Preparation method of salidroside and analogues thereof
A technology of salidroside and its analogues, which is applied in the direction of biochemical equipment and methods, microorganisms, and the use of vectors to introduce foreign genetic materials, etc., can solve problems such as unfavorable industrial production, achieve industrial production, reduce production costs, and solve Effects of Source Questions
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Embodiment 1
[0035] Design of keto decarboxylase gene synkdc and glycosyltransferase gene synyjic
[0036] Preferably, the Pichia pastoris GS115 keto decarboxylase gene and the Bacillus licheniformis glycosyltransferase gene have their amino acid sequences as shown in SEQ ID No. 01 and SEQ ID No. 02 in the sequence listing, respectively. Use JCAT online codon optimization software (http: / / www.jcat.de) combined with OPTIMIZER online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER / ), and use E. coli's preference for codons Optimize, design the full length synkdc gene and synyjic gene, as shown in the sequence table SEQ ID No.03 and SEQ ID No.04, respectively.
Embodiment 2
[0038] The lambda-red homologous recombination method integrates T7 RNA polymerase into the chromosome of the chassis strain SyBE-002447.
[0039] The detailed steps of the construction process of the chassis strain integrating T7 RNA polymerase into the chromosome of the chassis strain SyBE-002447 are as follows:
[0040] 1. Design primers T7-RNA F and T7-RNA R sequences as shown in the sequence table SEQ ID NO.06 and SEQ ID NO.07 respectively. T7RNA polymerase gene was cloned from Escherichia coli BL21(DE3) bacteria, the T7RNA polymerase The gene sequence is as shown in the sequence table SEQ ID NO.05.
[0041] 2. The sequences of primers T7F and T7R, T7-Chl F and T7-Chl R are as shown in the sequence listing SEQ ID NO.08, SEQ ID NO.09, SEQ ID NO.10, SEQ ID NO.11, and the overlap extension PCR preparation has chlorine T7 RNA polymerase fragment resistant to mycin.
[0042] 3. Introduce the pKD46 plasmid into the strain SyBE-002447 to obtain SyBE-002447 / pKD46. Inoculate the activate...
Embodiment 3
[0046] The lambda-red homologous recombination method knocks out the feaB gene and integrates the synkdc gene into the chromosome of the chassis strain SyBE-002447 (DE3).
[0047] The detailed steps of the construction process of the chassis strain that knock out the feaB gene and integrate the synkdc gene into the chromosome of the chassis strain SyBE-002447 (DE3) are as follows:
[0048] 1. The sequences of primers KdcF and Kdc R, Kan F and Kan R are as shown in the sequence table SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, respectively. Overlapping extension PCR is used to prepare kan resistant Kdc-Kan fragment.
[0049] 2. Introduce the pKD46 plasmid into the strain SyBE-002447(DE3) to obtain SyBE-002447(DE3) / pKD46, and inoculate the activated strain SyBE-002447(DE3) / pKD46 in 10ml LB liquid medium at 30℃ , 200rpm, cultivate to OD 600 Is 0.4-0.6. Add L-arabinose at a final concentration of 10 mM, continue culturing for 3 h, centrifuge at 4000 rpm at 4° C. for 8 min, ...
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