CN112458108A

CN112458108A - Construction method of synthetic path for generating glutamic acid by utilizing xylose in corynebacterium glutamicum

Info

Publication number: CN112458108A
Application number: CN202011331840.8A
Authority: CN
Inventors: 鲍杰; 金慈
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2021-03-09

Abstract

The invention belongs to the field of genetic engineering, and discloses a method for constructing a synthetic path for generating glutamic acid by utilizing xylose in corynebacterium glutamicum, which comprises the following steps: (1) expressing a heterologous pentose transporter in corynebacterium glutamicum; (2) expressing heterologous xylose isomerase and xylulokinase in C.glutamicum; (3) the generated 5-xylulose phosphate can generate alpha-ketoglutarate through a pentose phosphate pathway and a glycolysis pathway, and alpha-ketoglutarate dehydrogenase is subjected to attenuation expression so that the alpha-ketoglutarate flows to the synthesis of glutamic acid more; (4) the glutamic acid secretory protein is modified so that the corynebacterium glutamicum can not respond to the external biotin pressure any more and continuously secrete glutamic acid to the outside of cells. The synthesis path from xylose to glutamic acid constructed by the invention enables corynebacterium glutamicum to produce glutamic acid by using xylose in a conventional synthesis medium, and can synthesize the glutamic acid from xylose in the environment of high biotin such as lignocellulose.

Description

Construction method of synthetic path for generating glutamic acid by utilizing xylose in corynebacterium glutamicum

Technical Field

The invention belongs to the field of genetic engineering, and relates to a construction method of a synthetic path for producing glutamic acid by utilizing xylose in corynebacterium glutamicum, an engineering strain for producing the glutamic acid by utilizing the xylose in a high-biotin environment and application thereof.

Background

Glutamic acid is an important industrial amino acid, is widely used in the fields of food, feed and the like, and is also used as a monomer for producing biological polymerization materials such as polyglutamic acid and the like. In 2013, the annual yield of glutamic acid has reached 300 ten thousand tons, which indicates that the demand is huge. At present, glutamic acid is mainly produced by a microbial fermentation method, but most of raw materials used by the method are grain crops such as starch and the like, so that the problem of 'competing for grain with people' is caused. Therefore, it is urgent to find more inexpensive and widely available raw materials. Lignocellulose has wide sources, large supply quantity and low price, and is the best choice as glutamic acid fermentation raw material.

Lignocellulose is mainly composed of cellulose, hemicellulose and lignin. The abundant glucose in cellulose can be utilized by most microorganisms, but the xylose from hemicellulose also accounts for 30% of the total sugar of lignocellulose, and the utilization of the xylose for fermentation production is a precondition for high-efficiency utilization of lignocellulose.

Corynebacterium glutamicum has a wide substrate production potential and is used for synthesizing various amino acids, organic acids, biofuels and other bio-based chemicals. However, wild Corynebacterium glutamicum cannot utilize xylose, so that metabolic engineering to utilize xylose in lignocellulose is a prerequisite for lignocellulose fermentation. Meanwhile, lignocellulose contains a large amount of biotin, and the biotin can promote the synthesis of the cell wall of the corynebacterium glutamicum so as to inhibit the secretion of glutamic acid, so that the problem that the corynebacterium glutamicum secretes the glutamic acid in a high-biotin environment is also a necessary step.

At present, no report on the construction of xylose path of corynebacterium glutamicum so as to utilize glucose and xylose in lignocellulose for producing glutamic acid is available, so that the establishment of a corynebacterium glutamicum construction method for producing glutamic acid by co-fermenting glucose and xylose in wheat straw hydrolysate is very necessary for the industrial production of lignocellulose glutamic acid.

Disclosure of Invention

The purpose of the present invention is to provide a method for constructing a synthetic pathway for producing glutamic acid from xylose in Corynebacterium glutamicum.

Another purpose of the invention is to construct an engineering strain capable of producing glutamic acid by using xylose in a high biotin environment.

The starting strain is Corynebacterium glutamicum Glutaminocus mS9114 which is purchased from Shanghai Industrial microorganism institute (SIIM, Shanghai, China, http:// www.gsysiim.com /), and the Collection number is SIIM B460, and the strain is also collected in China Industrial microorganism strain Collection management Center (China Center of Industrial Culture Collection, CICC, http:// www.china-cic, org /) and the Collection number is CICC 20935. The modified strain has not been deposited

The technical scheme adopted by the invention for realizing the construction of the synthetic route from xylose to glutamic acid in corynebacterium glutamicum is as follows: firstly, heterologous pentose transporters are expressed in the corynebacterium glutamicum to help the corynebacterium glutamicum to absorb xylose into cells; then expressing heterologous xylose isomerase and xylulokinase in corynebacterium glutamicum, isomerizing xylose into xylulose, and then phosphorylating to xylulose-5-phosphate; the generated xylulose-5-phosphate can generate alpha-ketoglutarate through a pentose phosphate pathway and a glycolysis pathway of the corynebacterium glutamicum, and the alpha-ketoglutarate dehydrogenase is subjected to attenuation expression, so that the alpha-ketoglutarate flows to the synthesis of glutamic acid more; finally, the glutamic acid secretory protein is modified, so that the corynebacterium glutamicum can not respond to the pressure of external biotin any more and can secrete the glutamic acid out of the cells continuously.

The specific method for gene knockout and gene integration is as follows: the constructed knock-out plasmid or integrative plasmid is electrically transformed into corynebacterium glutamicum, and then coated on a plate containing kanamycin resistance, pcr identification is carried out on the grown colony, and a primary recombinant strain is determined. Culturing the bacterial strain which is subjected to the primary recombination in an LB culture medium for overnight culture, coating the bacterial strain on a sucrose plate, carrying out pcr verification on the grown bacterial colony again through sucrose marking and screening, and carrying out gene sequencing to obtain the correct bacterial strain which is the successfully constructed bacterial strain.

The construction method of the knockout plasmid comprises the following steps: taking a corynebacterium glutamicum genome as a template, obtaining upstream and downstream homology arms of a target gene to be knocked out by PCR, and then respectively connecting the upstream and downstream homology arms to pK18mobsacB plasmids by enzyme digestion connection.

The method for constructing the synthetic route for generating the glutamic acid by utilizing the xylose in the corynebacterium glutamicum specifically comprises the following steps:

(1) the xylAB expression cassette is integrated at the ldhA gene locus of c.glutamicum;

(2) modification of glutamate secretory channel protein;

(3) attenuating the expression of alpha-ketoglutarate dehydrogenase;

(4) expression of the heterologous pentose transporter araE.

Further, the step (1) of integrating the xylAB expression cassette into the ldhA gene site of c. Firstly, constructing an integration plasmid of xylAB, then transferring the integration plasmid pK 18-delta ldhA of xylAB into C.glutamicum in an electrotransfer mode, and further screening out a strain which generates correct homologous recombination in a pcr verification mode to obtain recombinant corynebacterium glutamicum, which is named as C.glutamicum XyltoGA 01;

the step (2) is modification of glutamic acid secretory channel protein: firstly constructing a knockout plasmid with 330 bases at the carbon end of MscCG, then transferring the knockout plasmid pK 18-delta MscG 330 into C.glutamicum XyltoGA01 in an electric transfer mode, and screening out a strain which generates correct homologous recombination in a pcr verification mode to obtain recombinant corynebacterium glutamicum, which is named as C.glutamicum XyltoGA 02;

said step (3) attenuating the expression of alpha-ketoglutarate dehydrogenase: firstly, constructing an integrated plasmid which replaces an original RBS sequence of odhA and is an RBS sequence with the transcription initiation strength of 0.1, then transferring the knocked-out plasmid into C.glutamicum xyltotA 02 in an electric transfer mode, and screening a strain which generates correct homologous recombination in a pcr verification mode to obtain recombinant corynebacterium glutamicum, which is named as C.glutamicum xyltotA 03;

the step (4) expresses a heterologous pentose transporter araE: firstly, constructing an integration plasmid of araE, then transferring the integration plasmid pK 18-delta ack of araE into C.glutamicum xyltotGA 03 in an electrotransfer mode, further screening a strain which generates correct homologous recombination in a pcr verification mode, and finally obtaining the recombinant corynebacterium glutamicum which is named C.glutamicum xyltotGA 04.

According to the construction method of the synthetic route for producing glutamic acid by utilizing xylose in corynebacterium glutamicum, a recombinant strain C.glutamicum xyltotaGA 04 is cultured and fermented, the culture medium is a high-biotin-concentration culture medium containing 60g/L glucose and 40g/L xylose, the temperature is 32 ℃, the pH is controlled to be 7.2 by ammonia water, the ventilation amount is 1.4vvm, and the rotation speed is 600 rpm.

The invention also provides the corynebacterium glutamicum which is constructed by the construction method of the synthetic pathway for producing the glutamic acid by utilizing the xylose in the corynebacterium glutamicum and can highly produce the glutamic acid by utilizing the xylose in a high-biotin environment.

The invention also provides application of the corynebacterium glutamicum for producing glutamic acid with high yield by using xylose in the environment with high biotin in lignocellulose fermentation.

The invention relates to a method for constructing a synthetic route for generating glutamic acid by utilizing xylose in corynebacterium glutamicum, which more particularly comprises the following steps:

1: integration of xylAB expression cassette into ldhA Gene site of C

Firstly, constructing an integrative plasmid of xylAB, wherein the specific construction method comprises the following steps: using a genome of C.glutamcums as a template, and amplifying by using a Peftu-F (shown as a Peftu-F sequence in a sequence table) primer and a Peftu-R (shown as a Peftu-R sequence in the sequence table) primer through a PCR method to obtain a Peftu promoter (shown as a Peftu sequence in the sequence table); taking a genome of E.coli BL21 as a template, and amplifying by using an xylAB _ BL21-F (shown as a xylAB _ BL21-F sequence in a sequence table) primer and a xylAB _ BL21-R (shown as a xylAB _ BL21-R sequence in the sequence table) primer in a PCR (polymerase chain reaction) manner to obtain a xylAB _ BL21 (shown as a xylAB _ BL21 sequence in the sequence table) fragment; obtaining a Peftu _ xylAB _ BL21 fusion fragment by taking Peftu and xylAB _ BL21 as templates and using Peftu-F and xylAB _ BL21-R primers in a mode of overlapping extension PCR; using a genome of Glutamicum as a template, and using ldhA-up-F (shown as ldhA-up-F sequence in the sequence table) and ldhA-up-R (shown as ldhA-up-R sequence in the sequence table) primers, amplifying by a PCR method to obtain an ldhA-up fragment (shown as ldhA-up sequence in the sequence table); using the genome of Glutamicum as a template, and using ldhA-down-F (shown as ldhA-down-F sequence in the sequence table) and ldhA-down-R (shown as ldhA-down-R sequence in the sequence table) primers, an ldhA-down fragment (shown as ldhA-down sequence in the sequence table) is obtained by PCR amplification; a. DELTA. ldhA:. xylAB fusion fragment was obtained by overlap extension PCR using ldhA-up-F and ldhA-down-R primers using ldhA-up-BL 21 and ldhA-down as templates, and was treated with EcoRI and HindIII endonucleases and inserted into pK18mob plasmid using T4 ligase to obtain pK 18-. DELTA. ldhA:. xylAB plasmid. Then, the integrated plasmid pK 18-delta ldhA is transferred into C.glutamicum by electrotransfer, and a strain which generates correct homologous recombination is screened out by pcr verification to obtain the recombinant corynebacterium glutamicum, which is named as C.glutamicum XyltoGA 01.

2: modification of glutamate secretory channel proteins

Firstly constructing a knockout plasmid with 330 bases at the carbon end of MscCG, wherein the specific construction method comprises the following steps: using a genome of C.glutamicum as a template, and amplifying by using MscCG-up-F (shown as an MscCG-up-F sequence in a sequence table) and MscCG-up-R (shown as an MscCG-up-R sequence in the sequence table) primers through a PCR (polymerase chain reaction) method to obtain an MscCG-up fragment (shown as an MscCG-up sequence in the sequence table); using a genome of C.glutamcum as a template, and amplifying by using MscG-down-F (shown as an MscCG-down-F sequence in a sequence table) and MscCG-down-R (shown as an MscCG-down-R sequence in the sequence table) primers through a PCR (polymerase chain reaction) method to obtain an MscG-down fragment (shown as an MscCG-down sequence in the sequence table); using MscCG-up and MscCG-down as templates, using MscCG-up-F and MscCG-down-R primers, obtaining a delta MscCG330 fusion fragment in a mode of overlapping extension PCR, using EcoRI and HindIII endonucleases to process the delta MscCG330 fusion fragment, and using T4 ligase to insert the delta MscCG330 fusion fragment into a pK18mob plasmid to obtain a plasmid of 18-delta MscCG 330. And then transferring the knock-out plasmid pK 18-delta MscCG330 into C.glutamicum XyltoGA01 in an electrotransfer mode, and screening a strain which generates correct homologous recombination in a pcr verification mode to obtain the recombinant corynebacterium glutamicum, which is named as C.glutamicum XyltoGA 02.

3: attenuation of expression of alpha-ketoglutarate dehydrogenase

An integration plasmid in which the original RBS sequence replacing odhA was an RBS sequence having a transcription initiation strength of 0.1 was constructed, specifically, as follows: using genome of Glutamicum as a template, and amplifying an odhA-up fragment (shown as the odhA-up sequence in the sequence table) by using odhA-up-F (shown as the odhA-up-F sequence in the sequence table) and odhA-up-R (shown as the odhA-up-R sequence in the sequence table) primers through a PCR method; using genome of Glutamicum as template, using odhA-down-F (shown as odhA-down-F sequence in sequence table) and odhA-down-R (shown as odhA-down-R sequence in sequence table) primers to amplify by PCR to obtain odhA-down fragment (shown as odhA-down sequence in sequence table); an odhABS0.1 fusion fragment was obtained by overlap extension PCR using the odhA-up-F and odhA-down-R primers using the odhA-up and odhA-down as templates, and was treated with EcoRI and HindIII endonucleases, and then inserted into the pK18mob plasmid using T4 ligase to obtain the pK 18-odhABS0.1 plasmid. And then transferring the knock-out plasmid into C.glutamicum xyltotaGA 02 in an electric transfer mode, and screening out a strain which generates correct homologous recombination in a pcr verification mode to obtain the recombinant corynebacterium glutamicum, which is named as C.glutamicum xyltotaGA 03.

4: expression of heterologous pentose transporter araE

Firstly, constructing an araE integration plasmid, wherein the specific construction method comprises the following steps: synthesizing a PH36 promoter (shown as a PH36 sequence in a sequence table) by Czeri; taking the genome of E.coli BL21 as a template, and amplifying by using araE-F (shown as an araE-F sequence in a sequence table) and araE-R (shown as an araE-R sequence in the sequence table) primers in a PCR (polymerase chain reaction) mode to obtain an araE-up (shown as an araE-up sequence in the sequence table) fragment; taking PH36 and araE as templates, and obtaining a PH36_ araE fusion fragment by a mode of overlap extension PCR by using PH36-F and araE-R primers; taking the genome of E.coli BL21 as a template, and amplifying by using ack-up-F (shown as ack-up-F sequence in a sequence table) and ack-up-R (shown as ack-up-R sequence in the sequence table) primers in a PCR mode to obtain an ack-up (shown as ack-up sequence in the sequence table) fragment; using E.coli BL21 genome as template, and using ack-down-F (shown as ack-down-F sequence in sequence table) and ack-down-R (shown as ack-down-R sequence in sequence table) primers to amplify by PCR method to obtain ack-down fragment (shown as ack-down sequence in sequence table); the fusion fragment of araE was obtained by overlap extension PCR using ack-up-F and ack-down-R primers using ack-up-F and ack-down-R as templates, and was treated with EcoRI and HindIII endonucleases, and then inserted into pK18mob plasmid using T4 ligase to obtain pK 18-aaack. Then, the integrated plasmid pK 18-delta ack is transferred into C.glutamicum xyltotA 03 by electrotransfer, and a strain which generates correct homologous recombination is screened out by a pcr verification mode, and the obtained recombinant corynebacterium glutamicum is named as C.glutamicum xyltotA 04. The recombinant strain C.glutamicum xyltotA 04 was also cultured in the environment described in example 1 for fermentation, and it was found that the xylose utilization capacity of the engineered strain was greatly increased, and 40.7g/L xylose could be utilized in 48 hours (see FIG. 1).

The invention realizes that the corynebacterium glutamicum utilizes glucose and xylose to generate the glutamic acid together internationally for the first time in the environment of high biotin.

The xylose metabolic pathway construction and glutamic acid secretion pathway construction method adopted by the invention is not limited to the construction in corynebacterium glutamicum, and can also be applied to the construction of xylose metabolic pathways and glutamic acid secretion pathways of other microorganisms.

Drawings

FIG. 1: all metabolically engineered strains were compared to the fermentation performance (a xylose consumption, b glucose consumption and c glutamate production) of the starting strain.

FIG. 2: the fermentation performance (a glucose utilization, b xylose utilization and c glutamic acid production) of the recombinant strain was compared with that of the control strain in a lignocellulose hydrolysate.

FIG. 3: the invention constructs a synthetic pathway from xylose to glutamic acid.

Detailed description of the preferred embodiments

One, the strain used in the invention

Coli DH5 α was used for the construction of expression and knock-out plasmids, and e.coli bl21 was used to provide the e.coli-derived xylose utilization gene. Glutamicum S9114 was used mainly as the starting strain. Pediococcus acidilactici DSM20284 is used to provide a lactate-derived xylose utilization gene.

II, reagent and culture medium

PrimeSTARHSDNA polymerase was purchased from Takara bioengineering; restriction enzymes and T4DNAligase were purchased from Fermentas; the genome extraction kit is purchased from Tiangen company; the plasmid mini-pump kit, the PCR product recovery kit and the DNA gel recovery kit are purchased from Shanghai Czeri bioengineering GmbH; the Megazyme D-/L-Lactic acid Kit is purchased from Megazyme; other medicines and reagents are purchased from Shanghai Lingfeng Chemicals company or Shanghai national medicine chemical reagent group without special instructions; primer synthesis was performed by Shanghai Czeri bioengineering, Inc.

LB culture medium: 10g/L sodium chloride, 10g/L peptone, 5g/L yeast extract.

An LK medium: 10g/L sodium chloride, 10g/L peptone, 5g/L yeast extract, 50. mu.g/L kanamycin.

Limiting biotin medium: 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 0.5g/L corn steep liquor, and optionally 60g/L glucose or xylose as carbon source.

Biotin-rich medium: 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 25g/L corn steep liquor, and optionally 60g/L glucose or xylose as carbon source.

Example 1: integration of xylAB expression cassette into ldhA Gene site of C

Firstly, constructing an integrative plasmid of xylAB, wherein the specific construction method comprises the following steps: using a genome of C.glutamcums as a template, and amplifying by using a Peftu-F (shown as a Peftu-F sequence in a sequence table) primer and a Peftu-R (shown as a Peftu-R sequence in the sequence table) primer through a PCR method to obtain a Peftu promoter (shown as a Peftu sequence in the sequence table); taking a genome of E.coli BL21 as a template, and amplifying by using an xylAB _ BL21-F (shown as a xylAB _ BL21-F sequence in a sequence table) primer and a xylAB _ BL21-R (shown as a xylAB _ BL21-R sequence in the sequence table) primer in a PCR (polymerase chain reaction) manner to obtain a xylAB _ BL21 (shown as a xylAB _ BL21 sequence in the sequence table) fragment; obtaining a Peftu _ xylAB _ BL21 fusion fragment by taking Peftu and xylAB _ BL21 as templates and using Peftu-F and xylAB _ BL21-R primers in a mode of overlapping extension PCR; using a genome of Glutamicum as a template, and using ldhA-up-F (shown as ldhA-up-F sequence in the sequence table) and ldhA-up-R (shown as ldhA-up-R sequence in the sequence table) primers, amplifying by a PCR method to obtain an ldhA-up fragment (shown as ldhA-up sequence in the sequence table); using the genome of Glutamicum as a template, and using ldhA-down-F (shown as ldhA-down-F sequence in the sequence table) and ldhA-down-R (shown as ldhA-down-R sequence in the sequence table) primers, an ldhA-down fragment (shown as ldhA-down sequence in the sequence table) is obtained by PCR amplification; a. DELTA. ldhA:. xylAB fusion fragment was obtained by overlap extension PCR using ldhA-up-F and ldhA-down-R primers using ldhA-up-BL 21 and ldhA-down as templates, and was treated with EcoRI and HindIII endonucleases and inserted into pK18mob plasmid using T4 ligase to obtain pK 18-. DELTA. ldhA:. xylAB plasmid.

The specific system of the above PCR experiment is as follows: template 1. mu.L, upstream and downstream primers 1. mu.L each, deionized water 22. mu.L and primeSTARmix (TAKARA, available from Haja, Shanghai) 25. mu.L, total 50. mu.L.

The amplification procedure for the above PCR experiment was as follows: the first step is at 95 ℃ for 10 minutes; the second step, at 95 ℃, 30 seconds; step three, the temperature is 55 ℃ and 30 seconds; a fourth step of 72 ℃ for 1 minute (time is adjusted depending on the size of the amplified fragment, generally calculated as 1 kb/min); then 30 cycles are carried out from the second step to the fourth step; the fifth step is that the temperature is 72 ℃ and the time is 10 minutes; and step six, 12 ℃ without time limitation.

The specific system of the above restriction enzyme cleavage is as follows: 50 μ L of template, 20 μ L of deionized water, 20 μ L of digestion buffer, 5 μ L each of the two restriction enzymes (both digestion buffer and restriction enzymes are brand name Thermo Fisher, purchased from Shanghai, Czert), and 100 μ L total.

The specific processes of the above connection, transformation and screening are as follows: firstly, 3.5 mu L of plasmid and 4 mu L of gene fragment after enzyme digestion are added into a 1.5mL centrifuge tube, then 7.5 mu L of ligase T4(Thermo Fisher, purchased from Shanghai, Jersey company) is added, and the mixture is placed into a low-temperature water bath kettle at 16 ℃ for half an hour after being gently mixed; then adding all the connecting liquid into 100 mu L of escherichia coli competence, gently mixing, and standing in ice water for half an hour; then, the transformation liquid is placed in a water bath kettle at 42 ℃ for heat shock for 1 minute and 30 seconds, then 0.9mL of LB culture medium is added into the transformation liquid, and the transformation liquid is placed in a shaking table at 37 ℃ for incubation for 1 hour; after the incubation, 100. mu.L of the transformation solution was spread on LK plates containing kanamycin resistance and cultured overnight in a 37 ℃ incubator; selecting colonies growing on the plate in an LK medium, taking gene upstream and downstream primers as verification primers, and screening out positive clones by a colony PCR (the PCR system and the amplification program are operated as in example 1); inoculating the positive clone into an LK culture medium for overnight culture, and pumping the plasmid every other day to a sequencing company for sequencing, wherein the plasmid with a correct sequencing result is the plasmid which is successfully connected.

Then, the integrated plasmid pK 18-delta ldhA is transferred into C.glutamicum by electrotransfer, and the strain which has the correct homologous recombination is screened out by pcr verification (same as the operation of example 1) to obtain the recombinant Corynebacterium glutamicum, which is named C.glutamicum XyltoGA 01.

The specific process of the electrical transformation is as follows: firstly, 20 mu L of plasmid is added into 100 mu L of corynebacterium glutamicum infected state, and the mixture is placed in ice water for half an hour after being mixed softly; then, adding all the conversion solution into an electric shock cup, and carrying out electric shock under the conditions of 2000V voltage and 200 omega resistance by an electric converter; sucking out all the conversion solution after electric shock, placing the conversion solution into a 1.5mL centrifuge tube, adding 0.8mL LB culture medium, and thermally shocking the mixture for 6 minutes in a water bath kettle at 46 ℃; then placing the heat-shocked transformation liquid in a shaking table at 30 ℃ for incubation for 2 hours; after the incubation, 100. mu.L of the transformation solution was applied to LK plates and incubated in an incubator at 30 ℃ for 48 hours.

And (3) carrying out fermentation comparison on the recombinant strain C.glutamicum XyltoGA01 and the original strain C.glutamicum in a 3L fermentation tank, wherein the culture medium is a high-biotin-concentration culture medium containing 60g/L glucose and 40g/L xylose, the temperature is 32 ℃, the pH is controlled to be 7.2 by ammonia water, the ventilation capacity is 1.4vvm, and the rotation speed is 600 rpm. As a result, it was found that the original strain could not utilize xylose, only glucose, and could not produce glutamic acid, whereas the recombinant strain c.

Example 2: modification of glutamate secretory channel proteins

Firstly constructing a knockout plasmid with 330 bases at the carbon end of MscCG, wherein the specific construction method comprises the following steps: using genome of C.glutamicum as a template, and using MscCG-up-F (shown as MscCG-up-F sequence in a sequence table) and MscCG-up-R (shown as MscCG-up-R sequence in the sequence table) primers to amplify by a PCR method (same with the operation of example 1) to obtain an MscCG-up fragment (shown as MscCG-up sequence in the sequence table); using genome of C.glutamcum as template, and using MscG-down-F (shown as MscCG-down-F sequence in sequence table) and MscCG-down-R (shown as MscCG-down-R sequence in sequence table) primers to amplify by PCR method (same as example 1 operation) to obtain MscCG-down fragment (shown as MscCG-down sequence in sequence table); using MscG-up and MscG-down as templates, Δ MscG 330 fusion fragments were obtained by overlap extension PCR using MscCG-up-F and MscCG-down-R primers (same as in example 1), and were treated with EcoRI and HindIII endonucleases (same as in example 1), and then inserted into pK18mob plasmid using T4 ligase to obtain pK18- Δ MscG 330 plasmid (same as in example 1). Then, the knock-out plasmid pK18- Δ MscCG330 was transferred into C.glutamicum XyltoGA01 by electrotransfer (same operation as example 1), and a strain which underwent correct homologous recombination was selected by pcr verification (same operation as example 1), so as to obtain a recombinant Corynebacterium glutamicum, which was named C.glutamicum XyltoGA 02. Recombinant strain c. glutamicum xyltota ga02 was also cultured in the environment described in example 1 and fermented, and it was found that the engineered strain could produce glutamic acid using glucose and xylose, but the synthesis of glutamic acid was less and only 10.0g/L of glutamic acid could be produced (see fig. 1).

Example 3: attenuation of expression of alpha-ketoglutarate dehydrogenase

An integration plasmid in which the original RBS sequence replacing odhA was an RBS sequence having a transcription initiation strength of 0.1 was constructed, specifically, as follows: using the genome of Glutamicum as a template, and using odhA-up-F (shown as the odhA-up-F sequence in the sequence table) and odhA-up-R (shown as the odhA-up-R sequence in the sequence table) primers, an odhA-up fragment (shown as the odhA-up sequence in the sequence table) is obtained by PCR (same as the procedure in example 1); (ii) amplifying an odhA-down fragment (shown as the odhA-down sequence in the sequence listing) by a PCR method (the same as in example 1) using an odhA-down-F (shown as the odhA-down-F sequence in the sequence listing) and an odhA-down-R (shown as the odhA-down-R sequence in the sequence listing) primer using the genome of Glutamicum as a template; an odhABRBS0.1 fusion fragment was obtained by overlap extension PCR using the odhA-up-F and odhA-down-R primers as templates (same procedure as in example 1), and was treated with EcoRI and HindIII endonucleases (same procedure as in example 1), and inserted into a pK18mob plasmid using T4 ligase to obtain a pK 18-odhABS0.1 plasmid (same procedure as in example 1). Then, the knock-out plasmid was transferred to c.glutamicum xyltota ga02 by electrotransfer (same as in example 1), and a strain that underwent correct homologous recombination was selected by pcr verification (same as in example 1), to obtain a recombinant corynebacterium glutamicum, which was named c.glutamicum xyltota ga 03. The recombinant strain C.glutamicum xyltota 03 was also cultured in the environment described in case 1 for fermentation, and it was found that the glutamic acid yield of the modified strain was greatly increased to 31.3g/L, but the xylose utilization rate was slow, and only 26.3g/L of xylose could be utilized in 48 hours (see FIG. 1).

Example 4: expression of heterologous pentose transporter araE

Firstly, constructing an araE integration plasmid, wherein the specific construction method comprises the following steps: synthesizing a PH36 promoter (shown as a PH36 sequence in a sequence table) by Czeri; taking the genome of E.coli BL21 as a template, and amplifying by using araE-F (shown as an araE-F sequence in a sequence table) and araE-R (shown as an araE-R sequence in the sequence table) primers in a PCR mode (the same operation as the example 1) to obtain an araE-up (shown as an araE-up sequence in the sequence table) fragment; using PH36 and araE as templates, and using PH36-F and araE-R primers to obtain a PH36_ araE fusion fragment by a mode of overlap extension PCR (the same operation as the example 1); taking the genome of E.coli BL21 as a template, and amplifying ack-up (shown as ack-up-F sequence in the sequence table) fragments by using ack-up-F (shown as ack-up-F sequence in the sequence table) and ack-up-R (shown as ack-up-R sequence in the sequence table) primers in a PCR mode (same as the operation of the example 1); using E.coli BL21 genome as template, using ack-down-F (as shown in ack-down-F sequence in sequence table) and ack-down-R (as shown in ack-down-R sequence in sequence table) primers to amplify by PCR (same as example 1) to obtain ack-down fragment (as shown in ack-down sequence in sequence table); A.DELTA.ack:: araE fusion fragment was obtained by overlap extension PCR using ack-up, PH36_ araE and ack-down as templates (same procedure as in example 1) and EcoRI and HindIII endonucleases (same procedure as in example 1), and inserted into pK18mob plasmid using T4 ligase to obtain pK 18-DELTA.ack:: araE plasmid (same procedure as in example 1). Subsequently, the integrated plasmid pK18- Δ ack was transferred into C.glutamicum xyltotA 03 by electrotransfer (same as the procedure in example 1), and the strain with correct homologous recombination was selected by pcr verification (same as the procedure in example 1), and the resulting recombinant C.glutamicum xyltotA 04 was named. The recombinant strain C.glutamicum xyltotA 04 was also cultured in the environment described in example 1 for fermentation, and it was found that the xylose utilization capacity of the engineered strain was greatly increased, and 40.7g/L xylose could be utilized in 48 hours (see FIG. 1).

Example 5: comparison of fermentation Performance of recombinant strains and control strains in lignocellulose hydrolysate

The collected wheat straws are subjected to dry dilute acid pretreatment, biological detoxification and enzyme hydrolysis, and finally separated to obtain wheat straw hydrolysate containing high-concentration glucose and xylose. The engineered strain C.glutamicum xyltoGA04 for xylose utilization and glutamic acid secretion constructed by the invention and a control strain C.glutamicum WJB which can not utilize xylose are fermented and compared in a 3L fermentation tank, wherein a culture medium is a wheat straw hydrolysate containing 120g/L glucose and 40g/L xylose, the temperature is 32 ℃, the pH is controlled at 7.2 by ammonia water, the ventilation amount is 1.4vvm, and the rotation speed is 600 rpm. As a result, it was found that the control strain produced only 49.2g/L of glutamic acid using glucose, whereas the recombinant strain produced 61.7g/L of glutamic acid using glucose and xylose in common (see FIG. 2). The results show that the engineering strain which has the functions of co-fermenting glucose and xylose and can produce glutamic acid in a high-biotin environment is successfully constructed through metabolic engineering modification.

The above describes the operation example of the technical solution of the present invention in detail, and is not to be considered as limiting the application of the present invention. Equivalent substitutions of operating conditions are within the scope of the invention.

Sequence listing

<110> university of east China's college of science

<120> a method for constructing a synthetic pathway for producing glutamic acid using xylose in Corynebacterium glutamicum

<160> 42

<170> SIPOSequenceListing 1.0

<210> 1

<211> 40

<212> DNA

<213> PH36-F

<400> 1

ttgacagctt atcatcaaaa gctgggtacc tctatctggt 40

<210> 2

<211> 40

<212> DNA

<213> PH36-R

<400> 2

tttgcgatca aataatgaca tggatcccat gctactccta 40

<210> 3

<211> 95

<212> DNA

<213> PH36

<400> 3

caaaagctgg gtacctctat ctggtgccct aaacggggga atattaacgg gcccagggtg 60

gtcgcacctt ggttggtagg agtagcatgg gatcc 95

<210> 4

<211> 39

<212> DNA

<213> Peftu-F

<400> 4

ttgacagctt atcatcgaaa agcaatttgc ttttcgacg 39

<210> 5

<211> 39

<212> DNA

<213> Peftu-R

<400> 5

ggtcaaaata ggcttgcatt gtatgtcctc ctggacttc 39

<210> 6

<211> 335

<212> DNA

<213> Peftu

<400> 6

cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60

tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120

ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180

aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240

tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300

agtcgtagcc accacgaagt ccaggaggac ataca 335

<210> 7

<211> 39

<212> DNA

<213> xylAB_BL21-F

<400> 7

gaagtccagg aggacataca atgcaagcct attttgacc 39

<210> 8

<211> 40

<212> DNA

<213> xylAB_BL21-R

<400> 8

ctcgaattcc atggtttacg ccattaatgg cagaagttgc 40

<210> 9

<211> 2849

<212> DNA

<213> xylAB_BL21

<400> 9

atgcaagcct attttgacca gctcgatcgc gttcgttatg aaggctcaaa atcctcaaac 60

ccgttagcat tccgtcacta caatcccgac gaactggtgt tgggtaagcg tatggaagag 120

cacttgcgtt ttgccgcctg ctactggcac accttctgct ggaacggggc ggatatgttt 180

ggtgtggggg cgtttaatcg tccgtggcag cagcctggtg aggcactggc gttggcgaag 240

cgtaaagcag atgtcgcatt tgagtttttc cacaagttac atgtgccatt ttattgcttc 300

cacgatgtgg atgtttcccc tgagggcgcg tcgttaaaag agtacatcaa taattttgcg 360

caaatggttg atgtcctggc aggcaagcaa gaagagagcg gcgtgaagct gctgtgggga 420

accgccaact gctttacaaa ccctcgctac ggcgcgggtg cggcgacgaa cccagatcct 480

gaagtcttca gctgggcggc aacgcaagtt gttacagcga tggaagcaac ccataaattg 540

ggcggtgaaa actatgtcct gtggggcggt cgtgaaggtt acgaaacgct gttaaatacc 600

gacttgcgtc aggagcgtga acaactgggc cgctttatgc agatggtggt tgagcataaa 660

cataaaatcg gtttccaggg cacgttgctt atcgaaccga aaccgcaaga accgaccaaa 720

catcaatatg attacgatgc cgcgacggtc tatggcttcc tgaaacagtt tggtctggaa 780

aaagagatta aactgaacat tgaagctaac cacgcgacgc tggcaggtca ctctttccat 840

catgaaatag ccaccgccat tgcgcttggc ctgttcggtt ctgtcgacgc caaccgtggc 900

gatgcgcaac tgggctggga caccgaccag ttcccgaaca gtgtggaaga gaatgcgctg 960

gtgatgtatg aaattctcaa agcaggcggt ttcaccaccg gtggtctgaa cttcgatgcc 1020

aaagtacgtc gtcaaagtac tgataaatat gatctgtttt acggtcatat cggcgcgatg 1080

gatacgatgg cactggcgct gaaaattgca gcgcgcatga ttgaagatgg cgagctggat 1140

aaacgcatcg cgcagcgtta ttccggctgg aatagcgaat tgggccagca aatcctgaaa 1200

ggccaaatgt cactggcaga tttagccaaa tatgctcagg aacataattt gtctccggtg 1260

catcagagtg gtcgccagga gcaactggaa aatctggtaa atcattatct gttcgacaaa 1320

taacggctaa ctgtgcagtc cgttggcccg gttatcggta gcgataccgg gcattttttt 1380

aaggaacgat cgatatgtat atcgggatag atcttggcac ctcgggcgta aaagttattt 1440

tgctcaacga gcagggtgag gtggttgctt cgcaaacgga aaagctgacc gtttcgcgcc 1500

cgcatccact ctggtcggaa caagacccgg aacagtggtg gcaggcaact gatcgcgcaa 1560

tgaaagctct gggcgatcag cattctctgc aggacgttaa agcattgggt attgccggcc 1620

agatgcatgg agcaacctta ctggatgctc aacaacgggt attgcgccct gccattttgt 1680

ggaacgacgg gcgctgtgcg caagagtgca ctttgctgga agcgagagtt ccgcaatcac 1740

gagtgattac cggcaacctg atgatgcccg gatttactgc gcctaaattg ctatgggttc 1800

agcggcatga gccggagata ttccgtcaaa tcgacaaagt attattaccg aaagattact 1860

tgcgtctgcg tatgacgggg gagtttgcca gcgatatgtc tgacgcagct ggcaccatgt 1920

ggctggatgt cgcaaagcgt gactggagtg acgtcatgct gcaggcttgc gacttatctc 1980

gtgaccagat gcccgcatta tacgaaggca gcgaaattac tggtgctttg ttacctgaag 2040

ttgcgaaagc gtggggtatg gcgacggtgc cagttgtcgc aggcggtggc gacaatgcag 2100

ctggtgcagt tggtgtggga atggttgatg ctaatcaggc aatgttatcg ctggggacgt 2160

cgggggtcta ttttgctgtc agcgaagggt tcttaagcaa gccagaaagc gccgtacata 2220

gcttttgcca tgcgctaccg caacgttggc atttaatgtc tgtgatgctg agtgcagcgt 2280

cgtgtctgga ttgggccgcg aaattaaccg gcctgagcaa tgtcccagct ttaatcgctg 2340

cagctcaaca ggctgatgaa agtgccgagc cagtttggtt tctgccttat ctttccggcg 2400

agcgtacgcc acacaataat ccccaggcga agggggtttt ctttggtttg actcatcaac 2460

atggccccaa tgaactggcg cgagcagtgc tggaaggcgt gggttatgcg ctggcagatg 2520

gcatggatgt cgtgcatgcc tgcggtatta aaccgcaaag tgttacgttg attgggggcg 2580

gggcgcgtag tgagtactgg cgtcagatgc tggcggatat cagcggtcag cagctcgatt 2640

accgtacggg aggggatgtg gggccagcac tgggcgcagc aaggctggcg cagatcgcgg 2700

cgaatccaga gaaatcgctc attgaattgt tgccgcaact accgttagaa cagtcgcatc 2760

taccagatgc gcagcgttat gccgcttatc agccacgacg agaaacgttc cgtcgcctct 2820

atcagcaact tctgccatta atggcgtaa 2849

<210> 10

<211> 37

<212> DNA

<213> araE-F

<400> 10

aggagtagca tgggatccat ggttactatc aatacgg 37

<210> 11

<211> 42

<212> DNA

<213> araE-R

<400> 11

ggtaccgagc tcgaattcca tggttcagac gccgatattt ct 42

<210> 12

<211> 1419

<212> DNA

<213> araE

<400> 12

atggttacta tcaatacgga atctgcttta acgccacgtt ctttgcggga tacgcggcgt 60

atgaatatgt ttgtttcggt agctgctgcg gtcgcaggat tgttatttgg tcttgatatc 120

ggcgtaatcg ccggagcgtt gccgttcatt accgatcact ttgtgctgac cagtcgtttg 180

caggaatggg tggttagtag catgatgctc ggtgcagcaa ttggtgcgct gtttaatggt 240

tggctgtcgt tccgcctggg gcgtaaatac agcctgatgg cgggggccat cctgtttgta 300

ctcggttcta tagggtccgc ttttgcgacc agcgtagaga tgttaatcgc cgctcgtgtg 360

gtgctgggca ttgctgtcgg gatcgcgtct tacaccgctc ctctgtatct ttctgaaatg 420

gcaagtgaaa acgttcgcgg taagatgatc agtatgtacc agttgatggt cacactcggc 480

atcgtgctgg cgtttttatc cgatacagcg ttcagttata gcggtaactg gcgcgcaatg 540

ttgggggttc ttgctttacc agcagttctg ctgattattc tggtagtctt cctgccaaat 600

agcccgcgct ggctggcgga aaaggggcgt catattgagg cggaagaagt attgcgtatg 660

ctgcgcgata cgtcggaaaa agcgcgagaa gaactcaacg aaattcgtga aagcctgaag 720

ttaaaacagg gcggttgggc actgtttaag atcaaccgta acgtccgtcg tgctgtgttt 780

ctcggtatgt tgttgcaggc gatgcagcag tttaccggta tgaacatcat catgtactac 840

gcgccgcgta tcttcaaaat ggcgggcttt acgaccacag aacaacagat gattgcgact 900

ctggtcgtag ggctgacctt tatgttcgcc acctttattg cggtgtttac ggtagataaa 960

gcagggcgta aaccggctct gaaaattggt ttcagcgtga tggcgttagg cactctggtg 1020

ctgggctatt gcctgatgca gtttgataac ggtacggctt ccagtggctt gtcctggctc 1080

tctgttggca tgacgatgat gtgtattgcc ggttatgcga tgagcgccgc gccagtggtg 1140

tggatcctgt gctctgaaat tcagccgctg aaatgccgcg atttcggtat tacctgttcg 1200

accaccacga actgggtgtc gaatatgatt atcggcgcga ccttcctgac actgcttgat 1260

agcattggcg ctgccggtac gttctggctc tacactgcgc tgaacattgc gtttgtgggc 1320

attactttct ggctcattcc ggaaaccaaa aatgtcacgc tggaacatat cgaacgcaaa 1380

ctgatggcag gcgagaagtt gagaaatatc ggcgtctga 1419

<210> 13

<211> 30

<212> DNA

<213> ldhA-up-F

<400> 13

ccggaattcg gaacaccatg cgattaaggt 30

<210> 14

<211> 38

<212> DNA

<213> ldhA-up-R

<400> 14

cgaaaagcaa attgcttttc gtttcgatcc cacttcct 38

<210> 15

<211> 943

<212> DNA

<213> ldhA-up

<400> 15

ggaacaccat gcgattaagg tgcgctgctt gaattgcaga attatgcaag atgcgccgca 60

acaaaacgcg atcggccaag gtcaaagtgg tcaatgtaat gaccgaaacc gctgcgatga 120

aactaatcca cggcggtaaa aacctctcaa ttaggagctt gacctcatta atgctgtgct 180

gggttaattc gccggtgatc agcagcgcgc cgtaccccaa ggtgccgaca ctaatgcccg 240

cgatcgtctc cttcggtcca aaattcttct gcccaatcag ccggatttgg gtgcgatgcc 300

tgatcaatcc cacaaccgtg gtggtcaacg tgatggcacc agttgcgatg tgggtggcgt 360

tgtaaatttt cctggatacc cgccggttgg ttctggggag gatcgagtgg attcccgtcg 420

ctgacgcatg ccccaccgct tgtaaaacag ccaggttagc agccgtaacc caccacggtt 480

tcggcaacaa tgacggcgag agagcccacc acattgcgat ttccgctccg ataaagccag 540

cgcccatatt tgcagggagg attcgcctgc ggtttggcga cattcggatc cccggaacca 600

gctctgcaat cacctgcgcg ccgagggaag cgaggtgggt ggcaggtttt agtgcgggtt 660

taagcgttgc caggcgagtg gtgagcagag acgctagtct ggggagcgaa accatattga 720

gtcatcttgg cagagcatgc acaattctgc agggcataga ttggttttgc tcgatttaca 780

atgtgatttt ttcaacaaaa ataacacatg gtctgaccac attttcggac ataatcgggc 840

ataattaaag gtgtaacaaa ggaatccggg cacaagctct tgctgatttt ctgagctgct 900

ttgtgggttg tccggttagg gaaatcagga agtgggatcg aaa 943

<210> 16

<211> 39

<212> DNA

<213> ldhA-down-F

<400> 16

gccattaatg gcgtaaatct ttggcgccta gttggcgac 39

<210> 17

<211> 31

<212> DNA

<213> ldhA-down-R

<400> 17

cccaagcttg tctgggacgt tgatgacgct g 31

<210> 18

<211> 959

<212> DNA

<213> ldhA-down

<400> 18

atctttggcg cctagttggc gacgcaagtg tttcattgga acacttgcgc tgccaacttt 60

ttggtttacg ggcaaaatga aactgttgga tggaatttaa agtgtttgta gcttaaggag 120

ctcaaatgaa tgagtttgac caggacattc tccaggagat caagactgaa ctcgacgagt 180

taattctaga acttgatgag gtgacacaaa ctcacagcga ggccatcggg caggtctccc 240

caacccatta cgttggtgcc cgcaacctca tgcattacgc gcatcttcgc accaaagacc 300

tccgtggcct gcagcaacgc ctctcctctg tgggagctac ccgcttgact accaccgaac 360

cagcagtgca ggcccgcctc aaggccgccc gcaatgttat cggagctttc gcaggtgaag 420

gcccacttta tccaccctca gatgtcgtcg atgccttcga agatgccgat gagattctcg 480

acgagcacgc cgaaattctc cttggcgaac ccctaccgga tactccatcc tgcatcatgg 540

tcaccctgcc caccgaagcc gccaccgaca ttgaacttgt ccgtggcttc gccaaaagcg 600

gcatgaatct agctcgcatc aactgtgcac acgacgatga aaccgtctgg aagcagatga 660

tcgacaacgt ccacaccgtt gcagaagaag ttggccggga aatccgcgtc agcatggacc 720

ttgccggacc aaaagtacgc accggcgaaa tcgccccagg cgcagaagta ggtcgcgcac 780

gagtaacccg cgacgaaacc ggaaaagtac tgacgcccgc aaaactgtgg atcaccgccc 840

acggctccga accagtccca gcccccgaaa gcctgcccgg tcgccccgct ctgccgattg 900

aagtcacccc agaatggttc gacaaactag aaatcggcag cgtcatcaac gtcccagac 959

<210> 19

<211> 36

<212> DNA

<213> ack-up-F

<400> 19

ctagtctaga atggctgccc accagctgct tgatca 36

<210> 20

<211> 35

<212> DNA

<213> ack-up-R

<400> 20

atagaggtac ccagcttttg tagctgcgtc ctcct 35

<210> 21

<211> 893

<212> DNA

<213> ack-up

<400> 21

atggctgccc accagctgct tgatcaagac atctgtgaca tcacgatcct gggcgatcca 60

gtaaagatca aggagcgcgc taccgaactt ggcctgcacc ttaacactgc atacctggtc 120

aatccgctga cagatcctcg cctggaggaa ttcgccgaac aattcgcgga gctgcgcaag 180

tcaaagggcg tcactatcga tgaagcccgc gaaatcatga aggatatttc ctacttcggc 240

accatgatgg tccacaacgg cgacgccgac ggaatggtat ccggtgcagc aaacaccacc 300

gcacacacca ttaagccaag cttccagatc atcaaaactg ttccagaagc atccgtcgtt 360

tcttccatct tcctcatggt gctgcgcggg cgactgtggg cattcggcga ctgtgctgtt 420

aacccgaacc caactgctga acagcttggt gaaatcgccg ttgtgtcagc aaaaactgca 480

gcacaatttg gcattgatcc tcgcgtagcc atcttgtcct actccactgg caactccggc 540

ggaggctcag atgtggatcg cgccattgac gctcttgcag aagcacgccg actcaaccca 600

gaactatgcg tcgatggacc acttcagttc gacgccgccg tcgacccggg tgtggcgcgc 660

aagaagatgc cagactctga cgtcgctggc caggcaaatg tgtttatctt ccctgacctg 720

gaggccggaa acatcggcta caaaactgca caacgcaccg gtcacgccct ggcagttggt 780

ccgattctgc agggcctgaa caaaccagtc aacgaccttt cccgtggtgc gacagtccct 840

gacatcgtca acactgtcgc catcaccgct attcaggcag gaggacgcag cta 893

<210> 22

<211> 40

<212> DNA

<213> ack-down-F

<400> 22

gttgagaaat atcggcgtct gaggccccta gacctcttgg 40

<210> 23

<211> 30

<212> DNA

<213> ack-down-R

<400> 23

acatgcatgc ttgtctgggc aaccaacccc 30

<210> 24

<211> 793

<212> DNA

<213> ack=down

<400> 24

ggcccctaga cctcttgggg ttgcgaattt tcgtccccac cgaacattaa aaggccggtt 60

ttggtcgaaa atttgctcta acaccttgct attatgcaaa tcttcgttcg atttaagcaa 120

atcccggcaa tctgtaatga gaagttgaac gggaaaccta cagtaacccc gcagaaatca 180

catcagcccc aattgtccca aaagtaacgc ccccggaatc gcttctaagg gcctaactcg 240

cccaaagcca aactagttgg acactggagc cacaaaggcc cttaaatcgc cacctaccaa 300

atagccccaa gccaaaacag ctagaaccaa ctcagtggcc gcaccgcatt cgccatatcc 360

actagtgcgt aacggtggtg ggggaaagga gcggaacggg cctgaatccg gagtgcctcg 420

gaaatgccgg tgcgcaggcc tttttgggag aacgggtatt caaacaaagg gtttgcggag 480

gcagaagctt tgagttttcg ttctcgaagc cagctgaggc ctgctgacat gatggcaatt 540

ttgatttggt tgaagcgggg ttcgttggtg gggatttcgc tgagtcggcg ggcggctcgg 600

cggatgcggg attcactcaa attggagctg accagcaaca agatggtggt gagggtggcc 660

attcggtagt gggtggatga ttgggggagt ttatctaggg cttggactgc gagttcgatt 720

tggttttcgg ccatgagttg gcgggcgagc ccgaacgcgg aggacacagt ggtggggttg 780

gttgcccaga caa 793

<210> 25

<211> 3184

<212> DNA

<213> Peftu_xylAB_BL21

<400> 25

cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60

tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120

ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180

aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240

tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300

agtcgtagcc accacgaagt ccaggaggac atacaatgca agcctatttt gaccagctcg 360

atcgcgttcg ttatgaaggc tcaaaatcct caaacccgtt agcattccgt cactacaatc 420

ccgacgaact ggtgttgggt aagcgtatgg aagagcactt gcgttttgcc gcctgctact 480

ggcacacctt ctgctggaac ggggcggata tgtttggtgt gggggcgttt aatcgtccgt 540

ggcagcagcc tggtgaggca ctggcgttgg cgaagcgtaa agcagatgtc gcatttgagt 600

ttttccacaa gttacatgtg ccattttatt gcttccacga tgtggatgtt tcccctgagg 660

gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 720

agcaagaaga gagcggcgtg aagctgctgt ggggaaccgc caactgcttt acaaaccctc 780

gctacggcgc gggtgcggcg acgaacccag atcctgaagt cttcagctgg gcggcaacgc 840

aagttgttac agcgatggaa gcaacccata aattgggcgg tgaaaactat gtcctgtggg 900

gcggtcgtga aggttacgaa acgctgttaa ataccgactt gcgtcaggag cgtgaacaac 960

tgggccgctt tatgcagatg gtggttgagc ataaacataa aatcggtttc cagggcacgt 1020

tgcttatcga accgaaaccg caagaaccga ccaaacatca atatgattac gatgccgcga 1080

cggtctatgg cttcctgaaa cagtttggtc tggaaaaaga gattaaactg aacattgaag 1140

ctaaccacgc gacgctggca ggtcactctt tccatcatga aatagccacc gccattgcgc 1200

ttggcctgtt cggttctgtc gacgccaacc gtggcgatgc gcaactgggc tgggacaccg 1260

accagttccc gaacagtgtg gaagagaatg cgctggtgat gtatgaaatt ctcaaagcag 1320

gcggtttcac caccggtggt ctgaacttcg atgccaaagt acgtcgtcaa agtactgata 1380

aatatgatct gttttacggt catatcggcg cgatggatac gatggcactg gcgctgaaaa 1440

ttgcagcgcg catgattgaa gatggcgagc tggataaacg catcgcgcag cgttattccg 1500

gctggaatag cgaattgggc cagcaaatcc tgaaaggcca aatgtcactg gcagatttag 1560

ccaaatatgc tcaggaacat aatttgtctc cggtgcatca gagtggtcgc caggagcaac 1620

tggaaaatct ggtaaatcat tatctgttcg acaaataacg gctaactgtg cagtccgttg 1680

gcccggttat cggtagcgat accgggcatt tttttaagga acgatcgata tgtatatcgg 1740

gatagatctt ggcacctcgg gcgtaaaagt tattttgctc aacgagcagg gtgaggtggt 1800

tgcttcgcaa acggaaaagc tgaccgtttc gcgcccgcat ccactctggt cggaacaaga 1860

cccggaacag tggtggcagg caactgatcg cgcaatgaaa gctctgggcg atcagcattc 1920

tctgcaggac gttaaagcat tgggtattgc cggccagatg catggagcaa ccttactgga 1980

tgctcaacaa cgggtattgc gccctgccat tttgtggaac gacgggcgct gtgcgcaaga 2040

gtgcactttg ctggaagcga gagttccgca atcacgagtg attaccggca acctgatgat 2100

gcccggattt actgcgccta aattgctatg ggttcagcgg catgagccgg agatattccg 2160

tcaaatcgac aaagtattat taccgaaaga ttacttgcgt ctgcgtatga cgggggagtt 2220

tgccagcgat atgtctgacg cagctggcac catgtggctg gatgtcgcaa agcgtgactg 2280

gagtgacgtc atgctgcagg cttgcgactt atctcgtgac cagatgcccg cattatacga 2340

aggcagcgaa attactggtg ctttgttacc tgaagttgcg aaagcgtggg gtatggcgac 2400

ggtgccagtt gtcgcaggcg gtggcgacaa tgcagctggt gcagttggtg tgggaatggt 2460

tgatgctaat caggcaatgt tatcgctggg gacgtcgggg gtctattttg ctgtcagcga 2520

agggttctta agcaagccag aaagcgccgt acatagcttt tgccatgcgc taccgcaacg 2580

ttggcattta atgtctgtga tgctgagtgc agcgtcgtgt ctggattggg ccgcgaaatt 2640

aaccggcctg agcaatgtcc cagctttaat cgctgcagct caacaggctg atgaaagtgc 2700

cgagccagtt tggtttctgc cttatctttc cggcgagcgt acgccacaca ataatcccca 2760

ggcgaagggg gttttctttg gtttgactca tcaacatggc cccaatgaac tggcgcgagc 2820

agtgctggaa ggcgtgggtt atgcgctggc agatggcatg gatgtcgtgc atgcctgcgg 2880

tattaaaccg caaagtgtta cgttgattgg gggcggggcg cgtagtgagt actggcgtca 2940

gatgctggcg gatatcagcg gtcagcagct cgattaccgt acgggagggg atgtggggcc 3000

agcactgggc gcagcaaggc tggcgcagat cgcggcgaat ccagagaaat cgctcattga 3060

attgttgccg caactaccgt tagaacagtc gcatctacca gatgcgcagc gttatgccgc 3120

ttatcagcca cgacgagaaa cgttccgtcg cctctatcag caacttctgc cattaatggc 3180

gtaa 3184

<210> 26

<211> 5086

<212> DNA

<213> ΔldhA::xylAB

<400> 26

ggaacaccat gcgattaagg tgcgctgctt gaattgcaga attatgcaag atgcgccgca 60

acaaaacgcg atcggccaag gtcaaagtgg tcaatgtaat gaccgaaacc gctgcgatga 120

aactaatcca cggcggtaaa aacctctcaa ttaggagctt gacctcatta atgctgtgct 180

gggttaattc gccggtgatc agcagcgcgc cgtaccccaa ggtgccgaca ctaatgcccg 240

cgatcgtctc cttcggtcca aaattcttct gcccaatcag ccggatttgg gtgcgatgcc 300

tgatcaatcc cacaaccgtg gtggtcaacg tgatggcacc agttgcgatg tgggtggcgt 360

tgtaaatttt cctggatacc cgccggttgg ttctggggag gatcgagtgg attcccgtcg 420

ctgacgcatg ccccaccgct tgtaaaacag ccaggttagc agccgtaacc caccacggtt 480

tcggcaacaa tgacggcgag agagcccacc acattgcgat ttccgctccg ataaagccag 540

cgcccatatt tgcagggagg attcgcctgc ggtttggcga cattcggatc cccggaacca 600

gctctgcaat cacctgcgcg ccgagggaag cgaggtgggt ggcaggtttt agtgcgggtt 660

taagcgttgc caggcgagtg gtgagcagag acgctagtct ggggagcgaa accatattga 720

gtcatcttgg cagagcatgc acaattctgc agggcataga ttggttttgc tcgatttaca 780

atgtgatttt ttcaacaaaa ataacacatg gtctgaccac attttcggac ataatcgggc 840

ataattaaag gtgtaacaaa ggaatccggg cacaagctct tgctgatttt ctgagctgct 900

ttgtgggttg tccggttagg gaaatcagga agtgggatcg aaacgaaaag caatttgctt 960

ttcgacgccc caccccgcgc gttttagcgt gtcagtaggc gcgtagggta agtggggtag 1020

cggcttgtta gatatcttga aatcggcttt caacagcatt gatttcgatg tatttagctg 1080

gccgttaccc tgcgaatgtc cacagggtag ctggtagttt gaaaatcaac gccgttgccc 1140

ttaggattca gtaactggca cattttgtaa tgcgctagat ctgtgtgctc agtcttccag 1200

gctgcttatc acagtgaaag caaaaccaat tcgtggctgc gaaagtcgta gccaccacga 1260

agtccaggag gacatacaat gcaagcctat tttgaccagc tcgatcgcgt tcgttatgaa 1320

ggctcaaaat cctcaaaccc gttagcattc cgtcactaca atcccgacga actggtgttg 1380

ggtaagcgta tggaagagca cttgcgtttt gccgcctgct actggcacac cttctgctgg 1440

aacggggcgg atatgtttgg tgtgggggcg tttaatcgtc cgtggcagca gcctggtgag 1500

gcactggcgt tggcgaagcg taaagcagat gtcgcatttg agtttttcca caagttacat 1560

gtgccatttt attgcttcca cgatgtggat gtttcccctg agggcgcgtc gttaaaagag 1620

tacatcaata attttgcgca aatggttgat gtcctggcag gcaagcaaga agagagcggc 1680

gtgaagctgc tgtggggaac cgccaactgc tttacaaacc ctcgctacgg cgcgggtgcg 1740

gcgacgaacc cagatcctga agtcttcagc tgggcggcaa cgcaagttgt tacagcgatg 1800

gaagcaaccc ataaattggg cggtgaaaac tatgtcctgt ggggcggtcg tgaaggttac 1860

gaaacgctgt taaataccga cttgcgtcag gagcgtgaac aactgggccg ctttatgcag 1920

atggtggttg agcataaaca taaaatcggt ttccagggca cgttgcttat cgaaccgaaa 1980

ccgcaagaac cgaccaaaca tcaatatgat tacgatgccg cgacggtcta tggcttcctg 2040

aaacagtttg gtctggaaaa agagattaaa ctgaacattg aagctaacca cgcgacgctg 2100

gcaggtcact ctttccatca tgaaatagcc accgccattg cgcttggcct gttcggttct 2160

gtcgacgcca accgtggcga tgcgcaactg ggctgggaca ccgaccagtt cccgaacagt 2220

gtggaagaga atgcgctggt gatgtatgaa attctcaaag caggcggttt caccaccggt 2280

ggtctgaact tcgatgccaa agtacgtcgt caaagtactg ataaatatga tctgttttac 2340

ggtcatatcg gcgcgatgga tacgatggca ctggcgctga aaattgcagc gcgcatgatt 2400

gaagatggcg agctggataa acgcatcgcg cagcgttatt ccggctggaa tagcgaattg 2460

ggccagcaaa tcctgaaagg ccaaatgtca ctggcagatt tagccaaata tgctcaggaa 2520

cataatttgt ctccggtgca tcagagtggt cgccaggagc aactggaaaa tctggtaaat 2580

cattatctgt tcgacaaata acggctaact gtgcagtccg ttggcccggt tatcggtagc 2640

gataccgggc atttttttaa ggaacgatcg atatgtatat cgggatagat cttggcacct 2700

cgggcgtaaa agttattttg ctcaacgagc agggtgaggt ggttgcttcg caaacggaaa 2760

agctgaccgt ttcgcgcccg catccactct ggtcggaaca agacccggaa cagtggtggc 2820

aggcaactga tcgcgcaatg aaagctctgg gcgatcagca ttctctgcag gacgttaaag 2880

cattgggtat tgccggccag atgcatggag caaccttact ggatgctcaa caacgggtat 2940

tgcgccctgc cattttgtgg aacgacgggc gctgtgcgca agagtgcact ttgctggaag 3000

cgagagttcc gcaatcacga gtgattaccg gcaacctgat gatgcccgga tttactgcgc 3060

ctaaattgct atgggttcag cggcatgagc cggagatatt ccgtcaaatc gacaaagtat 3120

tattaccgaa agattacttg cgtctgcgta tgacggggga gtttgccagc gatatgtctg 3180

acgcagctgg caccatgtgg ctggatgtcg caaagcgtga ctggagtgac gtcatgctgc 3240

aggcttgcga cttatctcgt gaccagatgc ccgcattata cgaaggcagc gaaattactg 3300

gtgctttgtt acctgaagtt gcgaaagcgt ggggtatggc gacggtgcca gttgtcgcag 3360

gcggtggcga caatgcagct ggtgcagttg gtgtgggaat ggttgatgct aatcaggcaa 3420

tgttatcgct ggggacgtcg ggggtctatt ttgctgtcag cgaagggttc ttaagcaagc 3480

cagaaagcgc cgtacatagc ttttgccatg cgctaccgca acgttggcat ttaatgtctg 3540

tgatgctgag tgcagcgtcg tgtctggatt gggccgcgaa attaaccggc ctgagcaatg 3600

tcccagcttt aatcgctgca gctcaacagg ctgatgaaag tgccgagcca gtttggtttc 3660

tgccttatct ttccggcgag cgtacgccac acaataatcc ccaggcgaag ggggttttct 3720

ttggtttgac tcatcaacat ggccccaatg aactggcgcg agcagtgctg gaaggcgtgg 3780

gttatgcgct ggcagatggc atggatgtcg tgcatgcctg cggtattaaa ccgcaaagtg 3840

ttacgttgat tgggggcggg gcgcgtagtg agtactggcg tcagatgctg gcggatatca 3900

gcggtcagca gctcgattac cgtacgggag gggatgtggg gccagcactg ggcgcagcaa 3960

ggctggcgca gatcgcggcg aatccagaga aatcgctcat tgaattgttg ccgcaactac 4020

cgttagaaca gtcgcatcta ccagatgcgc agcgttatgc cgcttatcag ccacgacgag 4080

aaacgttccg tcgcctctat cagcaacttc tgccattaat ggcgtaaatc tttggcgcct 4140

agttggcgac gcaagtgttt cattggaaca cttgcgctgc caactttttg gtttacgggc 4200

aaaatgaaac tgttggatgg aatttaaagt gtttgtagct taaggagctc aaatgaatga 4260

gtttgaccag gacattctcc aggagatcaa gactgaactc gacgagttaa ttctagaact 4320

tgatgaggtg acacaaactc acagcgaggc catcgggcag gtctccccaa cccattacgt 4380

tggtgcccgc aacctcatgc attacgcgca tcttcgcacc aaagacctcc gtggcctgca 4440

gcaacgcctc tcctctgtgg gagctacccg cttgactacc accgaaccag cagtgcaggc 4500

ccgcctcaag gccgcccgca atgttatcgg agctttcgca ggtgaaggcc cactttatcc 4560

accctcagat gtcgtcgatg ccttcgaaga tgccgatgag attctcgacg agcacgccga 4620

aattctcctt ggcgaacccc taccggatac tccatcctgc atcatggtca ccctgcccac 4680

cgaagccgcc accgacattg aacttgtccg tggcttcgcc aaaagcggca tgaatctagc 4740

tcgcatcaac tgtgcacacg acgatgaaac cgtctggaag cagatgatcg acaacgtcca 4800

caccgttgca gaagaagttg gccgggaaat ccgcgtcagc atggaccttg ccggaccaaa 4860

agtacgcacc ggcgaaatcg ccccaggcgc agaagtaggt cgcgcacgag taacccgcga 4920

cgaaaccgga aaagtactga cgcccgcaaa actgtggatc accgcccacg gctccgaacc 4980

agtcccagcc cccgaaagcc tgcccggtcg ccccgctctg ccgattgaag tcaccccaga 5040

atggttcgac aaactagaaa tcggcagcgt catcaacgtc ccagac 5086

<210> 27

<211> 26

<212> DNA

<213> MscCG-up-F

<400> 27

gatccgcgcg ggcgctgcga ttccgg 26

<210> 28

<211> 22

<212> DNA

<213> MscCG-up-R

<400> 28

ttccacagtc atgaccttaa at 22

<210> 29

<211> 997

<212> DNA

<213> MscCG-up

<400> 29

gatccgcgcg ggcgctgcga ttccggcaac cattgcgtca gctgccattg gtcttggtgc 60

gcagtcgatt gttgcggact tcttggccgg atttttcatc ctgacggaaa agcaattcgg 120

cgtgggtgac tgggtgcgtt ttgagggcaa cggcatcgtt gttgaaggca cagtcattga 180

gatcaccatg cgcgcgacca aaattcgcac gattgcacaa gagaccgtga ttatccccaa 240

ctccacggcg aaagtgtgca tcaacaattc taataactgg tcgcgtgcgg ttgtcgttat 300

tccgatcccc atgttgggtt ctgaaaacat cacagatgtc atcgcgcgct ctgaagctgc 360

gactcgtcgc gcacttggcc aggagaaaat cgcgccggaa attctcggtg aactcgatgt 420

gcacccagcc acggaagtca cgccgccaac ggtggtcggc atgccgtgga tggtaaccat 480

gcgtttcctc gtgcaagtca ccgccggcaa tcaatggctg gtcgaacgcg ccatccgcac 540

ggaaatcatc agcgaattct gggaagaata cggcagcgca accactacat cgggaaccct 600

cattgattcc ttacacgttg cgcatgaaga gccaaagacc tcgcttatcg acgcctcccc 660

ccaggctctt aaggaaccga agccggaggc tgcggcgacg gttgcatcgc tagctgcatc 720

gtctaacgac gatgcagaca atgcagacga ctcggtgatc aatgcaggca atccaaagaa 780

ggaacttgat tccgatgtgc tggaacaaga actctccagc gaagaaccgg aagaaacagc 840

aaaaccagat cactctctcc gaggcttctt ccgcactgat tactacccaa atcggtggca 900

aaagatcctg tcgtttggcg gacgtgtccg catgagcact tccctgttgt tgggtgcgct 960

gctcttgctg tcactattta aggtcatgac tgtggaa 997

<210> 30

<211> 22

<212> DNA

<213> MscCG-down-F

<400> 30

ctagagcaga cgctgattac ag 22

<210> 31

<211> 25

<212> DNA

<213> MscCG-down-R

<400> 31

tgggtgcatc tgccacaata tcgcc 25

<210> 32

<211> 1012

<212> DNA

<213> MscCG-down

<400> 32

ctagagcaga cgctgattac agacgtgtcc catttcttta ctactattgg aaattatgag 60

ttcagacgca gaaaaggcat ccgtggagct ttccgaaaaa tttcacccag aacgcaccca 120

tattttgggc gccgttgttt ttggcctgat ctcattatta gtcatcggcg cagcccctca 180

gtacctgttt tggctgctcg cactccctgt catcttcggt tactgggttc taaaatcatc 240

cacgatcgtt gatgaacagg gcatcaccgc aaactacgcc ttcaagggca aaaaggttgt 300

ggcctgggaa aacctcgcag gaatcggatt caagggtgcc cgcactttcg ctcgcaccac 360

ctctgatgca gaagtcaccc tccccggcgt caccttcaac tcccttcccc gccttgaagc 420

tgcttcccac ggccgcatcc ccgatgcgat caccgcaagc aaggaagcag ccgacggcaa 480

ggttgtagtc gttcaagaag acggctactc cgtgatgatg tccaaggaag agtacttgga 540

gcgccaaaag gcactgggca agccagttca gttgagcttc gatgacgaca ccgatgggaa 600

tacaacacaa acagaaagcg ttgaatccca agagaccgga caagccgcgt ctgaaacctc 660

acatcgtgat aaccctgcgt cacagcacta gagtgtaata agccgtccga accaaaggtc 720

cacacctctg cacgagtaga agctcaccca agttttcaaa gtgccgttga ttcttgacac 780

ccccccgccg ctccttagag cagatttgaa aagcgcatca tgatcccact tcgttcaaaa 840

gtcaccaccg tcggtcgcaa tgcagctggc gctcgcgccc tttggcgtgc caccggcacc 900

aaggaaaatg agttcggcaa gccaattgtt gccatcgtga actcctacac ccagttcgtg 960

cccggacacg ttcaccttaa gaacgtcggc gatattgtgg cagatgcacc ca 1012

<210> 33

<211> 2013

<212> DNA

<213> ΔMscCG330

<400> 33

gatccgcgcg ggcgctgcga ttccggcaac cattgcgtca gctgccattg gtcttggtgc 60

gcagtcgatt gttgcggact tcttggccgg atttttcatc ctgacggaaa agcaattcgg 120

cgtgggtgac tgggtgcgtt ttgagggcaa cggcatcgtt gttgaaggca cagtcattga 180

gatcaccatg cgcgcgacca aaattcgcac gattgcacaa gagaccgtga ttatccccaa 240

ctccacggcg aaagtgtgca tcaacaattc taataactgg tcgcgtgcgg ttgtcgttat 300

tccgatcccc atgttgggtt ctgaaaacat cacagatgtc atcgcgcgct ctgaagctgc 360

gactcgtcgc gcacttggcc aggagaaaat cgcgccggaa attctcggtg aactcgatgt 420

gcacccagcc acggaagtca cgccgccaac ggtggtcggc atgccgtgga tggtaaccat 480

gcgtttcctc gtgcaagtca ccgccggcaa tcaatggctg gtcgaacgcg ccatccgcac 540

ggaaatcatc agcgaattct gggaagaata cggcagcgca accactacat cgggaaccct 600

cattgattcc ttacacgttg cgcatgaaga gccaaagacc tcgcttatcg acgcctcccc 660

ccaggctctt aaggaaccga agccggaggc tgcggcgacg gttgcatcgc tagctgcatc 720

gtctaacgac gatgcagaca atgcagacga ctcggtgatc aatgcaggca atccaaagaa 780

ggaacttgat tccgatgtgc tggaacaaga actctccagc gaagaaccgg aagaaacagc 840

aaaaccagat cactctctcc gaggcttctt ccgcactgat tactacccaa atcggtggca 900

aaagatcctg tcgtttggcg gacgtgtccg catgagcact tccctgttgt tgggtgcgct 960

gctcttgctg tcactattta aggtcatgac tgtggaatgc tctagagcag acgctgatta 1020

cagacgtgtc ccatttcttt actactattg gaaattatga gttcagacgc agaaaaggca 1080

tccgtggagc tttccgaaaa atttcaccca gaacgcaccc atattttggg cgccgttgtt 1140

tttggcctga tctcattatt agtcatcggc gcagcccctc agtacctgtt ttggctgctc 1200

gcactccctg tcatcttcgg ttactgggtt ctaaaatcat ccacgatcgt tgatgaacag 1260

ggcatcaccg caaactacgc cttcaagggc aaaaaggttg tggcctggga aaacctcgca 1320

ggaatcggat tcaagggtgc ccgcactttc gctcgcacca cctctgatgc agaagtcacc 1380

ctccccggcg tcaccttcaa ctcccttccc cgccttgaag ctgcttccca cggccgcatc 1440

cccgatgcga tcaccgcaag caaggaagca gccgacggca aggttgtagt cgttcaagaa 1500

gacggctact ccgtgatgat gtccaaggaa gagtacttgg agcgccaaaa ggcactgggc 1560

aagccagttc agttgagctt cgatgacgac accgatggga atacaacaca aacagaaagc 1620

gttgaatccc aagagaccgg acaagccgcg tctgaaacct cacatcgtga taaccctgcg 1680

tcacagcact agagtgtaat aagccgtccg aaccaaaggt ccacacctct gcacgagtag 1740

aagctcaccc aagttttcaa agtgccgttg attcttgaca cccccccgcc gctccttaga 1800

gcagatttga aaagcgcatc atgatcccac ttcgttcaaa agtcaccacc gtcggtcgca 1860

atgcagctgg cgctcgcgcc ctttggcgtg ccaccggcac caaggaaaat gagttcggca 1920

agccaattgt tgccatcgtg aactcctaca cccagttcgt gcccggacac gttcacctta 1980

agaacgtcgg cgatattgtg gcagatgcac cca 2013

<210> 34

<211> 24

<212> DNA

<213> odhA-up-F

<400> 34

tcagacgatc cgatcctaga aaac 24

<210> 35

<211> 50

<212> DNA

<213> odhA-up-R

<400> 35

cctagttatt gaactcgtgg gtgagcttct tgagggttta ttgagctttg 50

<210> 36

<211> 822

<212> DNA

<213> odhA-up

<400> 36

tcagacgatc cgatcctaga aaacgtcacc gtgcagatcg agccaggaac cactgtcgct 60

gtggtgggcc ccaccggcgc tggaaaatcc accgtggtga aactcctttc ccgtctctac 120

gaccccaatg aaggtgccgt gaaagctggc gagatcgata tcaaggactt ccccaccgct 180

gattggcgcc gcacaatcgg taccgtgcca caagaagcac acttgtttag tggaagcatc 240

gccgacaaca ttggctacgg atgcagggag gcgtcgacaa gcaaaatcga agcggcagca 300

cgtcgcgtcg gagccttaaa cgccatcgcc gccatccctg atggtttcaa ccatcaagtc 360

ggtgaacgcg ggcgcaacct gtcatccgga cagcgccaac tgatcgcgct ggcgcgcgcc 420

gaactcaccg agccttccat catgcttctc gacgaagcca cctccaccct cgaccccgcc 480

accgaagccg ttatcctcaa cgcctccgat cgagtcacta agggacgcac cagcatcatc 540

gtcgcgcacc gcttggcaac cgctaaaagg gccgaccgta ttcttgttgt tgaacaagga 600

cgtatcattg aggacggatc tcacgacgcg ttgttgtctg ctaacggcac ctacgcccgc 660

atgtggcatt taatggcctg acacgttatt tttaggagaa ctgtcaacaa attaatgcta 720

caactggggc ttaggcataa tcagccaacg accagcgtta cagtggataa aacaaagctc 780

aataaaccct caagaagctc acccacgagt tcaataacta gg 822

<210> 37

<211> 47

<212> DNA

<213> odhA-down-F

<400> 37

ctcacccacg agttcaataa ctagggtgag cagcgctagt actttcg 47

<210> 38

<211> 21

<212> DNA

<213> odhA-down-R

<400> 38

cttggtcaga cgtgggacag a 21

<210> 39

<211> 848

<212> DNA

<213> odhA-down

<400> 39

ctcacccacg agttcaataa ctagggtgag cagcgctagt actttcggcc agaatgcgtg 60

gctggtagac gagatgttcc agcagttcca gaaggacccc aagtccgtgg acaaggaatg 120

gagagaactc tttgaggcgc agggggggac caaatactac ccccgctaca acagaagcac 180

agccttcagc gcccaaggag tctgcgaaac cagcaccaaa ggctgcccct gcagccaagg 240

cagcacctcg cgtagaaacc aagccggccg acaaggccgc ccctaaggcc aaggagtcct 300

cagtgccaca gcaacctaag cttccggagc caggacaaac cccaatcagg ggtattttca 360

agtccatcgc gaagaacatg gatatctccc tggaaatccc aaccgcaacc tcggttcgcg 420

atatgccagc tcgcctcatg ttcgaaaacc gcgcgatggt caacgatcag ctcaagcgca 480

cccgcggtgg caagatctcc ttcacccaca tcattggcta cgccatggtg aaggcagtca 540

tggctcaccc ggacatgaac aactcctacg acgtcatcga cggcaagcca accctgatcg 600

tgcctgagca catcaacctg ggccttgcca tcgaccttcc tcagaaggac ggctcccgcg 660

cacttgtcgt agcagccatc aaggaaaccg agaagatgaa cttctccgag ttcctcgcag 720

catacgaaga catcgtgaca cgctcccgca agggcaagct caccatggat gactaccagg 780

gcgttaccgt ttccttgacc aacccaggtg gcatcggtac ccgccactct gtcccacgtc 840

tgaccaag 848

<210> 40

<211> 1645

<212> DNA

<213> odhARBS0.1

<400> 40

tcagacgatc cgatcctaga aaacgtcacc gtgcagatcg agccaggaac cactgtcgct 60

gtggtgggcc ccaccggcgc tggaaaatcc accgtggtga aactcctttc ccgtctctac 120

gaccccaatg aaggtgccgt gaaagctggc gagatcgata tcaaggactt ccccaccgct 180

gattggcgcc gcacaatcgg taccgtgcca caagaagcac acttgtttag tggaagcatc 240

gccgacaaca ttggctacgg atgcagggag gcgtcgacaa gcaaaatcga agcggcagca 300

cgtcgcgtcg gagccttaaa cgccatcgcc gccatccctg atggtttcaa ccatcaagtc 360

ggtgaacgcg ggcgcaacct gtcatccgga cagcgccaac tgatcgcgct ggcgcgcgcc 420

gaactcaccg agccttccat catgcttctc gacgaagcca cctccaccct cgaccccgcc 480

accgaagccg ttatcctcaa cgcctccgat cgagtcacta agggacgcac cagcatcatc 540

gtcgcgcacc gcttggcaac cgctaaaagg gccgaccgta ttcttgttgt tgaacaagga 600

cgtatcattg aggacggatc tcacgacgcg ttgttgtctg ctaacggcac ctacgcccgc 660

atgtggcatt taatggcctg acacgttatt tttaggagaa ctgtcaacaa attaatgcta 720

caactggggc ttaggcataa tcagccaacg accagcgtta cagtggataa aacaaagctc 780

aataaaccct caagaagctc acccacgagt tcaataacta gggtgagcag cgctagtact 840

ttcggccaga atgcgtggct ggtagacgag atgttccagc agttccagaa ggaccccaag 900

tccgtggaca aggaatggag agaactcttt gaggcgcagg gggggaccaa atactacccc 960

cgctacaaca gaagcacagc cttcagcgcc caaggagtct gcgaaaccag caccaaaggc 1020

tgcccctgca gccaaggcag cacctcgcgt agaaaccaag ccggccgaca aggccgcccc 1080

taaggccaag gagtcctcag tgccacagca acctaagctt ccggagccag gacaaacccc 1140

aatcaggggt attttcaagt ccatcgcgaa gaacatggat atctccctgg aaatcccaac 1200

cgcaacctcg gttcgcgata tgccagctcg cctcatgttc gaaaaccgcg cgatggtcaa 1260

cgatcagctc aagcgcaccc gcggtggcaa gatctccttc acccacatca ttggctacgc 1320

catggtgaag gcagtcatgg ctcacccgga catgaacaac tcctacgacg tcatcgacgg 1380

caagccaacc ctgatcgtgc ctgagcacat caacctgggc cttgccatcg accttcctca 1440

gaaggacggc tcccgcgcac ttgtcgtagc agccatcaag gaaaccgaga agatgaactt 1500

ctccgagttc ctcgcagcat acgaagacat cgtgacacgc tcccgcaagg gcaagctcac 1560

catggatgac taccagggcg ttaccgtttc cttgaccaac ccaggtggca tcggtacccg 1620

ccactctgtc ccacgtctga ccaag 1645

<210> 41

<211> 1514

<212> DNA

<213> PH36_araE

<400> 41

caaaagctgg gtacctctat ctggtgccct aaacggggga atattaacgg gcccagggtg 60

gtcgcacctt ggttggtagg agtagcatgg gatccatggt tactatcaat acggaatctg 120

ctttaacgcc acgttctttg cgggatacgc ggcgtatgaa tatgtttgtt tcggtagctg 180

ctgcggtcgc aggattgtta tttggtcttg atatcggcgt aatcgccgga gcgttgccgt 240

tcattaccga tcactttgtg ctgaccagtc gtttgcagga atgggtggtt agtagcatga 300

tgctcggtgc agcaattggt gcgctgttta atggttggct gtcgttccgc ctggggcgta 360

aatacagcct gatggcgggg gccatcctgt ttgtactcgg ttctataggg tccgcttttg 420

cgaccagcgt agagatgtta atcgccgctc gtgtggtgct gggcattgct gtcgggatcg 480

cgtcttacac cgctcctctg tatctttctg aaatggcaag tgaaaacgtt cgcggtaaga 540

tgatcagtat gtaccagttg atggtcacac tcggcatcgt gctggcgttt ttatccgata 600

cagcgttcag ttatagcggt aactggcgcg caatgttggg ggttcttgct ttaccagcag 660

ttctgctgat tattctggta gtcttcctgc caaatagccc gcgctggctg gcggaaaagg 720

ggcgtcatat tgaggcggaa gaagtattgc gtatgctgcg cgatacgtcg gaaaaagcgc 780

gagaagaact caacgaaatt cgtgaaagcc tgaagttaaa acagggcggt tgggcactgt 840

ttaagatcaa ccgtaacgtc cgtcgtgctg tgtttctcgg tatgttgttg caggcgatgc 900

agcagtttac cggtatgaac atcatcatgt actacgcgcc gcgtatcttc aaaatggcgg 960

gctttacgac cacagaacaa cagatgattg cgactctggt cgtagggctg acctttatgt 1020

tcgccacctt tattgcggtg tttacggtag ataaagcagg gcgtaaaccg gctctgaaaa 1080

ttggtttcag cgtgatggcg ttaggcactc tggtgctggg ctattgcctg atgcagtttg 1140

ataacggtac ggcttccagt ggcttgtcct ggctctctgt tggcatgacg atgatgtgta 1200

ttgccggtta tgcgatgagc gccgcgccag tggtgtggat cctgtgctct gaaattcagc 1260

cgctgaaatg ccgcgatttc ggtattacct gttcgaccac cacgaactgg gtgtcgaata 1320

tgattatcgg cgcgaccttc ctgacactgc ttgatagcat tggcgctgcc ggtacgttct 1380

ggctctacac tgcgctgaac attgcgtttg tgggcattac tttctggctc attccggaaa 1440

ccaaaaatgt cacgctggaa catatcgaac gcaaactgat ggcaggcgag aagttgagaa 1500

atatcggcgt ctga 1514

<210> 42

<211> 3200

<212> DNA

<213> Δack::araE

<400> 42

atggctgccc accagctgct tgatcaagac atctgtgaca tcacgatcct gggcgatcca 60

gtaaagatca aggagcgcgc taccgaactt ggcctgcacc ttaacactgc atacctggtc 120

aatccgctga cagatcctcg cctggaggaa ttcgccgaac aattcgcgga gctgcgcaag 180

tcaaagggcg tcactatcga tgaagcccgc gaaatcatga aggatatttc ctacttcggc 240

accatgatgg tccacaacgg cgacgccgac ggaatggtat ccggtgcagc aaacaccacc 300

gcacacacca ttaagccaag cttccagatc atcaaaactg ttccagaagc atccgtcgtt 360

tcttccatct tcctcatggt gctgcgcggg cgactgtggg cattcggcga ctgtgctgtt 420

aacccgaacc caactgctga acagcttggt gaaatcgccg ttgtgtcagc aaaaactgca 480

gcacaatttg gcattgatcc tcgcgtagcc atcttgtcct actccactgg caactccggc 540

ggaggctcag atgtggatcg cgccattgac gctcttgcag aagcacgccg actcaaccca 600

gaactatgcg tcgatggacc acttcagttc gacgccgccg tcgacccggg tgtggcgcgc 660

aagaagatgc cagactctga cgtcgctggc caggcaaatg tgtttatctt ccctgacctg 720

gaggccggaa acatcggcta caaaactgca caacgcaccg gtcacgccct ggcagttggt 780

ccgattctgc agggcctgaa caaaccagtc aacgaccttt cccgtggtgc gacagtccct 840

gacatcgtca acactgtcgc catcaccgct attcaggcag gaggacgcag ctacaaaagc 900

tgggtacctc tatctggtgc cctaaacggg ggaatattaa cgggcccagg gtggtcgcac 960

cttggttggt aggagtagca tgggatccat ggttactatc aatacggaat ctgctttaac 1020

gccacgttct ttgcgggata cgcggcgtat gaatatgttt gtttcggtag ctgctgcggt 1080

cgcaggattg ttatttggtc ttgatatcgg cgtaatcgcc ggagcgttgc cgttcattac 1140

cgatcacttt gtgctgacca gtcgtttgca ggaatgggtg gttagtagca tgatgctcgg 1200

tgcagcaatt ggtgcgctgt ttaatggttg gctgtcgttc cgcctggggc gtaaatacag 1260

cctgatggcg ggggccatcc tgtttgtact cggttctata gggtccgctt ttgcgaccag 1320

cgtagagatg ttaatcgccg ctcgtgtggt gctgggcatt gctgtcggga tcgcgtctta 1380

caccgctcct ctgtatcttt ctgaaatggc aagtgaaaac gttcgcggta agatgatcag 1440

tatgtaccag ttgatggtca cactcggcat cgtgctggcg tttttatccg atacagcgtt 1500

cagttatagc ggtaactggc gcgcaatgtt gggggttctt gctttaccag cagttctgct 1560

gattattctg gtagtcttcc tgccaaatag cccgcgctgg ctggcggaaa aggggcgtca 1620

tattgaggcg gaagaagtat tgcgtatgct gcgcgatacg tcggaaaaag cgcgagaaga 1680

actcaacgaa attcgtgaaa gcctgaagtt aaaacagggc ggttgggcac tgtttaagat 1740

caaccgtaac gtccgtcgtg ctgtgtttct cggtatgttg ttgcaggcga tgcagcagtt 1800

taccggtatg aacatcatca tgtactacgc gccgcgtatc ttcaaaatgg cgggctttac 1860

gaccacagaa caacagatga ttgcgactct ggtcgtaggg ctgaccttta tgttcgccac 1920

ctttattgcg gtgtttacgg tagataaagc agggcgtaaa ccggctctga aaattggttt 1980

cagcgtgatg gcgttaggca ctctggtgct gggctattgc ctgatgcagt ttgataacgg 2040

tacggcttcc agtggcttgt cctggctctc tgttggcatg acgatgatgt gtattgccgg 2100

ttatgcgatg agcgccgcgc cagtggtgtg gatcctgtgc tctgaaattc agccgctgaa 2160

atgccgcgat ttcggtatta cctgttcgac caccacgaac tgggtgtcga atatgattat 2220

cggcgcgacc ttcctgacac tgcttgatag cattggcgct gccggtacgt tctggctcta 2280

cactgcgctg aacattgcgt ttgtgggcat tactttctgg ctcattccgg aaaccaaaaa 2340

tgtcacgctg gaacatatcg aacgcaaact gatggcaggc gagaagttga gaaatatcgg 2400

cgtctgaggc ccctagacct cttggggttg cgaattttcg tccccaccga acattaaaag 2460

gccggttttg gtcgaaaatt tgctctaaca ccttgctatt atgcaaatct tcgttcgatt 2520

taagcaaatc ccggcaatct gtaatgagaa gttgaacggg aaacctacag taaccccgca 2580

gaaatcacat cagccccaat tgtcccaaaa gtaacgcccc cggaatcgct tctaagggcc 2640

taactcgccc aaagccaaac tagttggaca ctggagccac aaaggccctt aaatcgccac 2700

ctaccaaata gccccaagcc aaaacagcta gaaccaactc agtggccgca ccgcattcgc 2760

catatccact agtgcgtaac ggtggtgggg gaaaggagcg gaacgggcct gaatccggag 2820

tgcctcggaa atgccggtgc gcaggccttt ttgggagaac gggtattcaa acaaagggtt 2880

tgcggaggca gaagctttga gttttcgttc tcgaagccag ctgaggcctg ctgacatgat 2940

ggcaattttg atttggttga agcggggttc gttggtgggg atttcgctga gtcggcgggc 3000

ggctcggcgg atgcgggatt cactcaaatt ggagctgacc agcaacaaga tggtggtgag 3060

ggtggccatt cggtagtggg tggatgattg ggggagttta tctagggctt ggactgcgag 3120

ttcgatttgg ttttcggcca tgagttggcg ggcgagcccg aacgcggagg acacagtggt 3180

ggggttggtt gcccagacaa 3200

Claims

1. A method for constructing a synthetic pathway for producing glutamic acid by xylose in Corynebacterium glutamicum, comprising the steps of:

(1) expressing a heterologous pentose transporter in C.glutamicum to help it take up xylose intracellularly;

(2) expressing heterologous xylose isomerase and xylulokinase in corynebacterium glutamicum, isomerizing xylose into xylulose, and then phosphorylating to xylulose-5-phosphate;

(3) the generated xylulose-5-phosphate can generate alpha-ketoglutarate through a pentose phosphate pathway and a glycolysis pathway owned by corynebacterium glutamicum, and alpha-ketoglutarate dehydrogenase is subjected to attenuation expression so that the alpha-ketoglutarate flows to the synthesis of glutamic acid more;

(4) the glutamic acid secretory protein is modified so that the corynebacterium glutamicum can not respond to the external biotin pressure any more and continuously secrete glutamic acid to the outside of cells.

2. The method according to claim 1, wherein the synthesis pathway for producing glutamic acid from xylose in Corynebacterium glutamicum comprises: the method specifically comprises the following steps:

(2) modification of glutamate secretory channel protein;

(3) attenuating the expression of alpha-ketoglutarate dehydrogenase;

(4) expression of the heterologous pentose transporter araE.

3. The method according to claim 2, wherein the synthesis pathway for producing glutamic acid from xylose in Corynebacterium glutamicum comprises:

the step (1) integration of xylAB expression cassette into ldhA gene locus of c.glutamcum: firstly, constructing an integration plasmid of xylAB, then transferring the integration plasmid pK 18-delta ldhA of xylAB into C.glutamicum in an electrotransfer mode, and further screening out a strain which generates correct homologous recombination in a pcr verification mode to obtain recombinant corynebacterium glutamicum, which is named as C.glutamicum XyltoGA 01;

4. The method according to claim 3, wherein the synthesis pathway for producing glutamic acid from xylose in Corynebacterium glutamicum comprises: culturing and fermenting the recombinant strain C.glutamicum xyltoGA04, wherein the culture medium is a high-biotin-concentration culture medium containing 60g/L glucose and 40g/L xylose, the temperature is 32 ℃, the pH is controlled to be 7.2 by ammonia water, the ventilation amount is 1.4vvm, and the rotation speed is 600 rpm.

5. Corynebacterium glutamicum capable of producing glutamic acid with xylose in a high biotin environment, constructed by the method according to any one of claims 1 to 4.

6. The use of C.glutamicum producing glutamic acid with xylose in a high biotin environment in a lignocellulosic fermentation process according to claim 5.