Method for producing short-and-medium-chain-length polyhydroxyalkanoate (PHA) and functional derivatives thereof

A technology of chain polyhydroxyalkanoates and derivatives, applied in the fields of biotechnology and materials, can solve the problems of easy contamination, consumption of a large amount, and difficulty in controlling the ratio of short and medium-chain PHA monomers.

Active Publication Date: 2020-06-05
TSINGHUA UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the production method of PHA (SCL-co-MCL PHA) of short-chain and medium-chain copolymerization has the following disadvantages: 1. The proportion of functional groups of functional derivatives is relatively low
Limited by the ability of the expression system and the enzymatic activity of PHA polymerase, the proportion of functional groups in the functional PHA currently produced is still low
2. The ratio of side chain monomers is difficult to control
Due to the existence or incomplete weakening of the β-oxidation cycle, after the medium and long-chain carbon source enters the cell, two carbon atoms will be lost in each round of the β-oxidation cycle, and multiple PHA units including C6, C8, C10, and C12 will be obtained. Therefore, it is difficult to control the monomer ratio of the short and medium chain PHA produced, and it is difficult to obtain stable material properties
3. Lack of suitable chassis strains
At present, the production of short-medium chain PHA and its functional derivatives is mainly based on traditional industrial production bacteria, such as Pseudomonas, recombinant Escherichia coli, etc., which are not suitable for modern times due to the problems of easy-to-contaminate bacteria, discontinuous growth, and the need to consume a large amount of fresh water. Biotechnology development needs

Method used

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  • Method for producing short-and-medium-chain-length polyhydroxyalkanoate (PHA) and functional derivatives thereof
  • Method for producing short-and-medium-chain-length polyhydroxyalkanoate (PHA) and functional derivatives thereof
  • Method for producing short-and-medium-chain-length polyhydroxyalkanoate (PHA) and functional derivatives thereof

Examples

Experimental program
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preparation example Construction

[0092] The preparation method of the seed liquid in the following examples is as follows: 1) strain activation: get the strain glycerol tube stored in the -80°C refrigerator, and inoculate it into the 60-LB medium containing 25mg / L chloramphenicol plate, cultured at 37°C for 24-36h. 2) First-class seeds: Pick a single colony from the plate that completed step 1, inoculate it in 20 mL of liquid 60-LB medium containing 25 mg / L chloramphenicol, and culture it with shaking at 37°C and 200 rpm for 24 hours. 3) Secondary seed: Take the primary seed liquid obtained in step 2, inoculate it into 20 mL liquid 60-LB medium containing 25 mg / L chloramphenicol according to the inoculum size of 1%, and culture it with shaking at 37° C. and 200 rpm for 8-10 hours.

[0093] The fermentation culture method in the following examples is specifically as follows: 1) Preparation of seed liquid: refer to the preparation method of the above-mentioned seed liquid. 2) Preparation of fermentation medium...

Embodiment 1

[0105] Example 1. Controllable production of short and medium chain PHA [P(3HB-co-3HHx)] using recombinant Halomonas

[0106] 1. Construction of Recombinant Halomonas

[0107] 1. Construction of endogenous phaC gene-deficient strain H. bluephagenesis TDCKO3

[0108] 1) Extract the genomic DNA of Halomonas H. bluephagenesis TD01, and use it as a template to clone homologous fragments phaC-HR-L-3 and phaC-HR-R-3 with a length of 500 bp, respectively. It is located upstream and downstream of the phaC gene of H. bluephagenesis TD01.

[0109] 2) Using pQ31 (construct the backbone plasmid for CRISPR / Cas9 knockout gRNA [Qin, Q., Ling, C., Zhao, Y.Q., Yang, T., Yin, J., Guo, Y.Y., and Chen, G.Q. (2018) CRISPR / Cas9 editing genome of extremophile Halomonas spp., Metab.Eng.47, 219-229.]) as a template, the two homologous fragments and the backbone fragment of pQ31 were amplified by PCR, and constructed by One-spot method pTDCKO3-donor plasmid, based on the correct pTDCKO3-donor plasmi...

Embodiment 2

[0162] Example 2. Controllable production of short and medium chain functional PHA [P(3HB-co-3HHxE)] using recombinant Halomonas

[0163] 1. Preparation of recombinant Halomonas H. bluephagenesis TDCKO3 (pSA3CJ-P porin -57) seed solution.

[0164] 2. Fermentation with glucose and different concentrations of hexenoic acid as dual carbon sources

[0165] Get 2.5mL of the recombinant Halomonas H.bluephagenesis TDCKO3 (pSA3CJ-P porin -57) Seed solution, inoculated in a 500 mL shake flask containing 47.5 mL of shake flask culture medium, cultured with shaking at 37° C. and 200 rpm for 48 hours.

[0166] Shake flask medium is any of the following:

[0167] Shake flask medium 10G+0.25HEA: 60-MM medium containing 25mg / L chloramphenicol, 10g / L glucose and 0.25g / L 5-hexenoic acid;

[0168] Shake flask medium 10G+0.5HEA: 60-MM medium containing 25mg / L chloramphenicol, 10g / L glucose and 0.5g / L 5-hexenoic acid;

[0169] Shake flask medium 10G+1HEA: 60-MM medium containing 25mg / L chlor...

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Abstract

The invention discloses a construction method of a recombinant bacterium for producing short-and-medium-chain-length PHA and functional derivatives thereof. The method comprises the following steps of: introducing a coding gene of specific PHA polymerase, a coding gene of a key protein of a PHA synthesis pathway, a promoter or a mutated promoter, related genes capable of enhancing carbon source utilization capacity of medium-chain and long-chain fatty acids, the ribosome binding site RCJ and the ribosome binding site RD into a starting strain from which an endogenous PHA polymerase gene is knocked out; and adjusting the proportions of all monomers in the short-and-medium-chain-length PHA and the functional derivatives thereof in the recombinant bacteria so as to realize controllable production of the short-and-medium-chain-length PHA and the functional derivatives thereof.

Description

technical field [0001] The invention belongs to the field of biotechnology and materials, in particular to a method for producing short-medium chain polyhydroxyalkanoate PHA and its functional derivatives. Background technique [0002] Polyhydroxyalkanoate (PHA) is a kind of biological linear polymer polyester, which is commonly found in microbial cells with unbalanced growth conditions, and it is widely used as a storage material for energy and carbon sources. Due to its good biodegradability, biocompatibility and many excellent properties, PHA is considered to be one of the most potential biomaterials to solve global plastic pollution. At present, a variety of PHAs have been successfully synthesized and put into industrial production, including poly 3-hydroxybutyrate (PHB), poly 3-hydroxybutyrate-3-hydroxyvalerate (PHBV), poly 3-hydroxybutyrate -3-Hydroxyhexanoate (PHBHHx), poly-3-hydroxybutyrate-4-hydroxybutyrate (P3HB4HB). Traditional PHA is relatively biologically ine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N9/88C12N15/60C12N9/10C12N15/54C12N1/21C12P7/62C12R1/01
CPCC12N9/1029C12N9/88C12N15/52C12N15/74C12P7/625C12Y203/01086C12Y402/01119
Inventor 陈国强俞林萍闫煦蒋笑然
Owner TSINGHUA UNIV
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