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A kind of engineering bacteria and its application in the production of medium and long-chain 3-hydroxy fatty acids

A technology of engineering bacteria and genes, applied in the field of genetic engineering and microbial fermentation, can solve the problems of complex process, large production investment, difficult separation, etc., and achieve the effect of broad application prospects.

Active Publication Date: 2015-09-30
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex process of these methods, large production investment, low product purity and difficulty in separation, the production cost is high

Method used

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  • A kind of engineering bacteria and its application in the production of medium and long-chain 3-hydroxy fatty acids
  • A kind of engineering bacteria and its application in the production of medium and long-chain 3-hydroxy fatty acids
  • A kind of engineering bacteria and its application in the production of medium and long-chain 3-hydroxy fatty acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the construction of engineering bacteria

[0042] 1. Construction of recombinant plasmid pALgSC (suicide plasmid for knocking out phaC1-phaZ-phaC2 in Pseudomonas entomophila LAC25)

[0043] 1. Synthesize the double-stranded DNA molecule shown in sequence 4 of the sequence listing, wherein the 10th to 510th nucleotides are upstream homologous fragments (named as H1 fragments), and the 517th to 1366th nucleotides are gentamicin sulfate Gm resistance gene (also known as Gm r gene, wherein the 523-1356 nucleotides are the open reading frame), and the 1372-1835 nucleotides are the downstream homologous fragments (named as H2 fragments).

[0044] 2. Digest the double-stranded DNA molecule in step 1 with restriction endonucleases HindIII and NheI, and recover the digested product.

[0045] 3. Digest the pK18mobsacB plasmid with restriction endonucleases HindIII and NheI, and recover the vector backbone (about 5.5kb).

[0046] 4. Ligate the digested product of ...

Embodiment 2

[0073] Example 2. Production of medium and long-chain 3-hydroxy fatty acids by recombinant engineering bacteria LAC31 (pSPH09)

[0074] 1. Production of 3-hydroxydecanoic acid by recombinant engineered bacteria LAC31 (pSPH09)

[0075] 1. Inoculate the recombinant engineered bacteria LAC31 (pSPH09) into 20 mL of LB medium (containing 50 μg / mL kanamycin sulfate), and culture at 30°C and 200 rpm for 12 hours to obtain the first-grade seed liquid.

[0076] 2. Inoculate the first-grade seed liquid obtained in step 1 to 4YLB medium containing 12g / L n-decanoic acid at an inoculation amount of 5% to obtain an initial system. In the initial system, the concentration of the recombinant engineered bacteria LAC31 (pSPH09) is OD600nm =0.45 (in practical applications, 0.3-0.6 is acceptable).

[0077] 3. Incubate the initial system obtained in step 2 at 30°C and 200 rpm for 48 hours to obtain the terminated system.

[0078] 4. Detect the dry weight of the bacterial cells in the termination...

Embodiment 3

[0095] Example 3. Production of medium and long-chain 3-hydroxy fatty acids by recombinant engineering bacteria LAC31 (pZGD01)

[0096] The recombinant engineered bacteria LAC31 (pZGD01) was used to replace the recombinant engineered bacteria LAC31 (pSPH09) and the others were the same as in Example 2.

[0097] Using n-decanoic acid as the carbon source, after the fermentation of the recombinant engineered bacteria LAC31 (pZGD01), the dry cell weight was 1.08±0.01g / L, the 3-HA concentration in the fermentation supernatant was 1.75±0.06g / L, and the 3-HA The component was detected as 3-HD by GC-MS. It proves that the method of the present invention can be used for the fermentation production of high-purity 3-HD.

[0098] Using n-dodecanoic acid as carbon source, after fermentation of recombinant engineering bacteria LAC31 (pZGD01), the dry cell weight was 0.90±0.09g / L, the concentration of 3-HA in the fermentation supernatant was 4.59±0.14g / L, and 3- The component of HA was de...

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Abstract

The invention discloses an engineering strain and application thereof to production of long-chain 3-hydroxy fatty acid. The engineering strain provided by the invention is a recombinant strain obtained by conducting the following modification on a starting strain: (a) inactivation of a PHA synthase gene; and (b) introduction of a thioesterase gene. The starting strain is bacteria producing polyhydroxyalkanoates. hen the engineering strain provided by the invention is used, and a middle to long-chain fatty acid is used as a single carbon source, a 3-hydroxy fatty acid product with high yield, high purity, and a structure highly consistent with that of the provided fatty acid carbon source can be obtained in a fermentation liquid. The method provided by the invention can be used for production of 3-hydroxy fatty acid with high yield and high purity, and has wide application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering and microbial fermentation, in particular to an engineering bacterium and its application in the production of medium and long-chain 3-hydroxy fatty acids (3-HA). Background technique [0002] 3-Hydroxy fatty acid (3-HA) is a common monomer for novel biomaterials polyhydroxyalkanoate (PHA). Due to its special chiral structure, 3-hydroxy fatty acids can be used as important precursors in the synthesis of drugs, antibiotics, vitamins, spices and pheromones. [0003] According to the length of the carbon chain skeleton, 3-hydroxy fatty acids can be divided into short chain and medium long chain: the former has a carbon chain length of 3-5, and the latter has a carbon chain length of 6-14. Among them, medium and long-chain 3-hydroxy fatty acids can be used as precursors for the synthesis of important drugs such as anticancer drugs. [0004] The traditional production method of 3-hydroxy fatty acid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P7/42C12R1/38
Inventor 陈国强郑美曾国栋
Owner TSINGHUA UNIV
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