High-yield fumaric acid Rhizopus delemar and application thereof
A technology of Rhizopus oryzae and Rhizopus oryzae, applied in the directions of application, introduction of foreign genetic material using vectors, biochemical equipment and methods, etc., can solve the problem of unclear details of carbon metabolism flow, weak genetic modification technology, and recombinant cells. Problems such as mitotic instability, to achieve the effect of promoting excessive accumulation
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Embodiment 1
[0026] The preparation of embodiment 1 germination spore
[0027] Wash the spores from the plate with sterile normal saline, filter through 6 layers of lens paper, and then wash with sterile normal saline 3 times, inoculate the washed spores in 30mL YEPD liquid medium, culture at 37°C, 200rpm for 4h to germinate the spores During the period, samples were taken every 1h to observe the spore morphology once. The germinated spores were collected by centrifugation at 8000 rpm at 4°C for 15 min, washed once with 20 mL of pre-cooled sterile saline, resuspended in 10 mL of YED medium, and incubated at 30°C at 150 rpm for 60 min. Collect the spores by centrifuging at 8000rpm at 4°C for 10 minutes, wash once with 10 mL of pre-cooled EB buffer, suspend the spores with 5 mL of pre-cooled EB buffer, aliquot them, and put them on ice for later use.
Embodiment 2
[0028] Example 2 Fusion PCR method to construct the target fragment ScPYC1 and the construction of the expression plasmid
[0029] Using Rhizopus oryzae pdcA non-translated promoter and terminator fragments as templates, PCR primers pdcProFPac I, pdcProR, pdcTerF, pdcTerRSalI (see Table 1 for details) were designed based on the R. oryzae AF282846 and AF282847 sequences published by NCBI, and thus amplified The pdcA promoter fragment with a length of 1219bp, and the terminator fragment with a length of 943bp, and two restriction sites of PacI and SalI were added to the 5' ends of the pdcProF and pdcTerR fragments respectively.
[0030] Using the NM_001180927.1 sequence published by NCBI, the PYC1 fragment of the coding region of Saccharomyces cerevisiae pyruvate carboxylase gene was synthesized by chemical total synthesis;
[0031] Under the action of pfu enzyme, through fusion PCR, the fusion fragment PYC1 of the three oligonucleotide chains of pdcAPro, pdcATer and PYC1 is amp...
Embodiment 3
[0036] Embodiment 3 fermentation produces the method for fumaric acid
[0037] Use Rhizopus oryzae R.delemar-pRS303H-PC as the starting strain, inoculate it into the spore-forming medium, and cultivate it at 30°C for 5-7 days until the conidia mature; wash the spores with sterile water, and dilute to 10 6 Individual / mL; transfer the spore suspension to the seed medium at a volume ratio of 4%, and cultivate at 30°C and 200rpm for 30h; cultivate the seeds before transfer with a volume ratio of 10% to the fermentation medium, and cultivate at 30°C and 200rpm for 72h.
[0038] Compared with the control bacteria, the recombinant bacteria: (1) the output of fumaric acid in the control bacteria was 46.87g / L, and the output of fumaric acid in the recombinant bacteria could reach 55.92g / L, which was 1.19 times that of the control bacteria; (2) after the fermentation, the control bacteria The body weight is 7.58g / L, and the weight of the recombinant bacteria is 7.41g / L, which is almost ...
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