Fucosyltransferase cysteine mutant, recombinant escherichia coli for synthesizing 2'-fucosyllactose by fucosyltransferase cysteine mutant and construction method
A technology for recombining Escherichia coli and fucosyllactose, which is applied in the field of genetic engineering, can solve the problems of low efficiency of 2′-FL, achieve the effects of simple construction method, promotion of synthesis, and good application prospects
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Embodiment 1
[0052] (1) Heterologous expression of α-1,2-fucosyltransferase derived from Helicobacter pylori
[0053] The whole gene synthesis of the α-1,2-fucosyltransferase gene futC sequence of Helicobacter pylori (Helicobacter pylori, ATCC No.26695) published on NCBI, the futC gene and the expression vector pMA09 were amplified by PCR, using DpnI The template plasmid was digested with enzymes, and the futC gene fragment and the linearized vector pMA09 fragment were purified and recovered. Then, the futC gene and the linearized vector pMA09 were recombined using a seamless cloning kit (Seamless Cloning Kit). After the recombinant reaction system was reacted at 50°C for 30 minutes, it was ice-bathed for 35 minutes, and then the recombinant system was transferred to Escherichia coli JM109 competent cells, recovered at 37°C for 1 hour, and spread on an ampicillin-resistant LB plate with a final concentration of 0.1 mM, 37 Cultivate at ℃ for 12h. Finally, a single colony on the ampicillin...
Embodiment 2
[0059] Construction of FutC Cysteine Combination Mutant S123C / I126C-I246C / S252C
[0060] Using the correctly sequenced FutC cysteine mutant vector pMA09-MCys-S123C / I126C in step (3) of Example 1 above as a template, linearize and amplify the mutant vector by inverse PCR, then digest the template plasmid with DpnI enzyme, and purify After recovering the linearized vector fragments, transform them into Escherichia coli JM109 competent cells, revive at 37°C for 1 h, spread the ampicillin-resistant LB plate with a final concentration of 0.1 mM, and incubate for 12 h. It was confirmed by sequencing whether the combined mutant S123C / I126C-I246C / S252C (SEQ ID NO.6) was constructed successfully.
Embodiment 3
[0062] Synthesis of 2′-FL by Shake Flask Fermentation Mutation Vector pMA09-MCys-S123C / I126C
[0063] Transform the Escherichia coli mutant plasmid pMA09-MCys-S123C / I126C with correct sequencing into 2′-FL synthetic Escherichia coli MG-26 competent cells to obtain recombinant Escherichia coli, recover at 37°C for 1 hour, and coat with a final concentration of 0.1mM Ampicillin-resistant LB plates were cultured at 37°C for 11 hours. A single colony was selected and cultured in LB medium (tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L) with a final concentration of 0.1 mM ampicillin for 9 h as the seed liquid for shake flask fermentation. Then the seed solution was inserted into a 250mL Erlenmeyer flask with 25mL fermentation medium in an inoculum size of 1%, while adding ampicillin with a final concentration of 0.1mM, the formula of the fermentation medium was: glycerol 5-6 g / L, tryptone 12g / L, yeast powder 24g / L, dipotassium hydrogen phosphate 12.54g / L, potassium dihydrogen...
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