Recombinant strain and application thereof in preparation of L-tryptophan
A technology of threonine and serine, applied in the field of genetic engineering, can solve the problems of inability to determine the intake of isomaltose into bacteria, inability to assimilate isomaltose, etc.
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Embodiment 1
[0049] Example 1: Overexpression of glvA / glvC gene protocol pTargetF vector and construction of full-length fragments
[0050] (1) Construction of pTargetF-glvA vector and P1-glvA full-length fragment of the overexpression glvA gene protocol
[0051] First, use gRNAglvAup-f1 / glvA-pF-r as primers and plasmid pTargetT as a template to amplify the N20 containing the speI restriction enzyme site in the 5' region and the SalI restriction enzyme site in the 3' region by PCR. and sgRNA fragments; secondly, the amplified fragments and pTargetT plasmid vector were treated with restriction enzymes speI and salI, and the fragment gel was recovered; then, the treated vector and fragment were ligated with T4 enzyme to construct pTargetF-glvA plasmid.
[0052] First, use glvAup-f1 / P1-glvAup-r1 as primers, and use the tryptophan strain MHZ-0800 genome as a template to amplify the upstream homology arm (fragment 1) of the glvA insertion site by PCR; secondly, use P1 -glvAdn-fl / P1-glvAdn-r1 a...
Embodiment 2
[0056] Embodiment 2: Construction of overexpression glvA and glvC gene strain
[0057] In this patent example, the parental strain is according to the method described in patent WO 87 / 01130 and Mascarenhas D etc. (Mascarenhas D et al, Deletion of pgi alters tryptophan biosynthesis inagenetically engineered strain of escherichia coli. , 1991) constructed tryptophan production strain MHZ-0800 (CGMCC NO.6863), its host strain SA01 is E.coli K-12 CICC 10303 tnaA serA derived from E.coli K-12 (CICC 10303), containing plasmid pMG43, derived from pBR322, contains the serA, and feedback repression removed aroG and trpEDCBA operons.
[0058] Take out the bacterial strain SA01 (without pMG43 plasmid) from the cryopreservation tube and activate it on the LB plate, cultivate it at 37°C for 16-24 hours, pick a single colony from the plate, and inoculate it into a test tube containing 5 mL of LB liquid medium. 37°C, shake culture at 150-240rpm for 2-3hr, until OD 600 If the value is betwe...
Embodiment 3
[0060] Example 3: Obtaining strains utilizing isomaltose by transformation plus evolution
[0061] Streak the above-mentioned SMW040 strain on an anti-resistant LB plate, culture at 37°C for 16-24hr, pick a single colony from the plate, and inoculate it into 5mL basic salt containing 2g / l isomaltose and 2g / l glucose In the test tube of the culture medium (the specific formula is as follows), culture to 24h, the test tube containing 2g / l isomaltose does not grow, the test tube containing 2g / l glucose grows normally, and the OD grows to 1.3, which proves that the strain is still unable to utilize isomaltose , the strain was subjected to directed evolution. The method is as follows: a single colony on the LB plate is inoculated into a test tube containing 5 mL of LB medium, diluted and spread on a double-antibody basic salt medium containing 2 g / L isomaltose, and the purity of isomaltose is 95%. Cultivate at 37°C for 70 hours, transfer the transformants to the streaked M9 plate ...
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