Method for efficiently producing D-xylosic acid by using engineering escherichia coli and using corn cob hydrolyzate as substrate
A corncob hydrolyzate, Escherichia coli technology, applied in microorganism-based methods, biochemical equipment and methods, and introduction of foreign genetic material using vectors, etc., can solve problems such as low production efficiency, non-single products, and excess corncob capacity.
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Embodiment 1
[0043] Embodiment 1: Construction of Escherichia coli XGL2 bacterial strain
[0044] (1) Knockout method: The gene knockout method used in the present invention is a one-step knockout technique (Datsenko KA et al., Proc. Natl. Acad. Sci. US A., 2000, 97:6640-6645). In this method, pKD4 (CGSC7632), pKD46 (CGSC7669), and pCP20 (CGSC14177) were purchased from Escherichia coli Gene Collection (New Haven, Connecticut, USA).
[0045] (2) Obtaining the mutant fragment: the nucleotide mutant fragment used in the above method has two homologous arms upstream and downstream of the knockout gene and a kanamycin resistance gene cassette. There are two ways to obtain the nucleotide mutation fragments used in the present invention. The nucleotide mutation fragments of xylA, ackA, adhE, poxB, ptsG and yjhG genes were directly amplified by PCR. The template was purchased from the Escherichia coli Gene Collection (New Haven, Connecticut, USA). Nucleotide mutation fragments of ldhA, yagF and...
Embodiment 2
[0081] Example 2: Construction of an expression vector containing xylose dehydrogenase gene xylB and xylonolactonase gene xylC
[0082] (1) Gene cloning: The original pET28a-xylBC vector carrying xylose dehydrogenase gene xylB gene and xylonolactonase gene xylC gene was synthesized by General Biosystems (Anhui) Co., Ltd. (Chuzhou, Anhui, China). The nucleotide sequence of xylose dehydrogenase gene xylB is shown in SEQ ID NO.1; the nucleotide sequence of xylonolactonase gene xylC is shown in SEQ ID NO.2. Primers are designed according to the nucleotide sequence of the gene, and synthetic primers are used to amplify the target fragment using the vector as a template, and the fragment contains xylB, xylC and restriction enzyme cutting sites EcoRI and HindIII. Amplification primer sequences are as follows:
[0083] The upstream primer 5'-ATATGAATTCATGTCCTCAGCCATCTATCCCAGCC-3' carries an EcoRI site;
[0084] The downstream primer 5'-ATCAAAGCTTTTAGACAAGGCGGACCTCATGCTGG-3' carries ...
Embodiment 3
[0088] Example 3: Using Escherichia coli XGL2 / pETPtac-xylBC to ferment and produce D-xylonic acid with corn cob hydrolyzate as substrate
[0089] (1) Plate culture: Streak the bacterial strain Escherichia coli XGL2 / pETPtac-xylBC onto an LB plate containing 1.5-1.8% agar by mass volume ratio and 50 μg / mL kanamycin, 37±1 Incubate at ℃ for 12±1 hours;
[0090] (2) First-class seeds: under sterile conditions, use a sterile toothpick to pick a single colony on the plate of step (1), and then inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin medium, 37±1°C shaker culture for 12±1 hours;
[0091] (3) Secondary seeds: under sterile conditions, take the bacterial solution cultivated in step (2), and inoculate 100 mL of LB liquid containing 50 μg / mL of kanamycin with an inoculation amount of 1 to 2% by volume In the culture medium, culture on a shaker at 37±1°C for 12±1 hours;
[0092] (4) Fermentation culture: under aseptic conditions, take the cultured bacter...
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