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Extreme halophilic archaea engineering bacteria for producing bioplastics PHBV by effectively utilizing carbon source

An extremely halophilic archaea, sequence technology, applied in the directions of microorganism-based methods, microorganisms, genetic engineering, etc., can solve the problems of increasing the viscosity of the culture medium, increasing the amount of foaming, and wasting carbon sources, and solve the problem of the culture medium becoming viscous. Effect

Active Publication Date: 2013-12-18
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The synthesis and accumulation of exopolysaccharides not only cause unnecessary waste of carbon sources, but also have some side effects on production, including increasing the viscosity of the culture medium, increasing the amount of foaming, and reducing dissolved oxygen.
Studies on exopolysaccharides of Mediterranean halobacteria are currently limited to their production optimization, physicochemical properties, physiological functions, and compositional structures, while there are no reports at the genetic level.

Method used

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  • Extreme halophilic archaea engineering bacteria for producing bioplastics PHBV by effectively utilizing carbon source
  • Extreme halophilic archaea engineering bacteria for producing bioplastics PHBV by effectively utilizing carbon source
  • Extreme halophilic archaea engineering bacteria for producing bioplastics PHBV by effectively utilizing carbon source

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1. Determination of key gene clusters for exopolysaccharide synthesis and acquisition of genes

[0059] The complete set of glycosyltransferases is summarized based on the whole genome sequence and annotations of Halobacterium mediterranei. After analysis, the gene HFX2145-2148 was considered as a possible key gene cluster for exopolysaccharide synthesis (eps). These four genes may share a promoter. Among them, HFX2145 is involved in the biosynthesis of the precursor substance UDP-N-acetylglucosamine, HFX2146 and HFX2147 are involved in the sequential transfer of sugar groups to the plasma membrane carrier dolicol phosphate, and HFX2148 is involved in the trisaccharide monosaccharide body transmembrane transport. The structure of the four coding genes and the arrangement of the upstream and downstream genes are as follows: figure 1 shown.

[0060] A pair of primers epsF1 / epsR1 was designed for PCR amplification of the nucleotide sequence including the key gen...

Embodiment 2

[0067] Example 2. Identification of eps function of the key gene cluster for exopolysaccharide synthesis

[0068] Knock out and complement eps by genetic engineering technology, and verify that it is necessary for exopolysaccharide synthesis by genetic methods.

[0069] 1. Construction of uracil synthesis-deficient exopolysaccharide synthesis key gene cluster (eps) knockout strain

[0070] Using the genetic operating system based on the pyrF gene, the structural diagram of the pyrF gene and its upstream and downstream sequences is shown in figure 2 As shown, using Haloferax mediterranei ΔpyrF (Haloferax mediterranei ΔpyrF is the Mediterranean haloferax mediterranei CGMCC 1.2087 as the starting strain, the uracil synthesis-deficient strain obtained by knocking out the pyrF gene is convenient for the screening of gene knockout and complementation, and improves the efficiency of knockout. Knockout efficiency; Liu,H.,J.Han,et al.(2011)."Development of pyrF-based gene knockout sy...

Embodiment 3

[0121] Example 3, Construction of eps knockout strain independent of exogenous addition of uracil growth

[0122] The eps knockout strain Haloferax mediterranei ΔpyrFΔeps, which is deficient in uracil synthesis, relies on exogenously added uracil. Even though it can efficiently use carbon sources to produce PHBV, it is difficult to be used as a strain for industrial production. Therefore, it is necessary to complement its missing gene pyrF in situ.

[0123] 1. In situ knock-in of deletion gene pyrF in strains deficient in uracil synthesis

[0124] Using the genetic operating system based on the pyrF gene, Haloferax mediterranei ΔpyrFΔeps was used as the starting strain, and the deleted gene pyrF was knocked in in situ to construct an eps deletion mutant that does not rely on exogenous addition of uracil. First construct the integrated plasmid pT-pyrFr for in situ knock-in of pyrF, and then undergo homologous recombination single crossover and homologous recombination double cr...

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Abstract

The invention discloses extreme halophilic archaea engineering bacteria for producing bioplastics PHBV (Poly-(HydroxyButyrate-co-Hydroxy Valerate)) by effectively utilizing a carbon source. The recombined extreme halophilic archaea is extracellular polysaccharide synthesis function-deficient engineering bacteria obtained by deleting at least one protein function expressed by an extracellular polysaccharide synthesis cluster in the genome of the extreme halophilic archaea Haloferax mediterranei. The extreme halophilic archaea has the advantages that infectious microbe is not easy to pollute, PHA (Poly Hydroxy Alkanoate) is convenient to extract, the PHBV from a non-correlated carbon source can be synthesized, and the like, and is considered as a highly preponderant PHBV producing strain. The extracellular polysaccharide synthesis function-deficient strain engineering bacteria are characterized in that the polyhydroxyalkanoate can be produced from various carbon sources such as glucose, starch and whey more efficiently in contrast with a wild type strain, the concentration of the PHBV is 20% higher than that of the wild type strain under the same fermentation conditions, and the problems such as sticking, lots of bubbles and dissolved oxygen reduction of a culture solution caused by extracellular polysaccharide accumulation are also solved.

Description

technical field [0001] The invention relates to an engineering bacterium of extreme halophilic archaea producing polyhydroxyalkanoate PHBV. Background technique [0002] Polyhydroxyalkanoate is a kind of high-molecular polyester accumulated in cells by microorganisms when nutrition is unbalanced. It exists widely in nature as a carbon source and energy reserve. Due to its thermoplasticity, biodegradability, and biocompatibility, PHA is widely considered as a potential substitute for petrochemically derived plastics that cause white pollution, and can be applied in applications including everyday items (e.g. plastic bags, containers) and biomedical materials (e.g. Artificial blood vessels, drug release carriers), and more and more attention from the scientific and commercial communities. [0003] At present, PHB, PHBV and P3HB4HB are the main bacteria that realize the industrial production of PHA. PHB is similar to thermoplastics in some properties, and its mechanical prope...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/54C12N15/31C12N15/11C12N1/21C12P7/62C12R1/01
Inventor 向华赵大贺
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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