Recombinant strain producing L-amino acids, constructing method therefor and method for producing L-amino acids

Abstract

The present invention relates to recombinant bacteria producing L-amino acid, in which the recombinant bacteria has reduced expression of the glucose-6-phosphate isomerase gene pgi and improved expression of the glucose-6-phosphate dehydrogenase gene -opcA than the starting bacteria, where the starting bacterium is a bacterial strain that can accumulate target amino acid(s) and preferably, the amino acid is L-histidine.

Claims

1. A recombinant bacteria producing L-amino acid(s), said recombinant bacteria has reduced expression of the glucose-6-phosphate isomerase gene pgi and improved expression of the glucose-6-phosphate dehydrogenase gene zwf-opcA than the starting bacteria, wherein: said starting bacterium is a bacterial strain that can accumulate target amino acid(s) and preferably, said amino acid is L-histidine, wherein, the gene pgi on the chromosome of the recombinant bacterium has been inactivated, preferably knocked out or the regulatory element of the gene pgi has been replaced with a regulatory element with low transcription or low expression activity, also said recombinant bacteria has two or more copied genes zwf-opcA, or the promoter of the operon tkt-tal-zwf-opcA-devB on the chromosome of said starting bacterium is replaced with a strong promoter, preferably, said strong promoter is the promoter P.sub.eftu, of the original bacteria.

2. The recombinant bacteria according to claim 1, wherein: said starting bacteria has enhanced expression of the genes hisEG and hisDCB of the operon for L-histidine synthesis than the original bacteria, preferably a strong promoter is used to replace the promoter of said genes hisEG and hisDCB, more preferably, the promoter P.sub.glyA on the chromosome of said original bacteria replaces respectively the promoters of the genes hisEG and hisDCB, further preferably, said starting bacteria has enhanced expression of PRPP synthetase PrsA than the original bacteria, more preferably, said starting bacteria has two or more copied gene prsA or a strong promoter replaces the promoter of the gene prsA, preferably, said strong promoter is the promoter P.sub.sod of said original bacteria.

3. The recombinant bacteria according to claim 2, wherein: said recombinant bacteria has enhanced expression of AICAR transmethylase/IMP ring hydrase PurH than said starting bacteria, preferably, said recombinant bacteria has two or more copied genes purH, or the promoter of the gene purH is replaced with a strong promoter, more preferably, said strong promoter is the promoter P.sub.eftu of said original bacteria.

4. The recombinant bacteria according to claim 3, wherein: said recombinant bacteria has weakened expression of the amidophosphoribosyl transterase PurF than said starting bacteria, preferably, the promoter of the gene purF is replaced with a weak promoter, more preferably, said weak promoter is the promoter P.sub.hom in said original bacteria.

5. The recombinant bacteria according to claim 4, wherein: said original bacterium is a bacterial strain selected from corynebacterium, dialister or brevibacterium, preferably, said bacteria of corynebacterium is a bacterial strain selected from Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes, Corynebacterium aminogenes, Corynebacterium lilium, Corynebacterium callunae and Corynebacterium herculis, said bacteria of dialister is a bacterial strain selected from Microbacterium ammoniaphilum, and said bacteria of brevibacterium is a bacterial strain selected from Brevibacteriaceae flvum, Brevibacteriaceae lactofermentum and Brevibacteriaceae ammoniagenes; more preferably, said original bacterium is the wild type of Corynebacterium glutamicum ATCC13032.

6. The recombinant bacteria according to claim 5, wherein: the chromosome of said starting bacteria has the promoter P.sub.glyA as shown by nucleotides 863-1038 in the 5′ end of the nucleotide sequence of SEQ ID NO: 7 used to replace respectively the promoters of the operons hisEG and hisDCB for L-histidine synthesis on the chromosome of said Corynebacterium glutamicum ATCC13032, and said starting bacteria can express the mutated ATP-phosphoribosyl transferase, said mutated ATP-phosphoribosyl transferase is the enzyme of ATP-phosphoribosyl transferase as shown by SEQ ID NO: 6 whose No. 215 asparagine is mutated to lysine, No. 231 leucine to phenylalanine and No. 235 threonine to alanine, preferably, the chromosome of said starting bacteria has a gene hisG.sup.fbr as shown by nucleotides 1007-1852 in the 5′ end of the nucleotide sequence of SEQ ID NO: 4 used to replace the gene hisG on the chromosome of said Corynebacterium glutamicum ATCC13032, preferably, the chromosome of said starting bacteria has the promoter P.sub.sod as shown by nucleotides 656-847 in the 5′ end of the nucleotide sequence of SEQ ID NO: 11 used to replace the promoter of the gene prsA on the chromosome of said Corynebacterium glutamicum ATCC13032, or, said starting bacteria has two or more copied genes prsA and hisG.sup.fbr, wherein, said gene prsA is, or at least 99% homologous with, the nucleotides 15-992 in the 5′ end of the nucleotide sequence as shown by SEQ ID NO: 4 in the sequence table.

7. The recombinant bacteria according to claim 6, wherein: said gene pgi is, or at least 99% homologous with, the nucleotide sequence as shown by SEQ ID NO: 13, said genezwf-opcA is, or at least 99% homologous with, the nucleotide sequence as shown by SEQ ID NO: 2 and said promoter Peftu is nucleotides 635-834 in the 5′ end of the nucleotide sequence of SEQ ID NO: 12.

8. The recombinant bacteria according to claim 7, wherein: said gene purH is, or at least 99% homologous with, the nucleotide sequence as shown by SEQ ID NO: 15.

9. The recombinant bacteria according to claim 8, wherein: said promoter P.sub.hom is nucleotides 736-865 in the 5′ end of the nucleotide sequence of SEQ ID NO: 18.

10. A method of constructing the recombinant bacteria capable of producing L-histidine, which comprises the following steps: reduce the expression of the glucose-6-phosphate isomerase Pgi in the starting bacteria and improve the expression of the glucose-6-phosphate dehydrogenase Zwf-OpcA in said starting bacteria to obtain said recombinant bacteria, wherein: said starting bacterium is a bacterial strain which can accumulate target amino acid(s), more preferably, said target L-amino acid.

11. The method according to claim 10, wherein: said reducing the expression of Pgi in starting bacterium is realized by means of the following A) or B): A) Inactivate the gene pgi of the chromosome of said starting bacteria; said inactivation is preferably knocking out, B) Replace the regulatory element of the gene pgi in said starting bacteria with a regulatory element of low transcription and low expression activity, and said improving the expression of Zwf-OpcA in said starting bacterium is realized by means of the following C) or D): C) Increase the copy number of the gene zwf-opcA in said starting bacteria, D) Replace the promoter of the operon tkt-tal-zwf-opcA-devB on the chromosome of said starting bacteria with a strong promoter, preferably, said strong promoter is the promoter P.sub.eftu on the chromosome of said original bacteria.

12. The method according to claim 10, wherein: obtaining said starting bacteria comprises the step(s) of replacing the promoter of the operon hisEG and hisDCB for L-histidine synthesis on the chromosome of starting bacteria respectively with a strong promoter, preferably, said strong promoter is the promoter P.sub.glyA on the chromosome of said original bacteria, preferably, obtaining said starting bacteria further comprises the step(s) of improving the expression of PRPP synthetase PrsA in said starting bacteria, more preferably, said improving the expression of PrsA in said starting bacterium is realized by means of the following E) or F): E) Increase the copy number of the gene prsA in said starting bacteria, F) Replace the promoter of the gene prsA on the chromosome of said starting bacteria with a strong promoter, preferably, said strong promoter is the promoter P.sub.sod on the chromosome of said original bacteria.

13. The method according to claim 12, wherein: said method further comprises the step(s) of improving the expression of AICAR transmethylase/IMP ring hydrase PurH in said recombinant bacteria, preferably, said improving the expression of PurH in said recombinant bacterium is realized by means of the following G) or H): G) Increase the copy number of the gene purH in said starting bacteria, H) Replace the promoter of the gene purH on the chromosome of said starting bacteria with a strong promoter, preferably, said strong promoter is the promoter P.sub.eftu on the chromosome of said original bacteria.

14. The method according to claim 13, wherein: said method further comprises the step(s) of weakening the expression of the amidophosphoribosyl transterase PurF in said recombinant bacteria, preferably, said weakening the expression of PurF in said recombinant bacterium is realized through replacing the promoter of the gene purF with a weak promoter, more preferably, the promoter of the gene purF on the chromosome in said starting bacterium is replaced with the promoter P.sub.hom on the chromosome in said original bacteria.

15. The method according to claim 14, wherein: the original bacteria used to obtain said starting bacterium is a bacterial strain selected from corynebacterium, dialister or brevibacterium, preferably, said bacteria of corynebacterium is a bacterial strain selected from Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes, Corynebacterium aminogenes, Corynebacterium lilium, Corynebacterium callunae and Corynebacterium herculis, said bacteria of dialister is a bacterial strain selected from Microbacterium ammoniaphilum, and said bacteria of brevibacterium is a bacterial strain selected from Brevibacteriaceae flvum, Brevibacteriaceae lactofermentum and Brevibacteriaceae ammoniagenes, more preferably said original bacterium is the wild type of Corynebacterium glutamicum ATCC13032.

16. The method according to claim 15, wherein: the following steps are comprised to obtain said starting bacteria: replace the promoter of the operon hisEG and hisDCB for L-histidine synthesis on the chromosome of Corynebacterium glutamicum ATCC13032 respectively with the promoter P.sub.glyA as shown by nucleotides 863-1038 in the 5′ the nucleotide sequence of SEQ ID NO: 7, and mutate No. 215 asparagine to lysine, No. 231 leucine to phenylalanine and No. 235 threonine to alanine on ATP-phosphoribosyl transferase expressed by said Corynebacterium glutamicum ATCC13032 as shown in SEQ ID NO: 6, preferably, the gene used to carry out the mutations as above is the gene hisG.sup.fbr as shown by nucleotides 1007-1852 in the 5′ end of the nucleotide sequence of SEQ ID NO: 4, preferably, obtaining said starting bacteria further comprises the following steps: replace the promoter of the gene prsA on the chromosome of said Corynebacterium glutamicum ATCC13032 with the promoter P.sub.sod as shown by nucleotides 656-847 in the 5′ end if the nucleotide sequence of SEQ ID NO: 11, or, the following steps are further comprised: Increase the copy number of the gene prsA in said Corynebacterium glutamicum ATCC13032 and increase the copy number the gene hisG.sup.fbr in said Corynebacterium glutamicum ATCC13032, wherein, said gene prsA is, or at least 99% homologous with, the nucleotides 15-992 in the 5′ end of the nucleotide sequence as shown by SEQ ID NO: 4 in the sequence table.

17. The method according to claim 16, wherein: said gene pgi, or at least 99% homologous with, the nucleotide sequence as shown by SEQ ID NO: 13, said gene zwf-opcA is, or at least 99% homologous with, the nucleotide sequence as shown by SEQ ID NO: 2 and said promoter Peftu is nucleotides 635-834 in the 5′ end of the nucleotide sequence of SEQ ID NO: 12, and said promoter P.sub.eftu is nucleotides 635-834 in the 5′ end of the nucleotide sequence of SEQ ID NO: 12.

18. A method according to claim 17, wherein: said gene purH is, or at least 99% homologous with, the nucleotide sequence as shown by SEQ ID NO: 15.

19. The method according to claim 18, wherein: said promoter P.sub.hom is nucleotides 736-865 in the 5′ end of the nucleotide sequence of SEQ ID NO: 18.

20. A method of constructing recombinant bacteria of claim 1, which comprises the step(s) of fermenting and culturing a recombinant bacteria.

Description

DESCRIPTION OF THE DRAWINGS

(1) The following drawings can be referred to help understand the solution and the beneficial effects according to the present invention.

(2) FIG. 1 is the schematic diagram of the recombinant plasmid pXMJ19-prsA-hisG.sup.fbr.

(3) FIG. 2 is the electro-phoretogram of PCR identification of the genome DNA of the bacterial strain CG161 (gene pgi is knocked out).

(4) FIG. 3 is the schematic diagram of the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr.

(5) FIG. 4 is the SDS-PAGE diagram of the expression protein of L-histidine engineering bacteria CG171.

(6) FIG. 5 is the diagram of determining enzyme activity of the glucose-6-phosphate dehydrogenase in L-histidine engineering bacteria CG171.

(7) FIG. 6 is the schematic diagram of the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr-purH.

(8) FIG. 7 is the electro-phoretogram of PCR identification of the plasmid DNA carried by the bacterial strain CG328.

(9) FIG. 8 is the electro-phoretogram of PCR identification of the genome DNA of the bacterial strain CG353 (gene purF is weakened).

DETAILED DESCRIPTION OF EMBODIMENTS

(10) The specific implementation ways according to the present invention are described in more detail in combination with the drawings and the embodiments in order to better understand the solution and the advantages in each aspect according to the present invention.

(11) Nevertheless, the specific implementation ways and the embodiments described below are just for the purpose of explanation rather than any limit on the present invention. Specifically, all the following descriptions use (the wild type of) Corynebacterium glutamicum as the example to explain and test the construction of the recombinant engineering bacteria and the production of L-histidine. Nevertheless, those skilled in the art may easily understand that the modification strategy to the metabolic pathway of amino acid according to the present invention can be used to other appropriate bacterial strains in order to construct the engineering bacteria to improve the yield of L-histidine.

(12) As mentioned in the background technologies, the glucose-6-phosphate isomerase encoded by the gene pgi is the key enzyme of the glycolytic pathway. The precursor PRPP for L-histidine synthesis is synthesized from the pentose phosphate pathway. Thus, it is assumed that knocking out the gene pgi will weaken the metabolic flux of the glycolytic pathway and guide the metabolic flux of central carbon to the pentose phosphate pathway so as to enhance the metabolic flux of L-histidine synthesis pathway. The strategy of modification through knocking out the gene pgito enhance the metabolic flux of the pentose phosphate pathway has been reported in both literature and patents (used to produce the products such as L-lysine, L-valine and nucleoside. Marx, A., Hans, S., Mockel, B., Bathe, B., de Graaf, A. A., McCormack, A. C., Stapleton, C., Burke, K., O'Donohue, M., Dunican, L. K., 2003. Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum. J Biotechnol. 104, 185-197; Blombach, B., Schreiner, M. E., Bartek, T., Oldiges, M., Eikmanns, B. J., 2008. Corynebacterium glutamicum tailored for high-yield I-valine production. Appl Microbiol Biotechnol. 79, 471-479; Peifer, S., Barduhn, T., Zimmet, S., Volmer, D., Heinzle, E., Schneider, K., 2012. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine. Microb Cell Fact. 11, 138; U.S. Pat. No. 6,586,214B1; EP1087015A2).

(13) However, in fact, after the studies by the inventor, it is found that: knocking out the gene pgi can result in over accumulation of intermediate metabolites of sugar metabolism and sugar metabolic stress and hence cause slow glucose metabolism and growth of bacteria. The inventor also finds that: after the gene pgi is knocked out, the yield of L-histidine produced by the engineering bacteria producing L-histidine is clearly reduced, instead of increasing. The main reason is that: the histidine obtains the precursor to synthesize its molecular skeleton through the pentose phosphate pathway whereas the lysine and the valine acquire the cofactor NADPH of their synthetase through the pentose phosphate pathway. In addition, the synthesis process of histidine needs to consume a large number of energy carriers ATP. Thus, if the strategy of increasing the histidine yield through weakening the expression of the gene pgi and enhancing the metabolic flux of the pentose phosphate pathway is to be adopted, the balance of metabolic fluxes between the pentose phosphate pathway and the glycolytic pathway needs to be kept in order to ensure the synthetic precursor and the energy supply.

(14) In regard to such problems, the present invention finds through the experiments that the over-expression of the gene zwf-opcA (this gene encodes the glucose-6-phosphate dehydrogenase and is the key rate-limiting enzyme of the pentose phosphate pathway) can enhance the ability of bacteria to metabolize sugar, relieve sugar metabolic stress and restore the ability of glucose metabolism and growth of bacteria as well as balance the metabolic fluxes between the pentose phosphate pathway and the glycolytic pathway and balance the supplies of the precursors PRPP and ATP for histidine synthesis so as to hence improve the yield of L-histidine.

(15) According to the present invention, with the modification strategy of weakening (such as knocking out) the gene pgi and simultaneously over-expression of the gene zwf-opcA, the bacterial strains of which the expression of the gene prsA and the expression of the operon gene for L-histidine synthesis are enhanced are recombined and modified to obtain the strain(s) which successfully improves the yield of L-histidine.

(16) The strategy of modification through both weakening the gene pgi and over-expression of the gene zwf-opcA according to the present invention can increase NADPH and also balance the metabolic fluxes between the pentose phosphate pathway and the glycolytic pathway, can smooth away the problems of slow glucose metabolism and growth of bacteria due to weakening of the gene pgi and hence can also improve further the yield of amino acids.

(17) On this basis, the present invention further proposes a strategy to couple the synthetic pathway of L-histidine and the synthetic pathway of nucleotide. During the synthetic process of L-histidine, the imidazole glycerol phosphate synthase encoded by the genes hisH and hisF catalyzes and produces the imidazole glycerol phosphate and the 5-phosphoribosyl-4-formamido-5-aminoimidazole(AICAR), wherein: the former finally synthesizes L-histidine along the synthetic pathway of histidine, but the latter can enter the purine synthetic pathway and finally produce the purine nucleotides (AMP, ATP, etc.). ATP is one of the precursor substances to synthesize histidine and also provides energy for histidine synthesis. The bi-functional enzyme encoded by the gene purH, AICAR transmethylase/IMP ring hydrase, catalyzes two steps of reaction from AICAR to produce IMP. The inventor finds that enhancing the expression of the gene purH in Corynebacterium glutamicum can facilitate obviously the accumulation of L-histidine and can further enhance the effect in combination with the above modification strategy.

(18) Moreover, the synthetic pathway of L-histidine and the synthetic pathway of purine nucleotide couple with each other at the metabolite AICAR and use the same precursor substance PRPP. The inventor finds that weakening the encoding gene purF of the enzyme (amidophosphoribosyl transterase) for the first reaction step catalyzing the synthesis of purine nucleotide can conduct the metabolic coupling between the synthetic pathway of nucleotide and the synthetic pathway of histidine, synthesize nucleotide from the by-product AICAR of histidine synthesis, increase the supply of the precursor substance PRPP for histidine synthesis and simultaneously promote the metabolic flux of the synthetic pathway of histidine so as to facilitate the accumulation of L-histidine. Such gene modification can also improve further the yield of L-histidine.

(19) As described above, the present invention can recombine and modify several target spots in the pathways related to histidine synthesis of microorganism and effectively realize the accumulation of L-histidine. In addition to modifying the synthetic pathway of histidine, the synthetic pathway of histidine and the synthetic pathway of nucleotide are coupled to effectively utilize the coupling node AICAR of histidine synthesis and nucleotide synthesis to form a pathway of purine nucleotide and save the synthetic precursor PRPP so as to provide more precursor substances PRPP and ATP for histidine synthesis and further increase the accumulation of L-histidine.

Definitions

(20) The term “starting bacteria” mentioned in this article (also referred to as “base bacteria” in this article) refers to the initial bacterial strain used in the strategy of gene modification according to the present invention. This strain can be naturally occurring or bred by means of mutation or genetic engineering modification. In order to construct the engineering bacteria used to produce some L-amino acid (for example, L-histidine), said starting bacterium is preferred to a bacterial strain which can accumulate this L-amino acid (for example, L-histidine).

(21) The term “original bacteria” mentioned in this article refers to bacterial strain which is not ever modified at all through any genetic engineering. It can be naturally occurring or bred by means of artificial mutation.

(22) The term “homology” mentioned in this article refers to the level of similarity between different nucleotide sequences of DNA or different amino acid sequences of protein. Also, the DNAs and their encoded proteins with (some degree of) homology mentioned in this article shall have the same or better activity at least when used in the function(s) according to the present invention. Similarly, the proteins with (some degree of) homology shall have the same or better activity at least when used in the function(s) according to the present invention. For example, the gene hisG has high similarity with the gene hisG.sup.fbr obtained through mutation on three loci, wherein: the former encodes the ATP-phosphoribosyl transferase and the latter encodes the ATP-phosphoribosyl transferase of which the feedback inhibitory regulation of histidine is removed. These two enzymes are somewhat different in functions and activities as a whole, but they are the same in the function of “the catalyzing enzyme for the first step of reaction of histidine synthesis” according to the present invention. Thus, the gene hisG and the gene hisG.sup.fbr as well as the enzymes encoded by them are DNAs and proteins with homology meaningfully according to the present invention. They are all covered by the protection scope of the present invention.

(23) The execution order of various steps of the methods mentioned in this article, unless otherwise specified, is not limited to those reflected by the text of this article. That is, the execution order of various steps can be subject to change and other step(s) can be inserted between any two steps as necessary.

(24) Below the specific embodiments will be used to further describe the present invention. Unless otherwise specified, the experiment methods used in the following embodiments are all conventional methods. Unless otherwise specified, the materials, reagents, etc used in the following embodiments can all be obtained commercially.

(25) Unless otherwise specified, the technological means employed in the embodiments are the conventional means well known by those skilled in the art. Please see “Molecular Cloning: A Laboratory Method (Rev. 3)” (China Science Press), “Microbiology Experiment (Rev. 4)” (China Higher Education Press) as well as the manufacturer's instructions of corresponding instruments and reagents, etc. The instruments, equipments and reagents used in the embodiments are commonly sold in the market. The quantitative tests in the following embodiments are all repeated three times to calculate the average value for the result.

Embodiment 1: Obtaining L-Histidine Base Engineering Bacteria CG160

(26) Based on the previous studies by the inventor, this embodiment carries out the modification of enhancing histidine synthesis to the wild type of Corynebacterium glutamicum ATCC13032 so as to obtain the base bacteria of the aforementioned multi-target modification according to the present invention. First, replace the promoter of hisEG and hisDCB (two operons of histidine synthetic gene) with the endogenous strong promoter P.sub.glyA of Corynebacterium glutamicum (as shown by No. 863-1038 nucleotide sequence of 5′ end in SEQ ID NO: 7 or as shown by No. 752-927 nucleotide sequence of 5′ end in SEQ ID NO: 8) (Zhang, Y., Shang, X., Lai, S., Zhang, G., Liang, Y., Wen, T., 2012. Development and application of an arabinose-inducible expression aystem by facilitating inducer uptake in Corynebacterium glutamicum. Appl Environ Microbiol. 78, 5831-5838.). Simultaneously, replace the ribosome binding site (RBS) of the genes hisEand hisD with the conserved RBS sequence (AAAGGAGGA) of the highly expressed gene of Corynebacterium glutamicum (as shown by No. 1039-1047 nucleotide sequence of 5′ end in SEQ ID NO: 7 or as shown by No. 928-936 nucleotide sequence of 5′ end in SEQ ID NO: 8), so as to remove the weakening regulation of transcription and translation of the two operons of histidine synthesis genes, and replace the initiation codon GTG of the gene hisE with ATG (as shown by No. 1053-1055 nucleotide sequence of 5′ end in SEQ ID NO: 7) to enhance its expression. Second, replace the encoding gene hisG of the key rate-limiting enzyme ATP-phosphoribosyl transferase (HisG as shown by SEQ ID NO: 6) of the histidine synthesis pathway with the gene hisG.sup.fbr containing three loci of amino acid mutation (as shown by No. 1007-1852 nucleotide sequence of 5′ end in SEQ ID NO: 4), so as to remove the feedback inhibitory regulation of histidine to this enzyme and enhance the catalytic activity of this enzyme (Zhang, Y., Shang, X., Deng, A., Chai, X., Lai, S., Zhang, G., Wen, T., 2012. Genetic and biochemical characterization of Corynebacterium glutamicum ATP phosphoribosyl transferase and its three mutants resistant to feedback inhibition by histidine. Biochimie. 94, 829-838.).

(27) 1.1 Replace the Promoter of the Operon for L-Histidine Synthesis in the Wild Type of Corynebacterium glutamicu ATCC13032 with the Strong Promoter P.sub.glyA

(28) The primers are designed separately according to the operon hisEG of Corynebacterium glutamicu ATCC13032 in Genbank, its upstream and downstream sequences and the P.sub.glyA promoter sequence.

(29) With the genome DNA of Corynebacterium glutamicu ATCC13032 as the template and P1 and P2 as the primers, the upstream homologous arm of the promoter of the hisEG operon is amplified through PCR; the promoter P.sub.glyA is amplified with P3 and P4 as the primers; the downstream homologous arm of the promoter hisEG is amplified with P5 nd P6 as the primers. Then the PCR product above is purified and used as the template, the technique of overlap extension PCR (SOE) is employed with P1 and P6 as the primers to carry out amplification and obtain PCR product of 1920 bp. It is the segment (SEQ ID NO: 7) containing the replacing promoter P.sub.glyA and the upstream and downstream homologous arms of the replaced promoter P.sub.glyA, wherein: No. 1-862 nucleotides of 5′ end in SEQ ID NO: 7 is the upstream homologous arm of the replaced promoter P.sub.hisEG, No. 863-1038 nucleotides of 5′ end in SEQ ID NO: 7 is the promoter P.sub.glyA, No. 1053-1920 nucleotides of 5′ end in SEQ ID NO: 7 is the downstream homologous arm of the replaced promoter P.sub.hisEG.

(30) After double-enzyme digestion by Xba I and BamH I, the PCR product of 1920 bp as above connects with the homologous recombinant vector pK18mobsacB (purchased from American Type Culture Collection-ATCC, product number: 87097) after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing Kanamycin (50 μg/mL). Then, after the transformant is sub-cultured three generations, P13 and P14 are used as the primers and the colony PCR is employed to identify the transformant and then obtain 2132 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by Xba I and BamH I to obtain 1920 bp being positive.

(31) The positive plasmid is sequenced and the result shows it is the recombinant plasmid obtained after the nucleotide as shown by SEQ ID NO: 7 in the sequence table is inserted into the vector pK18mobsacB and named as pK18mobsacB-P.sub.glyA::P.sub.hisEG.

(32) The same method is employed to construct the homologous recombinant plasmid pK18mobsacB-P.sub.glyA::P.sub.hisDCB with the specific requirements as follows: P7 and P8 are used as the primers to amplify the upstream homologous arm of the promoter of the operon hisDCB; P9 and P10 are used as the primers to amplify the promoter P.sub.glyA; P11 and P12 are used as the primers to amplify the downstream homologous arm of the promoter of hisDCB. P7 and P12 are used as the primers and the technique of overlap extension PCR (SOE) is employed to carry out amplification. The PCR product of 1694 bp is obtained. It is the long segment (SEQ ID NO: 8) containing the replacing promoter P.sub.glyA and the upstream and downstream homologous arms of the replaced promoter P.sub.hisDCB, wherein: No. 1-751 nucleotides of 5′ end in SEQ ID NO: 8 is the upstream homologous arm of the replaced promoter P.sub.hisDCB, No. 752-927 nucleotides of 5′ end of SEQ ID NO: 8 is the promoter P.sub.glyA, No. 942-1694 nucleotides of 5′ end of SEQ ID NO: 8 is the downstream homologous arm of the replaced promoter P.sub.hisDCB.

(33) After double-enzyme digestion by Hind III and BamH I, the PCR product of 1694 bp as above connects with the homologous recombinant vector pK18mobsacB after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing Kanamycin (50 μg/mL). After the transformant is sub-cultured three generations, P13 and P14 are used as the primers and the colony PCR is employed to identify the transformant with 1906 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by Hind III and BamH I to obtain 1694 bp being positive. The positive plasmid is sequenced and the result shows it is the recombinant plasmid obtained after the nucleotide as shown by SEQ ID NO: 5 in the sequence table is inserted into the vector pK18mobsacB and named as pK18mobsacB-P.sub.glyA::P.sub.hisDCB.

(34) The sequences of the aforementioned primers used are as follows:

(35) TABLE-US-00001 (SEQ ID NO: 21) P1:  GCTCTAGAGTATCGGCGTGGAGTTGTC (Xba I) (SEQ ID NO: 22) P2:  TAGTGGAGTAGCTTTATTTTGCGACACCTGCC (SEQ ID NO: 23) P3:  GTCGCA AAATAAAGCTACTCCACTAGTGTGATCG (SEQ ID NO: 24) P4:  GGTTCCTCCTTTGCGTAAGACCTCACTCGC (SEQ ID NO: 25) P5:  GAGGTCTTACGCAAAGGAGGAACCGAATGAAGACATTTGA (SEQ ID NO: 26) P6:  CGCGGATCCCAGGATCTGCTGCTCTGG (BamH I) (SEQ ID NO: 27) P7:  CCCAAGCTTCGAGGAAACCGTTGAGGA (Hind III) (SEQ ID NO: 28) P8:  TAGTGGAGTAGCTATGGATTTCACCTCTGTGAATG (SEQ ID NO: 29) P9:  TCTCCACTTTAGGTAAGCTACTCCACTAGTGTGATCG (SEQ ID NO: 30) P10: CGATCCTCCTTTGCGTAAGACCTCACTCGC (SEQ ID NO: 31) P11: GAGGTCTTACGCAAAGGAGGATCGCCATGTTGAATGTC (SEQ ID NO: 32) P12: CGCGGATCCGGCAGAGGCATCAGCAAG (BamH I) (SEQ ID NO: 33) P13: ATGTGCTGCAAGGCGATTAA (SEQ ID NO: 34) P14: TATGCTTCCGGCTCGTATGT (SEQ ID NO: 35) P15: TTTTATATATGGGTATCGGCGGTCTATGCT.

(36) The homologous recombinant plasmid pK18mobsacB-P.sub.glyA::P.sub.hisEG identified through sequencing is electronically transformed into the wild type of Corynebacterium glutamicum ATCC13032. The kanamycin resistance screening is employed to obtain the bacterial colony that the recombinant plasmid is integrated on the chromosome. The sugar screening is employed to obtain the positive bacterial colony after two homologous recombinations. The positive colony is identified through PCR amplification with P15 and P6 as the primers and 948 bp is obtained as the recombinant bacteria and named as Corynebacterium glutamicum WT P.sub.glyA::P.sub.hisEG.

(37) The homologous recombinant plasmid pK18mobsacB-P.sub.glyA::P.sub.hisEG identified through sequencing is electronically transformed into Corynebacterium glutamicum WT-P.sub.glyA::P.sub.hisEG. The kanamycin resistance positive screening is employed to obtain the bacterial colony that the recombinant plasmid is integrated on the chromosome. The sugar inverse screening is employed to obtain the positive bacterial colony after two homologous recombinations. The positive colony is identified through PCR amplification with P15 and P12 as the primers and 833 bp is obtained as the recombinant bacteria and named as Corynebacterium glutamicum CG158 (WT-P.sub.glyA::P.sub.hisEG-P.sub.glyA::P.sub.hisDCB).

(38) After the genome DNA of the recombinant bacterium is extracted and sequenced, the result proves that the promoters of the hisEG and hisDCB in the wild type of Corynebacterium glutamicum ATCC13032 have been replaced successfully with the endogenous strong P.sub.glyA in Corynebacterium glutamicum, the RBS of the genes hisE and hisD is replaced with the conserved RBS sequence (AAAGGAGGA) of the highly expressed gene of Corynebacterium glutamicum, the initiation codon GTG of the gene hisE is replaced with ATG of high expression intensity, thus Corynebacterium glutamicum CG158 (WT-P.sub.glyA::P.sub.hisEG-P.sub.glyA::P.sub.hisDCB) is constructed successfully.

(39) 1. 2. Obtaining L-Histidine Base Engineering Bacteria CG160 Through Site-Directed Mutation of Gene on the Chromosome

(40) The site-directed mutation of the gene hisG on chromosome goes through a procedure of two-step replacement in order to realize three simultaneous site-directed mutations on the gene. First, the homologous recombination is carried out between the long segment containing the chloramphenicol resistant gene Cm.sup.r and the upstream and downstream homologous arm of the mutation segment of the gene hisG as shown by SEQ ID NO: 9 in the sequence table, and CG158 to obtain the recombinant bacteria WT-P.sub.glyA:P.sub.hisEG-Cm.sup.r::hisG-P.sub.glyA::P.sub.hisDCB; then another homologous recombination is carried out between the long segment containing the 264 bp of segment at the end of the gene hisG with three point mutations and its upstream and downstream homologous arms as shown by SEQ ID NO: in the sequence table, and the recombinant bacteria WT-P.sub.hisEG-CM.sup.r::hisG-P.sub.glyA::P.sub.hisDCB to obtain CG160.

(41) The details are as follows:

(42) The genome DNA of Corynebacterium glutamicum ATCC13032 is used as template with P16 and P17 as the primers to carry out PCR amplification on the upstream homologous arms of the mutation segment of the gene hisG, P18 and P19 are used as the primers to amplify the downstream homologous arm of the mutation segment of the gene hisG; P20 and P21 are used as the primers with the plasmid pXMJ19 (purchased from Biovector Science Lab, Inc. Product number: SMD1168H) as the template to amplify the chloramphenicol resistant gene Cm.sup.r. Then the PCR product above is purified and used as the template with P16 and P21 as the primers to carry out amplification with the technique of overlap extension PCR (SOE) and obtain 1689 bp of long segment (SEQ ID NO: 9) containing the chloramphenicol resistant gene Cm.sup.r and the upstream and downstream homologous arms of the mutation segment of the gene hisG, wherein: No. 1-420 nucleotides of 5′ end in SEQ ID NO: 9 is the upstream homologous arm of the mutation segment of the gene hisG, No. 421-1281 nucleotides of 5′ end in SEQ ID NO: 9 is the chloramphenicol resistant gene Cm.sup.r, No. 1282-1689 nucleotides of 5′ end in SEQ ID NO: 9 is the downstream homologous arm of the mutation segment of the gene hisG.

(43) The genome DNA of Corynebacterium glutamicum is used as the template with P28 and P29 as the primers to amplify the segment of the gene hisG containing C645G (No. 215 asparagine is mutated to lysine) mutation locus, P30 and P31 are used as the primers to amplify the segment hisG containing the mutation loci A693C and A703G (No. 231 leucine is mutated to phenylalanine and No. 235 threonine is mutated to alanine). Then the PCR product above is purified and used as the template with P28 and P31 as the primers to carry out amplification with the technique of overlap extension PCR (SOE) and obtain 846 bp of the gene hisG containing three point mutations (No. 1007-1852 nucleotides of 5′ end of SEQ ID NO: 4). P16 and P22 are used as the primers to carry out PCR amplification on the upstream homologous arm of the site-directed mutation of the gene hisG, P25 and P21 are used as the primers to amplify the downstream homologous arm of the site-directed mutation of the gene hisG; P23 and P24 are used as the primers and the gene hisG containing three point mutations obtained as above is used as the template to amplify the 264 bp of segment at the end of the gene hisG containing three point mutations. Then the PCR product above is purified and used as the template with P16 and P21 as the primers to carry out amplification with the technique of overlap extension PCR (SOE) and obtain 1092 bp of long segment (SEQ ID NO: 10) containing 264 bp of segment at the end of the gene hisG with three point mutations and its upstream and downstream homologous arms, wherein: No. 1-420 nucleotides of 5′ end in SEQ ID NO: 10 is the upstream homologous arm, No. 421-684 nucleotides of 5′ end in SEQ ID NO: 10 is the 264 bp of segment at the end of the gene hisG containing three point mutations, No. 685-1092 nucleotides of 5′ end in SEQ ID NO: 10 is the downstream homologous arm.

(44) After double-enzyme digestion, two PCR products after extraction and purification connect with the knocking-out vector pK18mobsacB after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia Coli DH5α and the transformant is screened on LB plate containing kanamycin (50 μg/mL). After the transformant is sub-cultured three generations, P13 and P14 are used as the primers and the colony PCR is employed to identify the transformant and obtain the positive transformants of 1901 bp and 1304 bp separately containing two types of recombinant plasmids. The plasmids of the transformants after identified are extracted and identified through double-enzyme digestion by BamH I and EcoR I to obtain two recombinant plasmids respectively of 1689 bp and 1092 bp. After further verified through sequencing, the recombinant plasmids pK18mobsacB-Cm.sup.r::hisG and pK18mobsacB-hisG.sup.fbr::Cm.sup.r are constructed successfully.

(45) The pK18mobsacB-Cm.sup.r::hisG is the recombinant vector obtained through inserting the long segment (SEQ ID NO: 9) containing the chloramphenicol resistant gene Cm.sup.r and the upstream and downstream homologous arms of the mutation segment of the gene hisG into the vector pK18mobsacB.

(46) pK18mobsacB-hisG.sup.fbr::Cm.sup.r is the recombinant vector obtained through inserting the long segment (SEQ ID NO: 10) containing the 264 bp of segment at the end of the gene hisG with three point mutatios and its upstream and downstream homologous arms into the vector pK18mobsacB.

(47) The sequences of the primers used above are as follows:

(48) TABLE-US-00002 (SEQ ID NO: 36) P16: CGCGGATCCATCTACGTTGCTGGTGGC (BamH I) (SEQ ID NO: 37) P17: ACGGGCAACAGCTGCTGCTCTGGGGTGAC (SEQ ID NO: 38) P18: CAGAGCAGCAGCTGTTGCCCGTCTCACTGGT (SEQ ID NO: 39) P19: GGTAGTTAAAATTACGCCCCGCCCTGCCACT (SEQ ID NO: 40) P20: GCGGGGCGTAATTTTAACTACCCCCGAAAAT (SEQ ID NO: 41) P21: CCGGAATTCCGAATGAAATCTGGGACG (EcoR I) (SEQ ID NO: 42) P22: CGAAGCAGGATCTGCTGCTCTGGGGTGAC (SEQ ID NO: 43) P23: CAGAGCAGCAGATCCTGCTTCGCCGCATCCA (SEQ ID NO: 44) P24: GGTAGTTAAAACTAGATGCGGGCGATGCG (SEQ ID NO: 45) P25: CCCGCATCTAGTTTTAACTACCCCCGAAAAT (SEQ ID NO: 46) P26: TCCCAAACAAAGGCTCGC (SEQ ID NO: 47) P27: CAGTCGGCGGTTTGCTAA (SEQ ID NO: 48) P28: ATGTTGAAAATCGCTG (SEQ ID NO: 49) P29: TTACTGCAGTGGCAGCGTCCAGGTTGTCGCGGTCGACCTTGTAAT CCAGCAT (SEQ ID NO: 50) P30: ACCTGGACGCTGCCACTGCAGTAACCCCAGGCTTCTCCGGCCCAG CGGTATC (SEQ ID NO: 51) P31: CTAGATGCGGGCGATGCGG.

(49) The homologous recombinant plasmid pK18mobsacB-Cm.sup.r::hisG identified through sequencing is electronically transformed into Corynebacterium glutamicum CG158 and the kanamycin resistance screening is employed to obtain the bacterial colony that the recombinant plasmid is integrated on the chromosome and the sugar screening is employed to obtain the positive bacteria after two homologous recombinations. P26 and P27 are used as the primers to carry out PCR amplification and identification on the positive bacteria and obtain 1872 bp of recombinant bacteria WT-P.sub.glyA::P.sub.hisEG-Cm.sup.r::hisG-P.sub.glyA::P.sub.hisDCB.

(50) The homologous recombinant plasmid identified through sequencing pK18mobsacB-hisG.sup.fbr::Cm.sup.r is electronically transformed into the aforementioned constructed recombinant bacteria WT-P.sub.oyA::P.sub.hisEG-Cm.sup.r::hisG-P.sub.glyA::P.sub.hisDCB and the kanamycin resistance screening is employed to obtain the bacterial colony where the recombinant plasmid is integrated onto the chromosome and the sugar screening is employed to obtain the positive bacterial colony after two homologous recombinations. P26 and P27 are used as the primers to carry out PCR amplification and identification on the positive colony and obtain 1275 bp of the recombinant bacteria which is named as Corynebacterium glutamicum CG160 (WT-P.sub.glyA::P.sub.hisEG-hiSG.sup.fbr-P.sub.glyA::P.sub.hisDCB).

(51) After the genome DNA of the recombinant bacterium is extracted and sequenced, the result proves that the N215K/L231F/T235A of the gene hisG of the chromosome of Corynebacterium glutamicum CG158 have successful point mutation and Corynebacterium glutamicum CG160 (WT-P.sub.hisEG-SG.sup.fbr-P.sub.hisDCB) is constructed successfully.

(52) The point mutations of N215K/L231F/T235A of the gene hisGare to mutate No. 215 asparagine of ATP-phosphoribosyl transferase (HisG) encoded by the gene hisGto lysine, No. 231 leucine to phenylalanine and No. 235 threonine to alanine.

Embodiment 2: Construction of L-Histidine High-Yield Recombinant Bacteria CG171 Containing Plasmid

(53) In this embodiment, on the basis of the primary engineering bacteria obtained in Embodiment 1, the gene prsAis further over-expressed and the gene hisG.sup.fbr (No. 1007-1852 nucleotide sequence in SEQ ID NO: 4) is also over-expressed. Then knocking-out of the gene pgi (SEQ ID NO: 13) and over-expression of the gene zwf-opcA (SEQ ID NO: 2) are combined to obtain the high-yield engineering bacteria CG171.

(54) 2.1 Construction of L-Histidine Primary Engineering Bacteria CG176

(55) The gene prsA encodes the PRPP synthetase (PrsA as shown by SEQ ID NO: 5. PRPP is the precursor substance for histidine synthesis), enhances the expression of the gene prsA in order to increase the synthesis of the precursor PRPP for histidine synthesis and provides more precursor substances for histidine synthesis.

(56) On the basis of the base engineering bacteria CG160 obtained in Embodiment 1, both the gene prsA (as shown by No. 15-992 nucleotide sequence of 5′ end in SEQ ID NO: 4) and the gene hisG.sup.fbr (as shown by No. 1007-1852 nucleotide of 5′ end in SEQ ID NO: 4) are over-expressed in order to obtain the primary engineering bacteria CG176 with higher histidine yield. Thus, it will be convenient to implement the strategy according to the present invention and achieve a better performance. Of course, the skilled in the art may easily understand that the modification strategy according to the present invention shall not only be limited to recombination and modification on the primary engineering bacteria obtained in this embodiment, it can also be applied to other engineering bacteria of histidine.

(57) The genome DNA of the strain CG160 is used as the template with P32/P33 and P34/P35 respectively as the primers to carry out PCR amplification on the gene prsA (992 bp) and the gene hisG.sup.fbr (860 bp). The overlap extension PCR is employed to connect both genes and the amplified genes of hisG.sup.fbr and prsA are used as the template with P32 and P35 as the primers to carry out PCR amplification. The obtained 1852 bp of PCR product is the segment prsA-hisG.sup.fbr (SEQ ID NO: 4), wherein: No. 15-992 nucleotides of 5′ end in SEQ ID NO: 4 is prsA, No. 1007-1852 nucleotides of 5′ end in SEQ ID NO: 4 is hisG.sup.fbr (the gene hisG containing three point mutations).

(58) After double-enzyme digestion by Xba I and Sma I, the PCR product as above connects with the shuttle expression plasmid pXMJ19 of Corynebacterium glutamicum-Escherichia coli after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing chloramphenicol (20 μg/mL). After the transformant is sub-cultured three generations, P36 and P37 are used as the primers and the colony PCR is employed to identify the transformant and obtain 2054 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by Xba I and Sma I to obtain 1852 bp being positive.

(59) The pXMJ19-prsA-hisG.sup.fbr is further sequenced and analyzed. This plasmid is the vector pXMJ19-prsA-hisG.sup.fbr obtained through inserting the segment prsA-hisG.sup.fbr (SEQ ID NO: 4) between the enzyme digestion sites of Xba I and Sma I of the plasmid pXMJ19. It is named as the recombinant plasmid pWYE 1230 (as shown in FIG. 1).

(60) The plasmid pXMJ19-prsA-hisG.sup.fbr is transformed into the base engineering bacteria CG160 constructed as above. P36 and P37 are used as the primers and the colony PCR is employed to identify the transformant and obtain 2054 bp being the positive transformant. The plasmid of the transformant identified is extracted and identified to further confirm that the over-expressed plasmid is successfully transformed into the engineering bacteria and the L-histidine engineering bacteria CG176 WT-P.sub.glyA::P.sub.hisEG-hisG.sup.fbr-P.sub.glyA::P.sub.hisDCB/PXMJ19-prsA-hisG.sup.fbr) is constructed successfully.

(61) The sequences of the primers used above are as follows:

(62) TABLE-US-00003 (SEQ ID NO: 52) P32: GCTCTAGAAAAGGAGGATCCTCATGACTGCTCACTGG (Xba I) (SEQ ID NO: 53) P33: TTGTCCTCCTTTTTAGGCCTCGCCCTCGAA (SEQ ID NO: 54) P34: GGCGAGGCCTAAAAAGGAGGACAATCATGTTGAAAATCGCTG (SEQ ID NO: 55) P35: TCCCCCGGGCTAGATGCGGGCGATGCGG (Sma I) (SEQ ID NO: 56) P36: CAATTAATCATCGGCTCGTA (SEQ ID NO: 57) P37: ACCGCTTCTGCGTTCTGATT.
2.2 Obtaining L-Histidine Primary Engineering Bacteria CG161 and CG172

(63) The gene pgi encodes the glucose phosphate isomerase (Pgi as shown by SEQ ID NO: 14). On the basis of the base bacteria CG160 obtained as above, the gene pgi is knocked out (SEQ ID NO: 13) to obtain the primary engineering bacteria CG161. On the basis of CG161, both the gene prsA and the gene hisG.sup.fbr are over-expressed to obtain the engineering bacteria CG172 in which the pgi gene is knocked out.

(64) The primary engineering bacteria CG161 is obtained through knocking out the gene pgi (SEQ ID NO: 13) from the L-histidine base engineering bacteria CG160. The details are as follows:

(65) First, the primers are separately designed according to the gene pgi of the Corynebacterium glutamicum ATCC13032 and its upstream and downstream sequences in Genbank.

(66) The genome DNA of Corynebacterium glutamicum ATCC13032 is used as the template with P38 and P39 as the primers to carry out PCR amplification on the upstream homologous arm of the gene pgi; P40 and P41 are used as the primers to amplify the downstream homologous arm of the gene pgi. Then the PCR product as above is purified and used as the template with P38 and P41 as the primers to carry out amplification with the technique of overlap extension PCR (SOE). 1672 bp of segment containing the upstream and downstream homologous arms of the gene pgi to be knocked out is obtained (SEQ ID NO: 1), wherein: No. 1-834 nucleotides of 5′ end in SEQ ID NO: 1 is the upstream homologous arm of the gene pgi to be knocked out, No. 835-1672 nucleotides of 5′ end in SEQ ID NO: 1 is the downstream homologous arm of the gene pgi to be knocked out.

(67) After double-enzyme digestion by BamH I and EcoR I, the purified and extracted PCR product connects with the homologous recombinant vector pK18mobsacB after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing kanamycin (50 μg/mL). After the transformant is sub-cultured three generations, P13 and P14 are used as the primers and the colony PCR is employed to identify the transformant and obtain 1884 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by BamH I and EcoR I to obtain 1672 bp being positive. After further verified through sequencing, the recombinant plasmid pK18mobsacB-Δpgi is constructed successfully. It is the vector obtained through inserting the segment (SEQ ID NO: 1) containing the upstream and downstream homologous arms of the gene pgi to be knocked out between the enzyme digestion sites of BamH I and EcoR I of the vector pK18mobsacB.

(68) The sequences of the primers used are as follows:

(69) TABLE-US-00004 (SEQ ID NO: 58) P38: CGCGGATCCGCTCTTTCGGAGTGACCT (BamH I) (SEQ ID NO: 59) P39: TAAGCAAGCGAGAAAACTCCTTTATTGTCG (SEQ ID NO: 60) P40: TAAAGGAGTTTTCTCGCTTGCTTATAGGGTC (SEQ ID NO: 61) P41: CCGGAATTCTCGGGAAGCAGTTAGTGAAA (EcoR I) (SEQ ID NO: 62) P42: TTGACGACGCAAGAGCCA (SEQ ID NO: 63) P43: CACCATTACCGATGAGAAAC.

(70) The homologous recombinant plasmid pK18mobsacB-Δpgi identified through sequencing is electronically transformed into the Corynebacterium glutamicum CG160. The kanamycin resistance screening is employed to obtain the bacterial colony that the recombinant plasmid is integrated on the chromosome and the sugar screening is employed to obtain the bacterial colony after a second homologous recombination. P42 and P43 are used as the primers for PCR identification with the extracted genome DNA of the colony as the template so as to obtain 1759 bp being positive (FIG. 2). It is named as CG161 (WT-P.sub.glyA::P.sub.hisEG-hiSG.sup.fbr-P.sub.glyA::P.sub.hisDCB-ΔPgi).

(71) The CG161 (WT-P.sub.glyA::P.sub.hisEG-hiSG.sup.fbr-P.sub.glyA::P.sub.hisDCB-ΔPgi) is further sequenced and analyzed.

(72) The result shows that the gene pgi of the chromosome of the L-histidine base engineering bacteria CG160 is knocked out successfully and CG161 is constructed successfully.

(73) The engineering bacteria CG172 is the recombinant bacteria (WT-P.sub.glyA::P.sub.hisEG-hisG.sup.fbr-P.sub.glyA::P.sub.hisDCB-Δpgi/pXMJ19-prsA-hisG.sup.fbr) obtained through introducing the plasmid pXMJ19-prsA-hisG.sup.fbr into the engineering bacteria CG161. The specific operating methods are conventional and hence omitted here.

(74) 3. Construction of L-Histidine High-Yield Engineering Bacteria CG171 and Comparative Engineering Bacteria CG173

(75) The gene zwf-opcA encodes the glucose-6-phosphate dehydrogenase (Zwf-OpcA as shown by SEQ ID NO: 3 where No. 1-514 amino acids of 5′ end constitute Zwf subunit and No. 515-833 amino acids constitute OpcA subunit). The combinational modification through knocking out the gene pgi and over-expression the gene zwf-opcA (SEQ ID NO: 2) is carried out to obtain the high-yield engineering bacteria CG171. As comparison, the engineering bacteria CG173 whose gene pgi is not knocked out but the gene zwf-opcA is over-expressed is obtained.

(76) The primer is designed according to the gene sequence zwf-opcA of Corynebacterium glutamicum ATCC13032 in Genbank. The genome DNA of Corynebacterium glutamicum ATCC13032 is used as the template with the primers P44 and P45 as the primers to carry out PCR amplification on 2519 bp of the segment zwf-opcA (the initiation codon of the gene zwf is replaced from GTG to ATG in order to enhance its expression) (SEQ ID NO: 2). After double-enzyme digestion by Hind III and Xba I, it connects with the expression plasmid pXMJ19 after the same double-enzyme digestion to obtain the recombinant plasmid pXMJ19-zwf-opcA. The pXMJ19-zwf-opcA is further processed through double-enzyme digestion by XbaI and SmaI and then connects with 1852 bp of the segment prsA-hisG.sup.fbr obtained through double-enzyme digestion by Xba I and Sma I of the plasmid pXMJ19-prsA-hisG.sup.fbr prepared above.

(77) In the segment zwf-opcA, No. 1-1545 nucleotides of 5′ end in SEQ ID NO: 2 is the gene zwf and No. 1560-2519 nucleotides of 5′ end in SEQ ID NO: 2 is the gene opcA.

(78) The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing chloramphenicol (20 μg/mL). After the transformant is sub-cultured three generations, P36 and P37 are used as the primers and the colony PCR is employed to identify the transformant and obtain 4587 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by Xba I/Sma I and Hind III/Xba I to obtain separately 1852 bp and 2533 bp being positive.

(79) After verified through sequencing, the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr is constructed successfully and named as the recombinant plasmid pWYE 1229 (FIG. 3). It is the vector obtained through inserting the gene zwf-opcA (SEQ ID NO: 2) between the sites of Hind III and Xba I of pXMJ19 as well as inserting the segment prsA-hisG.sup.fbr (SEQ ID NO: 4) between the sites of Xba I and Sma I.

(80) TABLE-US-00005 (SEQ ID NO: 64) P44: CCCAAGCTTAAAGGAGGACCATCATGAGCACAAACACGACCCCCT (Hind III) (SEQ ID NO: 65) P45: GCTCTAGATTAGACGGTTTCCAGCTTG (Xba I)

(81) The recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr is electronically transformed respectively into the engineering bacteria CG160 without pgi deletion and the engineering bacteria CG161 with pgi deletion. P36 and P37 are used as the primers and the colony PCR is employed to identify the transformant. 4587 bp is obtained as the positive transformant. The plasmid of the identified transformant is extracted.

(82) The plasmid is sequenced and the result shows that the engineering bacteria CG173 of L-histidine (WT-P.sub.glyA::P.sub.hisEG-hisG.sup.fbr-P.sub.glyA::P.sub.hisDCB/pXMJ19-zwf-opcA-prsA-hisG.sup.fbr) contains the plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr. It is the bacteria obtained through introducing the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr into the engineering bacteria CG160.

(83) The CG171 (WT-P.sub.glyA::P.sub.hisEG-hiSG.sup.fbr-P.sub.glyA::P.sub.hisEG-hisG.sup.fbr-P.sub.glyA::P.sub.hisDCB-Δpgi/pXMJ19-zwf-opcA-prsA-hisG.sup.fbr) contains the plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr. It is the bacteria obtained through introducing the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr into the engineering bacteria CG161.

(84) The expression of the gene carried by the over-expressed plasmid is further verified. The cell lysis solution of CG171 is prepared to carry out SDS-PAGE test. The result is as shown in FIG. 4 where Lane 1 and 2 are the cell lysis solutions of CG171, Lane 3 is the cell lysis solution of ATCC13032/pXMJ19 (obtained through introducing the plasmid pXMJ19 into ATCC13032). After comparison, it shows that the genes zwf (57.5 kDa), opcA (34.8 kDa), prsA (35.6 kDa) and hisG.sup.fbr (30.2 kDa) carried by the over-expressed plasmid are expressed successfully in the engineering bacteria.

(85) The specific activity of glucose-6-phosphate dehydrogenase (Zwf-opcA) in the engineering bacterium is further determined. The reaction system for determination (0.5 mL) is as follows: 100 mmol/L Tris-HCl (pH 7.8), 200 mmol/L KCl, 1 mmol/L NADP, 10 mmol/L MgCl.sub.2, 5 mmol/L glucose-6-phosphate (G6P) and appropriate amount of cell lysis solution. The reaction is carried out at 30° C. for 5 minutes. The yield of NADPH is reflected through detecting the change of light absorbance at 340 nm. The enzyme activity unit (U) is defined as the amount of enzyme needed to produce 1nmol nicotinamide adenine dinucleotide phosphate (NADPH) in reduced form in every minute. The result is as shown in FIG. 5. Compared with the wild type of strains, the specific activity of glucose-6-phosphate dehydrogenase after over-expression of zwf-opcA through plasmid is improved by 34 times.

Embodiment 3: Construction of L-Histidine High-Yield Engineering Bacteria CG319 Containing Plasmid

(86) On the basis of the high-yield engineering bacteria CG171 obtained as above, in order to further over-express the gene purH encoding AICAR transmethylase/IMP ring hydrase (PurH as shown by SEQ ID NO: 16) and hence guide more by-product AICAR increased due to enhanced synthetic pathway of histidine to the synthetic pathway of purine nucleotides, the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr-purH is constructed and introduced into the primary bacteria CG161 to obtain the high-yield engineering bacteria.

(87) The primer is designed according to the gene sequence purH of Corynebacterium glutamicum ATCC13032 in Genbank. The genome DAN of ATCC13032 is used as the template and P46 and P47 are used as the primers to carry out PCR amplification on the gene purH (1563 bp) (SEQ ID NO: 15).

(88) After double-enzyme digestion of Sma I and EcoR I, the PCR product as above connects with the shuttle expression plasmid pXMJ19 of Corynebacterium glutamicum-Escherichia coli after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing chloramphenicol (20 μg/mL). After the transformant is sub-cultured three generations, P52 and P53 are used as the primers and the colony PCR is employed to identify the transformant and obtain 1779 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by XbaI and Sma I and obtain 1577 bp being positive and named as the recombinant plasmid pXMJ19-purH.

(89) The recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr is used as the template and P48/P49 and P50/P51 are respectively used as the primer to carry out PCR amplification on the segments zwf-opcA (2519 bp) and prsA-hisG.sup.fbr (1852 bp). The overlap extension PCR is employed to connect both segments to obtain 4385 bp of the segment zwf-opcA-prsA-hisG.sup.fbr (SEQ ID NO: 17), wherein: No. 15-2533 nucleotides of 5′ end in SEQ ID NO: 17 is zwf-opcA and No. 2534-4385 nucleotides of 5′ end in SEQ ID NO: 17 is prsA-hisG.sup.fbr.

(90) After double-enzyme digestion by Xba I and Sma I, the PCR product as above connects with the recombinant plasmid pXMJ19-purH after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing chloramphenicol (20 μg/mL). After the transformant is sub-cultured three generations, P52 and P53 are used as the primers and the colony PCR is employed to identify the transformant and obtain the 6164 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by Xba I and Sma I to obtain 4385 bp being positive and named as the recombinant plasmid pWYE1507 pXMJ19-zwf-opcA-prsA-hisG.sup.fbr-purH) (as shown in FIG. 6).

(91) The pXMJ19-zwf-opcA-prsA-hisG.sup.fbr-purH is further sequenced and analyzed. The result shows that this plasmid is the vector obtained through inserting the segment zwf-opcA-prsA-hisG.sup.fbr(SEQ ID NO: 17) between the enzyme digestion sites Xba I and Sma I of pXMJ19 as well as inserting purH between the enzyme digestion sites Sma I and EcoR I of the plasmid pXMJ19.

(92) The plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr-purH is transformed into the engineering bacteria CG161. P52 and P53 are used as the primers and the colony PCR is employed to identify the transformant and obtain 6164 bp being the positive transformant. The plamid of the transformant after identified is extracted and further confirmed that the over-expressed plasmid is successfully transformed into the engineering bacteria and the engineering bacteria of L-histidine CG319 (WT-P.sub.glyA::P.sub.hisEG-hisG.sup.fbr-P.sub.glyA::P.sub.hisDCB-Δpgi/pXMJ19-zwf-opcA-prsA-hisG.sup.fbr-purH) is constructed successfully.

(93) The sequences of the primers used above are as follows:

(94) TABLE-US-00006 (SEQ ID NO: 66) P46: TCCCCCGGGAAAGGAGGACCTTCATGAGCGATGATCGTAAG (Sma I) (SEQ ID NO: 67) P47: CCGGAATTCTTAGTGAGCGAAGTGTCGCG (EcoR I) (SEQ ID NO: 68) P48: GCTCTAGAAAAGGAGGACCATCATGAGCACAAACACGACCC (Xba I) (SEQ ID NO: 69) P49: AGTCATGAGGATCCTCCTTTTTAGACGGTTTCCAGCTTG (SEQ ID NO: 70) P50: TCAAGCTGGAAACCGTCTAAAAAGGAGGATCCTCATGACTGCTCA CTG (SEQ ID NO: 71) P51: TCCCCCGGGCTAGATGCGGGCGATGCGGATTTC(Sma I) (SEQ ID NO: 72) P52: CAATTAATCATCGGCTCGTA (SEQ ID NO: 73) P53: ACCGCTTCTGCGTTCTGATT

Embodiment 4: Construction of L-Histidine High-Yield Engineering Bacteria CG328 Containing Plasmid

(95) On the basis of CG139 obtained as above, in order to weaken the gene purF encoding the amidophosphoribosyl transterase (PurF, SEQ ID NO: 19) and increase the distribution of the precursor substance PRPP to the synthetic pathway of histidine, the promoter of purF in the primary engineering bacteria CG161 is replaced with P.sub.hom to obtain CG327 and then the plasmid is introduced to obtain the high-yield engineering bacteria CG328.

(96) The promoter of the gene purF of the primary engineering bacteria CG161 is replaced with P.sub.hom to obtain the engineering bacteria CG327. The details are as follows:

(97) First, the primers are designed separately according to the gene purF of Corynebacterium glutamicum ATCC13032 and its upstream and downstream sequences in Genbank.

(98) The genome DNA of Corynebacterium glutamicum ATCC13032 is used as the template with P54 and P55 as the primers to carry out PCR amplification on the upstream homologous arm of the gene purF; P56 and P57 are used as the primers to amplify the promoter P.sub.hom, P58 and P59 are used as the primers to amplify the downstream homologous arm of the gene purF. Then the PCR product as above is purified and used as the template with P54 and P59 as the primers to carry out amplification with the technique of overlay extension PCR (SOE) and obtain 1654 bp of the segment containing the promoter P.sub.hom and the upstream and downstream homologous arms of the promoter of the gene purF (SEQ ID NO: 18), wherein: No. 1-735 nucleotides of 5′ end in SEQ ID NO: 18 is the upstream of the promoter of the gene purF, No. 736-865 nucleotides of 5′ end in SEQ ID NO: 18 is the promoter P.sub.hom, No. 866-1654 nucleotides of 5′ end in SEQ ID NO: 18 is the downstream homologous arm of the promoter of the gene purF.

(99) After double-enzyme digestion by BamH I and EcoR I, the purified and extracted PCR product connects with the homologous recombinant vector pK18mobsacB after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing kanamycin (50 μg/mL). After the transformant is sub-cultured three generations, P13 and P14 are used as the primers and the colony PCR is employed to identify the transformant and obtain 1866 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by BamH I and EcoR I and obtain 1654 bp being positive. After further verified through sequencing, the recombinant plasmid pK18mobsacB-P.sub.hom::P.sub.purF is constructed successfully. It is the vector obtained through inserting the segment (SEQ ID NO: 18) containing the promoter P.sub.hom and the upstream and downstream homologous arms of the promoter to be replaced between the enzyme digestion sites BamH I and EcoR I of the vector pK18mobsacB.

(100) The sequences of the primers used above are as follows:

(101) TABLE-US-00007 (SEQ ID NO: 74) P54: CGCGGATCCTCCGCAGAAAGCACCTCA (BamH I) (SEQ ID NO: 75) P55: TTTAGTTTTCAACGGCTAAAGTTTGACCACTGG (SEQ ID NO: 76) P56: GTGGTCAAACTTTAGCCGTTGAAAACTAAAAAGC (SEQ ID NO: 77) P57: TCCGGTCCTCCTTTTACTTTGTTTCGGCCACCC (SEQ ID NO: 78) P58: GGCCGAAACAAAGTAAAAGGAGGACCGGAATGACCCAGGTAAACC AC (SEQ ID NO: 79) P59: CCGGAATTCAACCTTTGCGGGTTGTCT (EcoR I)

(102) The homologous recombinant plasmid pK18mobsacB-P.sub.hom::P.sub.purF after identified through sequencing is electronically transformed into Corynebacterium glutamicum CG161 and the kanamycin resistance screening is employed to obtain the bacterial colony that the recombinant plasmid is integrated on the chromosome and the sugar screening is employed to obtain the bacterial colony after a second homologous recombination. P56 and P59 are used as the primers to extract the genome DAN of the colony and carry out PCR amplification and identification and obtain 905 bp being positive. It is named as CG327 (WT-P.sub.glyA::P.sub.hisEG-hisG.sup.fbr-P.sub.glyA::P.sub.hisDCB-Δpgi::P.sub.hom::P.sub.purF). CG327 (WT-P.sub.glyA::P.sub.hisEG-hiSG.sup.fbr-P.sub.glyA::P.sub.hisDCB-Δpgi::P.sub.hom::P.sub.purF) is further sequenced and analyzed. The result shows that the promoter of the gene purF of the chromosome of the L-histidine primary engineering bacteria CG161 is replaced with P.sub.hom and CG327 is constructed successfully.

(103) The engineering bacteria CG328 is the recombinant bacteria WT-P.sub.glyA::P.sub.hisEG-hiSG.sup.fbr-P.sub.glyA::P.sub.hisDCB-Δpgi::P.sub.hom:P.sub.purF/pXMJ19-zwf-opcA-prsA-hisG.sup.fbr-purH) obtained through introducing the plasmid pXMJ19-zwf-opcA-prsA-hisG.sup.fbr-purH into the engineering bacteria CG327. The specific operating methods are similar to those preparing the engineering bacteria CG319 as above and conventional, so are not detailed here. PCR identification is carried out on the plasmid carried by the strain CG328. P52 and P53 are used as the primers to obtain 6164 bp of segment (FIG. 7). This DNA segment is sequenced and the result shows that it is the segment zwf-opcA-prsA-hisG.sup.fbr-purH and the strain CG328 is constructed successfully.

Embodiment 5: Construction of L-Histidine High-Yield Recombinant Bacteria CG351 Containing No Plasmid

(104) Carrying plasmid can impose metabolic burden on engineering bacteria and is not favorable to control the industrial fermentation of engineering bacteria and the safety of fermented products. Thus, in this embodiment, the expression of the gene carrying plasmid is increased on chromosome to construct a histidine engineering bacteria containing no plasmid so as to reduce the metabolic burden of engineering bacteria and realize the maximum conversion from the fermentation substrate(s) to the product(s).

(105) In this embodiment, further modification is carried out on the basis of the primary engineering bacteria CG161 whose the gene pgi is knocked out: use the promoter P.sub.sod to replace the promoter of the gene prsA in order to enhance the expression of PRPP synthetase (PrsA) and hence obtain CG350; moreover, use the promoter P.sub.eftu to replace the promoter of the operon tkt-tal-zwf-opcA-devB in order to improve the expression of glucose-6-phosphate dehydrogenase (Zwf-OpcA) and hence obtain CG351.

(106) The primers are designed separately according to the gene prsA of Corynebacterium glutamicum ATCC13032 in Genbank and its upstream and downstream sequences as well as the subsequence of the promoter P.sub.sod.

(107) The genome DNA of Corynebacterium glutamicum ATCC13032 is used as the template with P60 and P61 as the primers to amplify the upstream homologous arm of the promoter prsA; P62 and P63 are used as the primers to amplify the promoter P.sub.sod; P64 and P65 are used as the primers to amplify the downstream homologous arm of the promoter prsA. Then the PCR product as above is purified and used as the template. P60 and P65 are used as the primers and the technique of overlap extension PCR (SOE) is employed to carry out amplification and obtain 1455 bp of PCR product. It is the segment (SEQ ID NO: 11) containing the replacing promoter P.sub.sod and the upstream and downstream homologous arms of the replaced promoter P.sub.prsA; wherein: No. 1-655 nucleotides of 5′ end in SEQ ID NO: 11 is the upstream homologous arm of the replaced promoter P.sub.prsA, No. 656-847 nucleotides of 5′ end in SEQ ID NO: 11 is the promoter P.sub.sod, No. 848-1455 nucleotides of 5′ end in SEQ ID NO: 11 is the downstream homologous arm of the replaced promoter P.sub.prsA.

(108) After double-enzyme digestion by Hind III and BamH I, the PCR product of 1455 bp as above connects with the homologous recombinant vector pK18mobsacB after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing kanamycin (50 μg/mL). After the transformant is sub-cultured three generations, P13 and P14 are used as the primers and the colony PCR is employed to identify the transformant and obtain 1667 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by Hind III and BamH I to obtain 1455 bp being positive.

(109) The positive plasmid is sequenced and the result shows that this plasmid is the recombinant plasmid obtained through inserting the nucleotide as shown by SEQ ID NO:

(110) in the sequence table into the vector pK18mobsacB and named as pK18mobsacB-P.sub.sod::P.sub.prsA.

(111) The same method is employed to construct the homologous recombinant plasmid pK18mobsacB-P.sub.eftu::P.sub.tkt and the promoter of the operon tkt-tal-zwf-opcA-devB is replaced with the strong promoter P.sub.eftu. The details are as follows: use P66 and P67 as the primers to amplify the upstream homologous arm of the promoter of the operon tkt-tal-zwf-opcA-devB; use P68 and P69 as the primers to amplify the promoter P.sub.eftu; use P70 and P71 as the primers to amplify the downstream homologous arm of the promoter of the operon tkt-tal-zwf-opcA-devB. P66 and P71 are used as the primers and the technique is of overlap extension PCR (SOE) is employed to carry out amplification. The PCR product of 1512 bp is obtained and it is the long segment (SEQ ID NO: 12) containing the replacing promoter P.sub.etfu and the upstream and downstream homologous arms of the replaced promoter P.sub.tkt, wherein: No. 1-634 nucleotides of 5′ end in SEQ ID NO: 12 is the upstream homologous arm of the replaced promoter P.sub.tkt, No. 635-834 nucleotides of 5′ end in SEQ ID NO: 12 is the promoter P.sub.eftu, No. 835-1512 nucleotides of 5′ end in SEQ ID NO: 12 is the downstream homologous arm of the replaced promoter P.sub.tkt.

(112) After double-enzyme digestion by Hind III and BamH I, the PCR product of 1512 bp as above connects with the homologous recombinant vector pK18mobsacB after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing kanamycin (50 μg/mL). After the transformant is sub-cultured three generations, P13 and P14 are used as the primers and the colony PCR is employed to identify the transformant and obtain 1724 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by Hind III and BamH I to obtain 1512 bp being positive. The positive plasmid is sequenced and the result shows that this plasmid is the recombinant plasmid obtained through inserting the nucleotides as shown by SEQ ID NO: 12 in the sequence table into the vector pK18mobsacB and named as pK18mobsacB-P.sub.eftu::P.sub.tkt.

(113) The sequences of the primers used above are as follows:

(114) TABLE-US-00008 (SEQ ID NO: 80) P60: CCCAAGCTTTCCAGCAACCACCTGGAT (Hind III) (SEQ ID NO: 81) P61: AATTGGCAGCTATTAGCCTTCCTGGTTGTG (SEQ ID NO: 82) P62: CAGGAAGGCTAATAGCTGCCAATTATTCCG (SEQ ID NO: 83) P63: TTGTCCTCCTTTGGGTAAAAAATCCTTTCG (SEQ ID NO: 84) P64: GATTTTTTACCCAAAGGAGGACAACCATGACTGCTCACTGGAA (SEQ ID NO: 85) P65: CGCGGATCCCGCCATTGGGGCATCGCC(BamH I) (SEQ ID NO: 86) P66: CCCAAGCTTTCAACGATCACTGCCCAG(Hind III) (SEQ ID NO: 87) P67: GGGTAACGGCCAGTGTGTCTTAGAAAATG (SEQ ID NO: 88) P68: CTAAGACACACTGGCCGTTACCCTGCGAA (SEQ ID NO: 89) P69: TTGTCCTCCTTTTGTATGTCCTCCTGGACT (SEQ ID NO: 90) P70: GGAGGACATACAAAAGGAGGACAACCTTGACCACCTTGACGCTG (SEQ ID NO: 91) P71: CGCGGATCCAAGCGATCTCAGTGTTGT(BamH I) (SEQ ID NO: 92) P72: TGTGACCCGCTACCCGATAA (SEQ ID NO: 93) P73: CGTTACCCTGCGAATGTC

(115) The homologous recombinant plasmid pK18mobsacB-P.sub.sod::P.sub.prsA identified through sequencing is electronically transformed into L-histidine recombinant bacteria CG161. The kanamycin resistance screening is employed to obtain the bacterial colony that the recombinant plasmid is integrated on the chromosome and the sugar screening is employed to obtain the positive bacterial colony after two homologous recombinations. The positive bacterial colony is identified through PCR amplification with P72 and P65 as the primers to obtain 778 bp being the recombinant bacteria WT-P.sub.glyA::P.sub.hisEG-hiSG.sup.fbr-P.sub.glyA::P.sub.hisDCB-P.sub.sod::P.sub.prsA-Δpgi and named as CG350.

(116) The homologous recombinant plasmid pK18mobsacB-P.sub.eftu::P.sub.tkt identified through sequencing is electronically transformed into Corynebacterium glutamicum CG350 and the kanamycin resistance screening is employed to obtain the bacterial colony that the recombinant plasmid is integrated on the chromosome and the sugar screening is employed to obtain the positive bacterial colony after two homologous recombinations. The positive bacterial colony is identified through PCR amplification with P73 and P71 as the primers to obtain 874 bp of the recombinant bacteriaWT-P.sub.glyA::P.sub.hisEG-hisG.sup.fbr-P.sub.glyA::P.sub.hisDCB-P.sub.eftu::P.sub.tkt-P.sub.sod::P.sub.prsAΔpgi and name it as Corynebacterium glutamicum CG351.

(117) The genome DNA of the recombinant bacterium is extracted and sequenced. The result proves that the promoters of the operon tkt-tal-zwf-opcA-devB and the gene prsA in L-histidine recombinant bacteria CG161 are respectively replaced with the endogenous strong promoter P.sub.eftu and P.sub.sod in Corynebacterium glutamicum, L-histidine recombinant bacteria CG351 containing no plasmid (WT-P.sub.glyA::P.sub.hisEG-hisG.sup.fbr-P.sub.glyA::P.sub.hisDCB-P.sub.eftu:P.sub.tkt-P.sub.sod::P.sub.prsA-Δpgi) is constructed successfully.

Embodiment 6: Construction of L-Histidine High-Yield Recombinant Bacteria CG352 and CG353 Containing No Plasmid

(118) In this embodiment, on the basis of Embodiment 5 as above, the promoter of the gene purH is replaced with the strong promoter P.sub.eftu in order to enhance the expression of the bifunctional enzyme AICAR transmethylase I IMP ring hydrase (PurH, SEQ ID NO: 16) encoded by purH and hence further construct CG352; then the promoter of the gene purF is replaced with the promoter P.sub.hom in order to weaken the first enzyme in the synthetic pathway of nucleotide-amidophosphoribosyl transterase (PurF, SEQ ID NO: 19) and hence construct CG353.

(119) The method same as that in Embodiment 5 as above is employed to construct the homologous recombinant plasmid pK18mobsacB-P.sub.eftu::P.sub.purH and replace the promoter of the gene purH with the strong promoter P.sub.eftu. The primer is designed according to the upstream and downstream sequences of the gene purH of Corynebacterium glutamicum ATCC13032 in Genbank. The genome DN of Corynebacterium glutamicum ATCC13032 is used as the template with P74 and P75 as the primers to carry out amplification and obtain the promoter P.sub.etfu. P76 and P77 are used as the primers to carry out amplification and obtain the upstream homologous arm. P78 and P79 are used as the primers to carry out amplification and obtain the downstream homologous arm. Then the PCR product as above is purified and used as the template with P76 and P79 as the primers to carry out amplification with the technique of overlap extension PCR (SOE) and obtain 1473 bp of segment (SEQ ID NO: 20) containing the upstream and downstream homologous arms and the promoter P.sub.eftu, wherein: No. 1-633 nucleotides of 5′ end in SEQ ID NO: 20 is the upstream homologous arm, No. 634-833 nucleotides of 5′ end in SEQ ID NO: 20 is P.sub.eftu, No. 834-1473 nucleotides of 5′ end in SEQ ID NO: 20 is the downstream homologous arm.

(120) After double-enzyme digestion by Xba I and Sma I, the PCR product of 1473 bp as above connects with the homologous recombinant vector pK18mobsacB after the same double-enzyme digestion. The connection product is transformed through chemical method into Escherichia coli DH5α and the transformant is screened on LB plate containing kanamycin (50 μg/mL). After the transformant is sub-cultured three generations, P13 and P14 are used as the primers and the colony PCR is employed to identify the transformant and obtain 1685 bp being the positive transformant. The plasmid of the transformant after identified is extracted and identified through double-enzyme digestion by Xba I and Sma I and obtain 1473 bp being positive.

(121) The positive plasmid is sequenced and the result shows that this plasmid is the recombinant plasmid obtained through inserting the nucleotide as shown by SEQ ID NO: 20 in the sequence table into the vector pK18mobsacB and named as pK18mobsacB-P.sub.eftu::P.sub.purH.

(122) TABLE-US-00009 (SEQ ID NO: 94) P74: CTGGAGAGGCTAATGGCCGTTACCCTGCGAA (SEQ ID NO: 95) P75: ATCATCGCTCATTGTATGTCCTCCTGGACT (SEQ ID NO: 96) P76: GCTCTAGAATGATGGTTCCGAGGCCG (Xba I) (SEQ ID NO: 97) P77: GGGTAACGGCCATTAGCCTCTCCAGTTGAG (SEQ ID NO: 98) P78: GGAGGACATACAATGAGCGATGATCGTAAG (SEQ ID NO: 99) P79: TCCCCCGGGTGGTGCCGATCCAACCTG(Sma I)

(123) The homologous recombinant plasmid pK18mobsacB-P.sub.eftu::P.sub.purH identified through sequencing is electronically transformed into the L-histidine recombinant engineering bacteria CG351. The kanamycin resistance screening is employed to obtain the bacterial colony that the recombinant plasmid is integrated on the chromosome and the sugar screening is employed to obtain the positive bacterial colony after two homologous recombinations. The genome DNA of the positive colony is extracted and used as the template with P74 and P79 as the primers to carry out PCR amplification and obtain 840 bp being the positive clone. The sequencing verifies that the promoter of the gene purH in the L-histidine recombinant bacteria CG351 is successfully replaced with the endogenous strong promoter P.sub.eftu in Corynebacterium glutamicum, L-histidine recombinant bacteria CG352 containing no plasmid (WT-P.sub.glyA::P.sub.hisEG-hiSG.sup.fbr-P.sub.glyA::P.sub.hisDCB-P.sub.eftu::P.sub.tkt-P.sub.sod::P.sub.prsA-Δpgi-P.sub.etfu::P.sub.purH) is constructed successfully.

(124) The homologous recombinant plasmid pK18mobsacB-P.sub.hom::P.sub.purF identified through sequencing prepared in Embodiment 4 is electronically transformed into Corynebacterium glutamicum CG352. The Kanamycin resistance forward screening is employed to obtain the bacterial colony that the recombinant plasmid is integrated on the chromosome and the sugar inverse screening is employed to obtain the bacterial colony after a second homologous recombination. P56 and P59 are used as the primers to extract the genome DNA of the colony and carry out identification through PCR amplification. 905 bp is obtained to be positive (see FIG. 8) and named as CG353 (WT-P.sub.glyA::P.sub.hisEG-hiSG.sup.fbr-P.sub.glyA::P.sub.hisDCB-P.sub.eftu::P.sub.tkt-P.sub.sod::P.sub.prsA-Δpgi-P.sub.eftu::P.sub.purH-P.sub.hom::P.sub.purF). CG353 is further sequenced and analyzed. The result shows that the promoter of the gene purF of the chromosome of the engineering bacteria CG352 is replaced with P.sub.hom and CG353 is constructed successfully.

Embodiment 7: Application of L-Histidine Engineering Bacteria in Production of L-Histidine

(125) 1. Fermentation of High-Yield L-Histidine Recombinant Bacteria Containing Plasmid

(126) 1. Flask-Shaking Fermentation of High-Yield L-Histidine Recombinant Bacteria Containing Plasmid

(127) The details of fermentation medium used in flask shaking are as follows: glucose 40 g/L, (NH.sub.4).sub.2SO.sub.4 20 g/L, KH.sub.2PO.sub.4 0.5 g/L, K.sub.2HPO.sub.4.3H.sub.2O 0.5 g/L, MgSO.sub.4.7H.sub.2O 0.25 g/L, FeSO.sub.4.7H.sub.2O 0.01 g/L, MnSO.sub.4.H.sub.2O 0.01 g/L, ZnSO.sub.4.7H.sub.2O 0.001 g/L, CuSO.sub.4 0.0002 g/L, NiCl.sub.2.6H.sub.2O 0.00002 g/L, biotin 0.0002 g/L, pH 7.0-7.2, CaCO.sub.3 20 g/L. The glucose is autoclaved separately at 115° C. for 15 minutes. MgSO.sub.4.7H.sub.2O and inorganic ions are autoclaved separately at 121° C. for 20 minutes. The vitamin is sterilized through filtration by 0.22 μm sterile membrane. Other ingredients are autoclaved in 121° C. for 20 minutes.

(128) The details of seed medium are as follows: glucose 20 g/L, (NH.sub.4).sub.2SO.sub.4 5 g/L, K.sub.2HPO.sub.4.3H.sub.2O 1 g/L, MgSO.sub.4.7H.sub.2O 0.4 g/L, biotin 50 μg, Vitamin B.sub.1 1 mg, Angel yeast powder (FM802) 10 g/L, Angel peptone (FP318) 10 g/L.

(129) 1) Obtaining Seed Solution

(130) The engineering bacteria CG176, CG172, CG173 and CG171 prepared in Embodiment 2 as above are respectively inoculated into seed solution. The culture temperature of seed solution is 32° C. The rotation speed of shaker is 220 r/min. The incubation time is 8 h. OD.sub.600 is 20.

(131) 2) Fermentation

(132) The seed solution is inoculated by volume percentage as 3% into the fermentation medium containing chlorampenicol with final concentration of 10 μg/ml (30 mL solution in 500 mL baffled flask) and cultured at 32° C. and 220 r/min for 72 h. After fermented and cultured for 6 h, isopropyl-beta-D-thiogalactopyranoside (IPTG) with final concentration of 1 mmol/L is added to induce the expression of target gene. The strong ammonia water is added intermittently to control pH of fermentation solution to 7.0-7.2. As per the residual sugar, 400 g/L glucose mother liquid is added to control the residual sugar in fermentation solution to 5-10 g/L.

(133) Fermentation product is collected and centrifuged at 12000×g for 5 minutes and then the supernatant is collected.

(134) 3) Test the Content of L-Histidine

(135) HPLC is used and the details of test are as follows (2,4-DNFB pre-column derivation HPLC): take 50 μL the supernatant as above in 2 mL centrifuge tube and add 200 μL NaHCO.sub.3 aqueous solution (0.5 mol/L, pH 9.0) and 100 μL 1% of 2,4-DNFB-acetonitrile solution (volume ratio). Heat it in the dark in a water bath at 60° C. for 60 min, then cool it to 25° C. Add 650 μL KH.sub.2PO.sub.4 aqueous solution (0.01 mol/L, pH 7.2±0.05, adjust pH with NaOH aqueous solution). Keep still for 15 min and then filter before injection. The injection size is 15 μL.

(136) The chromatographic columns used are C18 columns (ZORBAX Eclipse XDB-C18, 4.6*150 mm, Agilent, USA); column temperature: 40° C.; UV detection wavelength: 360 nm; mobile phase A is 0.04 mol/L KH.sub.2PO.sub.4 aqueous solution (pH 7.2±0.05, adjust pH with 40 g/L KOH aqueous solution), mobile phase B is 55% acetonitrile aqueous solution (volume ratio). Flow rate of mobile phase is 1 mL/min. The elution process is as shown in Table 1 below:

(137) TABLE-US-00010 TABLE 1 Duration (min) Mobile A (%) Mobile B (%) 0 86 14 2 88 12 4 86 14 10 70 30 20 30 70 21 10 90 24 0 100

(138) With the wild type of strain C. glutamicum ATCC13032 as the control, the glucose consumption, OD.sub.600 and the final L-histidine yield during fermentation process are determined. The result is shown as in Table 2.

(139) Table 2 shows the glucose consumption, maximum OD.sub.600, specific growth rate and L-histidine yield by the L-histidine engineering bacteria CG160, CG176, CG172, CG173 and CG171 during flask-shaking test.

(140) TABLE-US-00011 TABLE 2 Glucose Maximum Specific growth L-histidine Strain consumption (g) OD.sub.600 rate (h.sup.−1) yield (g/L) CG160 3.8 62.67 0.131 0.03 CG176 3.8 60.97 0.127 1.18 CG172 1.2 46.67 0.073 0.77 CG173 3.8 59.07 0.120 1.50 CG171 1.8 53.83 0.108 2.40

(141) During the flask-shaking tests, after fermentation for 72 h, no L-histidine accumulation is measured for the wild type of C. glutamicum ATCC13032 and L-histidine yield by the base bacteria CG160 is 0.03 g/L. L-histidine yield by the base engineering bacteria CG176 that only L-histidine terminal metabolic pathway is modified is 1.18 g/L. On this basis, L-histidine yield by the engineering bacteria CG172 with only deletion of the gene pgi is 0.77 g/L and L-histidine yield by the engineering bacteria CG173 with only over-expression of zwf-opcA is 1.50 g/L. L-histidine yield by the engineering bacteria CG171 with both deletion of the gene pgi and over-expression of zwf-opcA is 2.40 g/L and increased by 2.1 times than that by the engineering bacteria CG172 with only deletion of the gene pgi, by 60% than that by the engineering bacteria CG173 with only over-expression of the gene zwf-opcA, and by 102% than that by the engineering bacteria CG176 that only the terminal metabolic pathway of L-histidine is modified.

(142) 2. L-Histidine Engineering Bacteria CG171, CG319 and CG328 Fermentation Tank Produce L-Histidine in Fermentor

(143) The details of the seed medium are as follows: glucose 20 g/L, (NH.sub.4).sub.2SO.sub.4 5 g/L, K.sub.2HPO.sub.4.3H.sub.2O 1 g/L, MgSO.sub.4.7H.sub.2O 0.9 g/L, biotin 50 μg, Vitamin B.sub.1 1 mg, Angel yeast powder (FM802) 2 g/L, Angel peptone (FP318) 2 g/L.

(144) The details of the fermentation medium are as follows: glucose 20 g/L, (NH.sub.4).sub.2SO.sub.4 5 g/L, K.sub.2HPO.sub.4 0.5 g/L, K.sub.2HPO.sub.4.3H.sub.2O 0.5 g/L, MgSO.sub.4.7H.sub.2O 0.25 g/L, FeSO.sub.4.7H.sub.2O 10 mg/L, MnSO.sub.4.H.sub.2O 10 mg/L, Vitamin B.sub.1 0.5 mg, Angel yeast powder (FM802) 5 g/L.

(145) 1) Obtaining Seed Solution

(146) The engineering bacteria CG171, CG319 and CG328 are inoculated into the seed medium. The seed solution is cultured at 32° C. for 8 h with the rotation speed of shaker being 220 r/min to give a seed solution of OD.sub.600 as 20.

(147) 2) Fermentation

(148) The seed solution is inoculated by volume percentage as 10% into the fermentation medium containing chlorampenicol with a final concentration of 10 μg/ml.

(149) 7.5 L fermentor is used (BioFlo115, NBS): it is builtin with constant-speed programmable-controlled bump to realize constant-speed feed supplement. During the process of fermentation, a peristaltic pump is used to supplement 600 g/L glucose and control the concentration of glucose in fermentation system to 5-10 g/L. 10 g/L Angel yeast powder (FM802) is dripped in simultaneously. Heating jacket and cooling water are used to control the fermentation temperature at 32° C. Air is introduced to supply dissolved oxygen which is controlled to 30% though cascade control between the rotation speed and dissolved oxygen signal. Strong ammonia water is added to regulate pH and keep it at about 6.9. The fermentation is run continuously for 52 h. When OD.sub.600=4-5, IPTG (isopropyl thiogalactopyranoside is added with final concentration of 0.5 mmol/L) to induce the expression of the gene carried by recombinant plasmid.

(150) The fermentation product is collected and centrifuged at 12000×g for 5 min to collect the supernatant.

(151) 3) Test the Content of L-Histidine

(152) L-histidine content in the supernatant is determined according to the method described in 3) of 1 above and the result is shown as follows: the highest yield of L-histidine by the engineering bacteria CG171 is 10.87 g/L, the production intensity is 0.21 g/L/h; the highest yield of L-histidine by the engineering bacteria CG319 is 14.15 g/L, the production intensity is 0.30 g/Uh; the highest yield of L-histidine by the engineering bacteria CG328 is 15.96 g/L and the production intensity is 0.32 g/L/h. See the result in Table 3.

(153) TABLE-US-00012 TABLE 3 Strain Histidine yield (g/L) Fermentation duration (h) CG171 10.87 52 CG319 14.15 47 CG328 15.96 50

(154) As shown in the table above, the fermentor tests show that CG171 achieves a very good result and the Histidine yield amounts to 10.87 g/L after fermentation for 52 h. Compared with CG171, the engineering bacteria CG319 with further over-expression of purH and the engineering bacteria CG328 with both further over-expression of purH and weakening purF respectively produce about 30% and 50% more histidine in shorter fermentation duration. That is, on the basis of weakening pgi and over-expression of zwf-opcA, the histidine synthetic pathway and the nucleotide synthetic pathway are coupled to promote the metabolic flux of histidine synthetic pathway and further improve the histidine yield greatly.

(155) 2. L-Histidine Engineering Bacteria CG350, CG351, CG352 and CG353 Containing No Plasmid Produce L-Histidine Through Flask-Shaking

(156) Obtaining seed solution of the engineering bacteria CG350, CG351, CG352 and CG353 as well as the flask-shaking fermentation method are the same as those described in 1 as above. The difference is that no chloramphenicol and inducer IPTG is needed during the fermentation process. The test of L-histidine content is the same as that described in 1 of this embodiment. The wild type of C. glutamicum ATCC13032 is used as the control. During the flask-shaking tests, after fermentation for 72 h, no accumulation of L-histidine is detected for the wild type of strain C. glutamicum ATCC13032. L-histidine yield by the engineering bacteria CG350 constructed through single deletion of the gene pgi is 0.65 g/L. On this basis, the engineering bacteria CG351 constructed with also improved expression quantity of zwf-opcA has a L-histidine yield of 1.86 g/L and increases by 186% than that by the engineering bacteria CG350 with single deletion of the gene pgi. L-histidine yield by the strain CG352 with further improved expression of the gene purH is 2.23 g/L and L-histidine yield by the strain CG353 with further decreased expression of the gene purF is 2.34 g/L. See the result in Table 4.

(157) TABLE-US-00013 TABLE 4 Strain Histidine yield (g/L) Wild type — CG350 0.65 CG351 1.86 CG352 2.23 CG353 2.34