Engineered yeast and construction method and application thereof
A yeast engineering and yeast technology, applied in the fields of biotechnology and bioenergy, can solve the problems of low tolerance and inability of lignin degradation products, and achieve the effects of broad industrial application prospects, improved yield and efficient transformation
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Embodiment 1
[0063] Example 1 Construction, screening and application of Rhodosporidium toruloides haploid engineering strain expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP
[0064] 1. Construction of Rhodosporidium toruloides haploid engineering strain expressing lignin multifunctional peroxidase VP
[0065] (1) Synthesis of lignin multifunctional peroxidase gene VP
[0066] The source of the VP gene is Pleurotus eryngii (GenBank ID: AF175710, the amino acid sequence of the encoded protein is shown in SEQ ID NO.1), and it is optimized according to the codon preference of Rhodosporidium toruloides haploid (the optimized VP gene sequence shown in SEQ ID NO.3), and the whole sequence was synthesized, and the synthesized gene was connected to the pUC57 vector to obtain the plasmid pUC57-VP.
[0067] (2) Construction of pZPK-VP vector
[0068] The target vector was constructed by RF cloning method. Plasmid pZPK-P PGK -HYG-P2A-BLE-T HSP (JiaoXiang, Zhang Qi, Z...
Embodiment 2
[0101] Example 2 Construction, Screening and Application of Hyphoderma Dermatoides Engineering Strain Expressing Lignin Multifunctional Peroxidase VP and Manganese Peroxidase MNP
[0102] 1. Construction and screening of engineering strains of Trichosporium dermatitis expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP
[0103] Trichosporon cutaneum (Trichosporon cutaneum) CGMCC No.2.571 was used as the starting strain to construct engineering strains of Trichosporon cutaneum overexpressing MNP and VP genes respectively. The construction method was as follows: the recombinant plasmids pZPK-MNP and pZPK-VP were transferred into The engineering strain obtained from the starting strain, wherein, the construction process of pZPK-MNP and pZPK-VP expression vectors, the ATMT transformation process is as described in Example 1, and the electric shock competent preparation and transformation steps are as follows:
[0104] (1) Pick a single colony of the yeast ...
Embodiment 3
[0118] Example 3 Construction, screening and application of L. stariae engineering strains expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP
[0119] 1. Construction and screening of engineering strains expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP Staria oleoyeast
[0120] Using Lipomyces starkeyi NRRL Y-11557 as the starting strain, the recombinant plasmids pZPK-MNP and pZPK-VP were transformed into L. starkeyi to obtain engineering strains of Lipomyces starkeyi overexpressing MNP and VP, respectively. The construction process of pZPK-MNP and pZPK-VP expression vectors, the ATMT transformation process are as described in Example 1, the electroporation transformation process is as described in Example 2, the engineering strain screening steps are as described in Example 1, and the obtained engineering strain is named LS -MNP and LS-VP.
[0121] 2. Fermentation of the engineered strains LS-MNP and LS-VP of Lepidoptera ...
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