Yarrowia lipolytica engineering bacteria resistant to ferulic acid and vanillic acid and its construction method
A technology of Yarrowia lipolytica and its construction method, which is applied in the field of Yarrowia lipolytica engineering bacteria and its construction, can solve the problem of hindering the stress response mechanism of yeast cell inhibitors, and deeply understand the development and tolerance mechanism of lignocellulosic biodiesel. less problems
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Embodiment 1
[0027] Example 1: Construction of pMCS-CEN1-E25201g overexpression strain
[0028] Primers were designed according to the gene sequence of the target gene YALI0_E25201g (GeneID: 2912710). The primers are shown in Table 1. by Y. lipolyticaXYL+ Genome is the template to amplify the target gene YALI0_E25201g. The PCR system used was EasyTaq DNA Polymerase, and the PCR program was: 94°C for 5 minutes, 94°C for 30s, 55°C for 30s, 72°C for 1-4 minutes (the time was determined by the gene length), and 72°C for 10 minutes. The PCR product was recovered and purified by gel, digested with SgsI and MssI, and then ligated with the pMCS-CEN1 vector that had undergone the same treatment. The ligation product is converted into E. coli TOP10 competent cells were cultured on LB selection medium overnight, single colonies were picked, and a small amount of plasmid was extracted for verification and DNA sequencing to obtain pMCS-CEN1-E25201g.
[0029] The pMCS-CEN1-E25201g plasmids were i...
Embodiment 2
[0032] Example 2: Construction of pMCS-CEN1-B18854g overexpression strain
[0033] Primers were designed according to the gene sequence of the target gene YALI0_B18854g (GeneID: 2906929). The primers are shown in Table 1. by Y. lipolytica The XYL+ genome was used as the template to amplify the target gene YALI0_B18854g. The PCR system used was EasyTaq DNA Polymerase, and the PCR program was: 94°C for 5 minutes, 94°C for 30s, 55°C for 30s, 72°C for 1-4 minutes (the time was determined by the gene length), and 72°C for 10 minutes. The PCR product was recovered and purified by gel, digested with SgsI and MssI, and then ligated with the pMCS-CEN1 vector that had undergone the same treatment. The ligation product was transformed into E. coli TOP10 competent cells, cultured on LB selective medium overnight, single colonies were picked, a small amount of plasmid was extracted for verification and DNA sequencing was performed to obtain pMCS-CEN1-B18854g.
[0034] The pMCS-CEN1-B1...
Embodiment 3
[0037] Example 3: Construction of pMCS-CEN1-F16731g overexpression strain
[0038] Primers were designed according to the gene sequence of the target gene YALI0_F16731g (GeneID: 2907989). The primers are shown in Table 1. by Y. lipolytica The XYL+ genome was used as the template to amplify the target gene YALI0_F16731g. The PCR system used was EasyTaq DNA Polymerase, and the PCR program was: 94°C for 5 minutes, 94°C for 30s, 55°C for 30s, 72°C for 1-4 minutes (the time was determined by the gene length), and 72°C for 10 minutes. The PCR product was recovered and purified by gel, digested with SgsI and MssI, and then ligated with the pMCS-CEN1 vector that had undergone the same treatment. The ligation product is converted into E. coli TOP10 competent cells were cultured on LB selection medium overnight, single colonies were picked, a small amount of plasmid was extracted for verification and DNA sequencing was performed to obtain pMCS-CEN1-F16731g.
[0039] The three pl...
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