Genetic engineering bacterium producing beta-ionone with high yield and construction method and application of genetic engineering bacterium
A technology of genetically engineered bacteria and ionones, applied in genetic engineering, microorganism-based methods, and other methods of inserting foreign genetic materials, etc., can solve the problem of less β-ionone biological heterologous synthesis and so on
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Embodiment 1
[0086] Embodiment 1 experimental scheme and primer design
[0087] The experimental scheme design of this embodiment is as follows:
[0088] Based on the β-ionone-producing genetically engineered bacteria disclosed in the patent "201810695855.9, a β-ionone-producing genetically engineered bacterium and its construction method and application", the Ura3 gene was used as a screening marker to select non-essential genes for yeast chromosomes As the integration site, through the integration of overexpression of 9 genes related to the mevalonate (MVA) pathway and the integration of a β-ionone expression cassette, and the introduction of fructose in the cytoplasm through a two-step reaction -6-phosphate conversion to phosphoketolase pathway of acetyl-CoA to increase the production of β-ionone in the original engineered bacteria. The assembly sequence of each gene module is as follows: Figure 2-4 shown. The CRISPR / cas9 operation vector, using pCAS1yl (ADDGENE No. 73226) as the st...
Embodiment 2
[0103] The construction of embodiment 2 genetically engineered bacteria
[0104] 2.1 Gene acquisition:
[0105] The β-ionone synthesis genes were selected from CarB and CarRP derived from Mucor circinolus and CCD1 derived from Petunia, which were codon-optimized and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; refer to Example 2 in 201810695855.9 In step 2.1, CarB, CarRP, and CCD1 gene fragments were respectively amplified; their sequences are respectively shown in SEQ ID NO: 7, 10, and 13 of the sequence listing in 201810695855.9.
[0106] The genes related to the MVA pathway were selected from the endogenous genes of Yarrowia lipolytica. Using the primers in Table 2 and using the yeast genome as a template, GGS1 (CP028451.1: 2084792-2085775) and tHMG1 (CP028452.1: 571078-572577)[When using this gene, the start codon ATG needs to be added to the 5′ end of the sequence in the annotation], ERG8(CP028452.1:746796-748052), ERG10(CP028449.1:1141697-1143297), ERG12 ...
Embodiment 3
[0135] Fermentation of embodiment 3 genetically engineered bacteria and the detection of β-ionone
[0136] 3.1 Fermentation
[0137] The seed liquid cultivation method is as follows: select the high-yield β-ionone genetically engineered bacteria finally constructed in Example 2, select a single clone, inoculate into YPD culture solution (50mL / 250mL Erlenmeyer flask), 28 ℃, 250rpm, cultivate to OD 600 3 to 5.
[0138]Shake flask fermentation culture method is as follows: inoculate seed solution in 20mL YPD medium (50mL shake flask), initial OD 600 The value is 0.1, the organic phase is n-dodecane (10% V / V), 20° C., 250 rpm, and cultured for 12 days.
[0139] The carbon source is glucose (D-Glucose), sucrose (Sucrose), starch (Starch), glycerol (Glycerol), oleic acid (Oil Acid), the concentration is 20g / L.
[0140] Nitrogen source selection tryptone (tryptone) and peptone (peptone).
[0141] When tryptone is used as the nitrogen source, the medium is named YPDm, and the conc...
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