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Construction and applications of nicotinamide adenine dinucleotide auxotroph escherichia coli

A nicotinamide adenine, auxotrophic technology, applied in the direction of bacteria, microbe-based methods, microbiological determination/inspection, etc., to achieve the effect of promoting production

Inactive Publication Date: 2013-02-13
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem of NAD transmembrane transport, the strategy adopted by the present invention is to introduce a protein expression vector capable of self-replication and carrying the NAD transport protein coding gene in the microorganism of the genus Escherichia

Method used

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  • Construction and applications of nicotinamide adenine dinucleotide auxotroph escherichia coli
  • Construction and applications of nicotinamide adenine dinucleotide auxotroph escherichia coli
  • Construction and applications of nicotinamide adenine dinucleotide auxotroph escherichia coli

Examples

Experimental program
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Effect test

Embodiment 1

[0020] (1) Transform the Red recombinase expression plasmid pKD46 (NCBI Accession No. AY048746) into BW25113 to obtain the strain BW25113 (pKD46).

[0021] (2) Construction of expression vector for NAD transporter NTT4:

[0022] With reference to the sequence of Chlamydia ntt4 (NCBI GeneID: 2780098), TaKaRa Company (Dalian, China) was commissioned to synthesize the ntt4 gene through total gene synthesis technology. Using the synthetic genetic material as a template, using ntt4-F / ntt4-R primers and PrimeSTAR TM HS DNA polymerase (TaKaRa, Dalian, China) was used to clone ntt4 by polymerase chain reaction. The amplified product and vector pET15b (Novagen, USA) were digested with NdeI and BamHII, and ligated with T4 ligase to obtain plasmid pET15b -NTT4. Using pET24b (Novagen, USA) as a template and kan-F / kan-R as primers, the kanamycin resistance coding gene kan was cloned. Then use RF cloning (van den Ent F and Lowe J, J Biochem Biophys Methods, 2006, 67, 67) method to replace kan...

Embodiment 2

[0045] (1) Construction of nadD knockout box:

[0046] NadD:cat-F / nadD:cat-R was used to amplify the knockout box nadD:cat with the chloramphenicol resistance gene cat from the template plasmid pKD3 (NCBI Accession No. AY048742) by PCR.

[0047] nadD: cat-F: 5’

[0048] -CGGGTTAGCTTTAAGGAAGTTTTGTCTTTTCTGTCTGGAGGGGTTCAatgggaattagccatggtcc-3’

[0049] nadD: cat-R: 5’

[0050] -CTTTTTCGCACAATCCAATATGTGCAAATTATTACTTTTTCCAGAAATCATCgtgtaggctggagctgcttc-3’

[0051] (The uppercase base in the primer indicates the homologous recombination sequence outside the nadD gene, and the lowercase part is the cat gene sequence).

[0052] (2) Knockout box transformation and screening

[0053] The knockout fragment nadE:cat was electrotransformed into the BW25113 (pKD46, pET15k-NTT4) constructed in Example 1, and spread on the SOB plate containing 50μg / mL kanamycin, 25μg / mL chloramphenicol and 500μM NAD Above, 37C, culture for 24 days. A single colony was selected and cultured on different plates at 37C in g...

Embodiment 3

[0056] (1) Transform the Red recombinase expression plasmid pKD46 (NCBI Accession No. AY048746) into MG1655 to obtain the strain MG1655 (pKD46).

[0057] (2) Construction of NAD transporter ScNDT1 expression vector:

[0058] Using S. cerevisiae S288C genomic DNA as template, using ScNDT1-F / ScNDT1-R primers and PrimeSTAR TM HS DNA polymerase (TaKaRa, Dalian, China) was used to clone ScNDT1 by polymerase chain reaction. The amplified product and vector pET15b (Novagen, USA) were digested with NdeI and BamHI, and ligated with T4 ligase to obtain plasmid pET15b -ScNDT1. Then use RF cloning (van den Ent F and Lowe J, J Biochem Biophys Methods, 2006, 67, 67) method to replace the kanamycin resistance coding gene kan with the ampicillin resistance coding gene b1a to obtain the vector pET15k-ScNDT1, Transform into E. coli MG1655 (pKD46) to obtain strain MG1655 (pKD46, pET15k-ScNDT1).

[0059] ScNDT1 cloning primer (underline indicates restriction site):

[0060] ScNDT1-F: 5’-GAG CATATG AC...

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Abstract

The present invention discloses construction and applications of nicotinamide adenine dinucleotide (NAD) auxotrophic Escherichia microbe, wherein NAD transporter protein is introduced to the Escherichia microbe, an intracellular NAD biosynthesis pathway is blocked, and the Escherichia engineered bacteria growing dependent on exogenous pyridine nucleotide coenzyme is constructed. The NAD auxotrophic Escherichia can be used in the field of bio-technology, and can be provided for screening inhibition drugs for pyridine nucleotide coenzyme metabolism, improving oxidation reduction biotransformation efficiency, and establishing non-antibiotic screening platforms for gene cloning and protein heterogenesis expression.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to the construction and application of a nicotinamide adenine dinucleotide (NAD) auxotrophic Escherichia (Escherichia) microorganism. More specifically, a carrier capable of expressing NAD transmembrane transporter is built into the cells of microorganisms of the genus Escherichia, and its natural NAD biosynthetic pathway is blocked, and growth depends on exogenous pyridine nucleotide coenzymes. The engineered bacteria are used in biocatalysis, NAD transmembrane transport protein inhibitor screening, metabolic regulation, non-antibiotic screening markers, and protein expression. Background technique [0002] Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are very important cofactors. NADH and NADPH are reduced forms of NAD and NADP, respectively, and they can be transformed into each other in the cell. NAD, NADH, NADP and NADPH are collectively...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12Q1/02C12R1/19
Inventor 赵宗保周雍进林心萍张素芳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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