CN109929855B - Polysaccharide pyrolysis monooxygenase LPMO9D encoding gene and enzyme, preparation and application - Google Patents

Abstract

The invention discloses a coding gene of polysaccharide pyrolysis monooxygenase LPMO9D derived from myceliophthora thermophila, and a preparation method and application of the enzyme. The polysaccharide-cleaving monooxygenase LPMO9D of the present invention is derived from myceliophthora thermophila (Thermomyces thermophila ATCC 42464). The invention also provides a method for preparing the novel polysaccharide pyrolysis monooxygenase, namely, a gene engineering technical method is utilized, the gene of the novel polysaccharide pyrolysis monooxygenase is cloned to a pichia pastoris expression vector to obtain a pichia pastoris recombinant strain capable of heterologously expressing the enzyme, and the polysaccharide pyrolysis monooxygenase LPMO9D prepared by heterologously expressing the strain can degrade PASC (microcrystalline cellulose treated by phosphoric acid) to generate cellooligosaccharide and cello-oligosaccharide acid. The polysaccharide pyrolysis monooxygenase LPMO9D provided by the invention can be widely applied to the fields of chemical industry, agriculture, feed addition, medicine and the like, and has great production potential and economic value.

Description

Polysaccharide pyrolysis monooxygenase LPMO9D encoding gene and enzyme, preparation and application

Technical Field

The invention relates to a gene sequence of polysaccharide pyrolysis monooxygenase, and a preparation method and application of the enzyme. The invention also provides a recombinant plasmid and a recombinant genetic engineering strain of the polysaccharide-cleaved monooxygenase. The polysaccharide pyrolysis monooxygenase LPMO9D can be widely applied to the fields of agriculture, food, feed additives, medicines and the like.

Background

With the rapid development of economy and the rapid growth of population, the traditional nonrenewable resources of fossil energy such as coal, petroleum and the like can not meet the economic development demand of the world today. Besides clean energy sources such as solar energy and wind energy, lignocellulose is the most abundant renewable resource in the world, and is widely concerned by various countries for developing and producing biomass fuels and chemicals by taking lignocellulose as a raw material. The reasonable utilization of such resources would have great significance in mitigating energy crisis. At present, two flows of pretreatment and saccharification are needed for the conversion of cellulose industrially, a chemical method and an enzymatic method are mainly adopted to degrade the cellulose in the whole process, the enzymatic method is mainly applied to degrading the cellulose by a glycoside hydrolase enzyme system consisting of endoglucanase, cellobiohydrolase and beta-1,4-D-glucosidase in the saccharification process, the degradation efficiency of the glycoside hydrolase enzyme system on a substrate crystallization area is low, the activity is easy to lose at high temperature, and the cost is high, so that the traditional cellulase system is difficult to meet the requirement of industrial application, and the efficient utilization of biomass such as cellulose is limited.

Polysaccharide lytic monooxygenases (LPMO) are oxidases that cleave biomass polysaccharides (cellulose, chitin, starch, etc.) by redox, which destroy crystalline regions of poorly soluble polysaccharide materials to produce more open substrate structures. The appearance of the polysaccharide improves the degradation efficiency of the insoluble polysaccharide, so that the high-efficiency conversion of biomass becomes possible. Sources of polysaccharide-cleaving monooxygenases are very abundant and are currently found in bacteria (e.g., streptomyces coelicolor A3 (2)), fungi (e.g., neurospora crassa OR 74A), and viruses (e.g., anomala cuprea entomopoxvirus CV 6M). There are 49 LPMO that have been reported so far, of which there are 26 AA9 families, 19 AA10 families, 1 AA11 family and 3 AA3 families; the polysaccharide pyrolysis monooxygenase LPMO9D is derived from myceliophthora thermophila, belongs to AA9 family, has high temperature resistance different from polysaccharide pyrolysis monooxygenases of other families, can improve reaction temperature, shorten saccharification time and reduce the pollution probability of a reactor when applied in the cellulose saccharification process, has the optimal action condition of an acid environment, is suitable for cellulose treatment after industrial acid pretreatment compared with other LPMO, is a novel LPMO, and lays a foundation for efficient degradation of subsequent cellulose through cloning expression.

Disclosure of Invention

The first purpose of the invention is to provide a novel polysaccharide-cleaving monooxygenase LPMO9D derived from myceliophthora thermophila (Thermomyces thermophila ATCC 42464) and a coding gene thereof.

It is a second object of the present invention to provide a process for the preparation of the novel polysaccharide-cleaving monooxygenase LPMO 9D.

The third purpose of the invention is to provide recombinant expression plasmid and recombinant gene engineering strain containing the polysaccharide-splitting monooxygenase lpmo9D gene.

It is another object of the present invention to provide the use of the novel polysaccharide-cleaving monooxygenase LPMO9D in the degradation of cellulose.

The polysaccharide pyrolysis monooxygenase LPMO9D provided by the invention is derived from myceliophthora thermophila (Thermoascus thermophila ATCC 42464), and the amino acid sequence of the polysaccharide pyrolysis monooxygenase LPMO9D has at least one of the following characteristics:

1) 1-303 or 20-303 amino acid residue sequence from amino terminal of SEQ ID NO.2 in the sequence table, wherein 1-19 is signal peptide, 20-303 is amino acid sequence of active polysaccharide-cleaving monooxygenase LPMO 9D.

2) The amino acid residue at the 1 st to 303 th or 20 th to 303 th position from the amino terminal of SEQ ID NO.2 in the sequence table is substituted, deleted or added with one or more than two amino acids to form the amino acid sequence with unchanged activity of the polysaccharide splitting monooxygenase.

The invention also provides a gene coding for the polysaccharide-cleaving monooxygenase LPMO9D (designated LPMO 9D) having at least one of the following nucleotide sequence features:

1) A deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in a sequence table;

2) A deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2 of the sequence list;

3) A deoxyribonucleic acid (DNA) sequence which has 80 percent of homology or more with the deoxyribonucleic acid (DNA) sequence defined by SEQ ID NO.1 and can code a protein for degrading cellulose.

3) A nucleotide sequence which is obtained by substituting, deleting or adding one or more than two nucleotides into a deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in a sequence table and codes the DNA sequence with the activity of the polysaccharide cleavage monooxygenase.

The amino acid sequence and the nucleotide coding sequence of the polysaccharide-cleaving monooxygenase LPMO9D can also be obtained by artificial synthesis according to the predicted amino acid sequence and the nucleotide coding sequence of LPMO 9D.

The method for preparing recombinase LPMO9D is to clone the coding gene LPMO9D into a recombinant expression vector, and introduce the recombinant expression vector into host cells to obtain the recombinant expressed polysaccharide-cleaved monooxygenase.

The expression vector of the recombinant expression polysaccharide pyrolysis monooxygenase LPMO9D can be an escherichia coli expression vector, a yeast expression vector, a bacillus subtilis expression vector, a lactic acid bacteria expression vector, a streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, a mammalian cell expression vector and the like.

Recombinant bacteria or transgenic cell lines for recombinantly expressed polysaccharide-cleaving monooxygenase LPMO9D may be E.coli host cells (e.g., escherichia coli BL21, escherichia coli J9D09, escherichia coli DH 5. Alpha. And the like), yeast host cells (e.g., saccharomyces cerevisiae, pichia pastoris, kluyveromyces lactis and the like), bacillus subtilis host cells (e.g., bacillus subtilis R25, bacillus subtilis 9920 and the like), lactic acid bacteria host cells (e.g., lactobacillus COCC101 and the like), actinomycete host cells (e.g., streptomyces spp. And the like), filamentous fungal host cells (e.g., trichoderma viride, trichoderma reesei, aspergillus niger, aspergillus nidulans and the like), insect cells (e.g., yeast, bacillus thuringiensis and the like), kidney cells (e.g., baby hamster ovary cells, CHO), and the like.

The polysaccharide-cleaving monooxygenase enzyme described above may be used in cellulose degradation, including one or both of the following;

1) The application in breaking cellulose glycosidic bond to obtain cello-oligosaccharide;

2) The application in oxidizing and degrading biomass such as lignin and the like to obtain cellooligosaccharide and cellooligosaccharide acid;

the polysaccharide pyrolysis monooxygenase LPMO9D can be applied to the aspects of synergistically degrading cellulose and preparing bioethanol after being mixed with a cellulase system.

The gene sequence of the polysaccharide pyrolysis monooxygenase LPMO9D is cloned from myceliophthora thermophila (Thermosaccharomyces thermophila ATCC 42464) by RT-PCR technology. The gene coding region is 912bp in length, 303 amino acids are coded, the theoretical molecular weight is 31.53KDa, and the gene belongs to the auxiliary activity enzyme AA9 family. The LPMO9D obtained by recombinant pichia pastoris expression can be used for oxidizing and degrading microcrystalline cellulose (PASC) after phosphoric acid treatment under the conditions of 45 ℃ and pH = 5.0. The polysaccharide pyrolysis monooxygenase LPMO9D is subjected to sequence comparison with LPMO which has been researched by the same family, and the result shows that the homology is only 44% at most, so that the polysaccharide pyrolysis monooxygenase LPMO can be widely applied to the fields of agriculture, food, feed addition, medicines and the like.

Drawings

FIG. 1 is an electrophoretic detection of the polysaccharide-cleaving monooxygenase lpmo9D gene;

the samples added in each lane are: lanes 1-BM 5000DNA Marker; lane 2-lpmo 9D PCR product

FIG. 2 is a polyacrylamide gel electrophoresis (SDS-PAGE) of the expression of the recombinant polysaccharide-cleaving monooxygenase LPMO 9D. The samples added in each lane are: lane 1-Prestained protein molecular weight standards; lane 2-total protein from supernatant after 96h induction of Pichia pastoris X-33/pPICZ. Alpha.A; lane 3-Pichia pastoris X-33/pPICZ α A-lpmo 9D Total protein after 96h induction.

FIG. 3: MALDI-TOF analysis spectrum of product of LPMO9D degradation PASC.

Detailed Description

EXAMPLE 1 full-Length polysaccharide-cleaving monooxygenase Gene cloning

The RNA of Thermomyces thermophilus strain Thermomyces thermophila ATCC 42464 was extracted according to the procedure of a fungal RNA extraction kit (Shanghai Producer, SK 8659), and cDNA was synthesized according to the procedure of a cDNA first strand synthesis kit (Code No.: 6210A) of TaKaRa Bio Inc. Designing a primer according to the nucleotide sequence of a target gene, wherein an upstream primer 5'-GACC1TCGAGAAAAGACACTACATCTTTCAGCAGCTG-3', a downstream primer: 5'-CTACAAGCACTGGCTGTAGTAGTC-3'. PCR was performed using the synthesized cDNA as a template and Primer Star HS DNA polymerase kit (Code No.: R010A) from TaKaRa Biometrics. The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 1min, 30 cycles in total, and extension at 72 ℃ for 5min. After the PCR product is analyzed by agarose gel electrophoresis, the target fragment recovered by cutting the gel is digested by double enzyme digestion, and the target gene is connected to an expression vector pPICZ alpha A by T4DNA ligase for sequencing.

Example 2 sequencing of the Polysaccaride lytic monooxygenase Gene

Sequencing results were analyzed using Basic Local Alignment Search Tool (BLAST) in the GenBank database, DNAMAN software for multiple sequence alignments, and VectorNTI 8.0 for sequence information. The obtained coding region of the polysaccharide splitting monooxygenase gene (named lpmo 9D) is 912bp in length, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1. lpmo9D encodes 303 amino acids and 1 stop codon, the amino acid sequence is shown in SEQ ID NO.2, the theoretical molecular weight of the protein is 31.53kDa, and the predicted isoelectric point is 6.16.SignalP analysis indicated that the amino acid sequence of LPMO9D had a signal peptide at positions 1-20. The domain characteristics of LPMO9D are more similar to those of AA9 family members, thereby indicating that LPMO9D is a new member of AA9 family. And (3) carrying out homologous modeling on the protein three-dimensional structure of the LPMO9D by using a SWISS-MODEL homologous modeling server (http:// swissmodel. Expasy. Org), and displaying that the QMEAN (QMEAN is the score for evaluating the quality of the MODEL) value of the constructed LPMO9D structure MODEL is in a credible range value, thereby indicating that the obtained LPMO9D three-dimensional structure MODEL is credible.

(1) Information of SEQ ID No:1 (see sequence Listing)

(a) Sequence characterization

* Length: 912 base pair

* Type (2): nucleic acids

* Chain type: double chain

* Topological structure: linearity

(b) Molecular type: cDNA

(c) Suppose that: whether or not

(d) Antisense: whether or not

(e) The initial sources were: myceliophthora thermophila Thermomyces thermophila

(2) Information of SEQ ID No:2 (see sequence Listing)

(a) Sequence characterization

* Length: 303 residue (iii)

* Type (2): amino acids

* Chain type: single strand

* Topological structure: linearity

(b) Molecular type: protein

SEQ ID NO.1

ATGAAGGGACTCCTCGGCGCCGCCGCCCTCTCGCTGGCCGTCAGCGATGTCTCGGCCCACTACATCTTTCAGCAGCTGACGACGGGCGGCGTCAAGCACGCTGTGTACCAGTACATCCGCAAGAACACCAACTATAACTCGCCCGTGACCGATCTGACGTCCAACGACCTCCGCTGCAATGTGGGTGCTACCGGTGCGGGCACCGATACCGTCACGGTGCGCGCCGGCGATTCGTTCACCTTCACGACCGATACGCCCGTTTACCACCAGGGCCCGACCTCGATCTACATGTCCAAGGCCCCCGGCAGCGCGTCCGACTACGACGGCAGCGGCGGCTGGTTCAAGATCAAGGACTGGGGTGCCGACTTTAGCAGCGGCCAGGCCACCTGGACCTTGGCGTCTGACTACACCGCCACGATTCCGGAATGTATTCCCCCCGGCGACTACCTGCTTCGCATCCAGCAACTCGGCATCCACAACCCTTGGCCCGCGGGCATCCCCCAGTTCTACATCTCTTGTGCCCAGATCACCGTGACTGGTGGCGGCAGTGCCAACCCCGGCCCGACCGTCTCCATCCCAGGCGCCTTCAAGGAGACCGACCCGGGCTACACTGTCAACATCTACAACAACTTCCACAACTACACCGTCCCTGGCCCAGCCGTCTTCACCTGCAACGGTAGCGGCGGCAACAACGGCGGCGGCTCCAACCCAGTCACCACCACCACCACCACCACCACCAGGCCGTCCACCAGCACCGCCCAGTCCCAGCCGTCGTCGAGCCCGACCAGCCCCTCCAGCTGCACCGTCGCGAAGTGGGGCCAGTGCGGAGGACAGGGTTACAGCGGCTGCACCGTGTGCGCGGCCGGGTCGACCTGCCAGAAGACCAACGACTACTACAGCCAGTGCTTGTAG

SEQ ID NO.2

MKGLLGAAALSLAVSDVSAHYIFQQLTTGGVKHAVYQYIRKNTNYNSPVTDLTSNDLRCNVGATGAGTDTVTVRAGDSFTFTTDTPVYHQGPTSIYMSKAPGSASDYDGSGGWFKIKDWGADFSSGQATWTLASDYTATIPECIPPGDYLLRIQQLGIHNPWPAGIPQFYISCAQITVTGGGSANPGPTVSIPGAFKETDPGYTVNIYNNFHNYTVPGPAVFTCNGSGGNNGGGSNPVTTTTTTTTRPSTSTAQSQPSSSPTSPSSCTVAKWGQCGGQGYSGCTVCAAGSTCQKTNDYYSQCL

Example 3lpmo 9D Gene recombinant expression in Pichia pastoris

The gene sequence (excluding the signal peptide gene) encoding the mature protein of polysaccharide-cleaving monooxygenase was amplified as described in example 1 using the genomic DNA of myceliophthora thermophila cDNA as a template and the designed upstream primer (5 '-GACC1TCGAGAAAAGACACTACATCTTTCAGCAGCTG-3', downstream primer: 5'-CTACAAGCACTGGCTGTAGTAGTC-3'.). Connecting the PCR amplification product with an expression vector pPICZ alpha A (Novagen company, USA) through T4DNA ligase, converting the ligation product into escherichia coli TOP10 competent cells, coating the competent cells on a low-salt Luria-Bertani culture medium solid plate containing 100 mu g/mL bleomycin, culturing at 37 ℃ for 12-16h, selecting a monoclonal, and performing colony PCR verification by using upstream and downstream primers to obtain an amplification product with correct size; inoculating the correct single clone into a liquid low-salt Luria-Bertani culture medium containing 100 mu g/mL bleomycin for culture, and extracting plasmids; then the recombinant plasmid is sent to Huada gene (Beijing) for sequencing, and the result shows that lpmo9D gene shown by SEQ ID NO 1 is inserted between Xho I enzyme cutting sites and Not I enzyme cutting sites, the insertion direction is correct, the construction success of the recombinant plasmid is proved, and the recombinant plasmid is named as pPICZ alpha A-lpmo 9D.

pPICZ α A-LPMO 9D was transformed into Pichia pastoris strain X-33 (Invitrogen, USA, V195-20) and induced expression of the polysaccharide cleaving monooxygenase LPMO9D was performed following the procedure provided by the company. The expression condition of the polysaccharide-cleaving monooxygenase is detected by polyacrylamide gel electrophoresis, and the result is shown in FIG. 2, wherein the polysaccharide-cleaving monooxygenase is obviously expressed under the induction of methanol.

Example 4 product analysis of the oxidative degradation of PASC by the polysaccharide-cleaving monooxygenase LPMO9D

5mg of PASC substrate was taken, polysaccharide-cleaving monooxygenase LPMO9D (1. Mu.M) and ascorbic acid (1 mM) were added thereto, the reaction was terminated after 24 hours at 50 ℃, insoluble substrate was removed by centrifugation, and the supernatant was taken and subjected to sevage method according to the reaction product: sevage reagent =4:1 and the products were detected by MALDI-TOF after centrifugation to remove protein from the products.

As shown in figure 3, the PASC is taken as a substrate, degradation products are mainly DP =2-7 oligosaccharide and DP =2-6 sugar acid, so that the enzyme can be used for preparing the oligosaccharide and the sugar acid, and can be used for degrading cellulose in cooperation with a cellulase system to improve the cellulose degradation efficiency and provide a new visual angle for the cellulose degradation.

Sequence listing

<110> institute of chemistry and physics, the university of Chinese academy of sciences

<120> polysaccharide pyrolysis monooxygenase LPMO9D encoding gene and enzyme, preparation and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 912

<212> DNA

<213> myceliophthora thermophila (Thermothermomyces thermophila)

<400> 1

atgaagggac tcctcggcgc cgccgccctc tcgctggccg tcagcgatgt ctcggcccac 60

tacatctttc agcagctgac gacgggcggc gtcaagcacg ctgtgtacca gtacatccgc 120

aagaacacca actataactc gcccgtgacc gatctgacgt ccaacgacct ccgctgcaat 180

gtgggtgcta ccggtgcggg caccgatacc gtcacggtgc gcgccggcga ttcgttcacc 240

ttcacgaccg atacgcccgt ttaccaccag ggcccgacct cgatctacat gtccaaggcc 300

cccggcagcg cgtccgacta cgacggcagc ggcggctggt tcaagatcaa ggactggggt 360

gccgacttta gcagcggcca ggccacctgg accttggcgt ctgactacac cgccacgatt 420

ccggaatgta ttccccccgg cgactacctg cttcgcatcc agcaactcgg catccacaac 480

ccttggcccg cgggcatccc ccagttctac atctcttgtg cccagatcac cgtgactggt 540

ggcggcagtg ccaaccccgg cccgaccgtc tccatcccag gcgccttcaa ggagaccgac 600

ccgggctaca ctgtcaacat ctacaacaac ttccacaact acaccgtccc tggcccagcc 660

gtcttcacct gcaacggtag cggcggcaac aacggcggcg gctccaaccc agtcaccacc 720

accaccacca ccaccaccag gccgtccacc agcaccgccc agtcccagcc gtcgtcgagc 780

ccgaccagcc cctccagctg caccgtcgcg aagtggggcc agtgcggagg acagggttac 840

agcggctgca ccgtgtgcgc ggccgggtcg acctgccaga agaccaacga ctactacagc 900

cagtgcttgt ag 912

Claims (6)

1. The application of recombinant polysaccharide-cleaved monooxygenase LPMO9D in cellulose degradation is characterized in that the encoding gene of the polysaccharide-cleaved monooxygenase LPMO9D is shown as SEQ ID No.1 in a sequence table; the amino acid sequence of the polysaccharide pyrolysis monooxygenase LPMO9D after the signal peptide is removed is shown as the 20 th to 303 th sites of SEQ ID NO. 2; the polysaccharide pyrolysis monooxygenase LPMO9D has oxidative degradation activity on a substrate PASC containing cellulose, and the product is oligosaccharide and oligosaccharide acid mixture.

2. The use of claim 1, wherein: cloning the coding gene of polysaccharide cleaved monooxygenase LPMO9D into an expression vector, and introducing into host cells to obtain the recombinant expressed polysaccharide cleaved monooxygenase.

3. Use according to claim 2, characterized in that: the expression vector for recombinant expression of the polysaccharide pyrolysis monooxygenase refers to one of an escherichia coli expression vector, a yeast expression vector, a bacillus subtilis expression vector, a lactic acid bacteria expression vector, a streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector.

4. The use of claim 2, wherein: the recombinant bacterium or transgenic cell line for recombinant expression of polysaccharide pyrolysis monooxygenase refers to one of Escherichia coli host cells, yeast host cells, bacillus subtilis host cells, lactic acid bacteria host cells, actinomycete host cells, filamentous fungi host cells, insect cells and mammalian cells.

5. The use of claim 1, wherein: comprises one or two of the following applications;

1) The application in breaking cellulose glycosidic bond to obtain cello-oligosaccharide;

2) The application of the xylooligosaccharide and cello-oligosaccharide acid in oxidative degradation of lignin biomass to obtain cello-oligosaccharide and cello-oligosaccharide acid;

3) When the lignin biomass is subjected to oxidative degradation, the enzyme has high temperature resistance, the optimal action temperature is 50 ℃, the enzyme can react for 24 hours at the temperature of 55 ℃, and the enzyme activity can still be kept above 80%.

6. The use of claim 1, wherein: the polysaccharide pyrolysis monooxygenase LPMO9D is mixed with a cellulase system and then applied to the aspects of synergistically degrading cellulose and preparing bioethanol.

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