A gene module for efficiently degrading feather and synthesizing artificial hemoglobin protein and application thereof

Abstract

The application artificially designs and creates a functional module SyKerMb with high efficient degradation of poultry feather waste and synthesis of new heme protein by using synthetic biology method. The application constructs a recombinant vector of the functional module, and assembles and expresses the same in a chassis microbial cell Escherichia coli. Experiments prove that the engineered strain integrated with the conversion functional module has the biological synthesis capacity of degrading animal waste feather and efficiently synthesizing artificial heme protein, and can be used in artificial protein preparation and future artificial food and other related industrial production.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and biotransformation technology, and relates to a highly efficient artificially designed biosynthetic system for degrading animal waste such as feathers and converting them into heme protein. Background Technology

[0002] Livestock waste, such as feathers, fur, hooves, and horns, has long been considered unusable agricultural organic waste. The large-scale accumulation of this waste not only poses a potential source of environmental pollution but also results in significant resource waste. However, these wastes are rich in protein and amino acids, representing potential protein and energy resources. Utilizing modern synthetic biology protein intelligent design methods to create highly efficient biotransformation cell factories for animal feather organic waste can accelerate the solution to the problem of massive livestock waste accumulation, achieving efficient utilization of waste reduction and resource recovery.

[0003] Synthetic biology, based on the intersection of multiple disciplines such as biology, chemistry, physics, computation, and engineering, redesigns or even synthesizes organisms from scratch in an engineered manner, which will overcome the limitations of natural evolution and create synthetic organisms that surpass the capabilities of natural life. Summary of the Invention

[0004] The purpose of this invention is to create a highly efficient biosynthetic system that utilizes synthetic biology methods to degrade animal waste, such as feathers, and convert them into heme proteins.

[0005] This invention creates a highly efficient biosynthetic system for converting animal waste into artificial heme protein, and provides a functional module capable of degrading poultry feather waste and synthesizing heme protein, named SyKerMb.

[0006] The SyKerMb module is a nucleotide sequence as shown in SEQ ID NO.3. The full-length nucleic acid sequence of SEQ ID NO.3 is 1635 bp in size, and this module encodes 544 amino acids.

[0007] The SEQ ID NO.3 is assembled from nucleotides of artificially designed sequences shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] Wherein, SEQ ID NO.1 is the sequence that was designed and synthesized for the first time in this invention.

[0009] The specific research work is as follows:

[0010] 1. Obtain the full-length nucleic acid sequence of the functional module SyKerMb.

[0011] The full-length nucleic acid sequence of the functional module SyKerMb was obtained through synthetic biology design and artificial chemical synthesis. It is 1635 bp in size and consists of 544 amino acids.

[0012] 2. Construct a recombinant engineered strain containing the functional module SyKerMb

[0013] 1) SyKerMb was cloned into the vector pJET to construct the recombinant cloning plasmid pJET-SyKerMb containing the complete functional module SyKerMb.

[0014] 2) The biosynthetic functional module SyKerMb was linked to the expression vector pET28t plasmid, which contains an inducible T7 promoter and can express the target module protein in the presence of the inducer IPTG. The recombinant plasmid pET28t-SyKerMb was constructed.

[0015] 3) The recombinant plasmid pET28t-SyKerMb containing the SyKerMb functional module was transformed into the recipient Escherichia coli BL21(DE3) to obtain the highly efficient biosynthetic engineered strain BL21-SyKerMb (see Example 1 for details);

[0016] 3. Functional verification of module SyKerMb

[0017] The following biodegradation and biosynthesis experiments were conducted using the engineered strain containing the SyKerMb module:

[0018] 1) Experiment on the degradation, transformation and biosynthesis of waste feathers

[0019] Experimental results showed that feathers began to decompose in large quantities in the culture medium of the engineered strain containing the module SyKerMb starting from 24 hours; after 96 hours of culture, the strain cells turned bright red and accumulated a large amount of heme protein inside the cells.

[0020] No degradation of feathers was observed in the culture medium of the control strain BL21-28, which did not contain SyKerMb (see Example 2 for details).

[0021] 2) Purification and molecular weight determination of the artificial protein obtained by transformation

[0022] The heme protein obtained above was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). After purification, the protein had a molecular weight of approximately 18 kDa.

[0023] This demonstrates that the engineered strain of the SyKerMb expression module can effectively degrade feather waste and synthesize artificial heme protein.

[0024] Sequence List Information

[0025] SEQ ID NO.1: A novel, artificially designed nucleotide sequence, one of the nucleotides that assemble SEQ ID NO.3;

[0026] SEQ ID NO.2: Another nucleotide for assembling SEQ ID NO.3;

[0027] SEQ ID NO.3: Nucleotide sequence of the functional module SyKerMb;

[0028] SEQ ID NO.4: Amino acid sequence of the protein encoded by SyKerMb. Attached Figure Description

[0029] Figure 1 The degradation and transformation effect of the engineered strain BL21-SyKerMb containing the module SyKerMb on feather substrate;

[0030] Figure 2 SDS-PAGE gel image of the engineered strain BL21-SyKerMb containing the SyKerMb module. 1. Supernatant of the total protein lysate of the strain after 24 h of induction; 2. Supernatant of the total protein lysate of the strain after 96 h of induction; 3. Total protein of the control strain. Detailed Implementation

[0031] The plasmids, strains, and microbial degradation targets mentioned in the following examples are only used to further illustrate the present invention and do not limit the substantive content of the invention. Unless otherwise specified, all experimental conditions were performed under conventional conditions well known to those skilled in the art or as recommended by the manufacturer. The sources of the plasmids and strains mentioned in the examples are as follows:

[0032] Cloning vector pJET: a commercially available product from ThermoFisher.

[0033] Shuttle plasmid pET-28t: preserved in our laboratory;

[0034] Escherichia coli BL21(DE3): A commercially available product from Beijing TransGen Biotech Co., Ltd.

[0035] Feathers: Chicken feathers were purchased from the market.

[0036] IPTG: Isopropyl-β-D-thiogalactoside.

[0037] Example 1: Construction of a recombinant engineered strain containing the transformation module SyKerMb

[0038] I. Experimental Materials

[0039] Cloning vector pJET: a commercially available product from ThermoFisher.

[0040] Expression plasmid pET-28t: preserved in our laboratory;

[0041] Escherichia coli BL21(DE3): A commercially available product from Beijing TransGen Biotech Co., Ltd.

[0042] II. Experimental Methods

[0043] 1. Using synthetic biology-designed modules, the full-length nucleic acid sequence of the functional module SyKerMb was obtained through artificial chemical synthesis. This module, consisting of 544 amino acids and 1635 bp in size, was cloned into the vector pJET to construct the recombinant cloning plasmid pJET-SyKerMb containing the complete functional module SyKerMb, which was then verified by sequencing. Subsequently, the functional module SyKerMb containing sticky ends and the expression vector pET-28t containing the T7 promoter were obtained by double digestion with Nde I / Xho I. The functional module SyKerMb was ligated into the pET-28t vector to construct the E. coli high-expression vector pET28t-SyKerMb, which was then transformed into E. coli BL21(DE3).

[0044] III. Experimental Results

[0045] The full-length nucleic acid sequence of the functional module SyKerMb was obtained using artificial chemical synthesis methods, and a recombinant E. coli strain expressing the SyKerMb functional module was successfully constructed. The inserted sequence was verified to be correct by PCR, enzyme digestion, and sequencing, and the strain was named BL21-SyKerMb. E. coli BL21(DE3) containing the pET-28t control empty plasmid was named BL21-28.

[0046] IV. Experimental Conclusions

[0047] Complete the construction of a recombinant Escherichia coli engineered strain expressing the SyKerMb functional module.

[0048] Example 2: Biotransformation Detection of Engineered Strains Containing the SyKerMb Module for High-Efficiency Biosynthesis

[0049] I. Experimental Materials

[0050] Recombinant engineered strain: The engineered strain BL21-SyKerMb expressing the SyKerMb functional module obtained in Example 1.

[0051] Control strain: BL21-28 strain containing an empty plasmid as described in Example 1.

[0052] II. Experimental Methods

[0053] 1. Experiment on the degradation of waste feathers

[0054] Rinse the chicken feathers with clean water and dry them for later use. Add 50 mL of inorganic salt medium, using the feathers as the sole carbon and nitrogen source, and sterilize at high temperature. Inorganic salt medium (m / v): NaCl 0.05%, K2HPO4 0.1%, KH2PO4 0.04%, MgCl2·7H2O 0.01%, CaCl2 0.006%, pH 7.5.

[0055] The seed culture of the engineered strain expressing the SyKerMb was transferred to an inorganic salt medium with feathers as the sole carbon and nitrogen source. IPTG (final concentration 0.1 mM) was added, and the medium was cultured in a shaker at 37°C. Feather degradation was observed every 12 hours.

[0056] 2. Expression and purification experiments of artificial heme protein

[0057] The engineered strain BL21-SyKerMb was cultured using feathers as the sole carbon and nitrogen source. IPTG (0.5 mM) was added as an inducer, and the strain was cultured in a shaker at 37°C. Centrifuged strains were collected every 24 hours. Cells were lysed using an ultrasonic cell disruptor, and the supernatant and precipitate were collected separately by centrifugation. A small sample was subjected to SDS-PAGE electrophoresis to detect and identify the protein expression of the engineered strain BL21-SyKerMb.

[0058] III. Experimental Results

[0059] Feather degradation experiments showed that strain BL21-SyKerMb began to degrade feathers extensively after 24 hours of culture; after 48 hours, the strain had grown significantly and turned pale pink; and after 96 hours, a large amount of artificial heme protein accumulated intracellularly, turning the strain cells bright red. Figure 1 SDS-PAGE results showed that after culturing the recombinant engineered strain for 48 hours, a large amount of artificial heme protein had been synthesized intracellularly; after 48 hours of culture, a large amount of intracellular sarcoplasmic protein accumulated. The molecular weight of artificial heme protein is approximately 18 kDa. Figure 2 The results showed that the engineered strains expressing the SyKerMb module were able to degrade feather waste to provide carbon and nitrogen sources for normal growth, while also synthesizing the target artificial heme protein intracellularly.

[0060] IV. Experimental Conclusions

[0061] Experiments show that the expression of the module SyKerMb enables the engineered strain to convert feather waste into the target artificial heme protein. sequence list <110> Institute of Biotechnology, Chinese Academy of Agricultural Sciences <120> A gene module for efficiently degrading feathers and synthesizing artificial heme protein and its application. <160> 4 <170> PatentIn version 3.1 <210> 1 <211> 564 <212> DNA <213> Artificial sequence <400> 1 atgggtctca gcgatgggga gtggcaattg gttctaaatg catggggcaa agtggaagca 60 aacattcctc agtactcagt ggtcttttat actagcatcc tagaaaagca cccagagacg 120 ctggaaaaat tcgataaatt taagcattta aaaacggaag cagaaatgaa ggcaaaccct 180 aaattagcgg gccacgctga aaaactcttt ggacttgtgc gtgatagtgc aatacaatta 240 cgtgccaaaa agaaggggca tcacgaagcg gaggacgctg ctttgggaag tattcatgca 300 caaaataagc ataaaatacc cgttaaatac ctcgaattca tatccgacgc aataattcat 360 gttctacatg ccaaacatcc ctcagatttt ggtgccgatg ctcaggctgc gatgtcgaaa 420 gctcttgaat tattcagaaa tgatatggct gcccagtata aagtacttgg gtttcatggt 480 gaaaacctgt atttccagtc gaaatacctg ctgccgaccg ctgctgctgg tctgctgctc 540 ctcgctgccc agccggcgat ggcc 564 <210> 2 <211> 1071 <212> DNA <213> Artificial Sequence <400> 2 attgccggtc aatatgtagt tgttttctct gagggtgcgt taggaaatga tcttagcgcg 60 caggatgcag ggtctctcat ccgttcgtta aacctcaacg cccagggtgt tgaaatccaa 120 aacatatatg cagcggcctt gcacggcttc agtgccaagc tgagttcgga aaatcttgcc 180 attctacgca gcgatcctcg agtgaaatat gtggagcaag acgcgattat gagaatgtcg 240 gcgacacaaa ccggcgcgac atgggggctt gacaggattg accagagaaa cctcccgctt 300 gacggttctt acacctacaa tagtacggcc agcggcgtaa aagtatatat cattgatacg 360 ggcataaaca ctgcgcatag caactttggc gggcgtgcaa tttggggaac caacactaca 420 [[ID=十八]]ggggacggaa acaactcaga ttgccaaggg catgggacgc acgtagccgg aacggtcggc 480 agctccactt acggggtagc taagggcgct accttggtag cggtaaaagt attaaactgc 540 cagggaagcg ggacgaactc aggcgtcatt gcaggtgtaa actgggctgt ctcgaacaaa 600<00001六六>ggatcagcaa cggcagttgc gaatatgagc ttagggggcg gagctagcca agcagttgat 660 It should be noted that there is an error in the numbering in the original text. The "十八" in the translation of line 18 is a correction based on the correct numbering. If this is not allowed, please let me know and I will adjust accordingly.gatgctgtga acaatgccgc ttcaaaaaac ctgattatgg cggttgcggc cggaaatgaa 720 aatcaaaacg catgtaatgt atcgcccgca cgggcggcct cagcgattac agtcggatct 780 acaacgaata cagatgcaag atcttccttt tcaaactacg gaacttgtgt tgacatattc 840 gctccgggct ccaacatcac atcaacatgg atcggaagta catccgctac caacacaatc 900 tcaggaacct cgatggcatc cccgcacgta gccggagcag cagctctact ggtggcggcc 960 ggcaatacga caacgtctgc tgtaacttct gcaatgatta ataatgccac ccccaacaaa 1020 gtaacaggtg ccggcaccgg gtcacctaac agattattat atacaggata a 1071 <210> 3 <211> 1635 <212> DNA <213> Artificial Sequence <400> 3 atgggtctca gcgatgggga gtggcaattg gttctaaatg catggggcaa agtggaagca 60 aacattcctc agtactcagt ggtcttttat actagcatcc tagaaaagca cccagagacg 120 ctggaaaaat tcgataaatt taagcattta aaaacggaag cagaaatgaa ggcaaaccct 180 aaattagcgg gccacgctga aaaactcttt ggacttgtgc gtgatagtgc aatacaatta 240 cgtgccaaaa agaaggggca tcacgaagcg gaggacgctg ctttgggaag tattcatgca 300 caaaataagc ataaaatacc cgttaaatac ctcgaattca tatccgacgc aataattcat 360 gttctacatg ccaaacatcc ctcagatttt ggtgccgatg ctcaggctgc gatgtcgaaa 420 gctcttgaat tattcagaaa tgatatggct gcccagtata aagtacttgg gtttcatggt 480 gaaaacctgt atttccagtc gaaatacctg ctgccgaccg ctgctgctgg tctgctgctc 540 ctcgctgccc agccggcgat ggccattgcc ggtcaatatg tagttgtttt ctctgagggt 600 gcgttaggaa atgatcttag cgcgcaggat gcagggtctc tcatccgttc gttaaacctc 660 aacgcccagg gtgttgaaat ccaaaacata tatgcagcgg ccttgcacgg cttcagtgcc 720 aagctgagtt cggaaaatct tgccattcta cgcagcgatc ctcgagtgaa atatgtggag 780 caagacgcga ttatgagaat gtcggcgaca caaaccggcg cgacatgggg gcttgacagg 840 attgaccaga gaaacctccc gcttgacggt tcttacacct acaatagtac ggccagcggc 900 gtaaaagtat atatcattga tacgggcata aacactgcgc atagcaactt tggcgggcgt 960 gcaatttggg gaaccaacac tacaggggac ggaaacaact cagattgcca agggcatggg 1020 acgcacgtag ccggaacggt cggcagctcc acttacgggg tagctaaggg cgctaccttg 1080 gtagcggtaa aagtattaaa ctgccaggga agcgggacga actcaggcgt cattgcaggt 1140 gtaaactggg ctgtctcgaa caaaggatca gcaacggcag ttgcgaatat gagcttaggg 1200 ggcggagcta gccaagcagt tgatgatgct gtgaacaatg ccgcttcaaa aaacctgatt 1260 atggcggttg cggccggaaa tgaaaatcaa aacgcatgta atgtatcgcc cgcacgggcg 1320 gcctcagcga ttacagtcgg atctacaacg aatacagatg caagatcttc cttttcaaac 1380 tacggaactt gtgttgacat attcgctccg ggctccaaca tcacatcaac atggatcgga 1440 agtacatccg ctaccaacac aatctcagga acctcgatgg catccccgca cgtagccgga 1500 gcagcagctc tactggtggc ggccggcaat acgacaacgt ctgctgtaac ttctgcaatg 1560 attaataatg ccacccccaa caaagtaaca ggtgccggca ccgggtcacc taacagatta 1620 ttatatacag gataa 1635 <210> 4 <211> 544 <212> PRT <213> Artificial sequence <400> 4 MET Gly Leu Ser Asp Gly Glu Trp Gln Leu Val Leu Asn Ala Trp Gly 1 5 10 15 Lys Val Glu Ala Asn Ile Pro Gln Tyr Ser Val Val Phe Tyr Thr Ser 20 25 30 Ile Leu Glu Lys His Pro Glu Thr Leu Glu Lys Phe Asp Lys Phe Lys 35 40 45 His Leu Lys Thr Glu Ala Glu MET Lys Ala Asn Pro Lys Leu Ala Gly 50 55 60 His Ala Glu Lys Leu Phe Gly Leu Val Arg Asp Ser Ala Ile Gln Leu 65 70 75 80 Arg Ala Lys Lys Lys Gly His His Glu Ala Glu Asp Ala Ala Leu Gly 85 90 95 Ser Ile His Ala Gln Asn Lys His Lys Ile Pro Val Lys Tyr Leu Glu 100 105 110 Phe Ile Ser Asp Ala Ile Ile His Val Leu His Ala Lys His Pro Ser 115 120 125 Asp Phe Gly Ala Asp Ala Gln Ala Ala MET Ser Lys Ala Leu Glu Leu 130 135 140 Phe Arg Asn Asp MET Ala Ala Gln Tyr Lys Val Leu Gly Phe His Gly 145 150 155 160 Glu Asn Leu Tyr Phe Gln Ser Lys Tyr Leu Leu Pro Thr Ala Ala Ala 165 170 175 Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala MET Ala Ile Ala Gly Gln 180 185 190 Tyr Val Val Val Phe Ser Glu Gly Ala Leu Gly Asn Asp Leu Ser Ala 195 200 205 Gln Asp Ala Gly Ser Leu Ile Arg Ser Leu Asn Leu Asn Ala Gln Gly 210 215 220 Val Glu Ile Gln Asn Ile Tyr Ala Ala Ala Leu His Gly Phe Ser Ala 225 230 235 240 Lys Leu Ser Ser Glu Asn Leu Ala Ile Leu Arg Ser Asp Pro Arg Val 245 250 255 Lys Tyr Val Glu Gln Asp Ala Ile MET Arg MET Ser Ala Thr Gln Thr 260 265 270 Gly Ala Thr Trp Gly Leu Asp Arg Ile Asp Gln Arg Asn Leu Pro Leu 275 280 285 Asp Gly Ser Tyr Thr Tyr Asn Ser Thr Ala Ser Gly Val Lys Val Tyr 290 295 300 Ile Ile Asp Thr Gly Ile Asn Thr Ala His Ser Asn Phe Gly Gly Arg 305 310 315 320 Ala Ile Trp Gly Thr Asn Thr Thr Gly Asp Gly Asn Asn Ser Asp Cys 325 330 335 Gln Gly His Gly Thr His Val Ala Gly Thr Val Gly Ser Ser Thr Tyr 340 345 350 Gly Val Ala Lys Gly Ala Thr Leu Val Ala Val Lys Val Leu Asn Cys 355 360 365 Gln Gly Ser Gly Thr Asn Ser Gly Val Ile Ala Gly Val Asn Trp Ala 370 375 380 Val Ser Asn Lys Gly Ser Ala Thr Ala Val Ala Asn MET Ser Leu Gly 385 390 395 400 Gly Gly Ala Ser Gln Ala Val Asp Asp Ala Val Asn Asn Ala Ala Ser 405 410 415 Lys Asn Leu Ile MET Ala Val Ala Ala Gly Asn Glu Asn Gln Asn Ala 420 425 430 Cys Asn Val Ser Pro Ala Arg Ala Ala Ser Ala Ile Thr Val Gly Ser 435 440 445 Thr Thr Asn Thr Asp Ala Arg Ser Ser Phe Ser Asn Tyr Gly Thr Cys 450 455 460 Val Asp Ile Phe Ala Pro Gly Ser Asn Ile Thr Ser Thr Trp Ile Gly 465 470 475 480 Ser Thr Ser Ala Thr Asn Thr Ile Ser Gly Thr Ser MET Ala Ser Pro 485 490 495 His Val Ala Gly Ala Ala Ala Leu Leu Val Ala Ala Gly Asn Thr Thr 500 505 510 Thr Ser Ala Val Thr Ser Ala MET Ile Asn Asn Ala Thr Pro Asn Lys 515 520 525 Val Thr Gly Ala Gly Thr Gly Ser Pro Asn Arg Leu Leu Tyr Thr Gly 530 535 540

Claims

1. A functional module for degrading feather and converting it into hemoprotein, consisting of DNA of the sequence shown in SEQ ID NO:

3.

2. Use of DNA of the sequence shown in SEQ ID NO: 3 in catalyzing biodegradation and conversion synthesis, which is a biosynthesis reaction for degrading and converting avian feather into artificial hemoprotein.

3. An amino acid sequence encoded by the functional module of the sequence shown in SEQ ID NO: 3, as shown in SEQ ID NO: 4.

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