construction of recombinant E.coli FG 13.

Strain FG10 was used to prepare competent cells, plasmid pSB1s-cgAT (Sol) -MhalklL with CaCl₂FG10 was transformed. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. The positive clone was selected and named recombinant E.coli FG 13.

(18) Construction of a plasmid overexpressing exogenous glutamic acid glyoxylate transaminase II gene cgAT and exogenous alkane uptake outer membrane protein gene MhalkL of sweet potato (Ipomoea nil).

Using the same method as in section (11) above, a plasmid pSB1s-cgaT (ini) -MhalkL was constructed in which the nucleotide sequence of the foreign glutamic acid glyoxylate transaminase II gene cgaT (ini) of the modified sweet potato (Ipomoea nil) is shown in SEQ ID No. 7. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that cgAT (Bvu) is changed to cgAT (Ini).

(19) Construction of recombinant E.coli FG 14.

Competent cells were prepared from strain FG10, plasmid pSB1s-cgAT (ini) -MhalklL with CaCl₂FG10 was transformed. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. The positive clone was selected and named recombinant E.coli FG 14.

(20) Construction of a plasmid overexpressing exogenous glutamic acid glyoxylate aminotransferase II gene cgAT of Chenopodium quinoa (Chenopodium quinoa) and exogenous alkane uptake outer membrane protein gene MhalklL.

Using the same method as in section (11) above, a plasmid pSB1s-cgaT (Cqu) -MhalkL was constructed in which the nucleotide sequence of a modified Chenopodium quinoa (Chenopodium quinoa) foreign glutamic acid glyoxylate transaminase II gene cgaT (Cqu) is shown in SEQ ID No. 8. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that cgAT (Bvu) is changed to cgAT (Cqu).

(21) Construction of recombinant E.coli FG 15.

Strain FG10 was used to prepare competent cells, plasmid pSB1s-cgAT (Cqu) -MhalklL with CaCl₂FG10 was transformed. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. The positive clone was selected and named recombinant E.coli FG 15.

(22) Construction of plasmid for overexpression of exogenous glutamate glyoxylate aminotransferase II gene cgAT and exogenous alkane uptake outer membrane protein gene MhalkL of tobacco (Nicotiana attenuata).

Using the same method as in section (11) above, plasmid pSB1s-cgaT (nat) -MhalkL was constructed in which the nucleotide sequence of the modified tobacco (Nicotiana attenuata) exogenous glutamate glyoxylate transaminase II gene cgaT (nat) is shown in SEQ ID No. 9. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that cgAT (Bvu) is changed to cgAT (nat).

(23) Construction of recombinant E.coli FG 16.

Strain FG10 was used to prepare competent cells, plasmid pSB1s-cgaT (nat) -Mhalkl with CaCl₂FG10 was transformed. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. The positive clone was selected and named recombinant E.coli FG 16.

(24) Construction of plasmid overexpressing exogenous glutamic acid glyoxylate transaminase II gene cgaT of maize (Zea mays) and exogenous alkane uptake outer membrane protein gene MhalkL.

Using the same method as in section (11) above, the nucleotide sequence of the modified maize (Zea mays) exogenous glutamate glyoxylate transaminase II gene cgaT (Zma) is shown in SEQ ID No.10, and plasmid pSB1s-cgaT (Zma) -MhalkL is constructed. The relevant primers are shown in Table 1, and the names of the primers are different from those in (2) only in that cgAT (Bvu) is changed to cgAT (zma).

(25) Construction of recombinant E.coli FG 17.

Preparing competent cells from strain FG10, and introducing plasmid pSB1s-cgAT (Zma) -MhalklL into CaCl₂Fangzhu (rotating shaft)FG10 is quantized. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. The positive clone was selected and named recombinant E.coli FG 17.

(26) Construction of recombinant E.coli FG 00.

Strain FG10 was used to prepare competent cells, plasmid pSB1s was CaCl₂FG10 was transformed. Spread on LB plates containing streptomycin and cultured overnight at 37 ℃. The positive clone was selected and named recombinant E.coli FG 00.

TABLE 1 primer sequences List

Example 2 production of Glycine from fatty acids Using recombinant E.coli strains FG11, FG12, FG13, FG14, FG15, FG16, FG17

(27) A medium component.

The solvent of the culture medium A is water, and the solutes and the final concentrations thereof are respectively as follows:

NaHPO₄：25mM

KH₂PO₄：25mM

NH₄Cl：50mM

Na₂SO₄：5mM

MgSO₄：2mM

glycerol: 0.5 percent

Yeast powder: 0.5 percent

Trace elements: 50 μ M FeCl₃，20μM CaCl₂，10μM MnCl₂，10μM ZnSO₄，2μM CoCl₂，2μM NiCl₂，2μM Na₂MO₄，2μM Na₂SeO₃And 2 μ M H₃BO₃。

(28) And B, medium components.

The component of the culture medium B is 0.5 percent of palmitic acid and 0.2 percent of polyoxyethylene ether Brij58 emulsifier on the basis of the component of the culture medium A.

(29) C medium component.

The component of the C culture medium is based on the component of the A culture medium, and also contains 0.5 percent of palmitic acid, 0.2 percent of polyoxyethylene ether Brij58 emulsifier, and NH₄Cl 10g/L。

(29) Culturing of the cells and induction of enzymes.

The engineering strains FG00, FG11, FG12, FG13, FG14, FG15, FG16 and FG17 which are cultured overnight are respectively inoculated into a shake flask containing 20mL of A culture medium by 1 percent of inoculum size, streptomycin is added into the culture medium, and the thalli are collected after the culture is carried out for 12h at 37 ℃. Transferred to a shake flask containing 20mL of B medium, cultured at 37 ℃ for 6 hours, and then induced for 24 hours by adding arabinose at a final concentration of 0.2%. And collecting the thallus.

(30) Whole cell catalysis of glycine.

The collected thallus is resuspended in a shake flask containing 20mL of C culture medium, cultured at 37 ℃ for 24h, and then a 300 μ L sample is centrifuged, supernatant is taken and filtered. Glycine content was checked by HPLC. The high performance liquid chromatography detection instrument and the detection conditions are as follows:

a detection instrument: SHIMADZU LC-20AT

Detection conditions are as follows: mobile phase A: 10mM KH₂PO₄(ii) a Mobile phase B: acetonitrile-methanol-water solution (volume ratio 45:45: 10); the detection wavelength is 360 nm; detection time: 35 min; detecting the temperature: 40 ℃; flow rate: 1 mL/min; a chromatographic column: Extnd-C18(5 μm, 4.6mm i.d.. times.250 mm); gradient elution: 1min 12% B; 2.5min 12% B; 2.51min 16% B; 13min 36% B; 13.01min 38% B; 25min 100% B; 28min 100% B; 28.01min 10% B; the 35min controller stops.

The results are shown in FIG. 1. The glycine yields of FG11, FG12, FG13, FG14, FG15, FG16 and FG17 were 1.96g/L, 1.54g/L, 1.64g/L, 1.67g/L, 3.17g/L, 2.34g/L and 1.41g/L, respectively.

Adding 200 mu L of 10% sulfuric acid-methanol solution into 300 mu L of sample precipitate, placing the precipitate in a water bath kettle for methyl esterification reaction at the reaction temperature of 70 ℃ for 20min, cooling to room temperature after the reaction is finished, adding 500 mu L of n-hexane, extracting and filtering, using the filtrate for gas chromatography analysis, detecting the content of palmitic acid, and calculating the mass conversion rate (the net yield of glycine divided by the net consumption of palmitic acid) multiplied by 100%. The gas chromatography detection instrument and the detection conditions are as follows:

a detection instrument: agilent HP-INNOWax

Detection conditions are as follows: gas-phase capillary column: (30 m.times.0.32 mm.times.0.25 μm, 19081N-113); carrier gas: nitrogen gas; a FID detector; column temperature, initial temperature 150 deg.C, keeping for 5 min; heating to 170 deg.C at 3 deg.C/min, maintaining for 5min, heating to 210 deg.C at 3 deg.C/min, and maintaining for 5 min; nitrogen flow rate: 45 mL/min; hydrogen flow rate: 40 ml/min; the split ratio is as follows: 10:1, sample size 1 uL.

The results show that: the residual amounts of FG11, FG12, FG13, FG14, FG15, FG16 and FG17 palmitic acid are all 0g/L, and the net consumption of palmitic acid is 5g/L, so the highest mass conversion rate of the 7 strains is FG15 strain, reaches 63.4 percent, and accounts for 27.06 percent of the theoretical mass conversion rate.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> construction and application of escherichia coli recombinant bacteria for synthesizing glycine by efficiently utilizing fatty acid

<160>10

<170>PatentIn version 3.5

<210>1

<211>1622

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gtctcgagaa tatcctcctt ataacttcgt ataatgtatg ctatacgaac ggtaagagcg 60

cttttgaagc tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc 120

cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa 180

tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttggaa ccccagagtc 240

ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg 300

cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat 360

cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga 420

tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg 480

tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg 540

gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc 600

gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat 660

caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa 720

ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg 780

cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata 840

gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa 900

gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct 960

gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca 1020

atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatcttgatc 1080

ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc 1140

caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg 1200

cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg 1260

cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct 1320

gcggactggc tttctacgtg ttccgcttcc tttagcagcc cttgcgccct gagtgcttgc 1380

ggcagcgtga ggggatcttt accgttcgta taatgtatgc tataccaagt tatgaagcta 1440

gcttatcaaa aagagtattg acataaagtc taacctatag ataattacag ccatcgagag 1500

ggacacggcg atttgctgtc accggatgtg ctttccggtc tgatgagtcc gtgaggacga 1560

aacagcctct acaaataatt ttgtttaaga attcaaaaga tcttttaaga aggagatata 1620

cc 1622

<210>2

<211>1443

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atgtctcata aaggcttaga ttatgaactg ctgaacgaaa atgttaaaaa atgtcagtat 60

gcagttcgtg gcgaactgta tctgcgtgca agcgaattgc agaaagaagg caagaagatc 120

atttttacca atgtgggtaa tccacacgcc ttaggtcaga aaccgctgac ctttccgcgt 180

caggttgttg cactgtgtca ggccccgttt ctgttagatg atccgaatgt gggcttagtg 240

tttccggcag atgcaattgc acgcgccaaa cattatctgg ccatgaccag cggtggcctg 300

ggtgcctata gcgatagtcg cggcattccg ggcgttcgca aagaaattgc agagtttatt 360

ggtcgtcgtg atggctatcc gagtgatccg gaactgattt ttctgaccga tggcgcctct 420

aaaggcgtta tgcagattct gaatgcaatt attcgcggtc agagcgatgg cattctggtt 480

ccggtgccgc agtatccgct gtatagcgcc accattagcc tgttaggcgg ctcactggtt 540

ccgtattatc tggaagaaac cgccaattgg ggcctggata ttaacaacct gcgtgattca 600

ctgcgtcagg caacctttaa aggcattacc gttcgcgcta tggtaattat taatccgggt 660

aatccgaccg gtcagtgtct gagcgaagcc aatctgaaag aaattgttaa tttttgtatt 720

caggaaaaac tggtgctgtt agccgatgaa gtgtatcagc agaatatata tcaggatgaa 780

cgcccgttta tttcagcccg gaaagtgctg atgaatatgg gcttaccgat gtctaaagaa 840

ttacagttag tgagctttca taccgtgagc aaaggctatt ggggcgaatg tggccagcgc 900

ggtggctatt ttgaaatgac caatattccg caggaatcag ttgatgaaat ctacaaaatt 960

gcaagtattg ccctgagccc gaatgtgccg ggtcagattt ttctgggcct gatggttaat 1020

ccgccgaaac cgggcgatat ttcatatctg cgctatgaac aggaatctaa aggcattctg 1080

gaaagtttac gcaaacgtgc acgcattatg accgatggct ttaatagttg tcgtaatgtg 1140

gtttgtaatt ttaccgaagg tgccatgtat agctttccgc agatttgtct gccgccgaaa 1200

gccattgaag atgccaaaaa agcaggtaaa catccggatg tgttttattg tctgaaactg 1260

ctggaagcaa ccggcattag taccgttccg ggtagcggtt ttggccagaa agaaggtgtg 1320

tttcacatgc gtaccaccat tctgcccgca gaagaagata tgccggcaat tatggatagc 1380

tttaaaaagt ttaatgatgc ctttatggaa cagtatgaag atcatcgcgg ctatagtcgc 1440

atg 1443

<210>3

<211>4792

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc 60

cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt 120

cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg 180

agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg 240

tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc 300

taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct 360

gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag 420

cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc 480

gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt 540

gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg 600

ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg 660

cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt 720

agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc 780

gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca 840

ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac 900

ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag 960

ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac 1020

attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta 1080

ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg 1140

tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg 1200

ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct 1260

ctactgtttc tccatacccg ttttttgggc taacaggagg aattaaccat gggtacctct 1320

catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagcctcga gggtagatct 1380

ggtactagtg gtgaattcgg tgagctcggt ctgcagctgg tgccgcgcgg cagccaccac 1440

caccaccacc actaatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 1500

tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 1560

gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 1620

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtcgaccaga cccgccataa 1680

aacgccctga gaagcccgtg acgggctttt cttgtattat gggtagtttc cttgcatgaa 1740

tccataaaag gcgcctgtag tgccatttac ccccattcac tgccagagcc gtgagcgcag 1800

cgaactgaat gtcacgaaaa agacagcgac tcaggtgcct gatggtcgga gacaaaagga 1860

atattcagcg atttgcccga gcttgcgagg gtgctactta agcctttagg gttttaaggt 1920

ctgttttgta gaggagcaaa cagcgtttgc gacatccttt tgtaatactg cggaactgac 1980

taaagtagtg agttatacac agggctggga tctattcttt ttatcttttt ttattctttc 2040

tttattctat aaattataac cacttgaata taaacaaaaa aaacacacaa aggtctagcg 2100

gaatttacag agggtctagc agaatttaca agttttccag caaaggtcta gcagaattta 2160

cagataccca caactcaaag gaaaaggtct agtaattatc attgactagc ccatctcaat 2220

tggtatagtg attaaaatca cctagaccaa ttgagatgta tgtctgaatt agttgttttc 2280

aaagcaaatg aactagcgat tagtcgctat gacttaacgg agcatgaaac caagctaatt 2340

ttatgctgtg tggcactact caaccccacg attgaaaacc ctacaaggaa agaacggacg 2400

gtatcgttca cttataacca atacgctcag atgatgaaca tcagtaggga aaatgcttat 2460

ggtgtattag ctaaagcaac cagagagctg atgacgagaa ctgtggaaat caggaatcct 2520

ttggttaaag gctttgagat tttccagtgg acaaactatg ccaagttctc aagcgaaaaa 2580

ttagaattag tttttagtga agagatattg ccttatcttt tccagttaaa aaaattcata 2640

aaatataatc tggaacatgt taagtctttt gaaaacaaat actctatgag gatttatgag 2700

tggttattaa aagaactaac acaaaagaaa actcacaagg caaatataga gattagcctt 2760

gatgaattta agttcatgtt aatgcttgaa aataactacc atgagtttaa aaggcttaac 2820

caatgggttt tgaaaccaat aagtaaagat ttaaacactt acagcaatat gaaattggtg 2880

gttgataagc gaggccgccc gactgatacg ttgattttcc aagttgaact agatagacaa 2940

atggatctcg taaccgaact tgagaacaac cagataaaaa tgaatggtga caaaatacca 3000

acaaccatta catcagattc ctacctacgt aacggactaa gaaaaacact acacgatgct 3060

ttaactgcaa aaattcagct caccagtttt gaggcaaaat ttttgagtga catgcaaagt 3120

aagcatgatc tcaatggttc gttctcatgg ctcacgcaaa aacaacgaac cacactagag 3180

aacatactgg ctaaatacgg aaggatctga ggttcttatg gctcttgtat ctatcagtga 3240

agcatcaaga ctaacaaaca aaagtagaac aactgttcac cgttagatat caaagggaaa 3300

actgtcgata tgcacagatg aaaacggtgt aaaaaagata gatacatcag agcttttacg 3360

agtttttggt gcatttaaag ctgttcacca tgaacagatc gacaatgtaa cagatgaaca 3420

gcatgtaaca cctaatagaa caggtgaaac cagtaaaaca aagcaactag aacatgaaat 3480

tgaacacctg agacaacttg ttacagctca acagtcacac atagacagcc tgaaacaggc 3540

gatgctgctt atcgaatcaa agctgccgac aacacgggag ccagtgacgc ctcccgtggg 3600

gaaaaaatca tggcaattct ggaagaaata gcgctttcag ccggcaaacc tgaagccgga 3660

tctgcgattc tgataacaaa ctagcaacac cagaacagcc cgtttgcggg cagcaaaacc 3720

cgcggccgcc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 3780

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagg gaagcggtga 3840

tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac 3900

cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca 3960

gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt 4020

tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg 4080

tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg 4140

aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca 4200

cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg 4260

taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc 4320

taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg 4380

tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg 4440

atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg 4500

aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt 4560

tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc aaataatgtc 4620

taacaattcg ttcaagccga ggggccgcaa gatccggcca cgatgacccg gtcgtcggtt 4680

cagggcaggg tcgttaaata gccgcttatg tctattgctg gtttaccggt ttattgacta 4740

ccggaagcag tgtgaccgtg tgcttctcaa atgcctgagg tttcaggcat gc 4792

<210>4

<211>686

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

caggaggaat taacatgaaa cctaaaatca ttagtaaagt gtcattagtt gcctttctgt 60

tactgtcact ggccgcaagt ttagcgaatg cacagagtga acctgtgtat agtcgcggcg 120

attgggttgt tggcttaaat gctactagag tgctgacgga tgaggacctg cgtagcgcct 180

cagccggctc agctccggtt ccgaactcaa atttgagcat caacaacgat acgaccgtga 240

gttttgatgt gagctatttt ctgagcaatc agttagcctt taatatcttt ggtggcatcc 300

ctgcgagtgc cgatctacag ggcgaagaat cactgagcgg cctgtttctg ggccagacag 360

attatggtcc ggttatcctg tctctacagt atcatgtgct gacgggtagc aattttagtc 420

catattttgg tgcaggtgtg ggtcgtatct tatttctgga tgaaaaagat cgcgcactga 480

ccgattttga tgtggaagat acatgggcac cagccgttca ggcaggcttt cgctggcgca 540

ttcataataa ctggagcgcc aattttgatg ttcgctatgc cccgtttgaa gcggatatta 600

ccggtaatct gggtccagct ccagttcagg ccaaagtgga agtagaccct accatcgtgt 660

ctatcggcgt tgcgtatcgc ttttaa 686

<210>5

<211>1446

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

atgccgccaa agccgctgga ttacggtagc atcaacgaaa acgtgaagaa aagccagtac 60

gcggttcgtg gtgagctgta tctgcgtgcg agcgaactgc aaaaagaggg taagaagatc 120

attttcacca acgtgggtaa cccgcacgcg ctgggtcaga aaccgctgag ctttccgcgt 180

caggtggttg cgctgtgcca agcgccgttc ctgctggacg atccgaacgt tggtctgctg 240

tttccggcgg atgcgattgc gcgtgcgaag cactatctga gcctgaccag cggtggcctg 300

ggtgcgtata gcgacagccg tggcctgccg ggtgtgcgta aagaggttgc ggaattcatc 360

ctgcgtcgtg atggttaccc gaccgacccg gaactgattt atctgaccga tggtgcgagc 420

aaaggcgtga tgcagatcct gaacaccatc attcgtggtc aagacgatgg cattctggtg 480

ccggttccgc agtacccgct gtatagcgcg accattgcgc tgctgggtgg caccctggtt 540

ccgtactatc tggaggaaac cgcgaactgg ggtctggacg tgaacgaact gcgtcagagc 600

gttgagcaag cgcgttttaa gggcattacc gtgaaagcga tggttatcat taacccgggt 660

aacccgaccg gccagtgcct gagcgaagcg aacctgcgtg aggtgctgca gttctgctac 720

caagaaaacc tggcgctgct gggtgatgag gtttaccaga ccaacatcta tcaagacgaa 780

cgtccgttta ttagcagccg taaggtgctg atggatctgg gtccgccgat cagcaaagag 840

gtgcagctga ttagcttcca cagcgttagc aagggctact atggtgaatg cggccaacgt 900

ggtggctact ttgagatgac caacatcccg ccggaaaccg tggacgagat ctataaagtt 960

gcgagcatta gcctgagccc gaacgtgccg gcgcagattt tcatgggtgt tatgctgcac 1020

ccgccgcaac cgggcgatat cagctacgac aagtttgttc gtgaaagcac cggtattctg 1080

gagagcctgc gtcgtcgtgc gcgtctgatg accgatggtt tcaacagctg ccgtaacgtg 1140

gtttgcaact tcaccgaagg cgcgatgtat agctttccgc agattcgtct gccgccgcgt 1200

gcgctggagg cggcgaagca agcgggcaaa gtgccggacg tttactattg cctgaagctg 1260

ctggaagcga ccggtattag caccgtgccg ggtagcggct tcggtcagaa agagggcgtt 1320

tttcacctgc gtaccaccat cctgccggcg gaggaagaca tgccggatat tatggacagc 1380

ttcaagaaat ttaacgacga gttcatggaa caatacgagg acgatcgtgg ttatagccgt 1440

ctgtaa 1446

<210>6

<211>1446

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

atgagcaagg gtctggacta cgaaaccctg aacgagaacg tgaagaaatg ccagtacgcg 60

gttcgtggtg agctgtatct gcgtgcgagc gaactgcaaa aagagggtaa gaagatcatt 120

ttcaccaacg tgggtaaccc gcacgcgctg ggtcagaaac cgctgacctt tccgcgtcag 180

gtggttgcgc tgtgccaagc gccgttcctg ctggacgatc cgaacgtggg tctggttttt 240

ccggcggatg cgattgcgcg tgcgaagcac tatctgagcc tgaccagcgg tggcctgggt 300

gcgtatagcg atagccgtgg cctgccgggt gttcgtaaag aaattgcgga gttcatcgcg 360

cgtcgtgacg gttatccgag cgatccggaa ctgatctttc tgaccgacgg tgcgagcaaa 420

ggcgtgatgc agattctgaa cgcggttatc ggtggccaaa gcgatggcat tctggtgccg 480

gttccgcagt acccgctgta tagcgcgacc atcagcctgc tgggtggcag cctggtgccg 540

tactatctgg aggaaaccgc gaactggggt ctggacatta ccaacctgcg tgatgcgatc 600

cgtcaagcga ccttcaaggg cattaaagtg cgtgcgatgg ttatcattaa cccgggtaac 660

ccgaccggcc agtgcctgag cgtggcgaac ctgcaagaaa ttgttaactt ctgcatcaag 720

gagaaactgg tgctgctggc ggacgaagtt taccagcaaa acatttatca ggatgagcgt 780

ccgtttatca gcgcgcgtaa ggtgctgatg gacatgggtc cgccgatgag caaagatctg 840

cagctggtta gcttccacac cgttagcaag ggctactggg gtgagtgcgg ccaacgtggt 900

ggctattttg aaatgaccaa catcccgcag caaagcgttg atgagatcta caaaattgcg 960

agcattgcgc tgagcccgaa cgtgccgggt cagattttcc tgggcctgat ggttaacccg 1020

ccgaagccgg gtgacatcag ctacctgaag tatgaacaag agagcaaagg cattctggaa 1080

agcctgcgta aacgtgcgcg tatcatgacc gatggtttta acagctgccg taacgtggtt 1140

tgcaacttca ccgagggcgc gatgtacagc tttccgcaga tttgcctgcc gccgaaggcg 1200

attgaagcgg cgaagaaagc gggcaagcac ccggacgttt tctattgcct gaaactgctg 1260

gaggcgaccg gtatcagcac cgtgccgggt agcggcttcg gtcaaaagga aggcgttttt 1320

cacatgcgta ccaccattct gccggcggag gaagagatgc cggcgatcat ggagagcttc 1380

aaaatgttta acgatgcgtt tatggaacag tacgacgatc aacgtgcggg ttatagccgt 1440

atgtaa 1446

<210>7

<211>1446

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

atgagcagca agccgctgga ttacgagaac ctgaacgaaa acgtgaagaa atgccagtac 60

gcggttcgtg gtgagctgta tctgcgtgcg agcgagctgc aaaaggaagg caagaaaatc 120

atttttacca acgtgggtaa cccgcacgcg ctgggtcaga aaccgctgac cttcccgcgt 180

caggttattg cgctgtgcca agcgccgttc ctgctggacg atccgaacgt gggtctggtt 240

tttccggcgg atgcgatcgc gcgtgcgaag caatttctga gcatgaccag cggtggcctg 300

ggtgcgtata gcgacagccg tggtatcccg ggtgtgcgta aagaggttgc ggaatttatt 360

ggtcgtcgtg atggctatcc gagcgacccg gaactgattt tcctgaccga tggtgcgagc 420

aaaggcgtga tgcagatcct gaacagcatc attcgtggtc cgaacgacgg cattctggtg 480

ccggttccgc aatacccgct gtatagcgcg accatcagcc tgctgggtgg cagcctggtt 540

ccgtacttcc tggaggaaac cgcgaactgg ggtctggaca tcaacgatct gcgtcgtagc 600

gtggcggatg cgcgtagcaa gggcctgacc attcgtgcga tggttatcat taacccgggt 660

aacccgaccg gccagtgcct gagcgaggcg aacctgaaag aaatcctgca attttgctat 720

cgtgagaacc tggtgctgct gggtgatgaa gtttaccagc aaaacattta tcaggacgag 780

cgtccgttcc tgagcagccg taaggtgctg ctggacatgg gtccgccgat cagcaaagaa 840

ctgcagctgg ttagctttca caccgttagc aagggctact ggggtgagtg cggccaacgt 900

ggtggctatt tcgaaatgac caacatcccg ccgcgtagcg tggaggaaat ctacaaggtt 960

gcgagcatta gcctgagccc gaacgtgccg ggtcagatct ttctgggcct gatggttaac 1020

ccgccgaaac cgggtgacat tagctatgat caattcgtgc gtgagagcaa gggcatcctg 1080

gaaagcctgc gtaaacgtgc gcgtattatg accgacggtt ttaacagctg ccgtaacgtg 1140

gtttgcaact tcaccgaggg cgcgatgtac agctttccgc agatccgtct gccgccgcgt 1200

gcgattgaaa ccgcgcaagg tctgggcaag gcgccggatg tttactattg cctgaaactg 1260

ctggaggcga ccggtatcag caccgtgccg ggtagcggct tcggtcagaa agaaggcgtt 1320

tttcacctgc gtaccaccat cctgccggcg gaggaagaca tgccggcgat tatggagagc 1380

ttcaagaaat ttaacgacga gttcatggat caatacgaag accaccgtgg ttatagccgt 1440

atgtaa 1446

<210>8

<211>1446

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

atgagcaagg gtctggacta cgaaggcctg aacgagaacg tgaagaaatg ccaatacgcg 60

gttcgtggtg agctgtatct gcgtgcgagc gaactgcaga aagagggtaa gaagatcatt 120

ttcaccaacg tgggtaaccc gcacgcgctg ggtcaaaaac cgctgacctt tccgcgtcag 180

gtggttgcgc tgtgccaagc gccgttcctg ctggacgatc cgaacgtggg tattgttttt 240

ccggcggatg cgattgcgcg tgcgaagcac tatctgagca tgaccagcgg tggcctgggt 300

gcgtatagcg atagccgtgg cattccgggt gttcgtaaag agattgcgga attcatcgag 360

cgtcgtgacg gttacccgag cgatccggaa ctgatctttc tgaccgacgg tgcgagcaaa 420

ggcgtgatgc aaattctgaa cgcggttatc ggtggccaga gcgatggcat tctggtgccg 480

gttccgcagt acccgctgta tagcgcgagc atcagcctgc tgggtggcag cctggtgccg 540

tactatctgg aggaaaccgc gaactggggt ctggacatta acaacctgcg tgatgcgatc 600

cagcaagcga ccttcaaggg cattaaagtg cgtgcgatgg ttatcattaa cccgggtaac 660

ccgaccggcc agtgcctgag cgtggcgaac ctgcaagaaa ttgttaactt ctgcatccag 720

gagaagctgg tgctgctggc ggacgaagtt taccagcaaa acatctatca agatgagcgt 780

ccgtttgtga gcgcgcgtaa ggttctgatg gacatgggtc cgccgatgaa caaagatctg 840

cagctggtta gcttccacac cgttagcaaa ggctactggg gtgagtgcgg ccaacgtggt 900

ggctattttg aaatgaccaa catcccgcag aagagcgttg atgagatcta caaaattgcg 960

agcattgcgc tgagcccgaa cgtgccgggt caaattttcc tgggcctgat ggttaacccg 1020

ccgaagccgg gtgacatcag ctatctgcgt tttgagcagg aaagcaaggg cattctggaa 1080

agcctgcgta aacgtgcgcg tatcatgacc gatggtttca acagctgccg taacgtggtt 1140

tgcaacttca ccgagggcgc gatgtacagc tttccgcaga tttgcctgcc gccgaaagcg 1200

gtggaagcgg cgaagaacgc gggtaaacac ccggacgtgt tctactgcct gaagctgctg 1260

gaggcgaccg gtatcagcac cgtgccgggt agcggcttcg gtcaaaaaga aggcgttttt 1320

cacatgcgta ccaccattct gccggcggag gaagatatgc cggcgatcat ggaaagcttc 1380

aagaaattta acgacgcgtt catggaacac tacgaggatc agcgtgcggg ttatagccgt 1440

atgtaa 1446

<210>9

<211>1446

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

atgagcagca agccgctgga ctacgataac ctgaacgaga acgtgaagaa atgccagtac 60

gcggttcgtg gtgaactgta tctgcgtgcg agcgagctgc aaaaggaagg caagaaaatc 120

attttcacca acgtgggtaa cccgcacgcg ctgggtcaga aaccgctgac cttcccgcgt 180

caggtgatcg cgctgtgcca agcgccgttt ctgctggacg atccgaacgt tggtttcctg 240

tttccggcgg atgcgattgc gcgtgcgaag cactatctgg cgatgaccag cggtggcctg 300

ggtgcgtata gcgatagccg tggcaccccg ggtgtgcgta aagaggttgc ggaattcatc 360

gagcgtcgtg acggttaccc gagcgatccg gagctgattt ttctgaccga cggtgcgagc 420

aaaggcgtga tgcaaatcct gaacagcgtt atccgtggtc cgagcgatgg catcctggtg 480

ccggttccgc agtacccgct gtatagcgcg agcattcaac tgctgggtgg cagcctggtg 540

ccgtactatc tggaggaaac cgcgaactgg ggtctggaca tcaacgatct gcgtcagagc 600

gttgcgcaag cgcgttataa gggcattacc gtgcgtgcga tggttatcat taacccgggt 660

aacccgaccg gccagtgcct gagcgtggcg aacctgaaac agatcattca attctgccac 720

caagagagcc tggtgctgct gggtgacgaa gtttaccagc aaaacatcta tcaggatgag 780

cgtccgttta ttagcgcgcg taaggttctg ctggacatgg gtccgccgat cagcaaagaa 840

ctgcagctgg ttagcttcca caccgttagc aagggctact ggggtgagtg cggccaacgt 900

ggtggctatt ttgaaatgac caacatcccg ccgaagagcg tggaggaaat ctacaaagtt 960

gcgagcatta gcctgagccc gaacgtgccg ggtcagattt tcatgggcct gatggttaac 1020

ccgccgaaac cgggtgacat cagctacgat caatatgttc gtgagagcaa gggcattctg 1080

gaaagcctgc gtaaacgtgc gcacatgatg accgatggtt ttaacagctg ccgtaacgtg 1140

gtttgcaact tcaccgaggg cgcgatgtac agctttccgc agattcgtct gccgccgcgt 1200

gcgattgaag cggcgaagaa actgggcaag gtgccggacg ttttctattg cctgcgtctg 1260

ctggaggcga ccggtatcag caccgtgccg ggtagcggct tcggtcagaa agaaggcgtt 1320

tttcacctgc gtaccaccat cctgccggcg gaggaagata tgccggcgat tatgagcagc 1380

ttcaagaaat ttaacgacga atttatggag caatacgaag atcacaaggg ttatagccgt 1440

atgtaa 1446

<210>10

<211>1437

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

atggcgcgta agccgctgga ctacgaggaa ctgaacgaaa acgtgaaacg tgttcagtac 60

gcggtgcgtg gtgagctgta tctgcgtgcg agcgaactgc aaaaagaggg taagaagatc 120

attttcacca acgttggtaa cccgcacgcg ctgggtcaga aaccgctgac ctttccgcgt 180

caggtggttg cgctgtgcca agcgccgttc ctgctggacg atccgcacgt gggtctgatg 240

tttccggcgg atgcgattgc gcgtgcgaag cactatctgg cgatggcgcc gggtggcctg 300

ggtgcgtata gcgacagccg tggtatcccg ggtattcgta aggaagtggc ggatttcatc 360

cacaaacgtg acggttaccc gagcgatccg gagctgattt atctgaccga cggtgcgagc 420

aaaggcgtta tgcagatcct gaacaccatc attcgtaacg aaatggatgg cattctggtg 480

ccggttccgc aatacccgct gtatagcgcg accatcagcc tgtacggtgg cagcctggtt 540

ccgtactatc tggaggaaga ggcgaactgg agcctggact tcgtgaacat tcgtcagacc 600

gttgcggagg cgcgtagcaa gggtatcagc gtgcgtgcga tggttatgat taacccgggt 660

aacccgaccg gccagtgcct gagcgaagcg aacatcaaag agctgctgca attttgctac 720

cacgaaaacc tggtgctgct ggcggacgag gtttaccagc aaaacatcta tcaagatgaa 780

cgtccgttca ttagcgcgcg taaggtgatg tttgacatgg gtccgccgat tagcagcgag 840

ctgcaggtgg ttagcttcca caccgttagc aaaggctact ggggtgaatg cggccaacgt 900

ggtggctatt ttgagatgac caacctgccg ccgaagaccg tggatgaaat ctacaaagtt 960

gcgagcattg cgctgagccc gaacgtgccg ggtcagatct tcatgggcgt gatggttaac 1020

ccgccgaagc cgggtgacat cagctatctg aagtttgcgg cggaaagcaa aagcattcac 1080

gagagcctgc gtcgtcgtgc gcgtatgatg accgatggtt tcaacagctg ccgtaacgtg 1140

gtttgcaact tcaccgaagg cgcgatgtac agctttccgc aaatccgtct gccgccgcgt 1200

gcgatcgaag cggcgcgtcg tgcgggcaag agcgcggatg tgttctactg cctgaaactg 1260

ctggaagcga ccggtatcag caccgtgccg ggtagcggct tcggtcagaa agagggcgtt 1320

tttcacctgc gtaccaccat cctgccggcg gaagaggact tcccggcgat tatgagcagc 1380

ttcaagaagt tcaacgatag cttcatggaa caatacgagg gttatagccg tctgtaa 1437

Claims (11)

1. A method for constructing a recombinant Escherichia coli, comprising the steps of carrying out (1)-(13) transformations to a recipient bacterium to obtain a recombinant Escherichia coli; (1) inhibiting the expression of the fadR gene in the recipient bacteria or/and inhibiting the activity of the protein encoded by the fadR gene in the recipient bacteria, that is, a fatty acid degradation transcription factor; (2) enhancing the expression of the sthA gene in the recipient bacteria or/and enhancing the activity of the protein encoded by the sthA gene in the recipient bacteria, namely pyridine nucleotide transhydrogenase; (3) inhibiting the expression of the pntA gene in the recipient bacteria or/and inhibiting the activity of the protein encoded by the pntA gene in the recipient bacteria, that is, the α subunit of pyridine nucleotide transhydrogenase; (4) enhancing the expression of the fadL gene in the recipient bacteria or/and enhancing the activity of the protein encoded by the fadL gene in the recipient bacteria, that is, the long-chain fatty acid uptake into the outer membrane protein; (5) enhancing the expression of genes involved in the fatty acid b oxidation pathway in the recipient bacteria or/and enhancing the activity of the proteins encoded by the genes involved in the fatty acid b oxidation pathway in the recipient bacteria; (6) enhancing the expression of genes involved in the short-chain fatty acid degradation pathway in the recipient bacteria or/and enhancing the activity of proteins encoded by the genes involved in the short-chain fatty acid degradation pathway in the recipient bacteria; (7) enhancing the expression of the exogenous alkL gene in the recipient bacteria or/and enhancing the activity of the protein encoded by the exogenous alkL gene in the recipient bacteria, that is, the intake of the exogenous alkL into the outer membrane protein; (8) inhibiting the expression of the iclR gene in the recipient bacteria or/and inhibiting the activity of the protein encoded by the iclR gene in the recipient bacteria, namely the glyoxylate pathway transcription inhibitor; (9) inhibiting the expression of the aceB gene in the recipient bacteria or/and inhibiting the activity of the malate synthase A protein encoded by the aceB gene in the receptor bacteria; (10) inhibiting the expression of the glcB gene in the recipient bacteria or/and inhibiting the activity of malate synthase G, the protein encoded by the glcB gene in the recipient bacteria; (11) enhancing the expression of the glyoxylate pathway aceA gene in the recipient bacteria or/and enhancing the activity of isocitrate lyase, the protein encoded by the glyoxylate pathway aceA gene in the recipient bacteria; (12) enhancing the expression of the exogenous cgaT gene in the recipient bacteria or/and enhancing the activity of the protein encoded by the exogenous cgaT gene in the recipient bacteria, namely glutamate glyoxylate aminotransferase II; The nucleotide sequences are shown in SEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9 or SEQ ID No. 10, 1-1399; (13) enhancing the expression of the gdhA gene in the recipient bacteria or/and enhancing the activity of the protein encoded by the gdhA gene in the recipient bacteria, namely glutamate dehydrogenase; The recipient bacteria is Escherichia coli. 2. The method according to claim 1, characterized in that: the genes involved in the fatty acid b oxidation pathway are selected from the following one or more genes: the gene fadD encoding fatty acyl-CoA synthase, the encoding fatty acyl-CoA dehydration Hydrogenase gene fadE, gene fadB encoding 3-hydroxyacyl-CoA dehydrogenase, gene fadA encoding 3-ketoacyl-CoA thiolase, gene fadI encoding 3-ketoacyl-CoA thiolase, The gene fadJ encoding 3-hydroxyacyl-CoA dehydrogenase and the gene fadK encoding short-chain fatty acyl-CoA synthase. 3. The method according to claim 1 or 2, wherein the gene involved in the short-chain fatty acid degradation pathway is selected from the genes contained in the following gene clusters: short-chain fatty acid degradation regulatory gene cluster atoSC, short-chain fatty acid degradation gene Cluster atoDAEB. 4 . The method according to claim 3 , wherein the short-chain fatty acid degradation regulatory gene cluster atoSC comprises the following genes: the gene atoC encoding a transcription activator of atoC, and the gene atoS encoding the atoS-sensing histidine kinase. 5 . 5. The method according to claim 3, wherein the short-chain fatty acid degradation gene cluster atoDAEB comprises the following genes: the gene atoA encoding the acetoacetyl-CoA transferase subunit, the gene encoding the acetoacetyl-CoA transferase subunit atoD, the gene atoE encoding the acetoacetate transporter, the gene atoB encoding the acetyl-CoA acetyltransferase. 6. The method according to claim 2, wherein the exogenous alkL gene is derived from Marinobacterhydrocarbonoclasticus or Pseudomonas putida . 7. The method according to claim 1 or 2, wherein the gdhA gene is derived from Escherichia coli or Bacillus subtilis . 8. The recombinant Escherichia coli constructed according to any one of claims 1-7. 9. The application of the recombinant Escherichia coli of claim 8 in the production of glycine. 10. A method for producing glycine, comprising the steps of: taking fatty acid as a substrate, using the recombinant Escherichia coli described in claim 8 to carry out biotransformation to synthesize glycine. 11. The method according to claim 10, wherein the fatty acid is stearic acid (C18), palmitic acid (C16), myristic acid (C14), lauric acid (C12), capric acid (C10) , caprylic acid (C8) or caproic acid (C6).

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