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Genetically engineered bacterium and application thereof

A genetically engineered bacteria and gene technology, applied in the field of genetic engineering, can solve the problems of reducing the mass transfer resistance of the substrate VD3, low calcifediol production, and increased calcifediol production, achieving low cost, high yield, and environmental protection. friendly effect

Pending Publication Date: 2021-10-12
弈柯莱生物科技(集团)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Patent US 8,148,119B2 reported that VDH (VD hydroxylase, sequence 2) derived from Pseudonocardia autotrophica can catalyze VD 3 Synthesize calcifediol, and use VDH and thcCD (ferredoxin gene thcC and ferredoxin reductase gene thcD) to co-express in Rhodococcus erythropolis or Escherichia coli to increase The production of calcifediol has been improved, but the production of calcifediol is still very low, as can be seen from its Figures 10 and 11 that the production should be lower than 250 μg / mL (according to VD 3 Conversion rate 50% calculation)
Tamura et al. (ChemBioChem 2013, 14, 2284-2291) modified the P450 enzyme derived from Pseudonocardia autotrophica (with mutations such as T107A), and performed heterologous expression in Rhodococcus, and increased the permeability of the cell membrane by adding nisin. Permeability, reducing the mass transfer resistance of the substrate VD3, and finally catalyzing VD3 in 2 hours to obtain the output of calcifediol can reach 573μg / mL, but the output of calcifediol needs to be improved

Method used

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  • Genetically engineered bacterium and application thereof
  • Genetically engineered bacterium and application thereof
  • Genetically engineered bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Screening of cytochrome P450 enzymes (CYP, Cytochrome P450 proteins)

[0078] 1.1 Acquisition and expression of CYP

[0079] According to the gene sequence SEQ ID NO.2, 4, 6, 8, 10, 12, 14, 16 whole gene synthesis of CYP genes. The gene synthesis company is Suzhou Jinweizhi Biotechnology Co., Ltd. (Floor C3, Bio-Nano Technology Park, No. 218, Xinghu Street, Suzhou Industrial Park).

[0080] Table 1

[0081]

[0082] Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 121°C for 20min, ready for use.

[0083] The synthesized CYP genes were respectively connected to the pET28a vector, and the restriction sites NdeI&HindIII were cut, and the enzyme-linked vectors were transformed into host E.coli BL21 (DE3) competent cells to obtain engineering strains containing CYP genes.

[0084] After the engineering bacteria containing the CYP gene were activated by streakin...

Embodiment 2

[0085] Example 2 Acquisition and expression of CYP electron transport chain genes

[0086] In the process of CYP reaction, NAD(P)H is required to provide electrons, and electron transport chain is required to transfer electrons. According to the gene sequence SEQ ID NO.18, 20, 22, 24, 26, 28 encoding the enzyme shown in the amino acid sequence of SEQ ID NO. Set of CYP electron transport chain genes. Wherein, Adx (adrenodoxin) is cortical ferredoxin, AdR (adrenodoxin reductase) is cortical ferredoxin reductase, Fdx (ferredoxin) is ferredoxin, and FdR (ferredoxin reductase) is ferredoxin reductase.

[0087] Table 2

[0088] Enzyme number gene source NCBI accession number amino acid sequence Nucleotide sequence boAdx Bos taurus BAA00362.1 SEQ ID NO.17 SEQ ID NO.18 wxya Bos taurus BAA11921.1 SEQ ID NO.19 SEQ ID NO.20 AciFdx Acinetobacter sp.OC4 BAE78451.1 SEQ ID NO.21 SEQ ID NO.22 wxya Acinetobacter sp.OC4 BAE7845...

Embodiment 3

[0091] Embodiment 3 Acquisition and expression of glucose dehydrogenase (GDH) gene

[0092] The glucose dehydrogenase gene was fully synthesized according to the glucose dehydrogenase gene sequence derived from Bacillus subtilis 168 (NCBI accession number: NP_388275.1).

[0093] Glucose dehydrogenase gene is connected to pET21a, restriction site NdeI&HindIII, and the enzyme-linked vector is transformed into host E.coli BL21(DE3) competent cells to obtain an engineering strain containing glucose dehydrogenase gene. After the engineered bacteria containing the glucose dehydrogenase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 ml LB liquid medium containing 100 μg / ml ampicillin, and cultured with shaking at 37°C for 12 hours. Transfer to 50ml fresh LB liquid medium also containing 100μg / ml ampicillin according to 2% (v / v) inoculum amount, and shake to OD at 37°C 600 When it reaches about 0.8, add IPTG to its final concentration of...

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Abstract

The invention discloses a genetically engineered bacterium. The genetically engineered bacterium is an engineered bacterium constructed by expressing a CYP gene, a ferredox protein gene, a ferredox protein reductase gene and a dehydrogenase gene in a cell; wherein an amino acid sequence coded by the CYP gene is as shown in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 9 or SEQ ID NO. 11. The invention further discloses application of the genetically engineered bacterium in preparation of calcifediol and / or calcitriol. When the engineering bacterium of the invention is applied to synthesis of the calcifediol and the calcitriol, the yield is remarkably increased, and when the engineering bacterium is applied to industrial production, the cost is lower, the reaction specificity is high, the reaction condition is mild, the engineering bacterium is environmentally friendly, and the industrial production requirements of the calcifediol and the calcitriol are met.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a genetically engineered bacterium and its application in synthesizing calcidiol and calcitriol. Background technique [0002] Calcitriol, also known as calcitriol, dihydroxycholecalciferol, is the vitamin D in the human body 3 The active form of β-β is also a hormone in the body, which plays an important role in regulating blood calcium and blood phosphorus concentrations. Calcitriol was developed by Roche, and was listed in Switzerland in 1978, and was first registered in my country in 1988. Calcitriol can increase the calcium level in the blood by increasing the absorption of calcium ions in the intestine, and can increase the calcium release in the bone to increase the blood calcium level. According to its principle of action, it can be used clinically to treat hypocalcemia, parathyroid gland Hypofunction (adults), osteomalacia, rickets (infants), chronic kidney...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/02C12R1/19
CPCC12N9/0042C12Y106/02004C12N9/0006C12N9/001C12Y103/07004C12N15/70C12P7/02
Inventor 吴燕陈宇恒冀勇良徐争田振华
Owner 弈柯莱生物科技(集团)股份有限公司
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