CN106929459B - A recombinant Escherichia coli and its construction method and a method for producing gluconic acid via metabolic engineering - Google Patents

A recombinant Escherichia coli and its construction method and a method for producing gluconic acid via metabolic engineering

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CN106929459B
CN106929459B CN201710200371.8A CN201710200371A CN106929459B CN 106929459 B CN106929459 B CN 106929459B CN 201710200371 A CN201710200371 A CN 201710200371A CN 106929459 B CN106929459 B CN 106929459B
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娄文勇
苏慧慧
宗敏华
徐培
杨继国
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South China University of Technology SCUT
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Abstract

The invention discloses recombinant escherichia coli, a construction method thereof and a method for producing glucaric acid through metabolic engineering, and belongs to the field of metabolic engineering. The invention realizes the production path of glucuronic acid by expressing inositol-1-phosphate synthase gene (Ino 1) and inositol oxidase gene (MIOX) from plants in escherichia coli, and simultaneously expresses aldehyde acid dehydrogenase gene (Udh) from agrobacterium tumefaciens (Agrobacterium tumefaciens), thereby successfully constructing the synthesis path of glucaric acid. Wherein, the recombinant escherichia coli generates 2.53G/L of glucaric acid by adding 10G/L of glucose into LB-G (LB culture medium) fermentation medium. The method for producing the glucaric acid by fermenting the recombinant escherichia coli has wide development prospect and lays a foundation for biologically synthesizing the glucaric acid.

Description

Recombinant escherichia coli, construction method thereof and method for producing glucaric acid through metabolic engineering
Technical Field
The invention belongs to the field of metabolic engineering, and in particular relates to recombinant escherichia coli, a construction method thereof and a method for producing glucaric acid through metabolic engineering.
Background
Glucaric acid (glucuric acid, SACCHARIC ACID), also known as glucaric acid, is a compound containing 4 chiral carbon atoms, usually in the form of the chiral compound D-glucaric acid, which spontaneously oxidizes in aqueous solution to form the mono-lactones D-glucaric acid-1, 4-lactone and D-glucaric acid-3, 6-lactone, and small amounts of the di-lactones D-glucaric acid-1, 4;3, 6-lactone.
Glucaric acid is a natural, non-toxic compound that is produced in small amounts in mammals, including humans, and in some plants, such as tomatoes, grapefruits and the like. Glucaric acid has important biological functions, and is identified by the U.S. department of energy as the "most valuable biorefinery". As with many nutrients, it can help and enhance immunity and body detoxification, thereby reducing human diseases, and it is known that glucaric acid has pharmacological action and can specifically and strongly inhibit activities of enzymes related to cancer occurrence, such as beta-glucuronidase. It can also reduce cholesterol, and its calcium salt is also a food additive. The glucaric acid and the derivatives thereof can obviously inhibit thrombin from inducing the peroxidation of arachidonic acid and play an obvious anti-inflammatory role, and the glucaric acid and the derivatives thereof influence the activation of platelets under the condition of oxidative stress and are helpful for preventing excessive platelet activation through an antioxidant mechanism, so that the glucaric acid and the derivatives thereof can be top health care products for preventing cardiovascular diseases, and some fruits and vegetables contain a large amount of glucaric acid and the derivatives thereof and can be used as diet for preventing cardiovascular diseases.
The glucaric acid has the advantages of no toxicity to human bodies, similar structure to that of monosaccharide, capability of entering the inside of cells through a sugar transport system of the human bodies, capability of being gathered on focus, rapid absorption by a small amount of ischemic tissues and the like, so that the glucaric acid can be used as an imaging agent for researching myocardial infarction and tumor. In addition, the glucaric acid can also be applied to the production of polymer monomers commonly used in living production, and the polymers are hydrolyzed into micromolecule monomers in nature and can be absorbed and utilized by plants and microorganisms, so that the glucaric acid has great potential economic value as a raw material for synthesizing various high-efficiency and environment-friendly emerging biomass energy sources.
The current preparation methods of glucaric acid mainly comprise a chemical method, an oxidation method with nitric acid and TEMPO as catalysts, and the like. The nitric acid oxidation method mainly oxidizes glucose into glucose and small molecular substances. The method produces a large amount of complex byproducts, and simultaneously has a large amount of harmful gases such as NO, NO 2 and the like which are discharged, so that the method pollutes the environment, and is gradually replaced by a new method. TEMPO oxidation refers to a process for synthesizing glucaric acid by electrochemical oxidation of glucose mediated by 2, 6-tetramethyl-1-piperidyl radical (TEMPO). The oxidation reactant metal destroyed by the catalyst participates in the reaction under mild reaction conditions, is selective to the product and can limit the products of unrecoverable byproducts, is convenient to operate, is a method commonly used at present, but is also an aspect of improvement due to the fact that the reaction temperature and pH value are controlled to be optimal in the reaction process and expensive catalysts are also needed.
For a long time, a common method for realizing efficient and environment-friendly production of organic acids by a microbial fermentation method. Compared with the chemical method, the microbial fermentation method has great improvement in the aspects of raw material loss, product purity and the like, and becomes a better synthesis method. Glucaric acid is found in mammals as a metabolic end product and requires at least 10 steps from glucose to glucaric acid in vivo. Prather et al obtained inositol-1-phosphate synthase (Ino 1) and inositol oxidase (MIOX) from Saccharomyces cerevisiae (Saccaromyces cerevisiae) and mice, and Pseudomonas syringae (Pseudomonas syringae) obtained aldehyde acid dehydrogenase (Udh), cloned into E.coli, and achieved biosynthesis of glucaric acid.
At present, no report of producing the glucaric acid by fermenting escherichia coli of recombinant plant-derived inositol oxidase is found, the content of the glucaric acid in plants is low, and the extraction, separation and purification are difficult.
Disclosure of Invention
The invention constructs recombinant bacteria by cloning and expressing inositol-1-phosphate synthase genes (Ino 1), inositol oxidase genes (MIOX) and aldehyde acid dehydrogenase genes (Udh) derived from agrobacterium tumefaciens into escherichia coli strains, wherein the recombinant bacteria can synthesize glucaric acid by taking glucose, glycerol, sucrose or inositol as substrates.
The invention is realized by the following technical scheme.
A recombinant E.coli which expresses both of inositol-1-phosphate synthase gene Ino1, inositol oxidase gene MIOX, and aldehyde acid dehydrogenase gene Udh.
Preferably, the inositol-1-phosphate synthase gene Ino1 is derived from any one of Saccharomyces cerevisiae (Saccaromyces cerevisiae), pichia pastoris (Pichia pastoris), tomato (Solanaceae Lycopersicon 1706), corn (Zea mays), and soybean (Glycine max).
Further preferred, the inositol-1-phosphate synthase gene Ino1, in one embodiment of the invention, is derived from tomato (Solanaceae Lycopersicon 1706,1706).
Preferably, the inositol oxidase gene MIOX is derived from any of Pichia pastoris (Pichia pastoris GS115,115), lycopersicon esculentum (Solanaceae Lycopersicon 1706,1706), corn (Zea mays), soybean (Glycine max).
Further preferred, the inositol oxidase gene MIOX, in one embodiment of the invention, is derived from tomato (Solanaceae Lycopersicon 1706,1706).
Preferably, the aldehyde acid dehydrogenase gene Udh is derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens GV 3103).
Preferably, the inositol-1-phosphate synthase gene Ino1, inositol oxidase gene MIOX, and aldehyde acid dehydrogenase gene Udh have nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, in one embodiment of the present invention.
Preferably, the amino acid sequences of the proteins expressed by the inositol-1-phosphate synthase gene Ino1, the inositol oxidase gene MIOX, and the aldehyde acid dehydrogenase gene Udh are the sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.
Preferably, the recombinant escherichia coli is a starting host bacterium of escherichia coli.
Preferably, the inositol-1-phosphate synthase gene Ino1, inositol oxidase gene MIOX, and aldehyde acid dehydrogenase gene Udh are expressed integrally.
The construction method of the recombinant escherichia coli comprises the following steps:
(1) Connecting inositol-1-phosphate synthase gene Ino1 and inositol oxidase gene MIOX to a binary expression vector, and transforming into an escherichia coli host strain to obtain recombinant escherichia coli 1;
(2) And (3) connecting the aldehyde acid dehydrogenase gene Udh with an expression vector, and then converting the gene Udh into the recombinant escherichia coli 1 obtained in the step (1) to obtain the recombinant escherichia coli.
Preferably, the method specifically comprises the following steps:
(1) Amplifying MIOX genes and Ino1 genes by taking tomato (Solanaceae Lycopersicon 1706) genome as a template, respectively adding proper enzyme cutting sites, connecting fragments to a binary expression vector pETDuet1, chemically converting DH5 alpha, screening to obtain correct recombinant plasmids, named pETDuet1-Ino1-MIOX, and converting the recombinant plasmids into host bacteria E.coli BL21 (DE 3) to obtain E.coli BL21 (DE 3)/Ino 1-MIOX;
(2) The Udh gene is amplified by taking the genome of agrobacterium tumefaciens (Agrobacterium tumefaciens GV 3103) as a template, and is connected to pET28a, and then the recombinant escherichia coli which is correctly screened from the expression host E.coli BL21 (DE 3)/Ino 1-MIOX constructed in the step (1) is converted by a chemical method, and the recombinant escherichia coli is named as E.coli BL21 (DE 3)/Ino 1-MIOX-Udh.
A method for producing glucaric acid by utilizing the recombinant escherichia coli comprises the step of catalyzing and synthesizing the glucaric acid by taking glucose, glycerol, sucrose or inositol as a substrate.
Preferably, the method specifically comprises inoculating recombinant E.coli seed solution into fermentation medium at an inoculum size of 2% -5%, adding IPTG with final concentration of 0.1-1mM and glucose with final concentration of 10g/L at 25-30 deg.C and 160-200 rpm, and culturing for 60-100 hr at OD600 of about 0.6.
Further preferably, the fermentation medium, in one embodiment of the present invention, has a carbon source of glucose and/or inositol.
Further preferably, in one embodiment of the invention, the seed culture is inoculated in an amount of 2% into 250ml shake flasks containing 50ml of fermentation medium, at an OD600 of about 0.6, with 0.1-1mM IPTG and 10g/L glucose, at a temperature of 30℃and a shaking table rotation of 180rpm, for a cultivation time of 100 hours.
Compared with the prior art, the invention has the following advantages:
(1) The invention provides a method for producing glucaric acid by fermenting heterologous plant source key enzyme gene recombinant escherichia coli, wherein 2.53g/L of glucaric acid is produced by the recombinant escherichia coli in an LB culture medium, and no glucaric acid is detected in a contrast escherichia coli BL21 (DE 3).
(2) The inositol-1-phosphate synthase gene and inositol oxidase gene derived from tomato used in the present invention are greatly different from the mouse-derived MIOX amino acids reported in the prior art, and there is no report on the fact that the Ino1 gene and MIOX gene derived from tomato can convert glucose into aldehyde acid.
(3) The recombinant escherichia coli of the invention synthesizes the glucaric acid, and has the advantages of low nutrition requirement, quick production, low cost in culture, stable heredity, high expression level and the like during culture.
(4) The invention adopts a metabolic engineering strategy, reforms a microorganism strain to synthesize the target product of the glucaric acid, and lays a solid foundation for the high-efficiency production of the glucaric acid by a biological method.
Drawings
FIG. 1 is a graph showing the increase in the production of recombinant E.coli shake-flask fermented glucaric acid with fermentation time;
FIG. 2a is an HPLC profile of a glucaric acid standard;
FIG. 2b is an HPLC profile of recombinant E.coli fermentation broth.
Detailed Description
The following description of the embodiments of the present invention will be made with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto, and the process parameters not specifically described may be performed with reference to conventional techniques.
The detection of glucaric acid was Waser (Binary HPLC Pump), 2414 time difference detector and 2487 UV detector. The preparation of the standard sample of the glucaric acid comprises accurately weighing 50mg of the glucaric acid, dissolving in 5mM H 2SO4, transferring the dissolved solution into a 10ml volumetric flask for constant volume, and the concentration is 100mg/L. Then diluted with 5mM H 2SO4 to 4mg/ml, 3mg/ml, 2mg/ml, 1mg/ml, respectively.
Sample preparation 1ml of fermentation broth was centrifuged at 12000rpm for 5min, the supernatant was filtered through a 0.22um filter membrane, and the filtrate was analyzed by liquid phase.
Analysis conditions:
Mobile phase 5mM H 2SO4, isocratic elution
Chromatographic column HPX-87H
Detectors 2414 time difference detector and 2487 ultraviolet detector
EXAMPLE 1 construction of recombinant E.coli BL21 (DE 3)/Ino 1-MIOX
The tomato (Solanaceae Lycopersicon, 1706) genome is used as a template to amplify the Ino1 gene and the MIOX gene, the Ino1-F (with the sequence shown as SEQ ID NO. 7) and the Ino1-R (with the sequence shown as SEQ ID NO. 8) are used as primers to amplify the Ino1 gene, and the MIOX-F (with the sequence shown as SEQ ID NO. 9) and the MIOX-R (with the sequence shown as SEQ ID NO. 10) are used as primers to amplify the MIOX gene. The obtained Ino1 gene and MIOX gene are connected to an expression vector pETDuet1 (purchased from Novegen company) with corresponding cuts through enzyme cutting sites at two ends of a primer and transformed into DH5 alpha (purchased from the whole-formula gold company), and under the condition of ensuring the correct reading frame, recombinant expression plasmids pETDuet1-Ino1-MIOX are identified, and the sequences are correct through sequencing comparison. The recombinant plasmid is transferred into an expression host E.coli BL21 (DE 3) (purchased from the full-scale gold company) by a chemical method, and the recombinant clone is correct through PCR verification and is named as E.coli BL21 (DE 3)/Ino 1-MIOX.
EXAMPLE 2 construction of recombinant E.coli BL21 (DE 3)/Ino 1-MIOX-Udh
The Udh gene is amplified by taking the agrobacterium tumefaciens (Agrobacterium tumefaciens GV 3103) genome as a template, udh-F (the sequence is shown as SEQ ID NO. 11) and Udh-R (the sequence is shown as SEQ ID NO. 12) are amplified by taking the primer, the Udh gene is connected to an expression vector pET28a (purchased from BioVector plasmid vector strain cytogene collection) with a corresponding notch through double enzyme cutting sites on the primer, and the expression vector pET28a is transformed into E.coli DH5 alpha (purchased from full-formula gold company), and recombinant expression plasmids pET28a-Udh are identified on the premise of ensuring the reading frame to be correct and are subjected to DNA sequencing comparison, so that the recombinant sequence is correct. The recombinant plasmid is transferred into an expression host E.coli BL21 (DE 3)/Ino 1-MIOX constructed in example 1 by a chemical method, and correct recombinant escherichia coli is screened and named as E.coli BL21 (DE 3)/Ino 1-MIOX-Udh.
Table 1 shows the primers used in examples 1 and 2.
TABLE 1
Primer(s) Sequence(s)
Udh-F CGCAAGCTTATGAAACGGCTTCTTGTTACC
Udh-R CGCTCGAGCGGTGTCGTCTCGGTTATAT
Ino1-F CGCGGATCCGATGTTTATTGAAAATTTTAAGGT
Ino1-R GCGCTGCAGGATTTGTATTCCAAAATCATG
MIOX-F CGGATATCGATGACTATTCTCATTGAGCAGCCT
MIOX-R CCGCTCGAGACCACCTCAGCTTTGTTGGAAAAT
EXAMPLE 3 recombinant E.coli fermentation to produce glucaric acid
Recombinant E.coli BL21 (DE 3)/Ino 1-MIOX-Udh was fermented. A monoclonal recombinant E.coli BL21 (DE 3)/Ino 1-MIOX-Udh was inoculated into 25ml of LB medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH 7) and cultured at 37℃and 180rpm for 16h. Then inoculating to 50ml (shake flask capacity 500 ml) fermentation medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10 g/L) according to 2% inoculum size, fermenting, adding inducer IPTG to make its concentration 0.2mM at OD600 of 0.6, adding glucose to make its concentration 10g/L, culturing at 30 ℃ and shaking table rotation speed 180rpm for 100 hours. After the completion of the cultivation, 1ml of the fermentation broth was centrifuged at 12000rpm for 5min, and the supernatant was filtered through a 0.22um filter membrane, and the product was detected by HPLC. FIG. 2a is an HPLC spectrum of a glucaric acid standard, and FIG. 2b is an HPLC spectrum of recombinant E.coli fermentation broth. From the standard, it can be seen that the peak time of glucaric acid was 10.058min, and glucaric acid having the same peak time could be detected in the fermentation broth of the recombinant strain.
FIG. 1 is a graph showing the increase of the production amount of the recombinant E.coli shake-flask fermented glucaric acid with the fermentation time, and as shown in FIG. 1, the production amount of glucaric acid was increased to 2.53g/L as the shake-flask fermentation time was increased.
SEQUENCE LISTING
<110> University of North China
<120> Recombinant E.coli, method for constructing the same and method for producing glucaric acid by metabolic engineering
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<160> 12
<170> PatentIn version 3.5
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<213> Artificial sequence
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Ala Thr Gly Thr Thr Thr Ala Thr Thr Gly Ala Ala Ala Ala Thr Thr
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Thr Thr Ala Ala Gly Gly Thr Gly Gly Ala Ala Ala Gly Cys Cys Cys
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Ala Ala Ala Thr Gly Thr Gly Ala Ala Gly Thr Ala Thr Ala Thr Thr
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Gly Ala Gly Ala Ala Thr Gly Ala Ala Ala Thr Thr Cys Ala Thr Thr
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Cys Thr Gly Thr Gly Thr Ala Thr Gly Ala Thr Thr Ala Thr Gly Ala
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Ala Ala Cys Cys Ala Cys Ala Gly Ala Gly Cys Thr Thr Gly Thr Thr
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Cys Ala Thr Gly Ala Ala Gly Ala Gly Ala Gly Ala Ala Ala Thr Gly
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Gly Ala Ala Cys Thr Thr Ala Thr Cys Ala Ala Thr Gly Gly Ala Thr
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Thr Gly Thr Thr Ala Ala Gly Cys Cys Thr Ala Ala Ala Ala Cys Thr
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Gly Thr Cys Ala Ala Ala Thr Ala Thr Gly Ala Ala Thr Thr Thr Ala
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Ala Ala Ala Cys Thr Gly Ala Thr Ala Cys Cys Cys Ala Thr Gly Thr
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Gly Cys Cys Ala Ala Ala Ala Thr Thr Gly Gly Gly Gly Gly Thr Thr
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Ala Thr Gly Cys Thr Thr Gly Thr Thr Gly Gly Ala Thr Gly Gly Gly
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Gly Ala Gly Gly Ala Ala Ala Cys Ala Ala Thr Gly Gly Thr Thr Cys
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Ala Ala Cys Ala Thr Thr Gly Ala Cys Thr Gly Gly Ala Gly Gly Thr
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Gly Thr Thr Ala Thr Thr Gly Cys Gly Ala Ala Thr Cys Gly Ala Gly
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Ala Ala Gly Gly Ala Ala Thr Thr Thr Cys Ala Thr Gly Gly Gly Cys
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Ala Ala Cys Gly Ala Ala Ala Gly Ala Ala Ala Ala Ala Gly Thr Gly
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Cys Ala Ala Cys Ala Ala Gly Cys Cys Ala Ala Thr Thr Ala Thr Thr
290 295 300
Thr Thr Gly Gly Gly Thr Cys Thr Cys Thr Thr Ala Cys Thr Cys Ala
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Gly Gly Cys Ala Thr Cys Ala Ala Cys Cys Ala Thr Thr Cys Gly Ala
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Gly Thr Thr Gly Gly Gly Thr Cys Thr Thr Thr Cys Ala Ala Thr Gly
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Gly Cys Gly Ala Ala Gly Ala Gly Ala Thr Cys Thr Ala Thr Gly Cys
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Ala Cys Cys Cys Thr Thr Cys Ala Ala Ala Ala Gly Cys Cys Thr Cys
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Cys Thr Thr Cys Cys Cys Ala Thr Gly Gly Thr Cys Ala Ala Cys Cys
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Cys Ala Gly Ala Cys Gly Ala Thr Gly Thr Ala Gly Thr Ala Thr Thr
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Thr Gly Gly Ala Gly Gly Ala Thr Gly Gly Gly Ala Cys Ala Thr Thr
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Ala Gly Cys Ala Ala Cys Ala Thr Gly Ala Ala Thr Thr Thr Gly Gly
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Cys Ala Gly Ala Thr Gly Cys Thr Ala Thr Gly Gly Thr Cys Ala Gly
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Gly Gly Cys Thr Ala Ala Gly Gly Thr Thr Thr Thr Cys Gly Ala Ala
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Gly Thr Thr Gly Ala Thr Cys Thr Gly Cys Ala Ala Ala Ala Gly Cys
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Ala Gly Cys Thr Gly Ala Gly Gly Cys Cys Cys Thr Ala Cys Ala Thr
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Gly Gly Ala Ala Thr Cys Cys Ala Thr Gly Gly Thr Thr Cys Cys Cys
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Cys Thr Thr Cys Cys Thr Gly Gly Thr Ala Thr Cys Thr Ala Thr Gly
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Ala Cys Cys Cys Thr Gly Ala Cys Thr Thr Cys Ala Thr Thr Gly Cys
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Gly Gly Cys Thr Ala Ala Cys Cys Ala Ala Gly Ala Ala Gly Cys Ala
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Cys Gly Thr Gly Cys Cys Ala Ala Cys Ala Ala Cys Gly Thr Gly Ala
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Thr Cys Ala Ala Ala Gly Gly Ala Ala Cys Cys Ala Ala Gly Ala Ala
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Ala Gly Ala Ala Cys Ala Ala Gly Thr Thr Gly Ala Ala Cys Ala Ala
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Ala Thr Thr Gly Thr Thr Ala Ala Ala Gly Ala Thr Ala Thr Thr Ala
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Gly Gly Gly Ala Gly Thr Thr Cys Ala Ala Gly Gly Ala Gly Ala Ala
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Gly Ala Ala Cys Ala Ala Gly Gly Thr Ala Gly Ala Cys Ala Ala Gly
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Ala Thr Ala Gly Thr Gly Gly Thr Thr Cys Thr Ala Thr Gly Gly Ala
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Cys Thr Gly Cys Cys Ala Ala Cys Ala Cys Cys Gly Ala Ala Ala Gly
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Gly Thr Ala Cys Ala Gly Thr Ala Ala Thr Gly Thr Gly Gly Thr Thr
705 710 715 720
Gly Thr Thr Gly Gly Cys Cys Thr Thr Ala Ala Thr Gly Ala Cys Ala
725 730 735
Cys Cys Ala Thr Gly Gly Ala Ala Ala Ala Cys Cys Thr Thr Thr Thr
740 745 750
Ala Gly Cys Thr Gly Cys Thr Gly Thr Gly Gly Ala Thr Ala Gly Ala
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Ala Ala Thr Gly Ala Gly Gly Cys Thr Gly Ala Ala Ala Thr Ala Thr
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Cys Thr Cys Cys Thr Thr Cys Thr Ala Cys Ala Thr Thr Gly Cys Ala
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Thr Gly Cys Thr Ala Thr Thr Gly Cys Thr Thr Gly Thr Ala Thr Thr
805 810 815
Ala Thr Gly Gly Ala Ala Ala Ala Thr Gly Thr Gly Cys Cys Thr Thr
820 825 830
Thr Cys Ala Thr Cys Ala Ala Cys Gly Gly Ala Ala Gly Cys Cys Cys
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Thr Cys Ala Ala Ala Ala Cys Ala Cys Thr Thr Thr Thr Gly Thr Thr
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Cys Cys Ala Gly Gly Thr Cys Thr Thr Ala Thr Thr Gly Ala Thr Thr
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Thr Gly Gly Cys Cys Ala Thr Ala Ala Ala Gly Ala Gly Ala Ala Ala
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Cys Ala Cys Thr Thr Thr Ala Ala Thr Thr Gly Gly Thr Gly Gly Thr
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Gly Ala Thr Gly Ala Cys Thr Thr Thr Ala Ala Gly Ala Gly Thr Gly
915 920 925
Gly Thr Cys Ala Ala Ala Cys Cys Ala Ala Gly Ala Thr Gly Ala Ala
930 935 940
Gly Thr Cys Cys Gly Thr Gly Thr Thr Gly Gly Thr Thr Gly Ala Thr
945 950 955 960
Thr Thr Cys Cys Thr Thr Gly Thr Thr Gly Gly Ala Gly Cys Cys Gly
965 970 975
Gly Thr Ala Thr Thr Ala Ala Gly Cys Cys Ala Ala Cys Gly Thr Cys
980 985 990
Ala Ala Thr Ala Gly Thr Gly Ala Gly Cys Thr Ala Cys Ala Ala Thr
995 1000 1005
Cys Ala Cys Thr Thr Gly Gly Gly Thr Ala Ala Cys Ala Ala Thr
1010 1015 1020
Gly Ala Thr Gly Gly Ala Ala Thr Gly Ala Ala Thr Cys Thr Thr
1025 1030 1035
Thr Cys Thr Gly Cys Thr Cys Cys Thr Cys Ala Ala Ala Cys Cys
1040 1045 1050
Thr Thr Cys Cys Gly Gly Thr Cys Thr Ala Ala Gly Gly Ala Gly
1055 1060 1065
Ala Thr Cys Thr Cys Ala Ala Ala Ala Ala Gly Thr Ala Ala Thr
1070 1075 1080
Gly Thr Thr Gly Thr Thr Gly Ala Thr Gly Ala Cys Ala Thr Gly
1085 1090 1095
Gly Thr Thr Gly Cys Thr Ala Gly Cys Ala Ala Cys Gly Cys Cys
1100 1105 1110
Ala Thr Thr Cys Thr Cys Thr Ala Thr Gly Ala Ala Thr Cys Thr
1115 1120 1125
Gly Gly Cys Gly Ala Gly Cys Ala Cys Cys Cys Thr Gly Ala Cys
1130 1135 1140
Cys Ala Thr Gly Thr Thr Gly Thr Cys Gly Thr Gly Ala Thr Cys
1145 1150 1155
Ala Ala Gly Thr Ala Thr Gly Thr Thr Cys Cys Ala Thr Ala Thr
1160 1165 1170
Gly Thr Thr Gly Gly Ala Gly Ala Cys Ala Gly Cys Ala Ala Gly
1175 1180 1185
Ala Gly Gly Gly Cys Ala Ala Thr Gly Gly Ala Thr Gly Ala Gly
1190 1195 1200
Thr Ala Cys Ala Thr Gly Thr Cys Ala Gly Ala Gly Ala Thr Thr
1205 1210 1215
Thr Thr Cys Ala Thr Gly Gly Gly Cys Gly Gly Ala Ala Ala Gly
1220 1225 1230
Ala Gly Cys Ala Cys Thr Ala Thr Ala Gly Thr Thr Ala Thr Gly
1235 1240 1245
Cys Ala Cys Ala Ala Cys Ala Cys Thr Thr Gly Thr Gly Ala Gly
1250 1255 1260
Gly Ala Cys Thr Cys Thr Cys Thr Thr Thr Thr Gly Gly Cys Ala
1265 1270 1275
Gly Cys Thr Cys Cys Ala Ala Thr Thr Ala Thr Cys Thr Thr Gly
1280 1285 1290
Gly Ala Thr Thr Thr Gly Gly Thr Cys Cys Thr Thr Cys Thr Cys
1295 1300 1305
Gly Cys Thr Gly Ala Ala Cys Thr Cys Ala Gly Cys Ala Cys Thr
1310 1315 1320
Cys Gly Cys Ala Thr Thr Cys Ala Gly Cys Thr Cys Ala Ala Ala
1325 1330 1335
Gly Cys Thr Gly Ala Ala Gly Gly Gly Gly Ala Gly Gly Gly Gly
1340 1345 1350
Ala Ala Gly Thr Thr Cys Cys Ala Cys Thr Cys Cys Thr Thr Cys
1355 1360 1365
Cys Ala Cys Cys Cys Thr Gly Thr Gly Gly Cys Thr Ala Cys Thr
1370 1375 1380
Ala Thr Thr Cys Thr Cys Ala Gly Cys Thr Ala Cys Cys Thr Cys
1385 1390 1395
Ala Cys Cys Ala Ala Gly Gly Cys Thr Cys Cys Cys Cys Thr Gly
1400 1405 1410
Gly Thr Ala Cys Cys Ala Cys Cys Ala Gly Gly Thr Ala Cys Thr
1415 1420 1425
Cys Cys Ala Gly Thr Gly Gly Thr Gly Ala Ala Thr Gly Cys Cys
1430 1435 1440
Cys Thr Thr Thr Cys Ala Ala Ala Gly Cys Ala Gly Ala Gly Gly
1445 1450 1455
Gly Cys Ala Ala Thr Gly Cys Thr Thr Gly Ala Gly Ala Ala Thr
1460 1465 1470
Ala Thr Ala Ala Thr Gly Ala Gly Ala Gly Cys Thr Thr Gly Thr
1475 1480 1485
Gly Thr Thr Gly Gly Thr Thr Thr Gly Gly Cys Ala Cys Cys Ala
1490 1495 1500
Gly Ala Gly Ala Ala Cys Ala Ala Cys Ala Thr Gly Ala Thr Thr
1505 1510 1515
Thr Thr Gly Gly Ala Ala Thr Ala Cys Ala Ala Ala Thr Gly Ala
1520 1525 1530
<210> 2
<211> 954
<212> PRT
<213> Artificial sequence
<400> 2
Ala Thr Gly Ala Cys Thr Ala Thr Thr Cys Thr Cys Ala Thr Thr Gly
1 5 10 15
Ala Gly Cys Ala Gly Cys Cys Thr Gly Ala Ala Thr Thr Thr Gly Gly
20 25 30
Ala Thr Cys Ala Cys Ala Ala Gly Thr Gly Gly Ala Gly Gly Ala Gly
35 40 45
Ala Ala Ala Ala Ala Ala Gly Thr Cys Thr Cys Ala Thr Thr Cys Ala
50 55 60
Ala Thr Gly Cys Cys Ala Ala Thr Gly Ala Ala Cys Thr Thr Ala Thr
65 70 75 80
Thr Thr Thr Gly Gly Ala Thr Gly Gly Thr Gly Gly Ala Thr Thr Thr
85 90 95
Ala Thr Gly Gly Thr Ala Cys Cys Ala Ala Ala Gly Ala Cys Ala Thr
100 105 110
Thr Gly Thr Cys Thr Thr Cys Thr Cys Ala Ala Gly Ala Thr Gly Ala
115 120 125
Ala Ala Thr Ala Thr Thr Thr Gly Ala Ala Gly Thr Gly Cys Cys Ala
130 135 140
Gly Ala Cys Ala Thr Ala Ala Ala Thr Gly Cys Ala Thr Thr Thr Gly
145 150 155 160
Gly Thr Cys Ala Ala Thr Cys Ala Thr Thr Thr Ala Gly Gly Gly Ala
165 170 175
Thr Thr Ala Thr Ala Ala Thr Gly Thr Ala Gly Ala Ala Ala Gly Thr
180 185 190
Gly Ala Gly Ala Gly Ala Cys Ala Ala Ala Ala Ala Thr Cys Ala Gly
195 200 205
Thr Gly Gly Ala Ala Gly Ala Ala Thr Thr Thr Thr Ala Thr Ala Gly
210 215 220
Gly Gly Thr Thr Cys Ala Ala Cys Ala Cys Ala Thr Thr Ala Ala Thr
225 230 235 240
Cys Ala Ala Ala Cys Ala Thr Ala Thr Gly Ala Cys Thr Ala Thr Gly
245 250 255
Thr Gly Ala Ala Ala Ala Ala Ala Ala Thr Gly Ala Gly Ala Ala Ala
260 265 270
Ala Gly Ala Ala Thr Ala Thr Gly Gly Ala Ala Ala Ala Thr Thr Gly
275 280 285
Ala Ala Cys Ala Ala Ala Ala Thr Thr Gly Ala Ala Ala Thr Gly Ala
290 295 300
Gly Thr Ala Thr Thr Thr Gly Gly Gly Ala Thr Thr Gly Thr Thr Gly
305 310 315 320
Thr Gly Ala Ala Cys Thr Thr Thr Thr Gly Ala Ala Thr Gly Ala Thr
325 330 335
Gly Thr Ala Gly Thr Thr Gly Ala Thr Gly Ala Thr Ala Gly Thr Gly
340 345 350
Ala Thr Cys Cys Thr Gly Ala Thr Thr Thr Gly Gly Ala Thr Gly Ala
355 360 365
Ala Cys Cys Ala Cys Ala Ala Ala Thr Thr Gly Ala Gly Cys Ala Thr
370 375 380
Thr Thr Gly Thr Thr Ala Cys Ala Ala Ala Cys Thr Gly Cys Thr Gly
385 390 395 400
Ala Ala Gly Cys Thr Ala Thr Thr Ala Gly Ala Ala Ala Ala Gly Ala
405 410 415
Thr Thr Ala Thr Cys Cys Ala Ala Ala Thr Gly Ala Ala Gly Ala Thr
420 425 430
Thr Gly Gly Cys Thr Thr Cys Ala Thr Thr Thr Gly Ala Cys Cys Gly
435 440 445
Gly Cys Cys Thr Cys Ala Thr Thr Cys Ala Cys Gly Ala Cys Cys Thr
450 455 460
Ala Gly Gly Thr Ala Ala Ala Gly Thr Ala Cys Thr Thr Cys Thr Thr
465 470 475 480
Cys Ala Thr Cys Cys Ala Ala Gly Thr Thr Thr Thr Gly Gly Ala Gly
485 490 495
Gly Gly Cys Thr Thr Cys Cys Thr Cys Ala Ala Thr Gly Gly Gly Cys
500 505 510
Thr Gly Thr Thr Gly Thr Thr Gly Gly Ala Gly Ala Cys Ala Cys Ala
515 520 525
Thr Thr Thr Cys Cys Thr Cys Thr Thr Gly Gly Thr Thr Gly Thr Gly
530 535 540
Cys Thr Thr Thr Thr Gly Ala Thr Gly Ala Ala Thr Cys Ala Ala Thr
545 550 555 560
Thr Gly Thr Thr Cys Ala Cys Cys Ala Cys Ala Ala Gly Thr Ala Thr
565 570 575
Thr Thr Thr Ala Ala Gly Gly Ala Ala Ala Ala Thr Cys Cys Ala Gly
580 585 590
Ala Cys Ala Thr Cys Ala Ala Cys Ala Ala Cys Ala Ala Thr Ala Thr
595 600 605
Thr Thr Ala Thr Ala Ala Thr Ala Cys Ala Ala Ala Ala Ala Ala Thr
610 615 620
Gly Gly Thr Gly Thr Ala Thr Ala Thr Gly Ala Ala Gly Ala Ala Gly
625 630 635 640
Gly Thr Thr Gly Thr Gly Gly Ala Cys Thr Thr Gly Ala Cys Ala Ala
645 650 655
Ala Gly Thr Thr Gly Thr Thr Ala Thr Gly Thr Cys Ala Thr Gly Gly
660 665 670
Gly Gly Ala Cys Ala Thr Gly Ala Thr Gly Ala Thr Thr Ala Thr Ala
675 680 685
Thr Gly Thr Ala Thr Thr Thr Ala Ala Thr Thr Gly Cys Ala Ala Ala
690 695 700
Gly Gly Ala Ala Ala Ala Thr Ala Ala Ala Ala Cys Thr Ala Cys Thr
705 710 715 720
Cys Thr Thr Cys Cys Thr Thr Cys Thr Gly Cys Thr Gly Cys Thr Thr
725 730 735
Thr Ala Thr Thr Thr Gly Thr Cys Ala Thr Ala Cys Gly Thr Thr Ala
740 745 750
Cys Cys Ala Cys Thr Cys Thr Thr Thr Cys Thr Ala Thr Gly Cys Ala
755 760 765
Thr Thr Ala Cys Ala Thr Ala Gly Ala Thr Cys Ala Gly Gly Ala Gly
770 775 780
Cys Ala Thr Ala Thr Ala Cys Ala Cys Ala Cys Thr Thr Gly Ala Thr
785 790 795 800
Gly Ala Ala Thr Gly Ala Gly Gly Ala Gly Gly Ala Cys Ala Ala Ala
805 810 815
Gly Ala Gly Ala Ala Cys Ala Thr Gly Ala Ala Gly Thr Gly Gly Cys
820 825 830
Thr Cys Ala Ala Cys Ala Thr Thr Thr Thr Thr Ala Ala Thr Ala Ala
835 840 845
Ala Thr Ala Thr Gly Ala Thr Thr Thr Ala Thr Ala Thr Ala Gly Cys
850 855 860
Ala Ala Gly Ala Gly Thr Ala Ala Ala Gly Thr Thr Cys Gly Ala Ala
865 870 875 880
Thr Thr Gly Ala Thr Gly Thr Gly Gly Ala Ala Ala Ala Ala Gly Thr
885 890 895
Cys Ala Ala Gly Cys Cys Ala Thr Ala Cys Thr Ala Thr Cys Thr Cys
900 905 910
Thr Cys Thr Cys Thr Thr Ala Thr Cys Gly Ala Ala Ala Ala Gly Thr
915 920 925
Ala Thr Thr Thr Thr Cys Cys Ala Ala Cys Ala Ala Ala Gly Cys Thr
930 935 940
Gly Ala Gly Gly Thr Gly Gly Thr Ala Ala
945 950
<210> 3
<211> 798
<212> PRT
<213> Artificial sequence
<400> 3
Ala Thr Gly Ala Ala Ala Cys Gly Gly Cys Thr Thr Cys Thr Thr Gly
1 5 10 15
Thr Thr Ala Cys Cys Gly Gly Thr Gly Cys Gly Gly Cys Gly Gly Gly
20 25 30
Cys Cys Ala Gly Cys Thr Thr Gly Gly Cys Cys Gly Cys Gly Thr Cys
35 40 45
Ala Thr Gly Cys Gly Cys Gly Ala Gly Cys Gly Thr Cys Thr Cys Gly
50 55 60
Cys Ala Cys Cys Gly Ala Thr Gly Gly Cys Gly Gly Ala Gly Ala Thr
65 70 75 80
Ala Cys Thr Gly Cys Gly Cys Cys Thr Thr Gly Cys Cys Gly Ala Thr
85 90 95
Cys Thr Cys Thr Cys Cys Cys Cys Gly Cys Thr Cys Gly Ala Cys Cys
100 105 110
Cys Gly Gly Cys Ala Gly Gly Gly Cys Cys Gly Ala Ala Cys Gly Ala
115 120 125
Ala Gly Ala Ala Thr Gly Cys Gly Thr Gly Cys Ala Ala Thr Gly Cys
130 135 140
Gly Ala Cys Cys Thr Thr Gly Cys Cys Gly Ala Thr Gly Cys Cys Ala
145 150 155 160
Ala Thr Gly Cys Cys Gly Thr Gly Ala Ala Thr Gly Cys Cys Ala Thr
165 170 175
Gly Gly Thr Cys Gly Cys Cys Gly Gly Thr Thr Gly Cys Gly Ala Cys
180 185 190
Gly Gly Thr Ala Thr Thr Gly Thr Thr Cys Ala Thr Cys Thr Cys Gly
195 200 205
Gly Cys Gly Gly Cys Ala Thr Cys Thr Cys Gly Gly Thr Gly Gly Ala
210 215 220
Gly Ala Ala Gly Cys Cys Cys Thr Thr Cys Gly Ala Ala Cys Ala Ala
225 230 235 240
Ala Thr Cys Cys Thr Thr Cys Ala Gly Gly Gly Cys Ala Ala Thr Ala
245 250 255
Thr Cys Ala Thr Cys Gly Gly Gly Cys Thr Thr Cys Ala Thr Ala Ala
260 265 270
Thr Cys Thr Cys Thr Ala Cys Gly Ala Gly Gly Cys Cys Gly Cys Cys
275 280 285
Cys Gly Cys Gly Cys Cys Cys Ala Thr Gly Gly Ala Cys Ala Gly Cys
290 295 300
Cys Ala Cys Gly Cys Ala Thr Cys Gly Thr Cys Thr Thr Thr Gly Cys
305 310 315 320
Cys Ala Gly Cys Thr Cys Cys Ala Ala Cys Cys Ala Cys Ala Cys Gly
325 330 335
Ala Thr Cys Gly Gly Cys Thr Ala Thr Thr Ala Thr Cys Cys Gly Cys
340 345 350
Ala Gly Ala Cys Cys Gly Ala Ala Cys Gly Gly Cys Thr Cys Gly Gly
355 360 365
Thr Cys Cys Gly Gly Ala Thr Gly Thr Thr Cys Cys Gly Gly Cys Gly
370 375 380
Cys Gly Gly Cys Cys Gly Gly Ala Cys Gly Gly Thr Cys Thr Thr Thr
385 390 395 400
Ala Cys Gly Gly Cys Gly Thr Cys Thr Cys Cys Ala Ala Ala Thr Gly
405 410 415
Thr Thr Thr Cys Gly Gly Cys Gly Ala Ala Ala Ala Cys Cys Thr Cys
420 425 430
Gly Cys Cys Cys Gly Cys Ala Thr Gly Thr Ala Thr Thr Thr Cys Gly
435 440 445
Ala Thr Ala Ala Ala Thr Thr Cys Gly Gly Gly Cys Ala Gly Gly Ala
450 455 460
Gly Ala Cys Gly Gly Cys Gly Cys Thr Gly Gly Thr Gly Cys Gly Cys
465 470 475 480
Ala Thr Cys Gly Gly Cys Thr Cys Cys Thr Gly Thr Ala Cys Gly Cys
485 490 495
Cys Gly Gly Ala Ala Cys Cys Cys Ala Ala Cys Ala Ala Thr Thr Ala
500 505 510
Cys Cys Gly Cys Ala Thr Gly Cys Thr Gly Thr Cys Cys Ala Cys Cys
515 520 525
Thr Gly Gly Thr Thr Thr Thr Cys Gly Cys Ala Cys Gly Ala Thr Gly
530 535 540
Ala Thr Thr Thr Cys Gly Thr Gly Thr Cys Gly Cys Thr Gly Ala Thr
545 550 555 560
Cys Gly Ala Gly Gly Cys Gly Gly Thr Gly Thr Thr Thr Cys Gly Cys
565 570 575
Gly Cys Gly Cys Cys Gly Gly Thr Gly Cys Thr Cys Gly Gly Cys Thr
580 585 590
Gly Cys Cys Cys Gly Gly Thr Cys Gly Thr Cys Thr Gly Gly Gly Gly
595 600 605
Gly Gly Cys Ala Thr Cys Gly Gly Cys Cys Ala Ala Thr Gly Ala Thr
610 615 620
Gly Cys Gly Gly Gly Cys Thr Gly Gly Thr Gly Gly Gly Ala Cys Ala
625 630 635 640
Ala Thr Thr Cys Gly Cys Ala Thr Cys Thr Thr Gly Gly Cys Thr Thr
645 650 655
Thr Cys Thr Gly Gly Gly Cys Thr Gly Gly Ala Ala Ala Cys Cys Gly
660 665 670
Gly Ala Gly Gly Ala Thr Ala Ala Thr Gly Cys Cys Gly Ala Gly Gly
675 680 685
Cys Cys Thr Thr Cys Cys Gly Gly Cys Gly Gly Cys Ala Thr Ala Thr
690 695 700
Ala Ala Cys Cys Gly Ala Gly Ala Cys Gly Ala Cys Ala Cys Cys Gly
705 710 715 720
Cys Cys Ala Cys Cys Gly Gly Ala Cys Cys Cys Gly Ala Ala Thr Gly
725 730 735
Ala Cys Gly Cys Gly Thr Thr Gly Gly Thr Gly Cys Gly Gly Thr Thr
740 745 750
Cys Cys Ala Gly Gly Gly Cys Gly Gly Thr Ala Cys Gly Thr Thr Thr
755 760 765
Gly Thr Cys Gly Ala Cys Ala Ala Cys Cys Cys Gly Ala Thr Cys Thr
770 775 780
Thr Cys Ala Ala Ala Cys Ala Gly Ala Gly Cys Thr Gly Ala
785 790 795
<210> 4
<211> 510
<212> PRT
<213> Artificial sequence
<400> 4
Met Phe Ile Glu Asn Phe Lys Val Glu Ser Pro Asn Val Lys Tyr Ile
1 5 10 15
Glu Asn Glu Ile His Ser Val Tyr Asp Tyr Glu Thr Thr Glu Leu Val
20 25 30
His Glu Glu Arg Asn Gly Thr Tyr Gln Trp Ile Val Lys Pro Lys Thr
35 40 45
Val Lys Tyr Glu Phe Lys Thr Asp Thr His Val Pro Lys Leu Gly Val
50 55 60
Met Leu Val Gly Trp Gly Gly Asn Asn Gly Ser Thr Leu Thr Gly Gly
65 70 75 80
Val Ile Ala Asn Arg Glu Gly Ile Ser Trp Ala Thr Lys Glu Lys Val
85 90 95
Gln Gln Ala Asn Tyr Phe Gly Ser Leu Thr Gln Ala Ser Thr Ile Arg
100 105 110
Val Gly Ser Phe Asn Gly Glu Glu Ile Tyr Ala Pro Phe Lys Ser Leu
115 120 125
Leu Pro Met Val Asn Pro Asp Asp Val Val Phe Gly Gly Trp Asp Ile
130 135 140
Ser Asn Met Asn Leu Ala Asp Ala Met Val Arg Ala Lys Val Phe Glu
145 150 155 160
Val Asp Leu Gln Lys Gln Leu Arg Pro Tyr Met Glu Ser Met Val Pro
165 170 175
Leu Pro Gly Ile Tyr Asp Pro Asp Phe Ile Ala Ala Asn Gln Glu Ala
180 185 190
Arg Ala Asn Asn Val Ile Lys Gly Thr Lys Lys Glu Gln Val Glu Gln
195 200 205
Ile Val Lys Asp Ile Arg Glu Phe Lys Glu Lys Asn Lys Val Asp Lys
210 215 220
Ile Val Val Leu Trp Thr Ala Asn Thr Glu Arg Tyr Ser Asn Val Val
225 230 235 240
Val Gly Leu Asn Asp Thr Met Glu Asn Leu Leu Ala Ala Val Asp Arg
245 250 255
Asn Glu Ala Glu Ile Ser Pro Ser Thr Leu His Ala Ile Ala Cys Ile
260 265 270
Met Glu Asn Val Pro Phe Ile Asn Gly Ser Pro Gln Asn Thr Phe Val
275 280 285
Pro Gly Leu Ile Asp Leu Ala Ile Lys Arg Asn Thr Leu Ile Gly Gly
290 295 300
Asp Asp Phe Lys Ser Gly Gln Thr Lys Met Lys Ser Val Leu Val Asp
305 310 315 320
Phe Leu Val Gly Ala Gly Ile Lys Pro Thr Ser Ile Val Ser Tyr Asn
325 330 335
His Leu Gly Asn Asn Asp Gly Met Asn Leu Ser Ala Pro Gln Thr Phe
340 345 350
Arg Ser Lys Glu Ile Ser Lys Ser Asn Val Val Asp Asp Met Val Ala
355 360 365
Ser Asn Ala Ile Leu Tyr Glu Ser Gly Glu His Pro Asp His Val Val
370 375 380
Val Ile Lys Tyr Val Pro Tyr Val Gly Asp Ser Lys Arg Ala Met Asp
385 390 395 400
Glu Tyr Met Ser Glu Ile Phe Met Gly Gly Lys Ser Thr Ile Val Met
405 410 415
His Asn Thr Cys Glu Asp Ser Leu Leu Ala Ala Pro Ile Ile Leu Asp
420 425 430
Leu Val Leu Leu Ala Glu Leu Ser Thr Arg Ile Gln Leu Lys Ala Glu
435 440 445
Gly Glu Gly Lys Phe His Ser Phe His Pro Val Ala Thr Ile Leu Ser
450 455 460
Tyr Leu Thr Lys Ala Pro Leu Val Pro Pro Gly Thr Pro Val Val Asn
465 470 475 480
Ala Leu Ser Lys Gln Arg Ala Met Leu Glu Asn Ile Met Arg Ala Cys
485 490 495
Val Gly Leu Ala Pro Glu Asn Asn Met Ile Leu Glu Tyr Lys
500 505 510
<210> 5
<211> 317
<212> PRT
<213> Artificial sequence
<400> 5
Met Thr Ile Leu Ile Glu Gln Pro Glu Phe Gly Ser Gln Val Glu Glu
1 5 10 15
Lys Lys Val Ser Phe Asn Ala Asn Glu Leu Ile Leu Asp Gly Gly Phe
20 25 30
Met Val Pro Lys Thr Leu Ser Ser Gln Asp Glu Ile Phe Glu Val Pro
35 40 45
Asp Ile Asn Ala Phe Gly Gln Ser Phe Arg Asp Tyr Asn Val Glu Ser
50 55 60
Glu Arg Gln Lys Ser Val Glu Glu Phe Tyr Arg Val Gln His Ile Asn
65 70 75 80
Gln Thr Tyr Asp Tyr Val Lys Lys Met Arg Lys Glu Tyr Gly Lys Leu
85 90 95
Asn Lys Ile Glu Met Ser Ile Trp Asp Cys Cys Glu Leu Leu Asn Asp
100 105 110
Val Val Asp Asp Ser Asp Pro Asp Leu Asp Glu Pro Gln Ile Glu His
115 120 125
Leu Leu Gln Thr Ala Glu Ala Ile Arg Lys Asp Tyr Pro Asn Glu Asp
130 135 140
Trp Leu His Leu Thr Gly Leu Ile His Asp Leu Gly Lys Val Leu Leu
145 150 155 160
His Pro Ser Phe Gly Gly Leu Pro Gln Trp Ala Val Val Gly Asp Thr
165 170 175
Phe Pro Leu Gly Cys Ala Phe Asp Glu Ser Ile Val His His Lys Tyr
180 185 190
Phe Lys Glu Asn Pro Asp Ile Asn Asn Asn Ile Tyr Asn Thr Lys Asn
195 200 205
Gly Val Tyr Glu Glu Gly Cys Gly Leu Asp Lys Val Val Met Ser Trp
210 215 220
Gly His Asp Asp Tyr Met Tyr Leu Ile Ala Lys Glu Asn Lys Thr Thr
225 230 235 240
Leu Pro Ser Ala Ala Leu Phe Val Ile Arg Tyr His Ser Phe Tyr Ala
245 250 255
Leu His Arg Ser Gly Ala Tyr Thr His Leu Met Asn Glu Glu Asp Lys
260 265 270
Glu Asn Met Lys Trp Leu Asn Ile Phe Asn Lys Tyr Asp Leu Tyr Ser
275 280 285
Lys Ser Lys Val Arg Ile Asp Val Glu Lys Val Lys Pro Tyr Tyr Leu
290 295 300
Ser Leu Ile Glu Lys Tyr Phe Pro Thr Lys Leu Arg Trp
305 310 315
<210> 6
<211> 265
<212> PRT
<213> Artificial sequence
<400> 6
Met Lys Arg Leu Leu Val Thr Gly Ala Ala Gly Gln Leu Gly Arg Val
1 5 10 15
Met Arg Glu Arg Leu Ala Pro Met Ala Glu Ile Leu Arg Leu Ala Asp
20 25 30
Leu Ser Pro Leu Asp Pro Ala Gly Pro Asn Glu Glu Cys Val Gln Cys
35 40 45
Asp Leu Ala Asp Ala Asn Ala Val Asn Ala Met Val Ala Gly Cys Asp
50 55 60
Gly Ile Val His Leu Gly Gly Ile Ser Val Glu Lys Pro Phe Glu Gln
65 70 75 80
Ile Leu Gln Gly Asn Ile Ile Gly Leu His Asn Leu Tyr Glu Ala Ala
85 90 95
Arg Ala His Gly Gln Pro Arg Ile Val Phe Ala Ser Ser Asn His Thr
100 105 110
Ile Gly Tyr Tyr Pro Gln Thr Glu Arg Leu Gly Pro Asp Val Pro Ala
115 120 125
Arg Pro Asp Gly Leu Tyr Gly Val Ser Lys Cys Phe Gly Glu Asn Leu
130 135 140
Ala Arg Met Tyr Phe Asp Lys Phe Gly Gln Glu Thr Ala Leu Val Arg
145 150 155 160
Ile Gly Ser Cys Thr Pro Glu Pro Asn Asn Tyr Arg Met Leu Ser Thr
165 170 175
Trp Phe Ser His Asp Asp Phe Val Ser Leu Ile Glu Ala Val Phe Arg
180 185 190
Ala Pro Val Leu Gly Cys Pro Val Val Trp Gly Ala Ser Ala Asn Asp
195 200 205
Ala Gly Trp Trp Asp Asn Ser His Leu Gly Phe Leu Gly Trp Lys Pro
210 215 220
Glu Asp Asn Ala Glu Ala Phe Arg Arg His Ile Thr Glu Thr Thr Pro
225 230 235 240
Pro Pro Asp Pro Asn Asp Ala Leu Val Arg Phe Gln Gly Gly Thr Phe
245 250 255
Val Asp Asn Pro Ile Phe Lys Gln Ser
260 265
<210> 7
<211> 33
<212> PRT
<213> Artificial sequence
<400> 7
Cys Gly Cys Gly Gly Ala Thr Cys Cys Gly Ala Thr Gly Thr Thr Thr
1 5 10 15
Ala Thr Thr Gly Ala Ala Ala Ala Thr Thr Thr Thr Ala Ala Gly Gly
20 25 30
Thr
<210> 8
<211> 30
<212> PRT
<213> Artificial sequence
<400> 8
Gly Cys Gly Cys Thr Gly Cys Ala Gly Gly Ala Thr Thr Thr Gly Thr
1 5 10 15
Ala Thr Thr Cys Cys Ala Ala Ala Ala Thr Cys Ala Thr Gly
20 25 30
<210> 9
<211> 33
<212> PRT
<213> Artificial sequence
<400> 9
Cys Gly Gly Ala Thr Ala Thr Cys Gly Ala Thr Gly Ala Cys Thr Ala
1 5 10 15
Thr Thr Cys Thr Cys Ala Thr Thr Gly Ala Gly Cys Ala Gly Cys Cys
20 25 30
Thr
<210> 10
<211> 33
<212> PRT
<213> Artificial sequence
<400> 10
Cys Cys Gly Cys Thr Cys Gly Ala Gly Ala Cys Cys Ala Cys Cys Thr
1 5 10 15
Cys Ala Gly Cys Thr Thr Thr Gly Thr Thr Gly Gly Ala Ala Ala Ala
20 25 30
Thr
<210> 11
<211> 30
<212> PRT
<213> Artificial sequence
<400> 11
Cys Gly Cys Ala Ala Gly Cys Thr Thr Ala Thr Gly Ala Ala Ala Cys
1 5 10 15
Gly Gly Cys Thr Thr Cys Thr Thr Gly Thr Thr Ala Cys Cys
20 25 30
<210> 12
<211> 28
<212> PRT
<213> Artificial sequence
<400> 12
Cys Gly Cys Thr Cys Gly Ala Gly Cys Gly Gly Thr Gly Thr Cys Gly
1 5 10 15
Thr Cys Thr Cys Gly Gly Thr Thr Ala Thr Ala Thr
20 25

Claims (7)

1.一种重组大肠杆菌，其特征在于，所述重组大肠杆菌同时表达肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX和醛酸脱氢酶基因Udh；所述肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX、醛酸脱氢酶基因Udh表达的蛋白质的对应的氨基酸序列分别是SEQ IDNO. 4、SEQ ID NO. 5、SEQ ID NO. 6所示的序列。1. A recombinant *Escherichia coli*, characterized in that the recombinant *Escherichia coli* simultaneously expresses the inositol-1-phosphate synthase gene Ino1, the inositol oxidase gene MIOX, and the aldehyde dehydrogenase gene Udh; the amino acid sequences corresponding to the proteins expressed by the inositol-1-phosphate synthase gene Ino1, the inositol oxidase gene MIOX, and the aldehyde dehydrogenase gene Udh are the sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively. 2.根据权利要求1所述的重组大肠杆菌，其特征在于，所述肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX及醛酸脱氢酶基因Udh为整合表达。2. The recombinant Escherichia coli according to claim 1, characterized in that the inositol-1-phosphate synthase gene Ino1, the inositol oxidase gene MIOX, and the aldehyde dehydrogenase gene Udh are integrated expression. 3.一种权利要求1所述重组大肠杆菌的构建方法，其特征在于，所述方法包括如下步骤：3. A method for constructing recombinant Escherichia coli according to claim 1, characterized in that the method comprises the following steps: （1）将肌醇-1-磷酸合成酶基因Ino1和肌醇氧化酶基因MIOX连接到双元表达载体上转化到大肠杆宿主菌种，得到重组大肠杆菌1；(1) The inositol-1-phosphate synthase gene Ino1 and the inositol oxidase gene MIOX were ligated into a binary expression vector and transformed into Escherichia coli host strains to obtain recombinant Escherichia coli 1; （2）将醛酸脱氢酶基因Udh连接到表达载体后转化到步骤（1）得到的重组大肠杆菌1中，得重组大肠杆菌。(2) The aldehyde dehydrogenase gene Udh was ligated into the expression vector and then transformed into the recombinant Escherichia coli 1 obtained in step (1) to obtain recombinant Escherichia coli. 4.根据权利要求3所述的构建方法，其特征在于，所述方法具体包括如下步骤：4. The construction method according to claim 3, characterized in that the method specifically includes the following steps: （1）以番茄（Solanaceae Lycopersicon）1706基因组为模板扩增肌醇-1-磷酸合成酶基因Ino1、肌醇氧化酶基因MIOX，并分别加上合适酶切位点，将片段连接到双元表达载体pETDuet1后，用化学法转化到DH5α中，筛选获得正确的重组质粒，命名为pETDuet1-Ino1-MIOX，将重组质粒转化到宿主菌E.coli BL21(DE3)中即得到E.coli BL21(DE3)/Ino1-MIOX；(1) Using the genome of tomato ( Solanaceae Lycopersicon ) 1706 as a template, the inositol-1-phosphate synthase gene Ino1 and the inositol oxidase gene MIOX were amplified and appropriate restriction sites were added. The fragments were ligated into the binary expression vector pETDuet1 and chemically transformed into DH5α. The correct recombinant plasmid was obtained by screening and named pETDuet1-Ino1-MIOX. The recombinant plasmid was transformed into the host bacterium E.coli BL21(DE3) to obtain E.coli BL21(DE3)/Ino1-MIOX; （2）以根癌农杆菌（Agrobacterium tumefaciens）GV3103基因组为模板扩增醛酸脱氢酶基因Udh，连接到pET28a后，用化学法转化到步骤（1）构建的表达宿主E.coli BL21(DE3)/Ino1-MIOX中，筛选正确的重组大肠杆菌，命名为E.coli BL21(DE3)/Ino1-MIOX-Udh。(2) Using the Agrobacterium tumefaciens GV3103 genome as a template, the aldehyde dehydrogenase gene Udh was amplified, ligated into pET28a, and then chemically transformed into the expression host E.coli BL21(DE3)/Ino1-MIOX constructed in step (1). The correct recombinant E. coli was screened and named E.coli BL21(DE3)/Ino1-MIOX-Udh. 5.一种利用权利要求1所述的重组大肠杆菌生产葡萄糖二酸的方法，其特征在于，该方法是以葡萄糖或者肌醇为底物催化合成葡萄糖二酸。5. A method for producing gluconic acid using the recombinant Escherichia coli of claim 1, characterized in that the method uses glucose or inositol as a substrate to catalyze the synthesis of gluconic acid. 6.根据权利要求5所述的方法，其特征在于，所述方法具体是将重组大肠杆菌种子液以2% - 5%的接种量接种到发酵培养基中，于25℃ - 30℃，160 rpm - 200 rpm下，培养60-100 h。6. The method according to claim 5, wherein the method specifically involves inoculating the recombinant Escherichia coli seed culture into the fermentation medium at an inoculation rate of 2%-5%, and culturing it at 25℃-30℃ and 160 rpm-200 rpm for 60-100 h. 7.根据权利要求6所述的方法，其特征在于，所述发酵培养基的碳源为葡萄糖和粗甘油中的一种或两种。7. The method according to claim 6, wherein the carbon source of the fermentation medium is one or both of glucose and crude glycerol.
CN201710200371.8A 2017-03-30 2017-03-30 A recombinant Escherichia coli and its construction method and a method for producing gluconic acid via metabolic engineering Active CN106929459B (en)

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