Engineering strain and application thereof to production of long-chain 3-hydroxy fatty acid

A technology of hydroxy fatty acid esters and engineering bacteria, which is applied in the field of genetic engineering and microbial fermentation, can solve the problems of complex process, high production cost, large production investment, etc., and achieve the effect of broad application prospects

Active Publication Date: 2013-03-06
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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  • Engineering strain and application thereof to production of long-chain 3-hydroxy fatty acid
  • Engineering strain and application thereof to production of long-chain 3-hydroxy fatty acid
  • Engineering strain and application thereof to production of long-chain 3-hydroxy fatty acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Construction of engineering bacteria

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

[0043] 1. Synthesize the double-stranded DNA molecule shown in sequence 4 of the sequence list, in which the 10th to 510th nucleotides are the upstream homologous fragment (named H1 fragment), and the 517 to 1366th nucleotides are Gentiana sulfate Resistance gene (also known as Gm r Genes, in which positions 523-1356 are open reading frames), and nucleotides 1372 to 1835 are downstream homologous fragments (named H2 fragments).

[0044] 2. Double-cut the double-stranded DNA molecule of step 1 with restriction enzymes HindIII and NheI, and recover the digested product.

[0045] 3. Double digest pK18mobsacB plasmid with restriction enzymes HindIII and NheI to recover the vector backbone (about 5.5kb).

[0046] 4. Connect the digested product of step 2 and the vector backbone of step 7 to obt...

Embodiment 2

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

[0074] 1. Use recombinant engineering bacteria LAC31 (pSPH09) to produce 3-hydroxydecanoic acid

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

[0076] 2. Inoculate the first-level seed solution obtained in step 1 into 4YLB medium containing 12g / L n-decanoic acid at 5% inoculum to obtain the initial system. In the initial system, the concentration of the recombinant engineered bacteria LAC31 (pSPH09) is OD600nm =0.45 (In actual applications, 0.3-0.6 can be used).

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

[0078] 4. Detect the dry weight of bacterial cells in the termination system and the content of 3-hydroxy fatty ...

Embodiment 3

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

[0096] Recombinant engineered strain LAC31 (pZGD01) was used instead of recombinant engineered strain LAC31 (pSPH09). Others are the same as in Example 2.

[0097] With n-decanoic acid as the carbon source, the dry cell weight of the recombinant engineered strain LAC31 (pZGD01) after fermentation is 1.08±0.01g / L, the 3-HA concentration in the fermentation supernatant is 1.75±0.06g / L, and 3-HA The component of GC-MS detected as 3-HD. It is proved 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 the carbon source, the dry cell weight of recombinant engineered strain LAC31 (pZGD01) was 0.90±0.09g / L, the 3-HA concentration in the fermentation supernatant was 4.59±0.14g / L, and 3-HA The HA component was detected as 3-HDD by GC-MS. It is proved that the method of the p...

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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 present invention relates to the fields of genetic engineering and microbial fermentation, in particular to an engineered bacteria and its application in the production of medium-long-chain 3-hydroxy fatty acids (3-HA). Background technique [0002] 3-Hydroxy fatty acid (3-HA) is a common monomer for new biomaterials, polyhydroxy fatty acid ester (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 backbone, 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, long-chain 3-hydroxy fatty acids can be used as precursors for the synthesis of important drugs such as anti-cancer drugs. [0004] The traditional production method of 3-hydroxy fatty acid is chemi...

Claims

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Application Information

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