Polyketide synthases EnPKS1 and EnPKS2 from Erythroxylum novogranatense, and gene and application of polyketide synthases EnPKS1 and EnPKS2

A technology of polyketide synthase and coca, applied in application, genetic engineering, plant gene improvement, etc., can solve the problem of no coding gene identification, etc., and achieve the effect of good industrialization prospects

Active Publication Date: 2021-11-09
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] To date, no polyketide synthases from coca are known in the art En PKS1 and En Identification of PKS2 and its encoding gene, and application of coca polyketide synthase

Method used

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  • Polyketide synthases EnPKS1 and EnPKS2 from Erythroxylum novogranatense, and gene and application of polyketide synthases EnPKS1 and EnPKS2
  • Polyketide synthases EnPKS1 and EnPKS2 from Erythroxylum novogranatense, and gene and application of polyketide synthases EnPKS1 and EnPKS2
  • Polyketide synthases EnPKS1 and EnPKS2 from Erythroxylum novogranatense, and gene and application of polyketide synthases EnPKS1 and EnPKS2

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Cloning of coca polyketide synthases (Polyketide synthases, PKS) gene.

[0046] (1) Extraction of total RNA from coca shoots and synthesis of first-strand cDNA.

[0047] Take an appropriate amount of coca leaf bud tissue, grind it in liquid nitrogen, and extract total RNA with the Biotek Polysaccharide and Polyphenol Plant Total RNA Rapid Extraction Kit according to the instructions. The concentration and quality of RNA were detected by Thermo Scientific NanoDrop spectrophotometer, and the quality of RNA was detected by agarose gel electrophoresis.

[0048] cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit reverse transcription kit from Novizyme, following the instructions of its product manual, using total RNA as a template.

[0049] (2) PKS Gene cloning.

[0050] Design specific primers, the specific primer sequences are as follows:

[0051] EnPKS1-F: 5'-atgaacggaaccgtcaagaaaatgaatg-3' (SEQ ID NO.1)

[0052] EnPKS1-R: 5'-tcattctgtaaccacag...

Embodiment 2

[0057] Prokaryotic expression verification PKS 1 and PKS 2 Gene function.

[0058] Introduced at the enzyme cleavage site EnPKS1 and EnPKS2 Gene. for EnPKS1 and EnPKS2 Gene Design Primers, PKS 1 forward primer with Bam The HI restriction site is shown in SEQ ID NO.9, and the reverse primer contains Sal I restriction site is as shown in SEQ ID NO.10; PKS 2 forward primers with Bam The HI restriction site is shown in SEQ ID NO.11, and the reverse primer contains xho The I restriction site is shown in SEQ ID NO.12. Amplified by PCR to obtain enzyme cleavage sites at both ends EnPKS1 and EnPKS2 After the gene, use the cohesive ends generated by these two restriction sites to separate the PKS 1 and PKS The complete sequence of the coding region of 2 was connected to the plasmid pET28a to obtain the recombinant expression vector pET28a- PKS 1 and pET28a- PKS 2.

[0059] The primer sequences are as follows:

[0060] BamHI-EnPKS1-F: 5'-cgggatccatga...

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Abstract

The invention discloses two polyketide synthases (PKS) EnPKS1 and EnPKS2 from Erythroxylum novogratin, as well as a coding gene and application of the two polyketide synthases. An amino acid sequence of the EnPKS1 is as shown in SEQ ID NO. 4, and a nucleotide sequence of the EnPKS1 is as shown in SEQ ID NO. 3; and an amino acid sequence of the EnPKS2 is as shown in SEQ ID NO. 8, and a nucleotide sequence of the EnPKS2 is as shown in SEQ ID NO. 7. A protein expressed by escherichia coli can be used for catalyzing the condensation of two molecules of malonyl-CoA (malonyl-CoA) to generate acetone dicarboxylic acid. The polyketide synthases can be applied to production of tropane alkaloids (such as hyoscyamine, scopolamine and cocaine) in synthetic biology and production of natural products of acetone dicarboxylic acid intermediates in a biosynthetic route; or the polyketide synthases are used for guiding molecular breeding of related medicinal plants.

Description

technical field [0001] The present invention relates to the field of plant molecular biology and discloses polyketide synthase from coca En PKS1 and En PKS2 and its gene, the identification of the gene encoding coca polyketide synthase, are also involved in the application of coca polyketide synthase. En PKS1 and En PKS2 can use malonyl-CoA as a substrate to catalyze the production of acetone dicarboxylic acid, which is an important intermediate in plant biosynthesis and in vitro chemical synthesis of tropane skeleton. The polyketide synthase of the present invention can be used as a component in the synthetic biology of tropine alkaloids, and in the synthetic metabolic engineering of natural products involving acetone dicarboxylic acid intermediates in the biosynthetic pathway, providing more synthetic biology Optional components. Background technique [0002] Tropic alkaloids (TAs), as an important class of natural products, are derived from Solanaceae, Erythroxylaceae...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P7/44C12R1/19
CPCC12N9/1029C12N15/70C12P7/44
Inventor 黄胜雄杨静田恬黄建萍王永江李洁
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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