Engineered yeast for fermentation production of sanguinarine and application of engineered yeast
A technology of yeast engineering and sanguinarine, applied in fermentation, application, genetic engineering, etc., can solve problems such as low catalytic activity, difficulty in heterologous expression, difficulty in series connection, etc., and achieve simplified separation and purification process, environmental friendliness, quality and safety sex assurance effect
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Embodiment 1
[0031] Cloning and expression of genes required for sanguinarine biosynthesis.
[0032](1) Most of the enzymes from plants or microorganisms required in the synthetic pathway of sanguinarine can find the corresponding amino acid sequence or Gene sequence (see Table 1). For the screening of high-efficiency argoline oxidase (BBE), tetrahydroprotoberberine-N-methyltransferase (TNMT), and protoopine 6-hydroxylase (P6H), through the China National Gene Bank ( CNGB) developed tBLASTn of The 1000 Plants Project (https: / / db.cngb.org / blast / ) and literature searches to identify different plant sources and corresponding gene sequences.
[0033] The obtained gene sequence was optimized by the codon optimization algorithm developed by GenScript Biotechnology Co., Ltd., and the codon optimization was carried out according to the corresponding yeast host, as shown in the sequence table. And directly synthesized under the universal plasmid pESC with galactose promoter (pGAL1 or pGAL10), the...
Embodiment 2
[0045] Construction of a chassis strain with high production of the common intermediate arachidine.
[0046] The CYP76AD5, DODC, NCS_N in embodiment 1 35 (The nucleotide sequence is shown in SEQ ID NO.2), 6OMT, CNMT, NMCH, 4'OMT corresponding gene sequence is integrated into the corresponding position on the Saccharomyces cerevisiae BY4741 genome, as figure 1 As shown, a chassis strain producing the common intermediate arcarline was thus constructed. Because the intermediate 4-hydroxyphenylacetaldehyde of the upstream central carbon metabolism is easily oxidized and reduced by the yeast's own Ehrlich pathway to generate by-products tyrosol and 4-hydroxyphenylacetic acid. To this end, seven oxidoreductases ARI1, AAD3, ADH6, HFD1, GRE2, YPR1 and YDR541C with redundant functions in Saccharomyces cerevisiae cells were used as knockout sites to optimize subsequent metabolic pathways.
[0047] In order to further enhance the metabolic flux of the upstream shikimate (Shikimate) pat...
Embodiment 3
[0052] N-terminal truncated signal peptide, MBP tag fusion expression and different organelle localization of arachiline oxidase BBE.
[0053] The length of the 5'-end fragment deletion was based on the Discovery studio software, and the crystal structure (PDB number 4EC3) of EcBBE (derived from the BBE gene of Eschscholzia californica) was used as a template on the protein database website PDB (www.rcsb.org), PsBBE (the BBE gene derived from Papaver somniferum), CjBBE (the BBE gene derived from Coptis japonica), McBBE (the BBE gene derived from Macleaya cordata), CmBBE (derived from the white The BBE gene of Chelidonium majus) and ScBBE (derived from the BBE gene of Sanguinaria canadensis) were used for homology modeling. At the same time, the Jaview software was used to compare the protein sequences of the above six sources of arachiline oxidase BBE. Combined with the results of homology modeling and sequence alignment, the length of the N-terminal signal peptide sequence o...
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