Phosphinothricin dehydrogenase mutant, genetically engineered bacteria and one-pot multi-enzyme synchronous directed evolution method

A technology of genetically engineered bacteria, glufosinate-ammonium, applied in genetic engineering, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem that the conversion rate cannot reach 100%, the raw material PPO cannot be completely converted, and the aspartic acid can not be fully converted. L-PPT separation is troublesome and other problems, to achieve the effect of high synergy efficiency, high space-time yield, and high total conversion number

Active Publication Date: 2020-09-04
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But utilize transaminase to prepare L-glufosinate-ammonium and there are two big defects, and one is that this is a reversible reaction, and raw material PPO can't be converted into L-PPT completely, and conversion rate can't reach 100%; -The direction of PPT needs to add at least 2 times more L-aspartic acid as the amino donor. Excessive aspartic acid brings great trouble to the separation of L-PPT

Method used

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  • Phosphinothricin dehydrogenase mutant, genetically engineered bacteria and one-pot multi-enzyme synchronous directed evolution method
  • Phosphinothricin dehydrogenase mutant, genetically engineered bacteria and one-pot multi-enzyme synchronous directed evolution method
  • Phosphinothricin dehydrogenase mutant, genetically engineered bacteria and one-pot multi-enzyme synchronous directed evolution method

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Experimental program
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Effect test

Embodiment 1

[0049] Construction of glufosinate-ammonium dehydrogenase and glucose dehydrogenase or formate dehydrogenase coupling expression vector and engineering bacteria.

[0050] Construction of expression vectors: The gene synthesis of glufosinate-ammonium dehydrogenase, glucose dehydrogenase and formate dehydrogenase described below was completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. The glufosinate-ammonium dehydrogenase gene (NCBI accession number WP_150701510.1) derived from Pseudomonas fluorescens was seamlessly cloned into SacI and NotI of the first multiple cloning site of pETDuet-1 vector by PCR In between, the glucose dehydrogenase gene (NCBI accession number: KM817194.1) derived from Exiguobacterium sibiricum or the formate dehydrogenase derived from Lactobacillus buchneri (NCBI accession number: WP_013726924.1) were seamlessly cloned into pETDuet- Between Bg1II and PacI of the second multiple cloning site of the 1 vector, obtain the expression vector pETDuet-PPTD...

Embodiment 2

[0054] Construction and screening of glufosinate-ammonium dehydrogenase and glucose dehydrogenase / formate dehydrogenase dual-enzyme coupled gene libraries.

[0055] 1) Establishment of high-throughput screening method

[0056]Prepare 50mL working solution (derivatization reagent): o-phthalaldehyde 0.013g, N-acetyl-L-cysteine ​​0.032g, dissolve with pH=9.8 boric acid buffer to 50mL, shake to fully dissolve, 4 Store in the refrigerator at ℃ for later use (no more than 4 days), as a high-throughput working solution, also known as a derivatization reagent. Draw 50 μL sample reaction solution and add 50 μL working solution to shake and react for 30 seconds, then add 100 μL ddH2O, at λ ex =340nm,λ em Fluorescence value was measured under the condition of =455nm.

[0057] 2) One-pot multi-enzyme simultaneous directed evolution

Embodiment 3

[0065] Site-directed saturation mutation of glufosinate-ammonium dehydrogenase in multi-enzyme coupling reaction system.

[0066] In order to further screen potential activity-promoting bacterial strains, two beneficial mutation sites of A164 and R205 of glufosinate-ammonium dehydrogenase obtained in Example 2 were first subjected to site-directed saturation mutation for further screening, and the PCR primers were designed as shown in Table 1 , PCR system (50 μL): 2*Phanta Max buffer 25 μL, dNTPs 1 μL, mutation primers 1 μL each, template (starting strain) 1 μL, Pfu DNA polymerase 0.5 μL, add ddH2O to 50 μL. The PCR conditions were: 95°C pre-denaturation for 3 minutes: 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 7 min 20 s, 30 cycles; 72°C final extension for 10 min. The PCR product was verified by DNA agarose gel electrophoresis, and after the template was digested by DpnI, the PCR product was transformed into Escherichia coli E.coli BL21 (DE3) com...

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Abstract

The invention discloses a phosphinothricin dehydrogenase mutant, genetic engineering bacteria and a one-pot multi-enzyme synchronous directed evolution method. The phosphinothricin dehydrogenase mutant is obtained by mutating the 164th amino acid from alanine to glycine, mutating the 205th arginine to lysine and mutating the 332nd threonine to alanine of phosphinothricin dehydrogenase derived fromPseudomonas fluorescens, and an amino acid sequence is as shown in SEQ ID No.1. The genetically engineered bacteria are obtained by introducing a gene of the phosphinothricin dehydrogenase mutant into a host cell. An encoding gene of glucose dehydrogenase or an encoding gene of formate dehydrogenase can also be introduced into the host cell to perform simultaneous directed evolution to overexpress the double genes. The one-pot multi-enzyme synchronous directed evolution method of the invention can screen out the genetically engineered bacteria with greatly improved activity. Compared with catalytic processes such as transaminase, the L-PPT preparation method of the invention has a relatively simple process, a high conversion rate of raw materials, a conversion rate of up to 100%, and highstereo-selectivity.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to a glufosinate-ammonium dehydrogenase mutant, a genetically engineered bacterium and a one-pot multi-enzyme synchronous directed evolution method. Background technique [0002] Glufosinate-ammonium (phosphinothricin, also known as glufosinate, referred to as PPT), the chemical name is 2-amino-4-[hydroxy (methyl) phosphono]-butyric acid, which is the second most resistant herbicide in genetically modified crops in the world. Developed and produced by Sturt (now owned by Bayer after several mergers), it is also known as glufosinate ammonium salt, Basta, Buster, etc. It is a phosphonic acid herbicide, and the non-selective (killing) contact herbicide is glutamine Synthetic enzyme inhibitors. [0003] Glufosinate-ammonium has two optical isomers, namely L-glufosinate-ammonium and D-glufosinate-ammonium. But only the L-type has physiological activity, and is easy to decompose in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12P13/04C12N1/21C12R1/39
CPCC12N9/0016C12Y104/01004C12N9/0006C12N9/0008C12Y101/01047C12Y102/01002C12P13/04C12N15/70C12N15/52C40B40/08C40B40/02C12N15/1058C12R2001/19C12R2001/39C12N1/20C12N2800/101
Inventor 薛亚平程峰李举谋李清华郑裕国
Owner ZHEJIANG UNIV OF TECH
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