1. Search and expression analysis of key gene sequences#
1) PDXA gene#
PdxA gene sequence
ATGGTTAAAACCCAACGTGTTGTGATCACTCCCGGCGAGCCCGCCGGGATTGGCCCGGACTTAGTTGTCCAGCTTGCACAGCGTGAGTGGCCGGTCGAACTGGTTGTTTGTGCCGATGCCACTCTCCTTACCAACCGGGCAGCGATGCTCGGTTTGCCGCTCACCCTCCGCCCTTATTCCCCCAACTCCCCTGCACAACCGCAAACTGCGGGCACATTAACGCTACTTCCTGTCGCGCTACGTGCACCTGTCACTGCGGGGCAGTTAGCGGTTGAAAATGGGCATTATGTGGTGGAAACGCTGGCGCGAGCGTGCGATGGTTGTCTGAACGGCGAATTTGCCGCGCTGATCACAGGTCCGGTGCATAAAGGCGTTATTAACGACGCTGGCATTCCTTTTACCGGTCATACCGAGTTTTTCGAAGAGCGTTCGCAGGCGAAAAAGGTGGTGATGATGCTGGCGACCGAAGAACTTCGCGTGGCGCTGGCAACGACGCATTTACCGCTGCGCGATATCGCAGACGCTATCACCCCTGCACTTTTGCACGAAGTGATTGCTATTTTGCATCACGATTTGCGGACCAAATTTGGTATTGCCGAACCGCGCATTCTGGTCTGCGGGCTGAATCCGCACGCGGGCGAAGGCGGTCATATGGGTACGGAAGAGATAGACACCATTATTCCGGTGCTCAATGAGCTGCGGGCGCAGGGGATGAAACTCAACGGGCCGCTGCCTGCCGATACCCTGTTTCAGCCGAAATATCTTGATAACGCCGACGCCGTGCTGGCGATGTACCACGATCAGGGTCTTCCCGTGCTAAAATACCAGGGCTTCGGGCGCGGTGTGAACATTACGCTGGGCCTGCCCTTTATTCGCACATCAGTGGACCACGGCACCGCGCTTGAACTGGCGGGACGTGGCAAAGCCGATGTCGGCAGTTTTATTACGGCGCTTAATCTCGCCATCAAAATGATTGTTAACACCCAATGA
Based on the above DNA sequence, codon is optimized and analyzed as the result shown in the figure below:
**PdxA amino acid expression codon: **
MVKTQRVVITPGEPAGIGPDLVVQLAQREWPVELVVCADATLLTNRAAMLGLPLTLRPYSPNSPAQPQTAGTLTLLPVALRAPVTAGQLAVENGHYVVETLARACDGCLNGEFAALITGPVHKGVINDAGIPFTGHTEFFEERSQAKKVVMMLATEELRVALATTHLPLRDIADAITPALLHEVIAILHHDLRTKFGIAEPRILVCGLNPHAGEGGHMGTEEIDTIIPVLNELRAQGMKLNGPLPADTLFQPKYLDNADAVLAMYHDQGLPVLKYQGFGRGVNITLGLPFIRTSVDHGTALELAGRGKADVGSFITALNLAIKMIVNTQ*
Amino acid sequence analysis revealed that the protein sequence contained no signal peptide and no transmembrane region and could be expressed normally.
2) PDXJ gene#
PDXJ sequence
ATGGCTGAATTACTGTTAGGCGTCAACATTGACCATATCGCTACGCTGCGCAACGCGCGCGGTACCGCTTACCCGGATCCGGTGCAGGCCGCGTTTATTGCCGAGCAGGCGGGAGCGGACGGCATTACCGTGCATTTACGTGAAGATCGCCGTCACATTACTGACCGCGACGTGCGCATCCTGCGTCAGACGCTGGATACCCGCATGAATCTGGAGATGGCGGTGACCGAAGAGATGCTGGCGATCGCCGTTGAGACGAAGCCACATTTTTGCTGCCTGGTACCGGAAAAGCGTCAGGAAGTAACAACCGAAGGCGGCCTGGATGTCGCAGGGCAGCGTGACAAAATGCGCGATGCCTGCAAACGTCTGGCAGATGCCGGGATTCAGGTTTCTCTGTTTATTGACGCCGATGAAGAGCAGATCAAAGCTGCGGCAGAGGTTGGCGCACCGTTTATCGAGATCCACACCGGTTGCTATGCTGATGCCAAAACTGACGCCGAACAGGCGCAAGAGCTGGCGCGTATCGCCAAAGCCGCGACCTTTGCCGCAAGCCTCGGTCTGAAAGTTAACGCCGGACACGGTCTGACCTATCACAACGTGAAAGCCATTGCCGCCATCCCTGAGATGCATGAACTGAATATCGGTCATGCCATTATTGGTCGTGCAGTGATGACCGGACTGAAAGATGCGGTGGCAGAAATGAAGCGTCTGATGCTGGAAGCGCGTGGCTAA
PdxJ amino acid codon
MAELLLGVNIDHIATLRNARGTAYPDPVQAAFIAEQAGADGITVHLREDRRHITDRDVRILRQTLDTRMNLEMAVTEEMLAIAVETKPHFCCLVPEKRQEVTTEGGLDVAGQRDKMRDACKRLADAGIQVSLFIDADEEQIKAAAEVGAPFIEIHTGCYADAKTDAEQAQELARIAKAATFAASLGLKVNAGHGLTYHNVKAIAAIPEMHELNIGHAIIGRAVMTGLKDAVAEMKRLMLEARG*
Amino acid sequence analysis revealed that the protein sequence was the same as that of PDXA, with no signal peptide and no transmembrane region in the amino acid sequence and could be expressed normally.
2. Construction and verification of conventional sufficient expression#
vectors for PDXA and PDXJ#
Construction of pRSFDuet-1-PdxA-PdxJ expression plasmid. The synthetic sequence was first digested by enzymes to obtain specific enzyme cleavage sites. The conjugate was transformed into E. coli BL21 (DE3) competent cells and screened with ampicillin to obtain a certain number of transformants. The plasmid was verified by enzyme digestion. The size of the digested target gene fragment meets the expected requirements, which are shown in the figure below.
Recombinant plasmid digestion assay#
3. Expression of target protein in E. coli#
In order to successfully implement the cell-free expression system, we first verified the traditional cell expression and obtained small lysates of the expression strain.GFP fluorescent protein detection#
The above results show that PDXA and PDXJ proteins were well expressed after transformation into E. coli and can be used for subsequent experimental research.
In order to further verify the expression results, we used SDS-PAGE to verify the target protein. The results are shown in the figure below, which are basically consistent with the expected results and well verify the expression of the protein.
4. Construction and verification of linear expression vector#
In order to perform expression and visualization detection of PDXA and PDXJ, we used GFP fluorescent protein as a reporter gene, which exhibited yellow-green fluorescence, and we constructed pRSFDuet-PGFP as a positive control.
We first connected the two genes, and added primer sequences at both ends. Subsequently, we performed PCR amplification and purification, obtained cDNA fragments containing known sequences. Finally, we introduced key sequences required for expression such as SP6 promoter sequence and 3-UTR sequence through primers. The results of gel electrophoresis verification are shown in the figure below.
We add the verified linear expression vector to the reaction system. The expression of the target gene will be determined by detecting fluorescence of the green fluorescent protein. As the figure shown below, after a period of reaction, the transfer part moves to another clean PCR tube easily, The precipitate is then separated by centrifugation, and the supernatant shows a more obvious yellow-green color, indicating that the target protein is expressed.
5. Research on target protein expression efficiency in cell-free systems#
In order to obtain the best PDXA and PDXJ protease yields, we optimized the cell-free expression system at different times and temperatures. First, a normal reaction was performed at 37°C, and the reaction solution at different time points was collected. The relative absorption value of fluorescence was detected using a spectrophotometer. The results showed that a relatively stable and high yield of target protein could be obtained in 3 hours. Among the incubations at different temperatures, we selected 15°C, 25°C and 37°C to compare the results.
6. Research on VB6 in vitro biosynthetic system#
After obtaining the optimal reaction conditions, we further explored the synthesis of VB6. Combining the experience of our predecessors, we focused on comparative studies of synthesis time and yield to determine the feasibility of VB6 in vitro biosynthesis, laying the foundation for subsequent green large-scale production.
The results in the figure above show that the peak of the reaction can be reached in vitro within 6 hours, and the expression level can reach 30 mg/L in the case of small-scale expression. Compared with intracellular (Escherichia coli expression system), the yield is significantly increased (p=0.0109, Figure 13 below), which is expected to develop a faster and better VB6 green production system.
In order to further verify the feasibility of this synthetic system, we also used high performance liquid phase to verify the reaction products.
Conclusion#
In this study, we first established a cell-free expression system, expressed and identified the key VB6 synthase, and verified the E. coli expression system; then, under optimized conditions, we explored the biosynthesis of VB6. The results showed that in our cell-free expression system, a higher VB6 concentration can be obtained than in conventional cell factories, and it has a certain potential for large-scale production and application. This method is relatively simple and convenient, and is very friendly to the green and pollution-free synthesis of vitamins, etc., and has important significance and potential promotion value.
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