Genetic engineering synechocystis capable of promoting regeneration of intracellular coenzyme NADPH and application of synechocystis

A technology of genetic engineering and Synechocystis, which is applied in the development field of genetic engineering cyanobacteria that promotes the regeneration of intracellular coenzyme NADPH, can solve the problems of low production efficiency, low asymmetric reduction activity, low coenzyme regeneration efficiency, etc.

Inactive Publication Date: 2016-05-11
WUHAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low efficiency of coenzyme regeneration in microalgae cells and the difficulty in regulating the regeneration metabolism process, there are generally problems such as low asymmetric reduction activity and low production efficiency in the biocatalysis process, which seriously restricts the development of microalgae catalytic technology.

Method used

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  • Genetic engineering synechocystis capable of promoting regeneration of intracellular coenzyme NADPH and application of synechocystis
  • Genetic engineering synechocystis capable of promoting regeneration of intracellular coenzyme NADPH and application of synechocystis
  • Genetic engineering synechocystis capable of promoting regeneration of intracellular coenzyme NADPH and application of synechocystis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] [Example 1] Construction of recombinant plasmid pKW-Ω-PpetE-petH

[0033] Using the total DNA of Synechocystis sp. PCC6803 as a template, petH (nucleotide sequence shown in SEQ ID NO.1) was obtained by PCR amplification, and then inserted into plasmid pHB1524 by homologous recombination to obtain recombinant plasmid pHB1524-petH; The recombinant plasmid pHB1524-petH was used as a template, the fragment Ω-PpetE-petH was amplified by PCR, and inserted into the plasmid pKW1188 by homologous recombination to obtain the recombinant plasmid pKW-Ω-PpetE-petH.

[0034] (1) Extraction of total DNA from Synechocystis sp. PCC6803

[0035] The DNA of Synechocystis PCC6803 was extracted by CTAB method and stored at -20°C for subsequent experiments.

[0036] (2) PCR amplification of petH and pHB1524 digestion

[0037] PCR amplification reaction system: Synechocystis sp. PCC6803 total DNA 1 μL, ePfuMix (1×) 20 μL, petH upstream primer 2 μL, petH downstream primer 2 μL, 25 μL in tota...

Embodiment 2

[0060] [Example 2] Construction and induced expression of Synechocystis sp. PCC6803::Ω-PpetE-petH

[0061] (1) Transformation of Synechocystis sp. PCC6803

[0062] Take 30mL culture to OD 730 =0.7-0.9 Synechocystis PCC6803 (WT) bacterial solution in a centrifuge tube, centrifuged at 5000rpm for 5min; collect the algal cells, suspend the sediment with fresh BG-11 medium, and distribute 0.1mL per tube into EP tubes; Put 5 μL of recombinant plasmid pKW-Ω-PpetE-petH in suspension, and incubate the mixture of algae cells and recombinant plasmid at 30°C and 2000 Lux light for 16 hours; On the 250mg / L BG-11 plate, under the conditions of 30°C and 2000Lux light, the transformants were screened and induced to grow out, and the transformants were identified by PCR method after subculture in liquid medium.

[0063] (2) Identification of Synechocystis sp. PCC6803::Ω-PpetE-petH

[0064] Such as Figure 4 As shown, two primers Ex1 and Ex2 in the slr0168 gene were used to perform PCR amp...

Embodiment 3

[0068] [Example 3] Detection of FNR crude enzyme liquid activity and NADPH concentration

[0069] (1) Extraction of FNR crude enzyme solution

[0070] Take 1mL of the algae liquid cultured at 400nmol / L copper ion, add the pre-cooled extract (50mM Tris-HC1, 0.1mMEDTA, 0.1mM β-mercaptoethanol (pH8.0), 1uMPMSF) and mix, ultrasonically break, 6 cycles, Centrifuge at 25,000×g for 30 minutes at 4°C for 45 seconds each time, and take the supernatant for detection.

[0071] (2) Activity detection of FNR crude enzyme solution

[0072] Take 1mL crude enzyme solution and 4mL reaction solution (0.5mMNADP + , 0.019mMDCPIP, 50mMTris-HCl (pH8.0)) mixed reaction, measured OD340nm every 30s, 2min. Define ferredoxin-NADP + Reductase (FNR) enzyme activity is one enzyme activity unit (U) required to obtain 1 μmol / L NADPH per minute, such as Figure 5 As shown, the FNR activity of Synechocystis PCC6803::Ω-PpetE-petH treated with 400nmol / L copper ions was doubled compared with that of Synechoc...

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Abstract

The invention discloses engineering synechocystis PCC6803 capable of promoting regeneration of an intracellular coenzyme NADPH and a construction method and application of the synechocystis. Homologous recombination is performed on a gene petH of ferredoxin-NADP reductase (FNR) which can catalyze regeneration of the coenzyme NADPH to obtain recombinant plasmids pKW-omega-PpetE-petH, the recombinant plasmids are transformed into the synechocystis PCC6803, the FNR gene is integrated in chromosomal DNA of the synechocystis through homologous recombination, and high-strength expression of the FNR can be regulated and controlled according to the concentration of Cu<2+>. The engineering synechocystis constructed through the method can promote overexpression of the FNR, improve the total enzyme activity of the intracellular FNR and greatly promote the regeneration efficiency of the intracellular coenzyme NADPH. The synechocystis obtained through the method can be applied to the biological catalysis and conversion process which has the large quantity demand on the coenzyme and the biotechnology field by promoting regeneration of the intracellular coenzyme NADPH of microalgae and has the wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the development and application of a genetically engineered cyanobacteria that promotes the regeneration of intracellular coenzyme NADPH. Background technique [0002] In biochemical engineering, coenzyme plays an important role. It provides the carrier required for redox for biosynthetic reactions and catabolic reactions, and plays an important role in the energy conversion process in cells. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a coenzyme that participates in anabolism, such as the synthesis of amino acids, lipids and nucleotides, and other cell components, and also plays a role in the normal growth and metabolism of cells. Important impact; in biotransformation, the large-scale production of unnatural amino acids, chiral alcohols and other fine chemicals such as carotenoids, biodegradable polymers and pigments by industrial microorganisms requi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/63C12P7/02C12P19/36
Inventor 杨忠华邓新星罗伟阮涛侯亚利周卫龚志伟黄皓
Owner WUHAN UNIV OF SCI & TECH
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