L-isoleucine genetic engineering production bacteria
A technology for producing isoleucine and bacteria, which is applied in the field of genetic engineering, can solve the problems that the production cannot meet the market demand and the low acid production capacity, and achieve wide industrial application prospects, effective accumulation, increase production and sugar-acid conversion rate Effect
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Embodiment 1
[0053] Example 1 : Construction of plasmid pMW-ilvGAM
[0054] 1.1. Construction of pMW-ilvGMEDA
[0055] The pMW119 vector (purchased from Nippon Gene Company) was digested with HindIII / EcoRI to recover a 4.1kb fragment; using the MG1655 genome as a template and ilvG119F / ilvG119R as primers, PCR amplified the ilvGMEDA fragment, about 9kb; the above fragment was GIBSON ligated ( Referring to Enzymatic assembly of DNA molecules up to several hundred kilobases, Daniel G Gibson, et al. Nature Methods. 2009), the pMW-ilvGMEDA plasmid was obtained.
[0056] 1.2. Construction of pMW-ilvGAM
[0057] Using the pMW-ilvGMEDA plasmid as a template and using PilvGMF / ilvAMF as primers, PCR amplified yifB and pMW119 vector fragments, about 7kb; using ilvGMR / ilvAMR as primers, PCR amplified part of the ilvGMEDA fragment, about 6.2kb; using ilvGMF / PilvGMR As a primer, a partial fragment of ilvG, about 1.1kb, was amplified by PCR; the above fragment was connected with GIBSON to obtain the ...
Embodiment 2
[0058] Example 2 : Obtaining of L-isoleucine expression strain
[0059] The pMW-ilvGAM plasmid obtained in Example 1.2 was transferred into the competent cells of the L-threonine-producing bacteria CCTCC M 2015556 (for specific methods of preparation and transformation of competent cells, refer to "Molecular Cloning Experiment Guide" (third edition), J. Sam Brook, edited by D.W. Russell (U.S.), translated by Huang Peitang, etc., Science Press, Beijing, 2002, page 96 of the first chapter), obtained the genetically engineered bacteria CIBTS1800 for producing L-isoleucine , whose genotype is MG1655(Δtdh,ΔthrL,thrA*(G433R),Ptac-rhtC,lysC*(T342I),ΔtdcC,ΔdacA::thrA*BC,ΔyihF::thrA*BCΔcadB::Thr,ΔyidJ::Thr ) / pMW-ilvGAM.
Embodiment 3
[0060] Example 3 : Fermentation of L-isoleucine genetically engineered bacteria to produce L-isoleucine
[0061] Utilize the L-isoleucine genetically engineered bacterium to produce the method for L-isoleucine by fermentation as follows:
[0062] A single colony was picked and inoculated in 400 μl of a 96-well plate containing ampicillin fermentation medium, and cultured at 37° C. and 290 rpm for 40 hours.
[0063] After the fermentation was finished, the L-isoleucine content in the supernatant of the fermentation broth was determined by HPLC, and the results are shown in Table 3.
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