Pseudouridine synthetase mutant, mutant gene and application of pseudouridine synthetase mutant in preparation of vitamin B2
A technology of synthesizing enzyme and pseudouridine, which is applied in the biological field to achieve the effect of great application value and improving ability
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Embodiment 1
[0036]Example 1: Confirmation and verification of ylyB gene mutation site
[0037] Since the ylyB gene is responsible for encoding pseudouridine synthase in Bacillus subtilis, which promotes the production of pseudouridine from uracil, the ylyB gene was selected for site-directed mutation.
[0038] The inventor used Jalview software to analyze the highly conserved region of the amino acid sequence of the pseudouridine synthase encoded by the ylyB gene. At the same time, the Swiss-Model tool was used to model the pseudouridine synthase of the ylyB gene, and the active center of the enzyme was predicted using HotSpot Wizard 2.0. It was found that the 239th position of the amino acid sequence of the pseudouridine synthase encoded by the ylyB gene was located near the active center and the highly conserved region.
[0039] The inventor screened out a high-yielding vitamin B strain in the early stage 2 Bacillus subtilis (CGMCC NO.4018), which can ferment and produce vitamin B 2 ...
Embodiment 2
[0042] Example 2: Construction of engineering strain 18-yB of ylyB back mutation
[0043] Using the Bacillus subtilis 168 chromosome as a template, use primers UPylyB-F, UPylyB-R (containing DR) to amplify the wild-type UPylyB (containing DR) fragment with the adapter, and use primers araR-F, araR-R (containing DR) amplifies the araR (containing DR) fragment with the adapter; with the pC194 plasmid as a template, use primers cat-F, cat-R to amplify the cat fragment with the adapter; with the chromosome of Bacillus subtilis 168 as a template, use The primers DNylyB-F and DNylyB-R amplify the downstream homology arm fragment DNylyB, wherein the nucleotide sequence of the ylyB gene is shown in SEQ ID No.1, and the encoded amino acid sequence is shown in SEQ ID No.3.
[0044] Using UPylyB fragment, cat fragment, araR fragment and DNylyB fragment as templates, primers UPylyB-F and DNylyB-R were used for fusion PCR to obtain the assembled fragment UCR-ylyB, which was detected correc...
Embodiment 3
[0052] Embodiment 3: Construct the engineering bacterial strain 19-yB containing ylyB mutant
[0053] Using the coding gene of the artificially synthesized pseudouridine synthase mutant (H239L) as a template, use primers UPylyB-F and UPylyB-R (containing DR) to amplify the UPylyB (containing DR) fragment with a point mutation and a linker, and use Primers araR-F, araR-R (containing DR) amplify the araR (containing DR) fragment with the linker; use the pC194 plasmid as a template, use primers cat-F, cat-R to amplify the cat fragment with the linker; The chromosome of Bacillus subtilis 168 was used as a template, and the downstream homology arm fragment DNylyB was amplified with primers DNylyB-F and DNylyB-R. Wherein, the nucleotide sequence of the pseudouridine synthase mutant (H239L) is shown in SEQ ID No.2, and the encoded amino acid sequence is shown in SEQ ID No.4.
[0054] Using UPylyB fragment, cat fragment, araR fragment and DNylyB fragment as templates, primers UPylyB-...
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