Compositions and methods for producing 3-hydroxypropionic acid
一种氨基酸、基因的技术，应用在生产3-羟基丙酸的组合物领域，能够解决耐受水平不足等问题
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Embodiment 1A
[0250] Example 1A: Selection of yeast host cells based on 3-HP resistance
[0251] A panel of wild-type yeast strains was tested for their ability to grow in the presence of 3-HP.
[0252] The concentration range of 3-HP used in the initial screening process was determined by evaluating the growth ability of 7 wild yeast strains (Table 1, group A) on media containing different levels of 3-HP. The 3-HP used in these experiments was obtained by chemical synthesis from aqueous acrylic acid (30%) and CO2 catalyst at 200 psi and 175°C as described in WO04 / 076398. Residual acrylic acid was removed by back extraction with isopropyl ether at room temperature (see WO05 / 021470).
[0253] Cells were streaked on YPD plates and cultured overnight. Cell plasma with an OD600 of about 4 was prepared in YPD medium at pH 3.0. This slurry was used to inoculate microtiter wells containing various concentrations of 3-HP (pH 3.9) to an OD600 of 0.05. Cover the plate with a gas-permeable membr...
Embodiment 1B
[0279] Example 1B: Mutagenesis and selection of mutant strains with 3-HP resistance
[0280] Yeast cells selected in Example 1A were mutagenized and exposed to selection pressure in order to identify mutants with high 3-HP resistance.
[0281] For example, yeast cells in fresh YP (yeast extract / peptone) + 20g / L glucose plate or liquid culture (OD600 of 1-4) are suspended in sterile water to an OD600 of around 10. Aliquots of 200 μl of this cell suspension were added dropwise to each tube and exposed to 3 μl of ethyl methanesulfonate (EMS) for about 1 hour, which killed about 65% of the cells. Higher EMS concentrations can be used to increase kill rates. After exposure, cells were neutralized with 5% sodium thiosulfate, washed in PBS buffer, recovered in nutrient-rich medium for about 4 hours, and cultured in selective medium. Mock samples were also performed (without EMS) to ensure conditions were selective. Alternatively, UV irradiation can be used to mutagenize the cell...
Embodiment 2A
[0283] Example 2A: Steps for Transforming DNA into the Host Genome
[0284] Transformation of DNA into the genome of a yeast host to generate the modified yeast strains described in the Examples below was carried out based on the specific steps described below.
[0285] Add 4 mL of YP+10% glucose medium to a 14 mL Falon tube, and use a sterile loop to inoculate the medium with the desired strain. Cultures were grown overnight (-16 hr) at 37°C with shaking at 250 rpm. 1 mL of the overnight culture was added to a 250 mL baffled flask containing 50 mL of liquid YP + 10% glucose medium. The flasks were incubated at 37°C with shaking at 250 rpm. Small aliquots of the culture were removed at approximately hourly intervals and the OD600 was measured. Cultures were grown to an OD600 of 0.6-1.0.
[0286] Cells were harvested by centrifugation at 2279 x g at room temperature, the pellet was resuspended in 25 mL of sterile water, and centrifuged at 2279 x g at room temperature. Resu...
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