CN111647572A

CN111647572A - Reductase derived from trichoderma reesei and coding gene and application thereof

Info

Publication number: CN111647572A
Application number: CN202010486390.3A
Authority: CN
Inventors: 赫荣琳; 贾文娣; 张东远
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2020-09-11

Abstract

The present invention discloses enzymes having the activity of-N- (L-glutaryl 2) -L-lysine reductase, which provide the function of catalyzing the formation of-N- (L-glutaryl 2) -L-lysine from 6-oxo-DL-hexanoic acid (L-alpha-aminoadipate6-semialdehyde) in the diaminopimelic acid pathway. The invention discloses a DNA molecule encoding the enzyme. The invention discloses a recombinant vector. The invention discloses a host cell. The invention discloses a construction method of lysine auxotroph mutant. The invention discloses an application of a DNA molecule as an auxotrophic marker gene of lysine. The invention discloses application of protein or DNA molecules in growth and development of trichoderma reesei and actual production optimization. The TrLys9 gene provided by the invention can be used as an auxotrophic marker gene of lysine and a marker gene for subsequent transgenic operation of Trichoderma reesei.

Description

Reductase derived from trichoderma reesei and coding gene and application thereof

Technical Field

The invention belongs to the field of related application of functional genes of filamentous fungi, and relates to a Saccharomyces reductase trys 9 cloned from the filamentous fungi Trichoderma reesei and a cDNA sequence thereof, and also provides a recombinant expression vector and a recombinant host cell of the Saccharomyces reductase cDNA sequence, and application of the recombinant expression vector and the recombinant host cell in influencing growth of Trichoderma reesei hyphae, production of conidiospores and the like.

Background

Lysine is an essential amino acid for humans and animals and can only be obtained from dietary proteins. Lysine can be synthesized in bacteria, fungi and some plants. Of the 20 common proteinogenic amino acids, only lysine has two distinct biosynthetic pathways. In bacteria, lower fungi (e.g. phycomycetes) and some plants, lysine is synthesized via the diaminopimelate pathway (DAP pathway); in higher fungi (whose cell wall contains chitin components) and euglena, the α -aminoadipate pathway (AAA pathway) is an important biochemical pathway for the synthesis of lysine and is also a pathway for the synthesis of lysine unique to the above-mentioned organisms. Aiming at the synthetic pathway, a series of biochemical and genetic researches are mainly carried out by taking Saccharomyces cerevisiae (Saccharomyces cerevisiae) as a model organism (Guo et al 1964; Winston et al 1987).

Trichoderma reesei (Trichoderma reesei) is an important industrial filamentous fungus, mainly used for the production of degrading lignocellulose enzyme lines. In addition, trichoderma reesei grows rapidly, branches to form typical fungal hyphae, produces large quantities of green conidia and releases. With the publication of trichoderma reesei genome database, the synthesis regulation mechanism of trichoderma reesei lignocellulose is studied systematically and deeply. It has also been found in their genome that under certain circumstances glycoside hydrolase genes cluster in the vicinity of many genes encoding some important metabolite biosynthetic pathways, and it is therefore speculated that these genes may influence the survival of trichoderma reesei in a competitive external environment (Martinez et al, 2008). However, there are few studies on how these metabolic pathways affect the competitive growth of trichoderma reesei, the growth and development of hyphae, and sporulation.

Lysine is synthesized in higher fungi primarily by the α -aminoadipic acid pathway. In recent years, with the growing genomic information, the understanding of the α -aminoadipate pathway to lysine has increased significantly at the molecular level, and through these studies, the biochemical problems that have not yet been solved in this pathway will be solved, and the evolution of various pathways with respect to the formation and degradation of lysine in different organisms can also be studied more thoroughly. Due to the uniqueness of the fungal lysine biosynthetic metabolic pathway, it is speculated that key enzymes in this pathway may be possible targets for selective fungal formulations. At the same time, the novelty of several genes in this pathway may make them molecular markers that facilitate the rapid identification of pathogenic fungi, such as the LYSF gene in Aspergillus fumigatus, the LYS2 gene in Magnaporthe oryzae, etc. (Weidner et al, 1997; Chen et al, 2014). Furthermore, AAA pathway intermediates of lysine biosynthesis are incorporated into secondary metabolites of fungi, and in Penicillium chrysogenum, blockade of the AAA pathway can increase penicillin production (Casqueiro et al, 1999). In the AAA synthetic pathway, seven enzymes are involved in a total of eight steps of reaction (Xu et al, 2006).

Disclosure of Invention

An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.

It is also an object of the present invention to provide an enzyme.

It is also an object of the invention to provide a DNA molecule, a recombinant vector and a host cell.

It is still another object of the present invention to provide a method for constructing lysine auxotrophic mutants.

It is a further object of the present invention to provide the use of the DNA molecule as a marker gene for lysine auxotrophy.

It is another object of the present invention to provide the use of the protein or DNA molecule in the growth and development of Trichoderma reesei and in the optimization of actual production.

Therefore, the technical scheme provided by the invention is as follows:

an enzyme having an activity of-N- (L-glutaryl 2) -L-lysine reductase (Saccharomyces reducase or L-Saccharomyces reducase) which provides a function of catalyzing 6-oxo-DL-hexanoate (L-alpha-aminoadipate6-semialdehyde) to form-N- (L-glutaryl 2) -L-lysine in a diaminopimelate pathway, the enzyme being a protein of the following (a) or (b):

(a) the amino acid sequence is shown as SEQ ID NO. 3;

(b) a protein derived from (a) having an amino acid sequence in (a) by substitution, deletion or addition of one or several amino acids and having an-N- (L-glutaryl-2) -L-lysine reductase activity.

A DNA molecule encoding the enzyme of claim 1.

Preferably, in the DNA molecule, the DNA molecule has a base sequence of (c), (d), (e) or (f) below:

(c) as shown in SEQ ID NO. 1;

(d) as shown in SEQ ID NO. 2;

(e) a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO. 3;

(f) hybridizing with the nucleotide sequence defined in (a) or (b) under strict hybridization conditions and coding the nucleotide sequence with the function of-N- (L glutaryl 2) -L-lysine reductase.

A recombinant vector comprising said DNA molecule and regulatory sequences for expression operably linked to said DNA molecule.

A host cell comprising said DNA molecule or said recombinant vector.

A method for constructing lysine auxotrophic mutants comprises the following steps:

taking wild Trichoderma reesei genome DNA as a template, carrying out PCR amplification by using a primer pair shown as SEQ ID NO. 6 and SEQ ID NO. 7 to obtain an upstream fragment of a TrLys9 gene, carrying out PCR amplification by using a primer pair shown as SEQ ID NO. 8 and SEQ ID NO. 9 to obtain a downstream fragment of a TrLys9 gene, and connecting the upstream fragment and the downstream fragment to a pBS-HPH vector in sequence to obtain a TrLys9 gene knockout vector;

and step two, transforming the TrLys9 gene knockout vector obtained in the step one into a host cell by a protoplast transformation method, and screening to obtain the lysine auxotrophic mutant.

Preferably, in the method for constructing the lysine auxotrophic mutant, in the first step, the upstream fragment obtained by amplification adopts Kpn I and Sal I restriction enzyme double-restriction enzyme sites, and the upstream fragment after double-restriction enzyme is connected to pBS-HPH using the same restriction enzyme sites;

connecting the amplified downstream fragment to a T vector, then adopting Hind III and Xba I restriction enzyme double restriction sites, simultaneously using Hind III and Xba I to cut the pBS-HPH vector connected with the upstream fragment, connecting the downstream fragment cut by the T vector to the pBS-HPH of the same cutting site, and constructing the TrLys9 gene knockout vector.

Preferably, in the method for constructing a lysine auxotrophic mutant, in the second step, the screening comprises the steps of: and (3) coating the strain on a CM plate containing hygromycin B resistance, culturing to obtain a transformant, selecting the transformant, culturing, and performing PCR amplification by using primer pairs such as SEQ ID NO. 10 and SEQ ID NO. 11, wherein the transformant without an amplified band is the lysine auxotrophic mutant.

The use of the DNA molecule as an auxotrophic marker gene for lysine.

The protein or the DNA molecule is used in the growth and development of the trichoderma reesei and the actual production optimization.

The invention at least comprises the following beneficial effects:

the invention researches the influence of the gene on the competitive growth and development of trichoderma reesei by analyzing the function of the saccharomyces reductase catalyzing the seventh step reaction. The TrLys9 gene knockout vector constructed by the invention contains upstream and downstream homologous recombination fragments of a TrLys9 gene DNA sequence, and completely avoids an open reading frame of the TrLys9 gene DNA, so that when gene knockout is carried out by using a recombinant plasmid, the TrLys9 gene DNA gene can be completely knocked out by a reporter gene. The knockout gene complementation vector is transformed into trichoderma reesei to obtain a recombinant transformant, namely a complementation mutant with the ethanolamine kinase coding gene TrLys9 gene function deletion.

Analysis of deletion mutant of TrLys9 gene shows that the deletion of the gene seriously affects colony morphology; under different culture medium conditions, the growth rate and sporulation quantity of the gene deletion mutant are sharply reduced; has no influence on the germination rate of conidiophores.

The TrLys9 knockout mutant has an impaired lysine synthesis pathway and does not grow on minimal medium without exogenous lysine, and growth is restored after lysine supplementation. Based on the lysine auxotrophy characteristics of the TrLys9 knockout mutant, the gene can be used as an auxotrophy selection marker to be applied to the gene manipulation of Trichoderma reesei.

In addition, the invention provides a method for searching for homologous proteins in other filamentous fungi with close relativity in NCBI databases by using the TrLys9 protein as an object, such as: penicillium chrysogenum (p. chrysogenum), aspergillus fumigatus (a. fumigates), aspergillus niger (a. niger), anthrax (Colletotrichum graminicola), and the like.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a phylogenetic tree analysis of the Lys9p protein sequence in Trichoderma reesei of the present invention with other filamentous fungi;

FIG. 2 is a diagram of a Southern blot for verifying whether the TrLys9 gene is successfully knocked out in one embodiment of the present invention;

FIG. 3 is a diagram of reverse transcription PCR gels from different strains according to one embodiment of the present invention;

FIG. 4 is a colony morphology of the QM9414 wild-type strain, Δ TrLys9-11 gene deletion mutant strain and 11HB gene complementation mutant strain on different media in one example of the present invention;

FIG. 5 shows the colony growth of the deletion mutant of Δ TrLys9-11 gene under the condition of different lysine concentrations in one embodiment of the present invention.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The present invention provides an enzyme having an activity of-N- (L-glutaryl 2) -L-lysine reductase (Saccharomyces reducase or L-Saccharomyces reducase) which provides a function of catalyzing 6-oxo-DL-hexanoic acid (L-alpha-aminoadipate6-semialdehyde) to form-N- (L-glutaryl 2) -L-lysine in the diaminopimelate pathway, the enzyme being a protein of the following (a) or (b):

(a) the amino acid sequence is shown as SEQ ID NO. 3;

The invention provides a DNA molecule encoding said enzyme.

In the above embodiment, the DNA molecule preferably has a base sequence of (c), (d), (e) or (f):

(c) as shown in SEQ ID NO. 1;

(d) as shown in SEQ ID NO. 2;

The invention also provides a recombinant vector comprising the DNA molecule and regulatory sequences for expression operably linked to the DNA molecule.

The invention also provides a host cell containing the DNA molecule or the recombinant vector of claim 3.

The invention also provides a construction method of the lysine auxotroph mutant, which comprises the following steps:

In the above scheme, preferably, in the first step, the upstream fragment obtained by amplification is subjected to double restriction sites with Kpn I and Sal I restriction enzymes, and the double-cleaved upstream fragment is linked to pBS-HPH subjected to the same restriction sites;

In the above scheme, preferably, in the second step, the screening includes the following steps: and (3) coating the strain on a CM plate containing hygromycin B resistance, culturing to obtain a transformant, selecting the transformant, culturing, and performing PCR amplification by using primer pairs such as SEQ ID NO. 10 and SEQ ID NO. 11, wherein the transformant without an amplified band is the lysine auxotrophic mutant.

The invention also provides the application of the DNA molecule as an auxotroph marker gene of lysine.

The invention also provides the application of the protein or the DNA molecule in the growth and development of the trichoderma reesei and the actual production optimization.

Meanwhile, the invention also provides a recombinant expression vector of the Saccharomyces reductase (-N- (L glutaryl 2) -L-lysine reductase gene) or the Saccharomyces reductase cDNA and a recombinant host cell of the recombinant expression vector.

The protein variants of the invention may be produced by genetic polymorphism or by human manipulation, such manipulations being generally known in the art. For example, amino acid sequence variants or fragments of transcription factors can be prepared by mutation of the DNA, wherein methods for mutagenizing or altering polynucleotides are well known in the art. Among these, conservative substitutions are substitutions of one amino acid residue for another with similar properties.

The invention connects the cloned TrLys9cDNA or the whole gene with an expression regulation element to construct a recombinant expression vector, and the recombinant expression vector is transformed into Trichoderma reesei to analyze the effect of Trichoderma reesei in growth and development. The recombinant expression vector or the expression cassette comprises a promoter, TrLys9cDNA or a whole gene and a terminator; among them, the TrLys9cDNA sequence or the whole gene is located downstream of the promoter, and the terminator is located downstream of the TrLys9cDNA sequence.

In order to confirm whether the cDNA or the whole gene of the coding gene TrLys9 of the Saccharomyces reductase separated by the invention has the activity of influencing the synthesis of lysine, the invention constructs a cDNA knockout vector of the coding gene TrLys9 of the Saccharomyces reductase and a complementary vector of the knockout vector.

The gene knockout vector or the expression cassette comprises an upstream 1000bp-2000bp length sequence of a Saccharomyces reductase coding gene TrLys9cDNA, a reporter gene and a downstream 1000bp-2000bp length sequence of the Saccharomyces reductase coding gene TrLys9 cDNA; wherein the reporter gene is positioned at the downstream of the upstream sequence of the TrLys9cDNA of the gene coding for the Saccharomyces reductase, and the downstream sequence of the TrLys9cDNA of the gene coding for the Saccharomyces reductase is positioned at the downstream of the reporter gene. The gene knockout vector is transformed into Trichoderma reesei to obtain a recombinant transformant, namely a mutant with the gene function of the Saccharomyces reductase coding gene TrLys9 being deleted.

The TrLys9 gene knockout vector constructed by the invention contains upstream and downstream homologous recombination fragments of a TrLys9 gene DNA sequence, and completely avoids an open reading frame of the TrLys9 gene DNA, so that when gene knockout is carried out by using a recombinant plasmid, the TrLys9 gene DNA gene can be completely knocked out by a reporter gene. The knockout gene complementation vector is transformed into trichoderma reesei to obtain a recombinant transformant, namely a complementation mutant with the ethanolamine kinase coding gene TrLys9 gene function deletion.

In order to make the technical solution of the present invention better understood by those skilled in the art, the following examples are now provided for illustration:

example 1 homology alignment and Gene of interest acquisition

1. Acquisition of Saccharomyces reductase encoding gene in Trichoderma reesei and homology comparison

Lys9p catalyzes the seventh reaction in the lysine synthesis pathway, i.e., catalyzes the formation of-N- (L glutaryl 2) -L-lysine from L-alpha-aminoadipate 6-semialdehyde. Comparing Lys9p in Saccharomyces cerevisiae in the Trichoderma reesei genome database (https:// mycocosm.jgi.doe.gov/pages/blast-query.jsf. Thus, protein ID123631 was named TrLys9 and its amino acid sequence is shown in SEQ ID NO 3. Meanwhile, the Lys9p protein sequence in Trichoderma reesei and other filamentous fungi are subjected to phylogenetic tree analysis, as shown in FIG. 1, Lys9p in Trichoderma reesei has strong conservation in filamentous fungi, such as Gibberella tritici (E-value0.0, 79% identity), anthrax (E-value0.0, 74% identity), Aspergillus niger (E-value0.0, 71% identity) and the like.

SEQ ID NO：1：

>DNA sequences

ATGGCTCCCCAACGTGCTCTGATGCTGTAAGTGCGACTCCCGGAAAGAAATACAATCAAAGAAAAGAGAAGAAAAGAAAAGAGGCGAAAAATGCCACCACCAGCTGCTGCCCCTCCCCCACTAACATATCCGGCTTCACGATAGCGGCTCCGGCTTCGTTACCAAGCCCACGCTCGACATTCTCAACGATGCCGGCGTTGAGGTTTCCGTTGGTGAGCACTGAAACAATCCCTGAAGAAGAGAGCCTTGAGCCTCTTCGAATATCATCTCCTCCTCCTCCTCAACCTCGAACCACCACCCCCCAAGCTAACAAACAAACCCCTTCCAGCCTGCCGAACCCTCGAAAGCGCAAAGAAGCTCTCTGCCGGCGTCAAGCTGGCCACGCCCATCTCCCTCGACGTCAACGACGACAAGGCCCTCGATGCCGCCGTTGCCCAGCACGACGTCGTCATCAGCCTCATCCCCTACACCTACCACGCCGCCGTCATCAAGTCCGCCATCCGCAACAAGAAGAATGTCGTCACCACCAGCTACGTCTCGCCCGCCATGATGGAGCTCGACCAGCAGGCCAAGGACGCCGGCATTACCGTCATGAACGAGATTGGTGTAAGTGGGAAGCCGACGCTGCTGCTATGCCCGTGTTCTGGGGACGATAACTGAGCGCATTTGTATAGCTTGATCCCGGTATCGACCACCTCAGTGCCGTCGAGACCATCTCCAACGTCCACGCTGCCGGAGGCAAGATTCTTTCCTTCAAGTCATTCTGCGGTGGACTGCCGGCGCCCGAGAACTCGGACAACCCTCTTGGCTACAAGTAAGCGAGACAACATCCTCAACTGCCGGCAATTGCTAACATTGTCTGCGTTCAGGTTCTCCTGGTCGTAAGTAGCCCGCAAGTCCATGTTCCGATGCAAGCGGGCGGCTCGTCAAGAGTTCGCATGCCGCTCACTTTTCGACATTGAGAGCGTGGTATTGCTGGAGCTCCATCTAACATCTGAAAAACAGATCCCGAGGCGTTCTCCTTGCCCTGCGAAACGCTGCCACCTTCTACGAGGACGGAAAGCTGGTCAACGTTGCCGGCCCTGGTAAGTAAATCAATGGACATGGCATGTCGATATATACAAACTAGACACCCTAACACATCTCATAGACCTGATGGCCACCGCCAAGCCTTACCACATCTACCCCGGTAAGCACATCCCAATATCAGGATTTCTATCAACCCCTCTGACCATTCCCAGGCTACGCCTTTGTCGCATACCCCAACCGCGACTCCAGCGTCTACAAGGAGCGCTACAACATCCCCGAGGCCCATACCATCATCCGCGGAGTAAGCAGAAGAGTTACAATCCACACAAGCAAACATTGAGAGACTAACGCATCGTACAGACCCTGCGCTACGCCGGATTCCCCGAGTTCATCAAGGTCCTCGTCGACACCGGCTTCCTCAACGACGAGGAGCAGGACTACTTCAAGCAGCCCATCCCCTGGAAGGAGGCCACCCAGAAGCTGCTCAACGCCCCTTCATCCAGCGAGGCCGACCTCGTCGCCGCCGTCTCGTCCAAGGCCACCTTTAAGGATGACGAGGAGAAGGCTCGTCTGATCGATGGTCTCAAGTGGAGTATGTCACACACCCCCCCCCCCCCCCCCCCCCCCCTTTTTTTTTTTTCCTCTCACCCAAACTTGATTGCCTACATCTTCGCTATTCCGTATCGTCCATGTTCATGGAAGCAAGAACGCTAATCTCGTCTGACAGTCGGCATCTTCTCCGACGCCACCATCACCCCCCGCGGCAACCCCCTCGACACCCTCTGCGCCACTCTCGAGGAGAAGATGCAGTACGAGGAGGGCGAGCGCGACTTTGTCGTAAGTGCTCTCTACCTTTCCCCTCCAACGCCCTCCCCAACACTTTCATTCACACAAACACACCCTTTTATACGCACCATAACATACCCACCCCAATGTATCAGAATCAAGCCAGCTGACCTTTATCCCCCCCTCCTGCAGATGCTCCAGCACAAGTTCGAAATCGAAAACAAGGACGGCTCCCGCGAGACGCGCACCTCCACCCTCGCCGAGTACGGCGCCCCCGTCGGCTCGGGCGGATACTCTGCCATGGCCCGCCTGGTCGGTGTGCCCGCCGGTGTTGGTAAGTCCAGCGAATCCCACGAGATCCCCTCTCAAGCCCATCCAACGTGACATGAATGGAGGAGTAAATGTTTGGAAAGAAGGAAGATGTGCTCACTGTTTTGTGTGTTCCGTACAATAGCCACCAAGCAGATCCTCGACGGAACCCTCTCCGAAAAGGGCGTCATCGCCCCAATGAGCCCCAAGATCAACGATCCTCTGATCAAGGAGCTTGCCAAGTACGGGTAAGTCTTACAAAGCCCCCCCCCTTTTCTCCATTCCCCTTTATTCTCTCTACCCTTTCTCCTTTCGTCCTTTGGGAGGTAAAGGAAGACAGAAAGAAAGAAAAAAAAAACTGATAGATATATGTCCGTATAGCATCGCTTTCAAGGAAAAGGTCATCAAGCACAGCGCTTAA

SEQ ID NO：2：

>cDNA sequences

ATGGCTCCCCAACGTGCTCTGATGCTCGGCTCCGGCTTCGTTACCAAGCCCACGCTCGACATTCTCAACGATGCCGGCGTTGAGGTTTCCGTTGCCTGCCGAACCCTCGAAAGCGCAAAGAAGCTCTCTGCCGGCGTCAAGCTGGCCACGCCCATCTCCCTCGACGTCAACGACGACAAGGCCCTCGATGCCGCCGTTGCCCAGCACGACGTCGTCATCAGCCTCATCCCCTACACCTACCACGCCGCCGTCATCAAGTCCGCCATCCGCAACAAGAAGAATGTCGTCACCACCAGCTACGTCTCGCCCGCCATGATGGAGCTCGACCAGCAGGCCAAGGACGCCGGCATTACCGTCATGAACGAGATTGGTCTTGATCCCGGTATCGACCACCTCAGTGCCGTCGAGACCATCTCCAACGTCCACGCTGCCGGAGGCAAGATTCTTTCCTTCAAGTCATTCTGCGGTGGACTGCCGGCGCCCGAGAACTCGGACAACCCTCTTGGCTACAAGTTCTCCTGGTCATCCCGAGGCGTTCTCCTTGCCCTGCGAAACGCTGCCACCTTCTACGAGGACGGAAAGCTGGTCAACGTTGCCGGCCCTGACCTGATGGCCACCGCCAAGCCTTACCACATCTACCCCGGCTACGCCTTTGTCGCATACCCCAACCGCGACTCCAGCGTCTACAAGGAGCGCTACAACATCCCCGAGGCCCATACCATCATCCGCGGAACCCTGCGCTACGCCGGATTCCCCGAGTTCATCAAGGTCCTCGTCGACACCGGCTTCCTCAACGACGAGGAGCAGGACTACTTCAAGCAGCCCATCCCCTGGAAGGAGGCCACCCAGAAGCTGCTCAACGCCCCTTCATCCAGCGAGGCCGACCTCGTCGCCGCCGTCTCGTCCAAGGCCACCTTTAAGGATGACGAGGAGAAGGCTCGTCTGATCGATGGTCTCAAGTGGATCGGCATCTTCTCCGACGCCACCATCACCCCCCGCGGCAACCCCCTCGACACCCTCTGCGCCACTCTCGAGGAGAAGATGCAGTACGAGGAGGGCGAGCGCGACTTTGTCATGCTCCAGCACAAGTTCGAAATCGAAAACAAGGACGGCTCCCGCGAGACGCGCACCTCCACCCTCGCCGAGTACGGCGCCCCCGTCGGCTCGGGCGGATACTCTGCCATGGCCCGCCTGGTCGGTGTGCCCGCCGGTGTTGCCACCAAGCAGATCCTCGACGGAACCCTCTCCGAAAAGGGCGTCATCGCCCCAATGAGCCCCAAGATCAACGATCCTCTGATCAAGGAGCTTGCCAAGTACGGCATCGCTTTCAAGGAAAAGGTCATCAAGCACAGCGCTTAA

SEQ ID NO：3：

>AA sequences

MAPQRALMLGSGFVTKPTLDILNDAGVEVSVACRTLESAKKLSAGVKLATPISLDVNDDKALDAAVAQHDVVISLIPYTYHAAVIKSAIRNKKNVVTTSYVSPAMMELDQQAKDAGITVMNEIGLDPGIDHLSAVETISNVHAAGGKILSFKSFCGGLPAPENSDNPLGYKFSWSSRGVLLALRNAATFYEDGKLVNVAGPDLMATAKPYHIYPGYAFVAYPNRDSSVYKERYNIPEAHTIIRGTLRYAGFPEFIKVLVDTGFLNDEEQDYFKQPIPWKEATQKLLNAPSSSEADLVAAVSSKATFKDDEEKARLIDGLKWIGIFSDATITPRGNPLDTLCATLEEKMQYEEGERDFVMLQHKFEIENKDGSRETRTSTLAEYGAPVGSGGYSAMARLVGVPAGVATKQILDGTLSEKGVIAPMSPKINDPLIKELAKYGIAFKEKVIKHSA*

Cloning of TrLys9 Gene

CM liquid medium: 1L of the culture medium comprises: 10g of glucose, 1g of yeast extract, 2g of peptone, 1g of tyrosine protein, 6g of sodium nitrate, 1.52g of monopotassium phosphate, 0.52g of potassium chloride, 0.52g of magnesium sulfate and pH6.5

OCM liquid medium: adding 1M sucrose into each liter of CM liquid culture medium to obtain OCM liquid culture medium.

Extraction of trichoderma reesei total RNA: 1mL of the solution was added to a concentration of 10⁷The strain/mL of Trichoderma reesei QM9414 spore suspension is inoculated into 100mL of CM liquid culture medium, and cultured for 16-18h at 30 ℃ and 180 rpm. The obtained mycelia were filtered through three layers of paper, blotted dry with absorbent paper, ground into powder with liquid nitrogen, and subjected to total RNA extraction using a plant total RNA extraction kit (TIANGEN, CHINA).

And (3) cDNA synthesis: cDNA synthesis is carried out using a reverse transcription kit, the details of which are described in the kit instructions (Firststrand cDNA synthesis kit, Fermentas).

Amplification of TrLys9 Gene cDNA sequence:

the primer sequences are as follows:

Lys9cDNA1:5’-ATGGCTCCCCAACGTGCTCTGA-3’(SEQ ID NO:4)

Lys9cDNA2:5’-TTAAGCGCTGTGCTTGATGACCTT-3’(SEQ ID NO:5)

and (3) PCR reaction system: mu.L of template cDNA, 0.4. mu.L of each primer (100mM), 25. mu.L of pfu Taq mix, and the balance of water to 50. mu.L.

And (3) PCR reaction conditions: a total of 35 cycles at 98 ℃ for 3 minutes, (94 ℃ for 30 seconds, 55 ℃ for 30 seconds, 68 ℃ for 1 minute), and 72 ℃ for 10 minutes.

The obtained PCR product is directly sent to a sample for sequencing after being recovered by glue, and the sequence is shown as SEQ ID NO:2 is shown in

Example 2 mutant acquisition

1. Construction of knockout vectors and complementary vectors

Extracting a trichoderma reesei genome: the culture conditions are the same as the above-mentioned RNA extraction culture conditions. Methods for extracting genomic DNA are described in (J Sambrook et al 2001).

Construction of the knockout vector: respectively carrying out PCR amplification on an upstream fragment and a downstream fragment of the TrLys9 gene, wherein each fragment is about 1200bp in length, the upstream fragment adopts a Kpn I restriction endonuclease double-restriction site and a Sal I restriction endonuclease double-restriction site, and the downstream fragment adopts a Hind III restriction endonuclease double-restriction endonuclease site and a Xba I restriction endonuclease double-restriction. The obtained PCR fragment is directly cut by enzyme, the upstream fragment is connected to pBS-HPH (Liuet al, 2007) with the same cutting site, then Hind III and Xba I are used for cutting the vector, the downstream fragment of the T vector after cutting by enzyme is connected to the cutting site, and the gene knockout vector is constructed.

Primer sequences are as follows (lower case letters indicate the sequence of the cleavage site):

LYS9Upp1:5’-TTggtaccCTACCCCGAGCCGAAAGATGG-3’(SEQ ID NO:6)

LYS9Upp2:5’-ATgtcgacTGGCGTATGGGAATTGTTGCTGTA-3’(SEQ ID NO:7)

LYS9DNp1:5’-TTaagcttTTACCGCGTGAAATACTGAAACC-3’(SEQ ID NO:8)

LYS9Dnp2:5’-TAtctagaACTAAGGGGCTGACGCTGGAGGAG-3’(SEQ ID NO:9)

construction of complementary vectors: and amplifying by using a high-fidelity long fragment PCR method and taking trichoderma reesei genome DNA as a template to obtain a DNA fragment containing the TrLys9 gene full length, a promoter region and a terminator region, recovering and purifying the fragment gel, and connecting the fragment gel to a pGEM-T vector to complete the construction of the complementary vector pGEM-HB. Plasmids of the vector pBS-SUR (Liu et al, 2007) containing the chlorimuron-ethyl resistance gene and the complementary vector pGEM-HB were co-transformed and screened.

The primer sequences are as follows:

LYS9HB1:5’-TTTCGGCGATAATGATGCTGTCC-3’(SEQ ID NO:10)

LYS9HB2:5’-TGTCCGTGAGGTTTCCGTTAGATT-3’(SEQ ID NO:11)

and (3) PCR reaction system: mu.L of template genomic DNA, 0.4. mu.L of each primer (100mM), 25. mu.L of pfu Taq mix, and the balance of water to 50. mu.L.

And (3) PCR reaction conditions: a total of 35 cycles at 98 ℃ for 3 minutes, (94 ℃ for 30 seconds, 55 ℃ for 30 seconds, 68 ℃ for 1-2 minutes), and 72 ℃ for 10 minutes.

2. Protoplast transformation

1mL of the solution was added to a concentration of 10⁷The strain/mL of Trichoderma reesei QM9414 spore suspension is inoculated into 100mL of CM liquid culture medium, and cultured for 16-18h at 30 ℃ and 180 rpm. The obtained mycelium was filtered through 3 layers of sterile gauze, washed 3 times with sterile water, squeezed to dryness and placed in a 100mL Erlenmeyer flask. The obtained mycelium is subjected to enzymolysis with lyase (i.e. collyases, concentration of 5mg/mL, 5mL enzyme solution per gram of mycelium) at 30 deg.C and 80-90rpm for 3-4 h. Filtering the enzymolysis solution with three-layer sterile filter paper, removing residue, centrifuging the filtrate at 4 deg.C and 3000-4000rpm for 10min, precipitating with STC (1.2M Sorbitol, 10mM Tris-HCl pH7.5, 20mM CaCl₂) Suspension, suspension 4 ℃, 3000-. The resulting protoplast pellet was suspended with STC to a final concentration of 10⁸one/mL.

Taking 150 μ L of 10⁸Adding 2 mu g of plasmid DNA into each/mL of protoplast in a 50mL centrifuge tube, gently mixing uniformly, and standing at 4 ℃ for 25 min; 1mL of PTC (60% PEG4000, 10mM Tris-HCl pH7.5, 20mM CaCl) was added dropwise₂) Mixing, and standing at 4 deg.C for 25 min; most preferablyThen 5mL of OCM liquid medium (Liu et al, 2007) was slowly added thereto, gently mixed, and then placed at 30 ℃ and 100rpm to resume growth for 2 to 3 hours. The protoplasts after growth recovery are spread on plates containing resistance and cultured in the dark for 3-5 days until transformants grow out.

3. Verification of transformants

And (3) verifying the knockout mutant: transformants selected on CM plates containing hygromycin B resistance (100. mu.g/mL hygromycin was added to CM solid medium) were picked, cultured in liquid, mycelium DNA was extracted and verified by PCR.

The primer sequences are as follows:

LYS9check1:5’-ACGTTGCCGGCCCTGGTAAGTAAA-3’(SEQ ID NO:12)

LYS9check2:5’-GGTCGGCCTCGCTGGATGAAG-3’(SEQ ID NO:13)

the PCR reaction conditions were as follows: 2 minutes at 94 ℃ (30 seconds at 94 ℃, 30 seconds at 55 ℃, 30 seconds at 68 ℃) for a total of 25 cycles, 3 minutes at 72 ℃.

And selecting transformants without amplified bands, picking out the transformants for liquid culture, extracting a large number of genomes, and preparing for hybridization verification. The results showed that the TrLys9 gene had been successfully knocked out (FIG. 2).

Verification of complementary mutants: transformants obtained by screening a CM plate containing chlorimuron-ethyl (100. mu.g/mL chlorimuron-ethyl is added into a CM solid culture medium) are picked out, liquid culture is carried out, mycelium RNA is extracted and is reversely transcribed into cDNA, and reverse transcription PCR verification is carried out (figure 3). As can be seen from FIG. 3, both wild-type QM9414 and the complementary mutant HB had the desired band amplified, while the mutant did not; indicating that the gene has been successfully complemented.

Example 3 Effect of TrLys9 Gene on growth and development of Trichoderma reesei

PDA solid medium: 200g/L of potato, 20g/L of glucose and 15-20g/L of agar powder.

MM solid medium: 20g/L glucose, 5g/L ammonium sulfate, 15g/L potassium dihydrogen phosphate, 0.6g/L magnesium sulfate, 0.6g/L calcium chloride, 0.005g/L ferrous sulfate, 0.016g/L manganese sulfate, 0.0014g/L zinc sulfate, 0.002g/L cobalt chloride, 15-20g/L agar powder, pH5.5

1. Observation of colony morphology

The strains were inoculated on CM, PDA and MM plates, respectively, and the colony morphology was observed after 3 days of incubation at 30 ℃. Wherein QM9414 is wild strain, Δ TrLys9-11 is gene deletion mutant strain, and 11HB is gene complementation mutant strain. As shown in FIG. 4, there was no difference in colony morphology between the respective strains on the CM medium, and the mutants grew slower in hyphae than the wild-type strains and thin in aerial hyphae on the PDA medium, whereas the mutants did not grow on the MM medium.

2. Spore yield and growth rate

The colony diameters of different strains were measured every 24h with a vernier caliper and the colony growth rate was calculated. Plates full of spores on different media were washed with 5ml of sterile water and filtered with three layers of lens-wiping paper, and the resulting spore suspensions were counted on a hemocytometer to calculate the spore yields.

As shown in Table 1, there was no difference in growth rate and spore yield between different strains on CM medium; the growth rate of the mutant on the PDA culture medium is reduced compared with that of the wild type, and conidium is not generated; mutants did not grow on MM medium.

TABLE 1 growth Rate and sporulation measurements

*ND＝not determined

EXAMPLE 4 restoration of growth of mutants at different lysine concentrations

Since knockout of the TrLys9 gene blocks the pathway of lysine synthesis in trichoderma reesei, in vitro lysine supplementation is required to satisfy the growth of trichoderma reesei. To determine the recovery of the growth of the mutant at different concentrations, the mutant strains were inoculated on MM plates containing lysine at different concentrations of 0.1mM, 0.5mM, 1mM, respectively, and incubated at 30 ℃ for observation. As can be seen from FIG. 5, the growth of the mutant was restored to various degrees under the conditions of different concentrations of lysine. At a lysine concentration of 1mM, the mutant grew in agreement with the wild type strain.

The results prove that the externally added lysine can restore the lysine synthesis pathway blocked by the TrLys9 gene deletion, so that the mutant can restore growth; on the other hand, the inability of the TrLys9 gene mutant to grow on a common MM medium suggests that the TrLys9 gene can be used as an auxotrophic marker gene for lysine, a marker gene for subsequent transgenic manipulation of Trichoderma reesei (the mutant is grown on a lysine-deficient medium as a screening method), and a homologous gene thereof in other filamentous fungi is also presumed to be used as an auxotrophic marker gene for transgenic manipulation.

The number of modules and the processing scale described herein are intended to simplify the description of the invention. The use, modification and variation of the Trichoderma reesei-derived-N- (L glutaryl 2) -L-lysine reductase of the present invention and its encoding gene and use will be apparent to those skilled in the art.

The invention discloses a Saccharomyces reductase derived from Trichoderma reesei, a coding gene and application thereof. The invention obtains Saccharomyces reductase cDNA separated from Trichoderma reesei (Trichoderma reesei), the nucleotide sequence of the cDNA is shown as SEQ ID No.2, and the amino acid sequence of the coded protein is shown as SEQ ID No. 3. The invention also provides a recombinant expression vector and a host cell containing the Saccharomyces reductase cDNA, and further provides the application of the Saccharomyces reductase cDNA in the growth and development of Trichoderma reesei and the optimization of the practical application of the strain.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> Saccharomyces cerevisiae amino acid reductase from Trichoderma reesei, and coding gene and application thereof

<130>2019

<160>13

<170>PatentIn version 3.5

<210>1

<211>2545

<212>DNA

<213>Trichoderma reesei

<400>1

atggctcccc aacgtgctct gatgctgtaa gtgcgactcc cggaaagaaa tacaatcaaa 60

gaaaagagaa gaaaagaaaa gaggcgaaaa atgccaccac cagctgctgc ccctccccca 120

ctaacatatc cggcttcacg atagcggctc cggcttcgtt accaagccca cgctcgacat 180

tctcaacgat gccggcgttg aggtttccgt tggtgagcac tgaaacaatc cctgaagaag 240

agagccttga gcctcttcga atatcatctc ctcctcctcc tcaacctcga accaccaccc 300

cccaagctaa caaacaaacc ccttccagcc tgccgaaccc tcgaaagcgc aaagaagctc 360

tctgccggcg tcaagctggc cacgcccatc tccctcgacg tcaacgacga caaggccctc 420

gatgccgccg ttgcccagca cgacgtcgtc atcagcctca tcccctacac ctaccacgcc 480

gccgtcatca agtccgccat ccgcaacaag aagaatgtcg tcaccaccag ctacgtctcg 540

cccgccatga tggagctcga ccagcaggcc aaggacgccg gcattaccgt catgaacgag 600

attggtgtaa gtgggaagcc gacgctgctg ctatgcccgt gttctgggga cgataactga 660

gcgcatttgt atagcttgat cccggtatcg accacctcag tgccgtcgag accatctcca 720

acgtccacgc tgccggaggc aagattcttt ccttcaagtc attctgcggt ggactgccgg 780

cgcccgagaa ctcggacaac cctcttggct acaagtaagc gagacaacat cctcaactgc 840

cggcaattgc taacattgtc tgcgttcagg ttctcctggt cgtaagtagc ccgcaagtcc 900

atgttccgat gcaagcgggc ggctcgtcaa gagttcgcat gccgctcact tttcgacatt 960

gagagcgtgg tattgctgga gctccatcta acatctgaaa aacagatccc gaggcgttct 1020

ccttgccctg cgaaacgctg ccaccttcta cgaggacgga aagctggtca acgttgccgg 1080

ccctggtaag taaatcaatg gacatggcat gtcgatatat acaaactaga caccctaaca 1140

catctcatag acctgatggc caccgccaag ccttaccaca tctaccccgg taagcacatc 1200

ccaatatcag gatttctatc aacccctctg accattccca ggctacgcct ttgtcgcata 1260

ccccaaccgc gactccagcg tctacaagga gcgctacaac atccccgagg cccataccat 1320

catccgcgga gtaagcagaa gagttacaat ccacacaagc aaacattgag agactaacgc 1380

atcgtacaga ccctgcgcta cgccggattc cccgagttca tcaaggtcct cgtcgacacc 1440

ggcttcctca acgacgagga gcaggactac ttcaagcagc ccatcccctg gaaggaggcc 1500

acccagaagc tgctcaacgc cccttcatcc agcgaggccg acctcgtcgc cgccgtctcg 1560

tccaaggcca cctttaagga tgacgaggag aaggctcgtc tgatcgatgg tctcaagtgg 1620

agtatgtcac acaccccccc cccccccccc cccccccttt tttttttttc ctctcaccca 1680

aacttgattg cctacatctt cgctattccg tatcgtccat gttcatggaa gcaagaacgc 1740

taatctcgtc tgacagtcgg catcttctcc gacgccacca tcaccccccg cggcaacccc 1800

ctcgacaccc tctgcgccac tctcgaggag aagatgcagt acgaggaggg cgagcgcgac 1860

tttgtcgtaa gtgctctcta cctttcccct ccaacgccct ccccaacact ttcattcaca 1920

caaacacacc cttttatacg caccataaca tacccacccc aatgtatcag aatcaagcca 1980

gctgaccttt atccccccct cctgcagatg ctccagcaca agttcgaaat cgaaaacaag 2040

gacggctccc gcgagacgcg cacctccacc ctcgccgagt acggcgcccc cgtcggctcg 2100

ggcggatact ctgccatggc ccgcctggtc ggtgtgcccg ccggtgttgg taagtccagc 2160

gaatcccacg agatcccctc tcaagcccat ccaacgtgac atgaatggag gagtaaatgt 2220

ttggaaagaa ggaagatgtg ctcactgttt tgtgtgttcc gtacaatagc caccaagcag 2280

atcctcgacg gaaccctctc cgaaaagggc gtcatcgccc caatgagccc caagatcaac 2340

gatcctctga tcaaggagct tgccaagtac gggtaagtct tacaaagccc cccccctttt 2400

ctccattccc ctttattctc tctacccttt ctcctttcgt cctttgggag gtaaaggaag 2460

acagaaagaa agaaaaaaaaaactgataga tatatgtccg tatagcatcg ctttcaagga 2520

aaaggtcatc aagcacagcg cttaa 2545

<210>2

<211>1359

<212>DNA

<213>Trichoderma reesei

<400>2

atggctcccc aacgtgctct gatgctcggc tccggcttcg ttaccaagcc cacgctcgac 60

attctcaacg atgccggcgt tgaggtttcc gttgcctgcc gaaccctcga aagcgcaaag 120

aagctctctg ccggcgtcaa gctggccacg cccatctccc tcgacgtcaa cgacgacaag 180

gccctcgatg ccgccgttgc ccagcacgac gtcgtcatca gcctcatccc ctacacctac 240

cacgccgccg tcatcaagtc cgccatccgc aacaagaaga atgtcgtcac caccagctac 300

gtctcgcccg ccatgatgga gctcgaccag caggccaagg acgccggcat taccgtcatg 360

aacgagattg gtcttgatcc cggtatcgac cacctcagtg ccgtcgagac catctccaac 420

gtccacgctg ccggaggcaa gattctttcc ttcaagtcat tctgcggtgg actgccggcg 480

cccgagaact cggacaaccc tcttggctac aagttctcct ggtcatcccg aggcgttctc 540

cttgccctgc gaaacgctgc caccttctac gaggacggaa agctggtcaa cgttgccggc 600

cctgacctga tggccaccgc caagccttac cacatctacc ccggctacgc ctttgtcgca 660

taccccaacc gcgactccag cgtctacaag gagcgctaca acatccccga ggcccatacc 720

atcatccgcg gaaccctgcg ctacgccgga ttccccgagt tcatcaaggt cctcgtcgac 780

accggcttcc tcaacgacga ggagcaggac tacttcaagc agcccatccc ctggaaggag 840

gccacccaga agctgctcaa cgccccttca tccagcgagg ccgacctcgt cgccgccgtc 900

tcgtccaagg ccacctttaa ggatgacgag gagaaggctc gtctgatcga tggtctcaag 960

tggatcggca tcttctccga cgccaccatc accccccgcg gcaaccccct cgacaccctc 1020

tgcgccactc tcgaggagaa gatgcagtac gaggagggcg agcgcgactt tgtcatgctc 1080

cagcacaagt tcgaaatcga aaacaaggac ggctcccgcg agacgcgcac ctccaccctc 1140

gccgagtacg gcgcccccgt cggctcgggc ggatactctg ccatggcccg cctggtcggt 1200

gtgcccgccg gtgttgccac caagcagatc ctcgacggaa ccctctccga aaagggcgtc 1260

atcgccccaa tgagccccaa gatcaacgat cctctgatca aggagcttgc caagtacggc 1320

atcgctttca aggaaaaggt catcaagcac agcgcttaa 1359

<210>3

<211>452

<212>PRT

<213>Trichoderma reesei

<400>3

Met Ala Pro Gln Arg Ala Leu Met Leu Gly Ser Gly Phe Val Thr Lys

1 5 10 15

Pro Thr Leu Asp Ile Leu Asn Asp Ala Gly Val Glu Val Ser Val Ala

20 25 30

Cys Arg Thr Leu Glu Ser Ala Lys Lys Leu Ser Ala Gly Val Lys Leu

35 40 45

Ala Thr Pro Ile Ser Leu Asp Val Asn Asp Asp Lys Ala Leu Asp Ala

50 55 60

Ala Val Ala Gln His Asp Val Val Ile Ser Leu Ile Pro Tyr Thr Tyr

65 70 75 80

His Ala Ala Val Ile Lys Ser Ala Ile Arg Asn Lys Lys Asn Val Val

85 90 95

Thr Thr Ser Tyr Val Ser Pro Ala Met Met Glu Leu Asp Gln Gln Ala

100 105 110

Lys Asp Ala Gly Ile Thr Val Met Asn Glu Ile Gly Leu Asp Pro Gly

115 120 125

Ile Asp His Leu Ser Ala Val Glu Thr Ile Ser Asn Val His Ala Ala

130 135 140

Gly Gly Lys Ile Leu Ser Phe Lys Ser Phe Cys Gly Gly Leu Pro Ala

145 150 155 160

Pro Glu Asn Ser Asp Asn Pro Leu Gly Tyr Lys Phe Ser Trp Ser Ser

165 170 175

Arg Gly Val Leu Leu Ala Leu Arg Asn Ala Ala Thr Phe Tyr Glu Asp

180 185 190

Gly Lys Leu Val Asn Val Ala Gly Pro Asp Leu Met Ala Thr Ala Lys

195 200 205

Pro Tyr His Ile Tyr Pro Gly Tyr Ala Phe Val Ala Tyr Pro Asn Arg

210 215 220

Asp Ser Ser Val Tyr Lys Glu Arg Tyr Asn Ile Pro Glu Ala His Thr

225 230 235 240

Ile Ile Arg Gly Thr Leu Arg Tyr Ala Gly Phe Pro Glu Phe Ile Lys

245 250 255

Val Leu Val Asp Thr Gly Phe Leu Asn Asp Glu Glu Gln Asp Tyr Phe

260 265 270

Lys Gln Pro Ile Pro Trp Lys Glu Ala Thr Gln Lys Leu Leu Asn Ala

275 280 285

Pro Ser Ser Ser Glu Ala Asp Leu Val Ala Ala Val Ser Ser Lys Ala

290 295 300

Thr Phe Lys Asp Asp Glu Glu Lys Ala Arg Leu Ile Asp Gly Leu Lys

305 310 315 320

Trp Ile Gly Ile Phe Ser Asp Ala Thr Ile Thr Pro Arg Gly Asn Pro

325 330 335

Leu Asp Thr Leu Cys Ala Thr Leu Glu Glu Lys Met Gln Tyr Glu Glu

340 345 350

Gly Glu Arg Asp Phe Val Met Leu Gln His Lys Phe Glu Ile Glu Asn

355 360 365

Lys Asp Gly Ser Arg Glu Thr Arg Thr Ser Thr Leu Ala Glu Tyr Gly

370 375 380

Ala Pro Val Gly Ser Gly Gly Tyr Ser Ala Met Ala Arg Leu Val Gly

385 390 395 400

Val Pro Ala Gly Val Ala Thr Lys Gln Ile Leu Asp Gly Thr Leu Ser

405 410 415

Glu Lys Gly Val Ile Ala Pro Met Ser Pro Lys Ile Asn Asp Pro Leu

420 425 430

Ile Lys Glu Leu Ala Lys Tyr Gly Ile Ala Phe Lys Glu Lys Val Ile

435 440 445

Lys His Ser Ala

450

<210>4

<211>22

<212>DNA

<213> Artificial sequence

<400>4

atggctcccc aacgtgctct ga 22

<210>5

<211>22

<212>DNA

<213> Artificial sequence

<400>5

atggctcccc aacgtgctct ga 22

<210>6

<211>29

<212>DNA

<213> Artificial sequence

<400>6

ttggtaccct accccgagcc gaaagatgg 29

<210>7

<211>32

<212>DNA

<213> Artificial sequence

<400>7

atgtcgactg gcgtatggga attgttgctg ta 32

<210>8

<211>31

<212>DNA

<213> Artificial sequence

<400>8

ttaagctttt accgcgtgaa atactgaaac c 31

<210>9

<211>32

<212>DNA

<213> Artificial sequence

<400>9

tatctagaac taaggggctg acgctggagg ag 32

<210>10

<211>23

<212>DNA

<213> Artificial sequence

<400>10

tttcggcgat aatgatgctg tcc 23

<210>11

<211>24

<212>DNA

<213> Artificial sequence

<400>11

tgtccgtgag gtttccgtta gatt 24

<210>12

<211>24

<212>DNA

<213> Artificial sequence

<400>12

tgtccgtgag gtttccgtta gatt 24

<210>13

<211>21

<212>DNA

<213> Artificial sequence

<400>13

ggtcggcctc gctggatgaa g 21

Claims

1. An enzyme having an activity of-N- (L glutaryl 2) -L-lysine reductase which provides a function of catalyzing 6-oxo-DL-alanine formation into-N- (L glutaryl 2) -L-lysine in the diaminopimelate pathway, said enzyme being a protein of the following (a) or (b):

(a) the amino acid sequence is shown as SEQ ID NO. 3;

2. A DNA molecule encoding the enzyme of claim 1.

3. The DNA molecule according to claim 2, wherein the DNA molecule has a base sequence of (c), (d), (e) or (f) below:

(c) as shown in SEQ ID NO. 1;

(d) as shown in SEQ ID NO. 2;

4. A recombinant vector comprising the DNA molecule of claim 2 and regulatory sequences for expression operably linked to said DNA molecule.

5. A host cell comprising the DNA molecule of claim 2 or the recombinant vector of claim 4.

6. A method for constructing a lysine auxotrophic mutant is characterized by comprising the following steps:

7. The method for constructing a lysine auxotrophic mutant according to claim 6, wherein in the first step, the upstream fragment obtained by amplification is subjected to double restriction sites with Kpn I and Sal I restriction enzymes, and the double-cleaved upstream fragment is ligated to pBS-HPH using the same restriction sites;

8. The method for constructing a lysine auxotrophic mutant according to claim 6, wherein the screening in the second step comprises the steps of: and (3) coating the strain on a CM plate containing hygromycin B resistance, culturing to obtain a transformant, selecting the transformant, culturing, and performing PCR amplification by using primer pairs such as SEQ ID NO. 10 and SEQ ID NO. 11, wherein the transformant without an amplified band is the lysine auxotrophic mutant.

9. Use of the DNA molecule of claim 2 as a marker gene for lysine auxotrophy.

10. Use of the protein of claim 1 or the DNA molecule of claim 2 in trichoderma reesei growth and development and in actual production optimization.