Engineering bacterium for producing hydroxytyrosol with high efficiency and application of engineering bacterium
A kind of genetically engineered bacteria and gene technology, applied in the field of engineering bacteria for efficient production of hydroxytyrosol, can solve the problems of toxicity of hydroxytyrosol bacteria, increase cost, increase the complexity of catalytic reactions, etc., and achieve good industrial application prospects and low price Inexpensive and readily available raw materials
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Embodiment 1
[0061] Screening of L-phenylalanine dehydrogenase: According to the random mutation high-throughput screening method of the aforementioned enzymes, error-prone PCR and high-throughput screening platforms were used to obtain three optimal mutants bspde1 and bspde2 for dopa dehydrogenation , bspde3, and sequenced. The amino acid sequences of bspde1, bspde2, and bspde3 are shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 respectively, and the nucleotide sequences of the genes encoding these three mutants are shown in SEQ ID NO:1 respectively , SEQ ID NO: 3, shown in SEQ ID NO: 5.
[0062] The specific enzymatic activities of the three mutants of L-phenylalanine dehydrogenase to levodopa dehydrogenation (that is, the conversion of 3,4-dihydroxyphenylpyruvate with levodopa dehydrogenation as a substrate) were 20.2 , 18.9, 6.4 U / mg. Taking L-phenylalanine dehydrogenase with the accession NO of the amino acid sequence on NCBI as BAA08816.1 as a control, the specific activity of t...
Embodiment 2
[0064] Recombinant Escherichia coli construction: first, the genes encoding L-phenylalanine dehydrogenase, α-ketoacid decarboxylase, and alcohol dehydrogenase were connected to the plasmid. The three-gene co-expression recombinant plasmid was obtained, and the plasmid was transformed into E. coli Escherichia coliBL21 (DE3), and the positive transformant was obtained by screening on an antibiotic plate, that is, the recombinant E. coli was obtained.
[0065] After the induction and expression of recombinant Escherichia coli is completed, the bacterial cells are collected. In a 100ml reaction system, the final concentration of cells is 200g / L, the concentration of NAD is 1g / L, the concentration of levodopa is 200g / L, pH 8.5, and react at 35°C. The shaking table rotates at a speed of 50 rpm, and the time is 36 hours. The yield of hydroxytyrosol was determined by liquid chromatography after conversion.
[0066] Table 1 Comparison of various recombinant bacteria
[0067]
Embodiment 3
[0069] During the experiment, it was found that the mutants of bspde had the ability to dehydrogenate other amino acids, so the transformation of some natural amino acids by several engineered bacteria was tested. The results are shown in Table 2, and all amino acids in the table are L-type.
[0070] Construct recombinant E. coli as in Example 2, collect the thalline after the recombinant E. coli is induced and expressed, in a 100ml reaction volume, the cell wet weight is 1g / L, NAD 0.5g / L, substrate concentration is 10g / L (related The substrate is shown in Table 2, the solubility of tyrosine is very small, and it can be reversed while dissolving), pH 8.5, reacted at 35°C, the shaker speed was 50 rpm, and the time was 48 hours. After the conversion, the yield and configuration of Danshensu were determined by liquid chromatography.
[0071] Transformation results of other substrates in table 2
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