Xylose isomerase mutant, dna molecule encoding the enzyme, recombinant strain introduced into the dna molecule and their application
A technology of xylose isomerase and DNA molecules, which is applied in the fields of protein engineering and genetic engineering, can solve problems such as the inability to meet actual needs, and achieve the effect of wide application value and high-efficiency sugar-alcohol conversion and fermentation
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Embodiment 1
[0056] Example 1: Preparation and Activity Determination of Xylose Isomerase Mutants
[0057]The xylose isomerase mutant described in the present invention is preferably obtained by the following method, but for those skilled in the art, the specific parameters in each operation can be adjusted appropriately according to actual needs, which is a matter for those skilled in the art to read It can be easily accomplished on the basis of the present invention.
[0058] 1. Obtaining the gene fragment encoding xylose isomerase mutant
[0059] Hereinafter, the xylose isomerase mutant Ala144Thr is taken as an example to describe in detail.
[0060] Invitrogen was entrusted to synthesize the gene fragment (SEQ ID No.14) encoding natural xylose isomerase, and EcoR I and Not I restriction sites were introduced at both ends of it for the purpose of cloning. After double digestion with EcoR I enzyme and Not I enzyme, it was cloned into the multiple cloning site of the pPIC9K vector that ...
Embodiment 2
[0067] The construction of embodiment 2 recombinant Saccharomyces cerevisiae strains
[0068] 1. Construction of the recombinant plasmid containing the XI gene expression cassette and the XK gene expression cassette
[0069] 1.1 Construction of plasmid containing XI gene expression cassette or XK gene expression cassette
[0070] Using the Golden Gate method, the XI gene (SEQ ID No.7 or SEQ ID No.14) and the TEF2p promoter (SEQ ID No.15) and CYC1t terminator (SEQ ID No.16) were assembled on the pUC19 vector to construct is the XI gene expression cassette.
[0071] Using the Golden Gate method, the XK gene (gene number 398366094), PGK1p promoter (SEQ ID No. 17) and PGK1t terminator (SEQ ID No. 18) were assembled on the pUC19 vector to construct an XK gene expression cassette.
[0072] 1.2 Construction of recombinant plasmids containing XI gene expression cassette and XK gene expression cassette
[0073] Using the Golden Gate method, the constructed XI gene expression cassett...
Embodiment 3
[0082] Adaptive evolution of embodiment 3 recombinant Saccharomyces cerevisiae strains in xylose medium
[0083] The selected strains were subjected to adaptive evolution in the YEPX medium, and the specific method was as follows: pick a single colony on the YEPX plate into a test tube containing 5ml YEPX, cultivate overnight at 30°C and 200rpm, and use the cultured bacterial solution with The absorbance at 600 nm was measured with a spectrophotometer. Take 200 μl of overnight culture liquid and inoculate it into a test tube containing fresh YEPX medium, measure the absorbance at 600 nm after overnight culture, and repeat the operation until the cell density of the recombinant Saccharomyces cerevisiae strain in xylose medium is stable. During the adaptive evolution process, the xylose utilization rate and sugar alcohol conversion rate of the recombinant Saccharomyces cerevisiae strain were detected.
[0084] The recombinant Saccharomyces cerevisiae strains that have evolved t...
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