Glutamic acid corynebacterium and construction method and application thereof
A Corynebacterium glutamicum and lysine technology, applied in the field of microorganisms, can solve the problems of low acid production rate, inability to meet large-scale industrial production, and poor fermentation performance of L-lysine, so as to increase production and enhance activity , the effect of increasing production
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Embodiment 1
[0036] Example 1: Construction of recombinant plasmid pK18mobsacB-lysE and addition of a copy of lysE in the starting strain MHZ-0912-1
[0037]Using the ATCC 13032 genome as a template, PCR amplification was performed with the lysE-1f / lysE-1r primer pair to obtain the upstream fragment lysE-up+lysE; using the ATCC 13032 genome as a template, PCR was performed with the lysE-2f / lysE-2r primer pair Amplify to obtain fragment lysE, use ATCC 13032 genome as a template, and perform PCR amplification with lysE-3f / lysE-3r primer pair to obtain fragment lysE-dn. The lysE-up+lysE, lysE, lysE-dn three-fragment mixture was used as a template, and the lysE-1f / lysE-3r primer pair was used for PCR amplification to obtain a lysE fragment with two copies. The fragment was double-digested with BamHI and PstI, and the vector pK18mobsacB was double-digested with the same enzymes. The two digested products were ligated with T4DNA Ligase, transformed into Trans1T1 competent cells, and the recombi...
Embodiment 2
[0039] Example 2: Recombinant plasmid pK18mobsacB-pyc P458S Construction of MHZ-0912-1 and the introduction of point mutations to enhance the activity of pyruvate carboxylase
[0040] Using the ATCC 13032 genome as a template, PCR amplification was performed with the pyc-1f / pyc-1r primer pair to obtain the upstream fragment pyc-up; using the ATCC 13032 genome as a template, PCR amplification was performed with the pyc-2f / pyc-2r primer pair , to get the downstream fragment pyc-dn. Using the mixture of pyc-up and pyc-dn fragments as a template, the pyc-1f / pyc-2r primer pair was used for PCR amplification to obtain the mutated pyc P458S fragment. pyc P458S The fragment was double-digested with SphI and NheI, and pK18mobsacB was double-digested with the same enzymes. The two digested products were ligated with T4DNA Ligase, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-pyc was obtained P458S .
[0041] Prepare MHZ-0912-1 competent cells a...
Embodiment 3
[0042] Example 3: Recombinant plasmid pK18mobsacB-pyc P458S Construction of MHZ-0913-1 and the introduction of point mutations to enhance the activity of pyruvate carboxylase
[0043] Construct recombinant plasmid pK18mobsacB-pyc according to the method described in Example 2 P458S : Using the ATCC13032 genome as a template, PCR amplification was performed with the pyc-1f / pyc-1r primer pair to obtain the upstream fragment pyc-up; using the ATCC 13032 genome as a template, PCR amplification was performed with the pyc-2f / pyc-2r primer pair , to get the downstream fragment pyc-dn. Using the mixture of pyc-up and pyc-dn fragments as a template, the pyc-1f / pyc-2r primer pair was used for PCR amplification to obtain the mutated pyc P458S fragment. pyc P458S The fragment was double-digested with SphI and NheI, and pK18mobsacB was double-digested with the same enzymes. The two digestion products were ligated with T4 DNALigase, transformed into Trans1T1 competent cells, and the re...
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