bdsf high-yield strain and its fermentation optimization method and application

A technology of high-yielding strains and strains, applied in biochemical equipment and methods, fermentation, and microbial-based methods, etc., can solve problems such as complex processing technology, high energy consumption, and increased BDSF production costs, and achieve high-efficiency production, increase yield, and yield Improved effect

Active Publication Date: 2020-11-06
周莲
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the temperature-controlled fermentation production process at 28°C will cause high energy consumption, which increases the production cost of BDSF
And Xcc is a plant pathogenic bacteria, and the post-fermentation treatment process is more complicated than that of E. coli

Method used

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  • bdsf high-yield strain and its fermentation optimization method and application
  • bdsf high-yield strain and its fermentation optimization method and application
  • bdsf high-yield strain and its fermentation optimization method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0041] The construction of embodiment 1BDSF high-yielding bacterial strain (BLSF)

[0042] 1. Amplify the RpfF protein coding gene rpfF necessary for BDSF biosynthesis.

[0043] First, primers were designed according to the rpfF sequence of the RpfF protein coding gene of the Xcc8004 strain in GenBank, and the nucleotide sequences of the primers were as follows:

[0044] Upstream primer (SEQ ID No.1):

[0045] 5'- CCGCTCGAG ATGTCTGCAGTTCAACCCTTCATTC-3';

[0046] Downstream primer (SEQ ID No.2):

[0047] 5'- CCGCTCGAG TCAGCCCGCGTCGAGCCCTG-3'.

[0048] The underline in the sequence is the cleavage site of restriction endonuclease XhoI. The primers were synthesized by Shanghai Sangon Bioengineering Technology Service Company. Then, using the Xcc8004 strain genomic DNA as a template, DNA polymerase Taq and designed primers were used to amplify the coding gene of BDSF biosynthesis. The amplified product was detected by 1% agarose gel electrophoresis, and the target band...

Embodiment 2B

[0057] The conventional fermentation culture of embodiment 2BDSF high-yield bacterial strain (BLSF)

[0058] Inoculate the genetically engineered strain BLSF on an LB medium plate, activate and culture it at 37°C for 16-24 hours; then inoculate a single colony of activated BLSF into 10mL liquid medium containing 100mg / L carbenicillin, with a volume of 250ml In a Erlenmeyer flask, shake culture at 37°C for 12 hours. Finally, it was transferred to 50 mL LB liquid medium containing 100 mg / L carbenicillin in a 250-mL Erlenmeyer flask for expanded fermentation, and cultured in a shaker at 37° C. at a speed of 220 rpm for 72 hours. The highest yield of the BLSF strain was 161.79 μM ( figure 2 ), the growth curve is as image 3 shown.

[0059] 1 liter of LB medium includes 10 grams of tryptone, 5 grams of yeast extract, 10 grams of sodium chloride, and 15 grams of agar, with a pH of 7.0-7.4. Extraction method of BDSF: Take 500 μL of fermentation broth in a 2 mL centrifuge tube...

Embodiment 3

[0060] Embodiment 3 optimizes BDSF high-yielding bacterial strain (BLSF) fermentation medium

[0061] 1. Selection of initial fermentation medium

[0062] By comparing the growth of engineering strain BLSF in four liquid media of LB, TB, SOB and M9 (OD 600 value) and the corresponding BDSF yield found that: under the same fermentation conditions (fermentation temperature: 37 ° C, shaker speed: 220 rpm; fermentation time: 72 hours), engineering strain BLSF BDSF in TB liquid medium medium The highest yield, up to 638.42μM ( Figure 4 A), and the biomass is the highest ( Figure 4 B). Therefore, in the future optimization experiments of fermentation conditions, TB medium was selected as the initial medium for the production of BDSF by engineering strain BLSF fermentation.

[0063] 2. Single factor test

[0064] On the basis of TB medium, different auxiliary carbon and nitrogen sources were added, and the final concentration of the added components was maintained at 40g / L, ...

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Abstract

The invention provides BDSF high-yield bacterial strain BL21(DE3)plysS / pET1b-rpfF(BLSF), and a construction method and applications thereof. According to the construction method, gene engineering method is adopted, cloning of RpfF protein coding gene rpfF into BL21(DE3)plysS is carried out so as to obtain the BDSF high-yield bacterial strain. The BDSF high-yield bacterial strain is capable of realizing stable and high efficiency production of BDSF. The invention also provides a culture medium and a fermentation method used for culturing the BDSF high-yield bacterial strain. BDSF yield can be increased obviously, the fermentation anaphase yield stability of the BDSF high-yield bacterial strain can be maintained in the optimized culture medium, and the highest yield ranges from 1937.75+-72.25<mu>M.

Description

technical field [0001] The invention relates to the technical field of genetic engineering strains and microbial fermentation optimization methods, in particular to a high-yielding cis-2-dodecenoic acid (BDSF) strain and its fermentation optimization method and application. Background technique [0002] Quorum Sensing (QS) is an important way for bacteria to perceive their own population density and communicate with each other. Specifically, in a specific environment, bacteria can produce and release one or more small signal molecules, that is, quorum sensing signal molecules. When the extracellular concentration of these signal molecules reaches a certain threshold, they can be activated by bacteria on the surface Or receptors in the cytoplasm induce expression of related genes and regulate specific biological functions. DSF (diffusible signal factor) is a new type of quorum sensing signal molecule first identified in Xanthomonas campestris in recent years. Its chemical st...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/64C12R1/19
CPCC12N9/00C12N9/16C12N15/70C12P7/6409C12Y301/02
Inventor 周莲杨丹丹何亚文
Owner 周莲
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