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
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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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