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Enzyme gene suitable for efficiently synthesizing NAD (Nicotinamide Adenine Dinucleotide) derivative in microorganism

A derivative, microbial cell technology, applied in the field of genetic engineering and bioengineering, can solve the problems of low yield and high cost

Active Publication Date: 2021-05-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]At present, NAD derivatives are mainly synthesized by enzymatic method or chemical synthesis, but the cost of these two methods is higher than that of microbial fermentation method
There are very few reports on the biosynthesis of NAD derivatives, and the detected yield is relatively low

Method used

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  • Enzyme gene suitable for efficiently synthesizing NAD (Nicotinamide Adenine Dinucleotide) derivative in microorganism
  • Enzyme gene suitable for efficiently synthesizing NAD (Nicotinamide Adenine Dinucleotide) derivative in microorganism
  • Enzyme gene suitable for efficiently synthesizing NAD (Nicotinamide Adenine Dinucleotide) derivative in microorganism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Whole Gene Synthesis of Key Genes for the Biosynthesis of NAD Derivatives

[0047] The genes shown in SEQ ID NO.1-7 were respectively synthesized.

[0048] Table 1 Biosynthetic genes of NAD derivatives from different sources

[0049] gene name gene source Nucleotide sequence TnPBEF Tetraodon nigroviridis SEQ ID NO.1 FtN Francisella tμLarensis SEQ ID NO.2 wxya Acinetobacter baumannii SEQ ID NO.3 VpNadV Vibrio bacteriophage KVP40 SEQ ID NO.4 Mm Nampt Mus musc μLus SEQ ID NO.5 wxya Haemophilus ducreyi SEQ ID NO.6 wxya Ralstonia solanacearum SEQ ID NO.7

Embodiment 2

[0050] Example 2: Construction of Gene Expression Vectors Related to the Synthetic Pathway of Nicotinamide Derivatives

[0051] (1) Construction of TnPBEF expression cassette

[0052] Using the TnPBEF synthetic sequence as a template, design a primer pair F1 / R1, use this primer pair to perform PCR amplification, select Phanta MasterMix (Vazyme Company) high-fidelity pfu enzyme, and perform pre-denaturation at 95°C for 3 minutes; 30 amplification stages Cycle according to 95°C, 15s, 58°C, 15s, 72°C, 1min; extend at 72°C, 5min. Purify the PCR product, use the vector pET28a as a template, design a primer pair F2 / R2, use this primer pair to perform PCR amplification, and select PhantaMasterMix (Vazyme Company) high-fidelity pfu enzyme for pre-denaturation at 95°C for 3 minutes; In the amplification phase, 30 cycles were performed according to 95°C, 15s, 58°C, 15s, 72°C, 3min; extension was 72°C, 5min. The PCR product was subjected to product purification. The fragment TnPBEF an...

Embodiment 3

[0059] Example 3: Strengthening the Construction of PRPP Synthetic Combination Plasmids

[0060] Nicotinamide phosphoribosyltransferase (Nampt / NadV) can catalyze the one-step synthesis of NAD derivatives from nicotinamide, which also requires another substrate, PRPP. In Escherichia coli, the PRPP synthase encoded by prs is the main source of PRPP, and the enzyme is regulated by the regulator encoded by purR. In order to strengthen the synthesis of PRPP, it is first necessary to overexpress the PRPP synthase gene prs through the plasmid, and connect the prs gene to the previously constructed recombinant plasmids pET28a+FtNadV, pET28a+RsNadV, pET28a+abNadV, pET28a+HdNadV, pET28a+MmNampt, and obtain Recombinant plasmids pET28a+BaPRS+FtNadV, pET28a+BaPRS+RsNadV, pET28a+BaPRS+abNadV, pET28a+BaPRS+HdNadV, pET28a+BaPRS+MmNampt.

[0061] Synthesize the PRPP synthase gene shown in SEQ ID NO.8. The gene BaPRS is derived from Bacillus amyloliquefaciens and has the ability to resist feed...

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Abstract

The invention discloses an enzyme gene suitable for efficiently synthesizing an NAD (Nicotinamide Adenine Dinucleotide) derivative in microorganisms, and belongs to the technical field of gene engineering and bioengineering. According to the invention, nicotinamide phosphoribosyltransferase gene VpNadV shown as any one of SEQ ID NO.1-7 is expressed in escherichia coli, and a pncC gene, a ushA gene, an nadR gene and a purR gene are knocked out, so that the accumulation amount of the NAD derivative in escherichia coli cells is increased, and fermentation conditions are optimized, so that the accumulation amount of the NAD derivative is further increased, the NAD derivative with the concentration of 1g / L or above can be obtained under a 5L fermentation system, and the enzyme gene has wide application prospects in the fields of foods, medicines, cosmetics, feeds and textiles.

Description

technical field [0001] The invention relates to an enzyme gene suitable for efficiently synthesizing NAD derivatives in microorganisms, applicable to such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, etc., and belongs to the technical field of genetic engineering and biological engineering. Background technique [0002] As early as the 1930s, people discovered that nicotinamide adenine dinucleotide (NAD) participated in many redox reactions in the form of a coenzyme and played an important role. At that time, Warburg et al. proved that NAD can participate in the redox process as a hydrogen acceptor and hydrogen donor. With the development of science and technology and the updating of technical means, NAD and its precursors, derivatives and metabolic enzymes have recently been proved to play a decisive role in a variety of biological functions, from classic oxidative phosphorylation and redox reactions to regulating genes Transcription, longevity, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/70C12N1/21C12P19/36C12R1/19C12R1/125C12R1/15
CPCC12N9/1077C12N9/1235C12N15/70C12P19/36C12Y204/02012C12Y207/06001
Inventor 周景文陈坚黄忠实曾伟主
Owner JIANGNAN UNIV
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