Method for producing L-amino acids by fermentation using bacteria having enhanced expression of xylose utilization genes
A technology of amino acids and bacteria, applied in the direction of bacteria, fermentation, and the use of vectors to introduce foreign genetic material, etc.
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Embodiment 1
[0143] Example 1: Cloning of the xylBFGHR locus from the chromosome of E. coli strain MG1655
[0144] According to the genome analysis of E.coli strain MG1655, the gene xylABFGHR can be cloned as an independent HindIII fragment (13.1kb) in all 556 HidIII chromosome fragments ( figure 1 ). For this purpose, a gene library was created with the vector pUC19, which contains an insert of this size, which is able to survive in E. coli.
[0145] To create such a library, the chromosomal DNA of MG1655 was restricted with HindIII and the pUC19 vector was restricted with Xbal. The strain MG1655 (ATCC47076, ATCC700926) can be obtained from the American Type Culture Collection (10801 University Boulevard, Manassas, VA., 20110-2209, U.S.A).
[0146] To prevent self-ligation, the cohesive ends of both DNA products were then filled in with Klenow fragments (two-base filling). After the ligation step, a library of recombinant pUC19 plasmids was obtained. The size of the library is great...
Embodiment 2
[0150] Example 2: Using L-histidine-producing bacteria to ferment L-histidine in a mixture of glucose and pentose
[0151] L-histidine producing E. coli strain 80 is a strain for the fermentation of a mixture of glucose and pentose sugars to produce L-histidine. E.coli strain 80 (VKPM B-7270) is described in detail in Russian patent RU2119536 and has been published on October 15, 1999 in the Russian National Collection of Industrial Microorganisms (Russia, 113545Moscow, 1 st Dorozhny proezd, 1) was deposited under accession number VRPMB-7270. Subsequently, it was transferred to an international deposit on January 12, 2004 in accordance with the provisions of the Budapest Treaty. Strain 80 was transformed with the pMW119mod-xylA-R plasmid by a conventional method to obtain strain 80 / pMW119mod-xylA-R.
[0152] To obtain seed cultures, both strains 80 and 80 / pMW119mod-xylA-R were prepared in 40 ml tubes containing 2 ml of L-broth containing 1 g / l streptomycin in 27° C. on ...
Embodiment 3
[0171] Example 3. Using L-threonine-producing bacteria to ferment and produce L-threonine in a mixture of glucose and pentose sugars
[0172] L-Threonine Production E. coli strain B-3996 was used to evaluate the fermentative production of L-histidine from a mixture of glucose and pentose sugars. respectively by using CaCl 2 The conventional method used pMW119mod-xylA-R plasmid and vector pMW119 to transform strain B-3996 to obtain strains 3996 / pMW119mod-xylA-R and 3996 / pMW119.
[0173] Both E. coli strains B-3996 and B-3996 / pMW119mod-xylA-R were grown on L-agar plates containing streptomycin (50mg / l) and ampicillin (150mg / l) at 37°C for 12-15 Hours. Subsequently, a loop of the strain was inoculated in a fermentation medium containing xylose (4%) as a carbon source. Fermentations were carried out in 2 ml of fermentation medium containing streptomycin (50 mg / l) in 20 x 20 mm tubes. Cells were grown for 65 hours at 32°C with shaking at 250 rpm.
[0174] After culturing, th...
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