Method for improving fermenting property of solventogenic clostridia

A technology of Solventogenic Clostridium and Clostridium acetobutylicum, applied in the direction of microorganism-based methods, fermentation, biochemical equipment and methods, etc., can solve problems that have not been explored and increase fermentation costs

Inactive Publication Date: 2017-05-17
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the use of biotin or natural substances containing biotin in the medium inevitably increases the cost of fermentation
Is it possible to overexpress biotin synthesis-related genes in solventogenic Clostridium to enhance its own biotin synthesis ability, thereby eliminating the dependence of solventogenic Clostridium on exogenous biotin or biotin-containing components in the process of growth and metabolism Woolen cloth? In addition, if the biotin synthesis ability of Solventogenic Clostridium is improved to achieve a continuous and sufficient supply of biotin in the body, will it be possible to further improve the fermentation performance of the strain and obtain better fermentation results than exogenous biotin addition? These questions have not been explored

Method used

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  • Method for improving fermenting property of solventogenic clostridia
  • Method for improving fermenting property of solventogenic clostridia
  • Method for improving fermenting property of solventogenic clostridia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1: adding biotin can strengthen the fermentation performance of Clostridium acetobutylicum

[0076] experimental method:

[0077] The wild-type Clostridium acetobutylicum was fermented using the P2 medium supplemented with different concentrations of biotin to determine the effect of biotin on the fermentation of Clostridium acetobutylicum.

[0078] Experimental steps:

[0079] 1) Add different concentrations of biotin (biotin concentration: 10, 15, 20, 25 and 30 μg / L) to the fermentation medium of Clostridium acetobutylicum (P2 medium without biotin) to construct different concentrations of biotin fermentation medium;

[0080] 2) Inoculate wild-type Clostridium acetobutylicum in the medium prepared in 1) at a ratio of 5% (v / v), and carry out static fermentation at 37°C in an anaerobic box; the specific fermentation conditions are as follows: P2 medium (30ml), 7% glucose (w / v), 5% inoculum size (v / v), biotin added concentrations: 10, 15, 20, 25 and 30 μg / L...

Embodiment 2

[0085] Example 2: Analysis and identification of biotin synthesis-related genes in Clostridium acetobutylicum and enhancement of their expression levels

[0086] experimental method:

[0087] Bioinformatics, comparative genomics and protein amino acid sequence homologous alignment were used to analyze and identify biotin synthesis genes in Clostridium acetobutylicum, and further improve the fermentation of Clostridium acetobutylicum by overexpressing biotin synthesis-related genes performance.

[0088] Experimental steps:

[0089] 1) According to the genome annotation in KEGG (http: / / www.genome.jp / kegg / ), the genes related to biotin synthesis in Clostridium acetobutylicum were analyzed;

[0090] 2) Using the amino acid sequence of biotin synthesis-related proteins in Bacillus subtilis as a template, search for potential biotin synthesis-related proteins in Clostridium acetobutylicum by using the amino acid sequence homologous alignment method (protein blast);

[0091] 3) En...

Embodiment 3

[0110] Embodiment 3: Determination of biotin content in BioYADB and EP strains

[0111] experimental method:

[0112] The content of biotin synthesized by Clostridium acetobutylicum was detected by biotin auxotrophic Lactobacillus plantarum E4.

[0113] Experimental steps:

[0114] 1) EP and BioYADB were anaerobically fermented in P2 medium without adding biotin, and samples were taken at 24h, 31h, 41h, 49h and 54h (4ml);

[0115] Fermentation conditions: P2 medium (400ml, no biotin added), 7% glucose (w / v), 5% inoculum size (v / v), erythromycin final concentration 10 μg / ml;

[0116] 2) Centrifuge the culture solution obtained in 1) to collect the bacteria, and collect the supernatant for determining the content of extracellular biotin;

[0117] 3) After washing the bacteria obtained in 2) three times with 0.9% NaCl (w / v), use 3M H 2 SO 4 Suspended bacteria were sterilized at 121°C for 1 hour to fully degrade the bacteria and release biotin in the body. After centrifugatio...

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Abstract

The invention provides a method for improving the fermenting property of solventogenic clostridia. Specifically, the invention provides a nucleic acid construct first, wherein the nucleic acid construct contains amino acid sequences shown as coding SEQ ID NO:1,2,3 and 4, or polynucleotide sequences or the complementary sequences thereof of amino acid sequences which are subjected to substitution, deletion or adding of one or multiple amino acids in the amino acid sequences shown as the SEQ ID NO:1,2,3 and 4, respectively maintain the biological activity of the SEQ ID NO:1,2,3 and 4 and are respectively derived by the SEQ ID NO:1,2,3 and 4, and an optional promoter sequence creatively connected with the above polynucleotide sequences or the complementary sequences thereof. The invention also discloses the solventogenic clostridia, a method for fermentation by the solventogenic clostridia and a method for improving the growing ability and / or the solvent production ability of the solventogenic clostridia.

Description

technical field [0001] The invention belongs to the field of fermentation of two important solventogenic clostridia, and specifically relates to a method for improving the fermentation performance of the solventogenic clostridia. Background technique [0002] Biotin, also known as vitamin H and coenzyme R, is a water-soluble sulfur-containing B vitamin. Biotin is an important trace element necessary for the growth of many microorganisms. It can be used as a coenzyme of carboxylase in bacteria to participate in various physiological activities of strains, such as amino acid metabolism, fatty acid metabolism, gluconeogenesis and toxin synthesis. Therefore, biotin is added to many microbial culture media. [0003] Solventogenic Clostridium can use various carbon sources such as glucose, sucrose, xylose, lactose, arabinose and carbon-containing gases (carbon monoxide and carbon dioxide) to ferment and produce acetone, ethanol and butanol. Among them, Clostridium acetobutylicum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N1/21C12P17/18C12R1/145
Inventor 顾阳杨云鹏杨晟姜卫红
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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