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Method for increasing yield of 2'-fucosyllactose in recombinant escherichia coli

A technology of fucosyllactose and Escherichia coli, which is applied in the field of genetic engineering, can solve the problems of high-efficiency expression transformation of FutC, unstable protein expression, inactive inclusion bodies, etc., and achieve the goal of simple construction method, promotion of synthesis, and improvement of transformation efficiency Effect

Pending Publication Date: 2021-02-05
JIANGNAN UNIV +1
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Problems solved by technology

Although Huang et al. investigated the efficiency of α-1,2-fucosyltransferase from different sources to catalyze the synthesis of 2'-FL, it was found that the α-1,2-fucosyltransferase from Helicobacter pylori synthesized 2'-FL has the highest efficiency, but it has not further modified the high-efficiency expression of FutC
On the other hand, in the currently constructed 2'-FL recombinant Escherichia coli, it is necessary to add the inducer isopropylthiogalactopyranoside (IPTG) to activate T 7 Promoter activity, thereby inducing the expression of α-1,2-fucosyltransferase, and IPTG reagent has a certain toxic effect on cell growth
[0007] Fusion protein tags provide an effective strategy for the soluble expression of foreign proteins in E. coli, but there are many factors that cause foreign proteins not to be expressed or expressed in low amounts in E. coli, such as formation due to incorrect folding during translation. Inactive inclusion bodies, or the formation of improperly paired disulfide bonds cause unstable protein expression, and different protein tags may have different effects on promoting the expression of foreign proteins in E. coli
There is currently no fusion protein tag that can improve the expression of α-1,2-fucosyltransferase in E. coli

Method used

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  • Method for increasing yield of 2'-fucosyllactose in recombinant escherichia coli
  • Method for increasing yield of 2'-fucosyllactose in recombinant escherichia coli
  • Method for increasing yield of 2'-fucosyllactose in recombinant escherichia coli

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Embodiment 1

[0031] (1) Heterologous expression of α-1,2-fucosyltransferase derived from Helicobacter pylori

[0032] The futC sequence of the α-1,2-fucosyltransferase gene of Helicobacter pylori (ATCC No. 26695) published on NCBI was fully synthesized, and the futC gene and the expression vector pMA09 were amplified by PCR, using The template plasmid was digested with DpnI enzyme, and the futC gene fragment and the linearized vector pMA09 fragment were purified and recovered. The futC gene and the linearized vector pMA09 were then recombinantly ligated using a Seamless Cloning Kit (Seamless Cloning Kit). The recombination reaction system was reacted at 50°C for 15-20min, then ice-bathed for 30-40min, then the recombinant system was transferred to E. coli JM109 competent cells, recovered at 37°C for 1h, and coated on ampicillin-resistant LB with a final concentration of 0.1mM. Plates and incubated at 37°C for 10-12h. Finally, single colonies on the ampicillin-resistant plate were selecte...

Embodiment 2

[0045] Synthesis of 2'-FL by Shake Flask Fermentation Recombinant Bacteria

[0046]The recombinant plasmids pMA09-TrxA-futC, pMA09-SUMO-futC, pMA09-MBP-futC and pMA09-NusA-futC that were sequenced correctly in the above examples and comparative examples were transferred into MG-26 competent cells, and recovered at 37°C for 1 h. The ampicillin-resistant LB plate with a final concentration of 0.1 mM was cultured at 37°C for 10-12 h to obtain pMA09-FP-futC fermented recombinant bacteria fused with different protein tags. Pick a single colony into LB medium (tryptone 10g / L, yeast powder 5g / L, NaCl 10g / L) with a final concentration of 0.1mM ampicillin for 8-10h, and use it as the seed solution for shake flask fermentation. Then the seed liquid was inserted into a 250mL conical flask containing 20-25mL fermentation medium at an inoculum amount of 1%, and ampicillin with a final concentration of 0.1mM was added at the same time. The formula of the fermentation medium was: glycerol 5-...

Embodiment 3

[0049] Knock-in TrxA-futC fusion protein gene

[0050] (1) Preparation of E. coli MG-26 competent with pCas9 plasmid

[0051] First, the pCas9 plasmid was transformed into Escherichia coli MG-26 and spread on a 50 μg / mL kanamycin plate. After culturing at 30°C for 12 h, a single colony on the plate was picked, inoculated into fresh LB medium, and the final concentration was added. 50 μg / mL kanamycin antibiotic, cultured overnight at 30°C and 220 r / min; transfer the overnight cultured bacterial solution to a 250 mL conical flask containing 50 mL of LB medium at an inoculum of 1%, and wait for the bacterial cells OD 600 When it reaches 0.2, add arabinose with a final concentration of 30-40mmol / L to induce pCas9 plasmid to express recombinase; continue to culture to OD 600 When the temperature is 0.6-0.7, the bacterial solution was ice-bathed for 20 min, and the bacterial cells were collected by centrifugation at 5000 r / min; the cells were washed twice with pre-cooled sterilize...

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Abstract

The invention discloses a method for increasing the yield of 2'-fucosyllactose in recombinant escherichia coli, and belongs to the technical field of gene engineering. According to the method, four different protein labels are fused at the N ends of alpha-1, 2-fucosyltransferase FutC respectively by adopting flexible Linker, and the constructed fusion protein FP-futC can improve the yield of 2'-FLcatalyzed and synthesized by alpha-1,2-fucosyltransferase to different extents. The yield of the 2'-FL synthesized by adopting the TrxA-futC fusion protein is the highest and reaches 2.94 g / L, the consumption of substrate lactose reaches 3.55 g / L, and the lactose conversion rate is 0.58 mol / mol. In the control group, the yield of the 2'-FL synthesized by the recombinant escherichia coli obtainedby plasmid pMA09-futC constructed without protein tags is only 1.71 g / L, the lactose consumption is 2.88 g / L, and the lactose conversion rate is only 0.42 mol / mol. Furthermore, the TrxA-futC fusion protein gene is integrated to a yjiP site on an escherichia coli MG1655 genome to obtain a plasmid-free 2'-FL genetically engineered strain MG-26 delta yjiP:: trxA-futC, the yield of the 2'-FL after shake-flask fermentation reaches 3.85 g / L, and the lactose conversion rate is 0.68 mol / mol.

Description

technical field [0001] The invention relates to a method for improving the yield of 2'-fucosyllactose in recombinant Escherichia coli, and belongs to the technical field of genetic engineering. Background technique [0002] Human milk oligosaccharides (HMOs) are a new type of functional sugar source that is second only to fat and lactose in breast milk. They can promote the growth of beneficial intestinal bacteria in newborns and enhance the intestinal barrier function. It plays an important role in the development of the neonatal immune system. HMOs have a variety of core monosaccharide structural units. According to the different spatial configurations, glycosyl sequences, chain lengths, etc. that constitute the monosaccharide structural units of HMOs, further fucosylation or sialylation at different sites of the sugar chain can form various About 200 HMOs have been identified, among which neutral fucosylated HMOs are the main ones. 2'-fucosyllactose (2'-FL) is the most ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/62C12P19/00C12R1/19
CPCC12N9/1051C12N15/70C12P19/00C07K2319/00Y02A50/30
Inventor 刘龙陈坚刘振民堵国成李江华吕雪芹苏米亚林璐房峻
Owner JIANGNAN UNIV
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