CN115896152A - Corynebacterium glutamicum and application thereof - Google Patents

Abstract

The invention discloses corynebacterium glutamicum and application thereof. The genetic engineering bacteria comprise an expression cassette containing a promoter and an xylAB gene, or an expression cassette combination of an expression cassette A containing the promoter and the xylAB gene and an expression cassette B containing the promoter and a PntAB gene, the development bacteria of the genetic engineering bacteria are corynebacterium glutamicum, and the genetic engineering bacteria over-express genes in the expression cassette or the expression cassette combination. The genetically engineered bacteria have a significantly better fermentation effect than genetically engineered bacteria expressing other lysine synthesis promoting genes or knocking out lysine synthesis inhibiting genes. When straw hydrolysate containing glucose and xylose is used, the yield of lysine of the genetically engineered bacteria is obviously improved compared with that of original bacteria.

Description

Corynebacterium glutamicum and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to corynebacterium glutamicum and application thereof.

Background

Lysine is an important amino acid, is widely used in the fields of food, feed and the like, and is also a polymerization monomer of nylon materials. The annual yield of lysine in 2013 reaches 200 ten thousand tons, and the demand is huge. The production of lysine by microbial fermentation using food starch raw materials is the most common lysine production mode at present, but the expensive starch raw materials greatly increase the production cost of lysine and hinder the commercial application of lysine. In order to reduce the raw material cost, some cheap carbon sources such as crude glycerol and silage juice have been tried for producing lysine, but the product concentration is too low to be practically used. Among the cheap carbon sources, lignocellulose is the only reliable renewable carbon source at present due to the advantages of large supply amount and wide source.

Lignocellulose is mainly composed of cellulose, hemicellulose and lignin, wherein the hemicellulose-derived xylose accounts for 30% of the total sugars of lignocellulose. The fermentation production of lysine by using xylose is one of the core steps for producing lysine by efficiently using lignocellulose biomass.

Corynebacterium glutamicum has a wide substrate production potential and is used for synthesizing various amino acids, organic acids, biofuels and other bio-based chemicals. Conventional C.glutamicum is unable to utilize xylose and is therefore a prerequisite for its lignocellulosic fermentation, by metabolic engineering and synthetic biological engineering, to be able to utilize xylose in lignocellulose.

The invention discloses a recombinant corynebacterium glutamicum for high yield of L-lysine and a construction method thereof with the application number of CN201811465343.X, and discloses a method for improving the yield of lysine by introducing a glyceraldehyde phosphate dehydrogenase encoding gene gapC into the genome of corynebacterium glutamicum CICC 23604. The high-yield lysine engineering strain constructed by the method can only utilize glucose but not xylose, so that the method is difficult to apply to the fermentation production of lysine by lignocellulose.

The invention entitled "method for producing L-amino acids by fermentation using bacteria with enhanced expression of xylose utilization genes" of application No. CN200510076242.X discloses a method for constructing a xylose-to-lysine synthesis pathway in Escherichia coli WC 196. Delta. CadA. Delta. Ldc by plasmid expression of the xylABFGHR locus derived from Escherichia coli MG 1655. The starting strain used in the method is escherichia coli, which has weak tolerance to lignocellulose-derived inhibitors and cannot normally grow and ferment lysine in a lignocellulose system.

Summarizing the above documents, it can be found that the biggest obstacle to the fermentative production of lysine by lignocellulose is the absence of a strain that can utilize glucose and xylose, while being more tolerant to lignocellulosic inhibitors. Therefore, no research report on the high yield of lysine by using glucose and xylose in lignocellulose exists at present.

Disclosure of Invention

Aiming at the defect that the prior art is lack of a genetic engineering bacterium for producing high-yield lysine, the invention provides an expression cassette capable of improving the yield of lysine of corynebacterium glutamicum and a genetic engineering bacterium capable of efficiently producing lysine by using straw hydrolysate. The genetic engineering bacteria can effectively utilize glucose and xylose in the straws in the straw hydrolysate.

In order to solve the above technical problems, one of the technical solutions provided by the present invention is: an expression cassette comprising a promoter and an xylAB gene, the promoter being Peftu, psod or PH36.

Preferably, the xylAB gene has a nucleotide sequence as set forth in SEQ ID NO. 4, or at least about 95% identity to SEQ ID NO. 4;

and/or the nucleotide sequence of the Peftu promoter is shown as SEQ ID NO. 1, the nucleotide sequence of the Psod promoter is shown as SEQ ID NO. 2, and the nucleotide sequence of the PH36 promoter is shown as SEQ ID NO. 3.

In order to solve the above technical problems, the second technical solution provided by the present invention is: an expression cassette combination comprising an expression cassette a as described in one of the claims and an expression cassette B comprising a promoter and a PntAB gene; the nucleotide sequence of the PntAB gene is shown in SEQ ID NO. 8, the promoter of the expression cassette B is Peftu with the nucleotide sequence shown in SEQ ID NO. 1, psod with the nucleotide sequence shown in SEQ ID NO. 2 or PH36 with the nucleotide sequence shown in SEQ ID NO. 3.

Preferably, the nucleotide sequence of the expression cassette A is shown as SEQ ID NO. 5, and the nucleotide sequence of the expression cassette B is shown as SEQ ID NO. 9.

In order to solve the technical problems, the third technical scheme provided by the invention is as follows: a recombinant vector comprising an expression cassette according to one of the claims or a combination of expression cassettes according to a second of the claims.

Preferably, when the recombinant vector comprises the expression cassette combination, the expression cassette a forms a recombinant integration vector with the backbone plasmid pK18 mob; and the expression cassette B and a skeleton plasmid pPeftumob form a recombinant expression vector.

In order to solve the above technical problems, the fourth technical solution provided by the present invention is: a genetically engineered bacterium comprising the expression cassette of claim one or the combination of expression cassettes of claim two, wherein the bacterium from which the genetically engineered bacterium originates is Corynebacterium glutamicum, and the genetically engineered bacterium overexpresses genes in the expression cassettes or the combination of expression cassettes.

Preferably, the genetically engineered bacterium does not express an ldh gene, e.g. the ldh gene is knocked out.

Preferably, after the expression cassette or the combination of expression cassettes has been introduced into the hairspray, the expression cassette a and/or the expression cassette B is integrated by homologous recombination on the genome of the hairspray or is present in the hairspray in a non-integrated form.

In a preferred embodiment of the present invention, the trichogen is c.glutamicum B253.

Preferably, when said genetically engineered bacterium comprises said expression cassette, said expression cassette is integrated on the genome of said producer; when the genetically engineered bacterium comprises the expression cassette combination, the expression cassette A is integrated on the genome of the outgoing bacterium, and the expression cassette B is present in the outgoing bacterium in a non-integrated form;

more preferably, when the genetically engineered bacterium comprises the expression cassette, a recombinant integration vector comprising the expression cassette is introduced into the outbreak bacterium, and the expression cassette is integrated into the ldh gene site on the genome; and/or, when the genetically engineered bacterium comprises the expression cassette combination, the expression cassette A is integrated into the ldh gene site on the genome, and the non-integrated form is: and transferring the recombinant expression vector containing the expression cassette B into the outbreak bacteria.

In order to solve the technical problems, the fifth technical scheme provided by the invention is as follows: a method for preparing lysine, the method comprising: and (3) fermenting the genetically engineered bacteria in the fourth technical scheme in a fermentation culture medium.

Preferably, the fermentation medium is a medium containing glucose and/or xylose, such as a straw hydrolysate, wherein the straw hydrolysate is a hydrolysate formed by degrading macromolecular carbohydrates such as cellulose, hemicellulose, lignin and the like in straws into micromolecular carbohydrates such as glucose and xylose after crop straws are subjected to enzymolysis and saccharification. The culture medium contains, for example, not less than 25g/L glucose and/or 25g/L xylose, such as 80-110g/L glucose and 25-40g/L xylose;

further, before the crop straws are subjected to enzymolysis and saccharification to prepare the hydrolysate, pretreatment, such as screening, impurity removal, dry acid pretreatment and/or biological detoxification, can be performed on the crop straws, so that the saccharification efficiency of the crop straws is improved, and the content of impurities such as acetic acid, furfural, 5-hydroxybenzaldehyde and the like in the crop straws is reduced.

And/or the fermentation conditions are: the temperature is 28-32 ℃, and/or the ventilation amount is 1.0-1.7vvm, and/or the pH is 6.8-7.2, and/or stirring is carried out during fermentation, and the rotation speed of the stirring is 400-800rpm.

In order to solve the technical problems, the fourth technical scheme provided by the invention is as follows: the use of the expression cassette according to the first aspect, the combination of expression cassettes according to the second aspect, the recombinant vector according to the third aspect, or the genetically engineered bacterium according to the fourth aspect in the preparation of lysine.

In order to solve the technical problems, the fifth technical scheme provided by the invention is as follows: the preparation method of the genetically engineered bacterium according to the fourth technical scheme comprises the following steps (without the sequence):

(1) Introducing the expression cassette according to the first technical scheme or the combination of the expression cassettes according to the second technical scheme into an outbreak bacterium, wherein the outbreak bacterium is C.glutamcum B253;

(2) Knocking out ldh gene to obtain the genetic engineering bacteria.

Preferably, in the preparation method, the expression cassette combination according to the second technical scheme is firstly introduced into a spawn of c.glutamicum B253, and then the ldh gene is knocked out, so as to obtain the genetically engineered bacterium.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the expression cassette provided by the invention has the advantages that the Peftu is used as the promoter of xylAB, so that the xylAB can be efficiently expressed, and the xylose utilization by the in-and-out bacteria is promoted. Besides high expression Peftu, the gene engineering bacteria preferably highly express PntAB gene, and obtain significantly better fermentation effect than gene engineering bacteria expressing other lysine synthesis promoting genes or knocking out lysine synthesis inhibiting genes. When straw hydrolysate containing glucose and xylose is used, the yield of lysine of the genetically engineered bacteria is obviously improved compared with that of original bacteria. The genetically engineered bacterium provided by the invention can effectively utilize agricultural wastes such as straws and the like to ferment, and has good application prospect.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. Experimental procedures without specifying specific conditions in the following examples were selected in accordance with conventional procedures and conditions, or in accordance with commercial instructions.

1. Strains used in the present invention

Coli E.coli BL21 was used to provide xylose utilization genes from E.coli. Glutamicum B253 is a strain for lysine production, purchased from the Shanghai industrial microbiology institute (SIIM, shanghai, china). Glutamicum B253 in this experiment was used mainly as the starting strain.

2. Reagents and culture media

The cellulase CTec 2.0 is used for hydrolyzing cellulose and hemicellulose in lignocellulose and is purchased from Novestin (China) company of Beijing, china. The cellulase had a filter paper enzyme activity of 203.2FPU/mL as measured by the method in the NREL LAP-006 guidelines, a cellobiase activity of 4900.0CBU/mL, and a protein concentration of 87.3mg/mL as measured by the Bradford method. Restriction enzymes were used to cleave plasmids or gene fragments to generate cohesive ends, and were purchased from Thermo Scientific (Wilmington, DE, USA). DNA polymerase is used to amplify gene fragments, and DNA ligase is used to ligate cleaved gene fragments with plasmid vectors, both of which are available from Takara (Otsu, japan). The seamless cloning kit is used for connecting gene fragments containing homologous fragments with plasmid vectors, and is purchased from Nanjing, china. Plasmid extraction kit, PCR product purification recovery kit and gel recovery kit were all purchased from Shanghai, strapdown Biotech (Shanghai, china). Other reagents were purchased from local suppliers.

The culture medium used for culturing the escherichia coli is Luria-Bertani (LB) culture medium, and the specific components are as follows: 10.0g/L sodium chloride, 10.0g/L peptone and 5.0g/L yeast extract.

The culture medium for culturing Corynebacterium glutamicum comprises the following components:

(1) Seed culture medium: 25g/L glucose, 1.5g/L potassium dihydrogen phosphate, 2.5g/L urea, 0.6g/L magnesium sulfate, and 25g/L corn steep liquor.

(2) Fermentation medium: 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 20g/L corn steep liquor, and optionally glucose and/or xylose as carbon source.

Example 1: starting strain C.glutamicum B253

Wheat straws are subjected to acid pretreatment, detoxification and enzyme hydrolysis to obtain wheat straw hydrolysate, and multiple corynebacterium glutamicum strains are cultured in the corn straw hydrolysate, so that C.glutamicum B253 (corynebacterium glutamicum B253) shows the highest cell growth and lysine production capacity. Glutamicum B253 can be fermented with straw hydrolysate and c glutamicum B253 can tolerate inhibitors (acetic acid, furfural, 5-hydroxybenzaldehyde, etc.) in straw hydrolysate for normal growth and lysine production.

Example 2: integrating the Peftu _ xylAB expression cassette into the ldh gene locus of C.glutamcum B253, and knocking out the ldh gene

Firstly, constructing an integration plasmid of xylAB (xylose isomerase and xylulokinase coding gene cluster), wherein the specific construction method comprises the following steps: using C.glutamcum genome as a template, and amplifying by using a PCR method by using a Peftu-F (shown as SEQ ID NO: 13) primer and a Peftu-R (shown as SEQ ID NO: 14) primer to obtain a Peftu promoter (shown as SEQ ID NO: 1); taking the genome of E.coli BL21 (Escherichia coli BL 21) as a template, and amplifying by PCR using xylAB-F (shown as SEQ ID NO: 19) and xylAB-R (shown as SEQ ID NO: 20) primers to obtain a xylAB fragment (shown as SEQ ID NO: 4); using Peftu and xylAB1 as templates, and using Peftu-xylAB-F (shown as SEQ ID NO: 21) and Peftu-xylAB-R (shown as SEQ ID NO: 22) primers to obtain a Peftu _ xylAB fusion fragment (shown as SEQ ID NO: 5) in a mode of overlapping extension PCR; using genome of C.glutamicum as a template, and amplifying by a PCR method by using ldh-up-F (shown as SEQ ID NO: 23) and ldh-up-R (shown as SEQ ID NO: 24) primers to obtain an ldh-up fragment (shown as SEQ ID NO: 6); using genome of C.glutamcum as a template, and amplifying by a PCR method by using ldh-down-F (shown as SEQ ID NO: 25) and ldh-down-R (shown as SEQ ID NO: 26) primers to obtain ldh-down fragment (shown as SEQ ID NO: 7); a ldh-up fragment, a Peftu _ xylAB fragment and a ldh-down fragment are used as templates, an ldh-up-F primer and an ldh-down-R primer are used to obtain a delta ldh:: xylAB fusion fragment by means of overlap extension PCR, ecoRI and HindIII endonucleases are used to treat the delta ldh:: xylAB fusion fragment, and the delta ldh:: xylAB fusion fragment is inserted into a pK18mob plasmid (the purchase route is shown in http:// www.biol.net/product/1089. Html) by using T4 ligase to obtain the pK 18-delta ldh:: xylAB plasmid. In the meantime, successful plasmids for ligation can be selected using LK plates containing kanamycin resistance.

And then transferring the integrated plasmid pK 18-delta ldh: xylAB into C.glutamicum in an electrotransfer 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 XyltoLy01.

Example 3: overexpression of transhydrogenase PntAB in engineered strains by plasmids

Firstly, constructing PntAB expression plasmid, and the specific construction method is as follows: taking the genome of E.coli BL21 as a template, and amplifying by a PCR mode by using PntAB-F (shown as SEQ ID NO: 27) and PntAB-R (shown as SEQ ID NO: 28) primers to obtain a PntAB (shown as SEQ ID NO: 8) fragment; ecoRI and HindIII endonuclease are used for processing, and then T4 ligase is used for inserting the EcoRI and HindIII endonuclease into pTRCmob expression plasmid (the purchase route is shown in http:// www.bioon.com.cn/reagent/show _ product.Rapid = 4926211), so as to obtain the expression plasmid of pPeftu-PntAB, wherein the nucleotide sequence of the Peftu-PntAB is shown as SEQ ID NO: 9. And then transferring the expression plasmid pPeftu-PntAB into C.glutamicum Xylly 01 in an electric transfer mode, and screening a recombinant strain containing the expression plasmid pPeftu-PntAB in a PCR verification mode to obtain the recombinant corynebacterium glutamicum named as C.glutamicum Xylly 02.

Example 4 fermentation of genetically engineered bacteria constructed with different promoters

The preparation of the genetically engineered bacteria was the same as in examples 2 and 3, except that the promoters were replaced with Psod (nucleotide sequence shown in SEQ ID NO: 2) and PH36 (nucleotide sequence shown in SEQ ID NO: 3), respectively, and the fermentation process was: the fermentation medium comprises 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 20g/L corn steep liquor, 40g/L xylose serving as a unique carbon source and 25 mu g/mL kanamycin. The fermentation was carried out in 250mL shake flasks at 30 ℃ at 200rpm for 96 hours. The fermentation results are shown in table 1:

TABLE 1 xylose utilization Gene and expression promoter screening

It can be seen that Peftu works best as a promoter for xylAB.

Example 5 fermentation of genetically engineered bacteria of different promoters

The preparation of the genetically engineered bacteria is the same as in examples 2 and 3, except that: the scheme for overexpression of dapA (shown as SEQ ID NO: 10) is to introduce the dapA gene in place of the PntAB gene into an expression plasmid; the pck knockout scheme is to introduce pck-up and pck-down genes into the knockout plasmid instead of ldh-up and ldh-down genes, without introducing xylAB genes.

The fermentation process comprises the following steps: fermentation medium: 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 20g/L corn steep liquor, and 25g/L xylose and 25g/L glucose. The fermentation was carried out in 250mL shake flasks at 30 ℃ and 200rpm, the pH of the medium was adjusted to 7 every 6 hours with 5M NaOH, and the fermentation time was 96 hours. The results are shown in Table 2. It can be seen that the PntAB-constructed plasmid has the best fermentation effect after being introduced into the genetically engineered bacteria obtained in example 2.

TABLE 2 expression of different genes to promote lysine production

Example 6: modified strain is fermented by lysine in lignocellulose hydrolysate

Crushing wheat straws, screening by using a screen with the diameter of 10 mm, washing the screened straws with water to remove impurities such as soil, stones, metal and the like, drying in a 105 ℃ drying oven to constant weight, and storing in a closed plastic bag for later use. And separating to obtain wheat straw hydrolysate containing 95.4g/L glucose and 34.7g/L xylose after dry acid pretreatment, biological detoxification and enzymolysis saccharification. Adding 20g/L ammonium sulfate, 5g/L methionine and threonine into the hydrolysate, culturing the transformed xylose utilization strain C.glutamicum XyltoLy02 and the starting strain C.glutamicum B253 in the wheat straw hydrolysate, and performing fermentation comparison, wherein the fermentation temperature is 30 ℃, the pH is controlled at 7.0 by ammonia water, the ventilation amount is 1.4vvm, the rotation speed is 600rpm, and the fermentation time is 72h.

The result shows that the original strain can only utilize glucose but not xylose in the wheat straw hydrolysate, 24.1g/L lysine is produced after fermentation is finished, about 34g/L of xylose is not utilized, and the glucose is completely consumed.

The transformed strain C.glutamicum XyltoLy02 can utilize glucose and xylose in wheat straw hydrolysate until fermentation is finished, so that 33.1g/L of lysine is produced, and the glucose and the xylose are completely consumed. Compared with the original strain, the yield of lysine is improved by 37.3 percent. The fermentation result shows that the modified strain obtained by the invention has stronger inhibitor tolerance and high-efficiency lysine production capacity, and has good application prospect.

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

SEQUENCE LISTING

<110> Shanghai Kaiser Biotech Ltd

CIC Energy Center

<120> Corynebacterium glutamicum and application thereof

<130> P21016188C

<160> 34

<170> PatentIn version 3.5

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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> 6

<211> 943

<212> DNA

<213> artificial sequence

<220>

<223> ldh-up

<400> 6

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> 7

<211> 959

<212> DNA

<213> artificial sequence

<220>

<223> ldh-down

<400> 7

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> 8

<211> 2932

<212> DNA

<213> artificial sequence

<220>

<223> PntAB

<400> 8

atgcgaattg gcataccaag agaacggtta accaatgaaa cccgtgttgc agcaacgcca 60

aaaacagtgg aacagctgct gaaactgggt tttaccgtcg cggtagagag cggcgcgggt 120

caactggcaa gttttgacga taaagcgttt gtgcaagcgg gcgctgaaat tgtagaaggg 180

aatagcgtct ggcagtcaga gatcattctg aaggtcaatg cgccgttaga tgatgaaatt 240

gcgttactga atcctgggac aacgctggtg agttttatct ggcctgcgca gaatccggaa 300

ttaatgcaaa aacttgcgga acgtaacgtg accgtgatgg cgatggactc tgtgccgcgt 360

atctcacgcg cacaatcgct ggacgcacta agctcgatgg cgaacatcgc cggttatcgc 420

gccattgttg aagcggcaca tgaatttggg cgcttcttta ccgggcaaat tactgcggcc 480

gggaaagtgc caccggcaaa agtgatggtg attggtgcgg gtgttgcagg tctggccgcc 540

attggcgcag caaacagtct cggcgcgatt gtgcgtgcat tcgacacccg cccggaagtg 600

aaagaacaag ttcaaagtat gggcgcggaa ttcctcgagc tggattttaa agaggaagct 660

ggcagcggcg atggctatgc caaagtgatg tcggacgcgt tcatcaaagc ggaaatggaa 720

ctctttgccg cccaggcaaa agaggtcgat atcattgtca ccaccgcgct tattccaggc 780

aaaccagcgc cgaagctaat tacccgtgaa atggttgact ccatgaaggc gggcagtgtg 840

attgtcgacc tggcagccca aaacggcggc aactgtgaat acaccgtgcc gggtgaaatc 900

ttcactacgg aaaatggtgt caaagtgatt ggttataccg atcttccggg ccgtctgccg 960

acgcaatcct cacagcttta cggcacaaac ctcgttaatc tgctgaaact gttgtgcaaa 1020

gagaaagacg gcaatatcac tgttgatttt gatgatgtgg tgattcgcgg cgtgaccgtg 1080

atccgtgcgg gcgaaattac ctggccggca ccgccgattc aggtatcagc tcagccgcag 1140

gcggcacaaa aagcggcacc ggaagtgaaa actgaggaaa aatgtacctg ctcaccgtgg 1200

cgtaaatacg cgttgatggc gctggcaatc attctttttg gctggatggc aagcgttgcg 1260

ccgaaagaat tccttgggca cttcaccgtt ttcgcgctgg cctgcgttgt cggttattac 1320

gtggtgtgga atgtatcgca cgcgctgcat acaccgttga tgtcggtcac caacgcgatt 1380

tcagggatta ttgttgtcgg agcactgttg cagattggcc agggcggctg ggttagcttc 1440

cttagtttta tcgcggtgct tatagccagc attaatattt tcggtggctt caccgtgact 1500

cagcgcatgc tgaaaatgtt ccgcaaaaat taaggggtaa catatgtctg gaggattagt 1560

tacagctgca tacattgttg ccgcgatcct gtttatcttc agtctggccg gtctttcgaa 1620

acatgaaacg tctcgccagg gtaacaactt cggtatcgcc gggatggcga ttgcgttaat 1680

cgcaaccatt tttggaccgg atacgggtaa tgttggctgg atcttgctgg cgatggtcat 1740

tggtggggca attggtatcc gtctggcgaa gaaagttgaa atgaccgaaa tgccagaact 1800

ggtggcgatc ctgcatagct tcgtgggtct ggcggcagtg ctggttggct ttaacagcta 1860

tctgcatcat gacgcgggaa tggcaccgat tctggtcaat attcacctga cggaagtgtt 1920

cctcggtatc ttcatcgggg cggtaacgtt cacgggttcg gtggtggcgt tcggcaaact 1980

gtgtggcaag atttcgtcta aaccattgat gctgccaaac cgtcacaaaa tgaacctggc 2040

ggctctggtc gtttccttcc tgctgctgat tgtatttgtt cgcacggaca gcgtcggcct 2100

gcaagtgctg gcattgctga taatgaccgc aattgcgctg gtattcggct ggcatttagt 2160

cgcctccatc ggtggtgcag atatgccagt ggtggtgtcg atgctgaact cgtactccgg 2220

ctgggcggct gcggctgcgg gctttatgct cagcaacgac ctgctgattg tgaccggtgc 2280

gctggtcggt tcttcggggg ctatcctttc ttacattatg tgtaaggcga tgaaccgttc 2340

ctttatcagc gttattgcgg gtggtttcgg caccgacggc tcttctactg gcgatgatca 2400

ggaagtgggt gagcaccgcg aaatcaccgc agaagagaca gcggaactgc tgaaaaactc 2460

ccattcagtg atcattactc cggggtacgg catggcagtc gcgcaggcgc aatatcctgt 2520

cgctgaaatt actgagaaat tgcgcgctcg tggtattaat gtgcgtttcg gtatccaccc 2580

ggtcgcgggg cgtttgcctg gacatatgaa cgtattgctg gctgaagcaa aagtaccgta 2640

tgacatcgtg ctggaaatgg acgagatcaa tgatgacttt gctgataccg ataccgtact 2700

ggtgattggt gctaacgata cggttaaccc ggcggcgcag gatgatccga agagtccgat 2760

tgctggtatg cctgtgctgg aagtgtggaa agcgcagaac gtgattgtct ttaaacgttc 2820

gatgaacact ggctatgctg gtgtgcaaaa cccgctgttc ttcaaggaaa acacccacat 2880

gctgtttggt gacgccaaag ccagcgtgga tgcaatcctg aaagctctgt aa 2932

<210> 9

<211> 3295

<212> DNA

<213> artificial sequence

<220>

<223> Peftu_PntAB

<400> 9

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 atacaaccat ggaattcgag ctcggtaccc 360

gggatgcgaa ttggcatacc aagagaacgg ttaaccaatg aaacccgtgt tgcagcaacg 420

ccaaaaacag tggaacagct gctgaaactg ggttttaccg tcgcggtaga gagcggcgcg 480

ggtcaactgg caagttttga cgataaagcg tttgtgcaag cgggcgctga aattgtagaa 540

gggaatagcg tctggcagtc agagatcatt ctgaaggtca atgcgccgtt agatgatgaa 600

attgcgttac tgaatcctgg gacaacgctg gtgagtttta tctggcctgc gcagaatccg 660

gaattaatgc aaaaacttgc ggaacgtaac gtgaccgtga tggcgatgga ctctgtgccg 720

cgtatctcac gcgcacaatc gctggacgca ctaagctcga tggcgaacat cgccggttat 780

cgcgccattg ttgaagcggc acatgaattt gggcgcttct ttaccgggca aattactgcg 840

gccgggaaag tgccaccggc aaaagtgatg gtgattggtg cgggtgttgc aggtctggcc 900

gccattggcg cagcaaacag tctcggcgcg attgtgcgtg cattcgacac ccgcccggaa 960

gtgaaagaac aagttcaaag tatgggcgcg gaattcctcg agctggattt taaagaggaa 1020

gctggcagcg gcgatggcta tgccaaagtg atgtcggacg cgttcatcaa agcggaaatg 1080

gaactctttg ccgcccaggc aaaagaggtc gatatcattg tcaccaccgc gcttattcca 1140

ggcaaaccag cgccgaagct aattacccgt gaaatggttg actccatgaa ggcgggcagt 1200

gtgattgtcg acctggcagc ccaaaacggc ggcaactgtg aatacaccgt gccgggtgaa 1260

atcttcacta cggaaaatgg tgtcaaagtg attggttata ccgatcttcc gggccgtctg 1320

ccgacgcaat cctcacagct ttacggcaca aacctcgtta atctgctgaa actgttgtgc 1380

aaagagaaag acggcaatat cactgttgat tttgatgatg tggtgattcg cggcgtgacc 1440

gtgatccgtg cgggcgaaat tacctggccg gcaccgccga ttcaggtatc agctcagccg 1500

caggcggcac aaaaagcggc accggaagtg aaaactgagg aaaaatgtac ctgctcaccg 1560

tggcgtaaat acgcgttgat ggcgctggca atcattcttt ttggctggat ggcaagcgtt 1620

gcgccgaaag aattccttgg gcacttcacc gttttcgcgc tggcctgcgt tgtcggttat 1680

tacgtggtgt ggaatgtatc gcacgcgctg catacaccgt tgatgtcggt caccaacgcg 1740

atttcaggga ttattgttgt cggagcactg ttgcagattg gccagggcgg ctgggttagc 1800

ttccttagtt ttatcgcggt gcttatagcc agcattaata ttttcggtgg cttcaccgtg 1860

actcagcgca tgctgaaaat gttccgcaaa aattaagggg taacatatgt ctggaggatt 1920

agttacagct gcatacattg ttgccgcgat cctgtttatc ttcagtctgg ccggtctttc 1980

gaaacatgaa acgtctcgcc agggtaacaa cttcggtatc gccgggatgg cgattgcgtt 2040

aatcgcaacc atttttggac cggatacggg taatgttggc tggatcttgc tggcgatggt 2100

cattggtggg gcaattggta tccgtctggc gaagaaagtt gaaatgaccg aaatgccaga 2160

actggtggcg atcctgcata gcttcgtggg tctggcggca gtgctggttg gctttaacag 2220

ctatctgcat catgacgcgg gaatggcacc gattctggtc aatattcacc tgacggaagt 2280

gttcctcggt atcttcatcg gggcggtaac gttcacgggt tcggtggtgg cgttcggcaa 2340

actgtgtggc aagatttcgt ctaaaccatt gatgctgcca aaccgtcaca aaatgaacct 2400

ggcggctctg gtcgtttcct tcctgctgct gattgtattt gttcgcacgg acagcgtcgg 2460

cctgcaagtg ctggcattgc tgataatgac cgcaattgcg ctggtattcg gctggcattt 2520

agtcgcctcc atcggtggtg cagatatgcc agtggtggtg tcgatgctga actcgtactc 2580

cggctgggcg gctgcggctg cgggctttat gctcagcaac gacctgctga ttgtgaccgg 2640

tgcgctggtc ggttcttcgg gggctatcct ttcttacatt atgtgtaagg cgatgaaccg 2700

ttcctttatc agcgttattg cgggtggttt cggcaccgac ggctcttcta ctggcgatga 2760

tcaggaagtg ggtgagcacc gcgaaatcac cgcagaagag acagcggaac tgctgaaaaa 2820

ctcccattca gtgatcatta ctccggggta cggcatggca gtcgcgcagg cgcaatatcc 2880

tgtcgctgaa attactgaga aattgcgcgc tcgtggtatt aatgtgcgtt tcggtatcca 2940

cccggtcgcg gggcgtttgc ctggacatat gaacgtattg ctggctgaag caaaagtacc 3000

gtatgacatc gtgctggaaa tggacgagat caatgatgac tttgctgata ccgataccgt 3060

actggtgatt ggtgctaacg atacggttaa cccggcggcg caggatgatc cgaagagtcc 3120

gattgctggt atgcctgtgc tggaagtgtg gaaagcgcag aacgtgattg tctttaaacg 3180

ttcgatgaac actggctatg ctggtgtgca aaacccgctg ttcttcaagg aaaacaccca 3240

catgctgttt ggtgacgcca aagccagcgt ggatgcaatc ctgaaagctc tgtaa 3295

<210> 10

<211> 939

<212> DNA

<213> artificial sequence

<220>

<223> dapA

<400> 10

atggcttccg caactttcac cggcgtgatc ccacccgtaa tgaccccact ccacgccgac 60

ggcagcgtag atgtagaaag cctccgcaag ctcgttgacc acctcatcaa tggtggcgtc 120

gacggacttt tcgcactggg ctcctcaggc gaagcggcat tcctcaccca cacccagcgc 180

aaacttgcac tgacgactat catcgagcac accgcaggcc gcgttcctgt aactgctgat 240

gtcattgaaa ccaccactgc tcgcgtgatt gagctcgtgg aagatgccct ggaagctggt 300

gccgaaggcc tcattgccac cgcacctttc tacacccgca cccacgatgt ggaaattgaa 360

gaacacttcc gcaagatcca cgccgtcgct ccagagctcc cactgtttgc ctacaacatc 420

ccagtgtcgg tgcactccaa cctcaaccca gtcatgcttt tgacgctggc caaggatggc 480

gttctcgcag gcaccaaaga ttccagtggc aatgatggcg caatccgctc actgatcgaa 540

gctcgtgatg atgctggact cactgagcag ttcaagatcc tcaccggcag cgaaaccacc 600

gttgatttct cctaccttgc tggtgccgat ggagttgtcc caggcctagg caatgttgat 660

cctgcagcat acgcagcttt agcaaaactc tgcctcgatg gaaagtgggc agaagctgct 720

gctttgcaga agcgcatcaa ccacctcttc cacatcgtct tcgtgggaga cacctcccat 780

atgtccggat ccagcgctgg tttgggcggt ttcaagacag cactcgcaca ccttggcatt 840

attgaatcca atgcgatggc agttcctcac cagagcctca gcgacgaaga aactgctcgc 900

attcacgcca ttgttgatga attcctgtac accgcttaa 939

<210> 11

<211> 897

<212> DNA

<213> artificial sequence

<220>

<223> pck-up

<400> 11

gtagcttttg gtcgaagagg gagtgggcat gcccattact ttaagccttt ggggcagtga 60

aaccgctaaa tgggggcgtt gtgcgctcga tcactggtct agacctttga gctccaagag 120

ttgcaatttc gcgaatactt caacacttgt ttgcaatgtt tgttaataaa tgggttcgcc 180

agtggattct gtcgttagta ctggccgtcg tggtgagatc atgtatttag gtagggcaaa 240

gttaagttca gggcaccttt tgatacgatt aactggatat aacctctggg gtaatgtggg 300

gatgtgtgtg agtaattttc aaagtattca aaagggggat ctagggtaaa aatttggctt 360

caagtacata cctttagttc ggtagttgag ggcgggtggt gacagtgcga gcatgcatgt 420

gggtgtaaat gttgttttaa aaaggggtgt actgacagtg ggccggtttg tgctggtcgg 480

ccactagcgg agtgcttgga ttgtgatggc agagtaaggg aaagggatta ccagtaccgc 540

tgttcttggc gttttgttgc ctattgtccg aatgttaagt gttaatggtc ggaaatctgg 600

gaaagttgtc tcctggaatg tgtgagaatt gcccaaatct gaacccaatg gccatggacg 660

gggaatgaac tgtcagagaa cggttgaggt taattcttga aaccaccccc aaaataggct 720

atttaaacgg gtgctctcat attaaagaaa gtgtgtagat gcgtgtgggc agggggtagg 780

tccactggta atgacaaatg tgtccgttgt ctcacctaaa gttttaacta gttctgtatc 840

tgaaagctac gctagggggc aagaactctg tcgaatgaca caaaatctgg agaagta 897

<210> 12

<211> 945

<212> DNA

<213> artificial sequence

<220>

<223> pck-down

<400> 12

agttcacgct taagaactgc taaataacaa gaaaggctcc caccgaaagt gggagccttt 60

cttgtcgtta agcgatgaat tcctcaaaac ctcagtgctt tttaaacacc aacaccaagt 120

tacttaccgc gaattcttgg agcactggga ctttaaccat ccaccaggcc caatacgggt 180

ggtagcgggg aaaagcggca accaattccg cattgcccac ggaggctccc cattccagcc 240

cctcccggca ggacacatta aacagtgact ccccgaaaac gttcttgggt ggatgcccgt 300

gtttcttcgt gtagcgatcg cgggcaaatt ctccgccaac gtagtgttcc cacagtccgg 360

tttcatggcc gccgaagggc cctaaccaaa tggtgtagct caggattgcc aggccgccgc 420

tgcgggtgac gcggagcatt tcttctccca attcccacgg tgcggagaca tgttctgcaa 480

cgttggagga gtacaccacg tcaaaggaat cgggaagaaa cggcaggtcg aggccggatc 540

cgcggactga tccgtggacg tcgatgccag ctgcggacat ttcgccaacg tcgggttcga 600

cggagaagta ggtggcgccc agtgtctcaa aggcttcggc aaagtatccg ggtccgccac 660

cgacgtcgag aactttcagg tcatttaatc cggcgccaga aatatcttca gacaaagccg 720

ccaccagact cgaggtatcg agggccaggt ttccgtaaaa gatgtcaggt cgggtttgtt 780

cgtatttgaa atcagacagt aaaccccacg acctgcccaa ggtagccagg cgacgaagag 840

ccggaagctc cggaaatgat gccatttatg cgcgggtcca gttgaggtcg cggatgtctt 900

cgccgttcat ccatcgcaaa atggtggtga tggcatcgtc gatgg 945

<210> 13

<211> 44

<212> DNA

<213> artificial sequence

<220>

<223> Peftu-F

<400> 13

ctggtttgac agcttatcat cgaaaagcaa tttgcttttc gacg 44

<210> 14

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> Peftu-R

<400> 14

ccgagctcga attccatggt tgtatgtcct cctggacttc gt 42

<210> 15

<211> 41

<212> DNA

<213> artificial sequence

<220>

<223> Psod-F

<400> 15

ctggtttgac agcttatcat agcggtaacc atcacgggtt c 41

<210> 16

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> Psod-R

<400> 16

ccgagctcga attccatggt gggtaaaaaa tcctttcgta 40

<210> 17

<211> 42

<212> DNA

<213> artificial sequence

<220>

<223> PH36-F

<400> 17

ctggtttgac agcttatcat caaaagctgg gtacctctat ct 42

<210> 18

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> PH36-R

<400> 18

ccgagctcga attccatggt ggatcccatg ctactcctac 40

<210> 19

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> xylAB-F

<400> 19

tcccccggga tgcaagccta ttttgacca 29

<210> 20

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> xylAB-R

<400> 20

ctagtctaga ttacgccatt aatggcagaa gt 32

<210> 21

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> Peftu_xylAB-F

<400> 21

aatcaggaag tgggatcgaa acgaaaagca atttgctttt cga 43

<210> 22

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> Peftu_xylAB-R

<400> 22

ccaactaggc gccaaagatt tacgccatta atggc 35

<210> 23

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> ldh-up-F

<400> 23

tcccccgggg gaacaccatg cgattaaggt gc 32

<210> 24

<211> 43

<212> DNA

<213> artificial sequence

<220>

<223> ldh-up-R

<400> 24

caaattgctt ttcgtttcga tcccacttcc tgatttccct aac 43

<210> 25

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> ldh-down-F

<400> 25

ttaatggcgt aaatctttgg cgcctagttg gc 32

<210> 26

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> ldh-down-R

<400> 26

ctagtctaga gtctgggacg ttgatgacgc t 31

<210> 27

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> PntAB-F

<400> 27

tcccccggga tgcgaattgg cataccaag 29

<210> 28

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> PntAB-R

<400> 28

ctagtctaga ttacagagct ttcaggattg 30

<210> 29

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> dapA-F

<400> 29

tcccccggga tggcttccgc aactttcac 29

<210> 30

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> dapA-R

<400> 30

ctagtctaga ttaagcggtg tacaggaatt 30

<210> 31

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> pck-up-F

<400> 31

tcccccgggg tagcttttgg tcgaagaggg agtg 34

<210> 32

<211> 47

<212> DNA

<213> artificial sequence

<220>

<223> pck-up-R

<400> 32

gttcttaagc gtgaacttac ttctccagat tttgtgtcat tcgacag 47

<210> 33

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> pck-down-F

<400> 33

caaaatctgg agaagtaagt tcacgcttaa gaactgctaa ataacaag 48

<210> 34

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> pck-down-R

<400> 34

ctagtctaga ccatcgacga tgccatcacc a 31

Claims (10)

1. An expression cassette comprising a promoter and an xylAB gene, the promoter being Peftu, psod or PH36;

preferably, the xylAB gene has a nucleotide sequence as set forth in SEQ ID NO. 4, or at least about 95% identity to SEQ ID NO. 4;

and/or the nucleotide sequences of the Peftu, the Psod and the PH36 are respectively shown as SEQ ID NO. 1-3.

2. An expression cassette combination, characterized in that it comprises an expression cassette a as defined in claim 1 and an expression cassette B comprising a promoter and a PntAB gene; the nucleotide sequence of the PntAB gene is shown as SEQ ID NO. 8, the promoter of the expression cassette B is Peftu with the nucleotide sequence shown as SEQ ID NO. 1, psod with the nucleotide sequence shown as SEQ ID NO. 2 or PH36 with the nucleotide sequence shown as SEQ ID NO. 3;

preferably, the nucleotide sequence of the expression cassette A is shown as SEQ ID NO. 5, and the nucleotide sequence of the expression cassette B is shown as SEQ ID NO. 9.

3. A recombinant vector comprising the expression cassette of claim 1 or the combination of expression cassettes of claim 2;

preferably, when said recombinant vector comprises said expression cassette combination, said expression cassette a forms a recombinant integrating vector with the backbone plasmid pK18 mob; and the expression cassette B and a framework plasmid pTRCmob form a recombinant expression vector.

4. A genetically engineered bacterium comprising the expression cassette of claim 1 or the combination of expression cassettes of claim 2, wherein the microorganism of the genetically engineered bacterium is Corynebacterium glutamicum, and wherein the genetically engineered bacterium overexpresses the genes in the expression cassettes or the combination of expression cassettes.

5. The genetically engineered bacterium of claim 4, wherein the genetically engineered bacterium does not express an ldh gene, e.g., the ldh gene is knocked out; and/or the presence of a gas in the gas,

after the expression cassette or the combination of expression cassettes has been introduced into the hairline, the expression cassette a and/or the expression cassette B is integrated by homologous recombination on the genome of the hairline or is present in the hairline in a non-integrated form;

preferably, the trichogen is c.glutamicum B253.

6. The genetically engineered bacterium of claim 5, wherein when said genetically engineered bacterium comprises said expression cassette, said expression cassette is integrated into the genome of said producer; when the genetically engineered bacterium comprises the expression cassette combination, the expression cassette A is integrated on the genome of the outgoing bacterium, and the expression cassette B is present in the outgoing bacterium in a non-integrated form;

preferably, when the genetically engineered bacterium comprises the expression cassette, a recombinant integration vector comprising the expression cassette is introduced into the starter bacterium, such that the expression cassette is integrated into the genomic ldh gene site; and/or, when the genetically engineered bacterium comprises the expression cassette combination, the expression cassette A is integrated into the ldh gene site on the genome, and the non-integrated form is: and transferring the recombinant expression vector containing the expression cassette B into the development bacteria.

7. A method for preparing lysine, which is characterized by comprising the following steps: fermenting the genetically engineered bacterium of any one of claims 4-6 in a fermentation medium.

8. The method of claim 7, wherein the fermentation medium is a glucose and/or xylose containing medium such as straw hydrolysate, e.g. containing not less than 25g/L glucose and/or 25g/L xylose, e.g. 80-110g/L glucose and 25-40g/L xylose;

and/or the fermentation conditions are as follows: stirring at 28-32 deg.C, ventilation amount of 1.0-1.7vvm, pH of 6.8-7.2 during fermentation, and rotation speed of 400-800rpm.

9. Use of the expression cassette of claim 1, the combination of expression cassettes of claim 2, the recombinant vector of claim 3, or the genetically engineered bacterium of any one of claims 4-6 for the production of lysine.

10. The method for preparing genetically engineered bacteria of any one of claims 4 to 6, comprising the steps of:

(1) Introducing the expression cassette of claim 1 or the combination of expression cassettes of claim 2 into an outbreak of c.glutamicum B253;

(2) Knocking out ldh gene to obtain the genetic engineering bacteria.