Recombinant engineering bacterium as well as construction method and application thereof
A technology of recombinant engineering bacteria and recombinant vectors, applied in the field of genetic engineering, can solve the problems of increased purification difficulty, poor appearance quality, difficult to guarantee product quality, etc., to improve selectivity, reaction efficiency and product conversion rate, and is suitable for large-scale Industrial production, avoiding the effect of cyclic racemization
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Embodiment 1
[0137] Example 1 Construction of multi-enzyme co-expression recombinant engineering bacteria
[0138] 1. Design and synthesis of the target gene
[0139] Step 1, the nucleotide sequence of the L-pantolactone dehydrogenase coding gene derived from Humibacter sp.BT305 (actinomycetes) is codon-optimized according to the codon preference of Escherichia coli (E.coli) , and adding XhoI and NdeI restriction sites to obtain the modified gene sequence of L-pantolactone dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO.1;
[0140] Step 2, the nucleotide sequence of the ketopantolide reductase coding gene derived from Candida magnolia is codon-optimized according to the codon preference of Escherichia coli (E.coli), and SacI and NotI are added Restriction sites are obtained to obtain the modified gene sequence of D-ketopantolactone, the nucleotide sequence of which is shown in SEQ ID NO.2.
[0141] Step 3: Codon-optimize the nucleotide sequence encoded by the glucos...
Embodiment 2
[0173] Example 2 Construction of recombinant engineering bacteria expressing D-pantoate hydrolase
[0174] 1. Design and synthesis of the target gene
[0175] The nucleotide sequence of the D-pantolactone hydrolase coding gene derived from FusariummoniliformeCGMCC0536 was codon-optimized according to the codon preference of Escherichia coli (E.coli), and XhoI and NdeI restriction sites were added, Obtain the modified gene sequence of D-pantolactone hydrolase, the nucleotide sequence of which is shown in SEQ ID NO.4;
[0176] 2. Construction of recombinant engineered bacteria
[0177] Step 1: Use the DNA molecule of the target genome 4 as a template, use the primer pair HYD-for and HYD-rev to perform PCR amplification, separate the PCR products by 1% agarose gel electrophoresis, and recover the target genome 4 with a gel recovery kit gene fragments.
[0178] The primer sequence is as follows: (the underline is the restriction site)
[0179] HYD-for: GGAATTC CATATG ATGGCTAA...
Embodiment 3
[0186] Example 3 Induced Expression of Recombinant Engineering Bacteria E-lpldh-kpr-GDH
[0187] The recombinant engineered bacteria E-lpldh-kpr-GDH prepared in Example 1 was inoculated in 5 mL of LB medium, and it was placed at 37 ° C and cultivated overnight at 200 rpm;
[0188] The seeds were inoculated into 100mL TB medium according to the inoculum amount of 2%, and cultured at 37°C and 200rpm for 2h, then the culture temperature was adjusted to 30°C, and the culture was continued for 15h;
[0189] Centrifuge at 6600rpm for 3min, discard the supernatant, wash twice with 0.2moL / L pH6.5 phosphate buffer, and collect the bacteria.
[0190] Among them, the composition of LB medium is as follows: ampicillin 50ug / mL, kanamycin 100mg / L, yeast powder 5g / L, tryptone 10g / L, sodium chloride 10g / L.
[0191] The composition of TB medium is as follows: tryptone 20g / L, yeast powder 5g / L, sodium chloride 5g / L, glucose 2g / L, lactose 0.1-2.0g / L, ampicillin 50mg / L, kanamycin 100mg / L.
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