Method for simultaneously transferring multiple genes into microbial genome
A technology of microorganisms and recombinant microorganisms, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of difficult transformation, difficult high-efficiency expression of foreign genes, and difficult recombination of strains, etc., to achieve the effect of avoiding trouble
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Embodiment 1
[0028] Example 1. Construction of xylose isomerization pathway in Saccharomyces cerevisiae
[0029] In this example, xylose isomerase gene (XI gene) and xylulokinase gene (XK gene) are expressed in S. The integration site is a multi-copy 18srDNA integration site.
[0030] 1. Obtaining the xylose isomerase gene expression cassette
[0031] Artificially synthesized xylose isomerase gene expression cassette (also known as C1 fragment, 3047bp), which is the double-stranded DNA molecule shown in sequence 1 of the sequence table (in sequence 1, the 1st to 766th nucleotides from the 5' end are Homologous sequence A of Saccharomyces cerevisiae 18srDNA, the 767th to 1444th nucleotides are the GPD promoter, the 1445th to 2758th nucleotides are the XI gene, and the 2759th to 3047th nucleotides are the CYC terminator). Wherein, the XI gene encodes the protein shown in sequence 4 of the sequence listing.
[0032] 2. Acquisition of Xylulokinase Gene Expression Cassette
[0033]Artificia...
Embodiment 2
[0046] Embodiment 2, expression NADH oxidase gene in Saccharomyces cerevisiae
[0047] In this embodiment, the NADH oxidase gene (noxE gene) is expressed in the recombinant bacteria obtained in Example 1 to reduce the production of xylitol in xylose fermentation; in order to increase the copy number, this case selects 26srDNA in Saccharomyces cerevisiae as gene integration site.
[0048] 1. Construction of NADH oxidase gene expression cassette
[0049] Artificially synthesized NADH oxidase gene expression cassette (also known as C4 fragment, 3460bp), which is the double-stranded DNA molecule shown in sequence 7 of the sequence table (the 1st to 1180th nucleotides from the 5' end of sequence 7 are Saccharomyces cerevisiae 26srDNA Homologous sequence A, the 1181st to 1778th nucleotide is the PGK promoter, the 1779th to 3119th nucleotide is the noxE gene, and the 3120th to 3460th nucleotide is the AOX terminator). Wherein, the noxE gene encodes the protein shown in sequence 9 o...
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