Genetically engineered bacterium with high yield of squalene, construction method and application thereof
A technology of genetically engineered bacteria and construction methods, applied in the field of high-yield squalene genetically engineered bacteria and its construction, to achieve efficient transformation and integration, shorten the construction cycle, and increase production
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[0062] Example 1
[0063] Construction of high-anglerallene genetic engineering GS-A3-S4:
[0064] Step S1, the construction of the THMG1 module
[0065] TCYC1_THMG1_PGAL10PGAL1
[0066] 1) The PCR reaction was carried out with Tcyc1-F and Tcyc1-R, THMG1-F and THMG1-R, THMG1-F and THMG1-R, THMG1-F and THMG1-R, PGAL1PGAL10-F and PGAL1 PGAL10-R, and THMG1-F and THMG1-R, THMG1-F, and THMG1-R, respectively.
[0067] 2) The three DNA fragments Tcyc1, THMG1, and PGAL10 PGAL1 obtained by step S1 are obtained by overlapping the PCR reaction by overlapping the PCR by the primers Tcyc1-F and PGAL1PGAL10-R, which is joined together, i.e., the TCYC1_THMG1_PGAL10PGAL1 module.
[0068] Step S2, the construction of the expression of ERG20-Linker-ERG9 module
[0069] ERG20_LINKER_ERG9_TERG20
[0070] 1) With the Saccharomyces 3000B genomic DNA as a template, the PCR reaction was performed with ERG20-F and ERG20-Linker-S-R primers, and the DNA fragment ERG20_Linker was obtained.
[0071] 2) PCR re...
Example Embodiment
[0092] Example 2
[0093] Construction of GS-A3-S5 in Gene Engineering:
[0094] The construct method is in Example 1, wherein the Linker selected in step S3 is Linker2, and the built-in galactose regulatory protein GAL80 gene expression DNA fragment is:
[0095] GAL80LEFT_HEG_TCYC1_THMG1_PGAL10PGAL1_ERG20_LINKER2_ERG9_TERG20_GAL80Right;
[0096] The restructed plasmid vector PSZ200 is:
[0097] PCZ200ΔGal80 :: hyg_tcyc1_thmg1_pgal10pgal1_erg20_linker2_ERG9_TERG20;
[0098] Construction of the engineered bacteria in the knockout galactose regulatory protein GAL80 gene expression of KAL80 genes for GS-A3-S2.
Example Embodiment
[0099] Example 3
[0100] Construction of GS-A3-S6 in Gene Engineering:
[0101] The construction method is in Example 1, wherein the Linker selected in step S3 is Linker3, and the built-in galactose regulatory protein GAL80 gene expression DNA fragment is:
[0102] GAL80LEFT_HYG_TCYC1_THMG1_PGAL10PGAL1_ERG20_LINKER3_ERG9_TERG20_GAL80RIGH;
[0103] The recombinant plasmid vector PSZ300 constructed is:
[0104] PCZ300ΔGal80 :: hyg_tcyc1_thmg1_pgal10pgal1_erg20_linker3_ERG9_TERG20;
[0105] Construction of engineered bacteria for knockout galactose regulatory protein GAL80 gene expression of GAL80 genes for GS-A3-S3.
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