CN113621531A - Yeast engineering bacterium and construction method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biotechnology and biological energy, in particular to a yeast engineering bacterium and a construction method and application thereof. The invention provides a yeast engineering bacterium, which expresses lignin multifunctional peroxidase and/or manganese peroxidase. The strain can grow, synthesize and accumulate microbial oil by using lignin degradation products as carbon sources, can realize the efficient conversion of lignin biomass raw materials into oil, provides a core technology for the full resource utilization and efficient conversion of lignocellulose biomass raw materials, and has wide industrial application prospects.

Description

Yeast engineering bacterium and construction method and application thereof

Technical Field

The invention relates to the technical field of biotechnology and biological energy, in particular to a yeast engineering bacterium and a construction method and application thereof.

Background

The oil is an important bulk product indispensable to human life, and can replace fossil resources to be used as a basic processing raw material of chemical industry and renewable energy industry. In recent years, the global demand for fats and oils has been increasing, resulting in a shortage of fat and oil resources and a large fluctuation in price. Microbial oils are intracellular products synthesized by oleaginous microorganisms under specific conditions and are of great interest because their production is not limited by climate, season and site. Research on the biorefinery of lipids using inexpensive and abundant lignocellulosic biomass resources as raw materials has received a great deal of attention in recent years and is considered to be a new approach for producing fats and oils with potential.

Lignocellulose is the main component of plant cell walls, mainly comprises cellulose (40-50%), hemicellulose (25-35%) and lignin (15-20%), and lignocellulose biomass such as straw, wheat straw, corn straw, bagasse and the like has high annual output, so that huge development and utilization space exists, and biotransformation is one of important transformation modes. Wherein, the cellulose and the hemicellulose can obtain the mixed sugar solution which can be directly utilized by microorganisms under the action of the compound glycosidase. Because three components of lignocellulose are interwoven to form a compact reticular structure, the effective utilization of the lignocellulose generally needs three steps of raw material pretreatment (structure depolymerization, exposure of cellulose and hemicellulose), enzyme hydrolysis (hydrolysis of the cellulose and the hemicellulose into monosaccharides or oligosaccharides) and product fermentation (product synthesis by microbial conversion of sugars), the lignocellulose is converted into grease or other high-value chemicals, and the conversion and the utilization of the cellulose and the hemicellulose are mainly focused.

The lignin is a renewable aromatic compound, can not be converted and utilized by oil-producing microorganisms, and can release more lignin degradation products with inhibiting effect on subsequent enzymolysis and microbial fermentation, such as micromolecular lignin-based phenolic compounds, organic acids and the like, in an extreme raw material pretreatment process (high temperature and high pressure, strong acid and strong alkali). Especially, in order to improve the product yield, the lignocellulose hydrolysate is usually concentrated to increase the sugar concentration in the fermentation broth, so that the concentration of lignin degradation products in the lignocellulose hydrolysate is correspondingly increased, and the high concentration of the lignin degradation products seriously inhibits the growth metabolism and the oil synthesis of the oil-producing microorganisms. Therefore, how to remove the inhibition of the lignin degradation products generated in the lignocellulose hydrolysis process on the growth of oil-producing microorganisms and the oil synthesis and further realize the biotransformation of the lignin degradation products is a bottleneck problem to be solved urgently for the efficient transformation of lignocellulose biomass into oil.

The oil-producing yeast can convert biomass resources to synthesize oil and lipid metabolic products, has important significance for biomass energy and advanced biological manufacturing, and is widely concerned. Natural oleaginous yeast cannot biologically convert lignin degradation products and has low tolerance to lignin degradation products. At present, no report is available for improving the tolerance of the oleaginous yeast to the lignin degradation products by using a metabolic engineering method to realize the bioconversion of the oleaginous yeast to the lignin degradation products.

Disclosure of Invention

The first purpose of the invention is to provide a yeast engineering bacterium capable of converting lignin degradation products.

The second purpose of the invention is to provide a construction method of the yeast engineering bacteria.

The third purpose of the invention is to provide the application of the yeast engineering bacteria.

The fourth purpose of the invention is to provide a method for producing microbial oil by using the yeast engineering bacteria.

According to the invention, lignin multifunctional peroxidase and manganese peroxidase are introduced into yeast, so that the tolerance and degradation capability of the yeast to lignin-reduced products are improved, and the yeast is endowed with the properties of growth and grease accumulation by using lignin-degraded products.

Specifically, the invention provides the following technical scheme:

in a first aspect, the invention provides an engineered yeast strain expressing lignin multifunctional peroxidase and/or manganese peroxidase; the yeast engineering bacteria belong to the genera Lipomyces (Lipomyces), Trichosporon (Trichosporon), Cryptococcus (Cryptococcus), Saccharomyces (Rhodosporidium) or Rhodotorula (Rhodotorula).

The yeast engineering bacteria provided by the invention can respectively express peroxidase and manganese peroxidase, and can also simultaneously express peroxidase and manganese peroxidase.

The invention screens the sources of lignin multifunctional peroxidase and manganese peroxidase, and finds that the lignin multifunctional peroxidase from pleurotus eryngii and the manganese peroxidase from phanerochaete chrysosporium can better exert the lignin degradation function in yeast compared with enzymes from other sources, so that the lignin reduction performance of the yeast is obviously improved, and the growth and oil accumulation performance of the yeast in lignin degradation products are improved.

Preferably, the lignin multifunctional peroxidase is derived from Pleurotus eryngii (Pleurotus eryngii). The manganese peroxidase is derived from Phanerochaete chrysosporium (Phanerochaete chrysosporium).

Among the enzymes of the above origin, those having specifically the following amino acid sequences are preferably used: the amino acid sequence of the lignin multifunctional peroxidase is shown as SEQ ID NO. 1. The amino acid sequence of the manganese peroxidase is shown as SEQ ID NO.2.

Compared with manganese peroxidase, the lignin multifunctional peroxidase has an obviously better effect on the growth of yeast in lignin degradation products and the improvement of oil production performance.

In order to ensure that the two enzymes are efficiently expressed in the yeast, the coding genes of the two enzymes are optimized according to the codon preference of the yeast.

After codon optimization, it is preferred to use a coding gene having the following nucleotide sequence: the sequence of the coding gene of the lignin multifunctional peroxidase is shown as SEQ ID NO. 3. The sequence of the coding gene of the manganese peroxidase is shown as SEQ ID NO. 4.

The expression mode of lignin multifunctional peroxidase and manganese peroxidase can adopt a mode of connecting the coding genes thereof to episomal plasmids for expression, and can also adopt a mode of integrating the coding genes thereof to the chromosome of yeast for expression.

In a preferred embodiment of the present invention, the genes encoding the two enzymes are integrated into the yeast chromosome for expression.

Based on the above, the yeast engineering bacteria of the invention contain one or two of lignin multifunctional peroxidase expression cassettes and manganese peroxidase expression cassettes on chromosomes.

Wherein the lignin multifunctional peroxidase expression cassette contains P_pgkA promoter (a promoter of the Rhodotorula glutinis phosphoglycerate kinase gene PGK), a lignin-multifunctional peroxidase-encoding gene VP, and a transcription terminator. Manganese peroxidase expression cassette containing P_pgkA promoter, a manganese peroxidase-encoding gene MNP and a transcription terminator.

In the above-mentioned expression cassette, the transcription terminator preferably used is T_HSP(terminator of the Rhodotorula yeast Heat shock protein Gene HSP).

Optionally, the expression cassette further comprises a resistance selection marker.

As a preferable scheme of the invention, the sequence of the lignin multifunctional peroxidase expression cassette is shown as SEQ ID NO.5, and the sequence of the manganese peroxidase expression cassette is shown as SEQ ID NO. 6. Wherein the sequence of the Ppgk promoter is 6068-th-7569 th site of the sequence shown in SEQ ID NO.5, and T_HSPThe terminator has the sequence of the 9783-10222 of the sequence shown in SEQ ID NO. 5.

The promoter and terminator can be obtained by PCR amplification using a yeast genome as a template or by artificial gene synthesis.

The expression cassette can enable the lignin multifunctional peroxidase and the manganese peroxidase to be efficiently and adaptively expressed in the yeast, and is beneficial to improving the capability of the yeast for growing and synthesizing oil by using lignin degradation products.

For the method of integrating the above two enzymes into the yeast chromosome, a yeast genetic engineering method which is conventional in the art can be employed, for example: the target gene is integrated on the chromosome by using a yeast integration type vector by using a non-homologous end joining or homologous recombination method.

In the invention, the starting strain of the yeast engineering bacteria is a yeast strain which does not express lignin multifunctional peroxidase and manganese peroxidase per se and is used as a receptor thallus to introduce coding genes of the lignin multifunctional peroxidase and the manganese peroxidase. Preferably, the oleaginous yeast capable of synthesizing and accumulating lipid and lipid metabolites using biomass resources may be selected from any one of strains belonging to the genera Lipomyces (Lipomyces), Trichosporon (trichosporin), Cryptococcus (Cryptococcus), saccharomyces (Rhodosporidium) or Rhodotorula (Rhodotorula).

In one embodiment of the present invention, the starting strain of the engineered yeast is any one selected from Rhodotorula toruloides (Rhodosporidium toruloides) with the collection number of CGMCC No.2.1389, Rhodotorula toruloides (Rhodosporidium toruloides) haploid NP11 with the collection number of GDMCC No.2.224, Trichosporon cutaneum (trichosporium toruloides) with the collection number of CGMCC No.2.571, Lipomyces stakkeri (lipomyceliophthora stakeyi) with the collection number of NRRL Y-11557, Rhodotorula glutinis (Rhodotorula glutinis) with the collection number of ATCC 204091, and Cryptococcus curvatus (Cryptococcus curvatus) with the collection number of ATCC 20509.

After the starting strain is integrated with the expression cassettes of lignin multifunctional peroxidase and manganese peroxidase, the lignin utilization capacity of the starting strain is remarkably improved, and lignocellulose biomass can be used for efficiently growing and accumulating grease.

Preferably, the yeast engineering bacteria contains 3 copies of the lignin multifunctional peroxidase expression cassette or 2 copies of the manganese peroxidase expression cassette on the chromosome.

As a preferred embodiment of the present invention, the chromosomal insertion sites of the 3 copies of the lignin multifunctional peroxidase expression cassette are: the non-coding region at 366954bp of the Scaffold 14 of the rhodosporidium toruloides is inserted into 2 expression cassettes, the 2 expression cassettes are sequentially connected, and the non-coding region at 317063bp of the Scaffold 22 is inserted into 1 expression cassette. The chromosome insertion sites of the 2 copies of the manganese peroxidase expression cassette are respectively: 1 expression cassette is inserted into 40034bp of Scaffold 46 and the intron region of autophagy protein gene (ATG4), and 1 expression cassette is inserted into 58108bp of Scaffold1 and the exon region of hypothetical protein RHTO _ 00026.

The engineering yeast strain with the chromosome integration copy number and the integration position has better effect of improving the capability of utilizing lignin, can utilize lignocellulose biomass to efficiently grow and synthesize oil, and particularly has better effect on rhodosporidium toruloides haploid NP11 with the preservation number of GDMCC No. 2.224.

In a second aspect, the invention provides a method for constructing the engineered yeast strain, which comprises the following steps: and (3) introducing the lignin multifunctional peroxidase expression cassette and/or the manganese peroxidase expression cassette into an original strain, and screening to obtain the strain expressing the lignin multifunctional peroxidase and/or the manganese peroxidase.

In the above-described method, a genetic engineering means that is conventional in the art may be employed to introduce the lignin multifunctional peroxidase expression cassette and/or the manganese peroxidase expression cassette into the starting strain.

Specifically, the expression cassette is connected to a yeast episomal vector or an integrative vector, and the episomal vector or the integrative vector is introduced into the starting strain.

As an embodiment of the present invention, the lignin multifunctional peroxidase VP gene and the manganese peroxidase MNP gene are ligated to the vector pZPK-P, respectively_PGK-HYG-P2A-HYG-T_HSPP of_PGKAnd introducing the plasmid into a yeast starting strain through an agrobacterium-mediated transformation method at the downstream of the promoter, and screening to obtain a strain with an expression cassette integrated into a yeast chromosome.

Wherein the vector pZPK-P_PGK-HYG-P2A-HYG-T_HSPIs prepared from plasmid pZPK-P_PGK-HYG-P2A-BLE-T_HSPAmplifying by primer P2A-HYG-F (5'-CTGGCGACGTCGAAGAGAACCCTGGCCCTATGCCGGAGCTCACGGCGAC-3') and HYG-THSP-R (5'-TGCATGCTGCAGGTCGA CTCTAGAGGATCCCGCGCACTTCTCTGCACTGC-3') to obtain P2A-HYG-THSP fragment as template, and integrating the fragment into the template again by RF cloning method to obtain vector pZPK-P_PGK-HYG-P2A-HYG-T_HSP。

In a third aspect, the invention provides any one of the following applications of the yeast engineering bacteria:

(1) the application in the production of microbial oil;

(2) use in increasing the tolerance of yeast to lignin degradation products;

(3) the application of the yeast in improving the growth performance of the yeast in raw materials containing lignin degradation products and the yield of microbial oil;

(4) application in breeding of yeast production strains;

(5) the application in the degradation of lignin biomass.

In a fourth aspect, the present invention provides a method for producing microbial oils, the method comprising: culturing the yeast engineering bacteria, and separating to obtain the microbial oil.

Preferably, the culture medium contains lignin degradation products.

The production method of the microbial oil specifically comprises the following steps: culturing the yeast engineering bacteria in a seed culture medium to obtain a seed solution, inoculating the seed solution into a fermentation culture medium containing lignin degradation products for fermentation, and separating and extracting after the fermentation is finished to obtain the microbial oil.

The lignin degradation product can be a hydrolysate (including concentrated hydrolysate) prepared by hydrolyzing lignin-containing biomass (such as straw, wheat straw, corn straw, bagasse and the like), or a mixture of one or more compounds (such as furfural, hydroxymethyl furfural, vanillin, p-hydroxybenzaldehyde, syringaldehyde, vanillic acid, p-hydroxybenzoic acid, vanillyl alcohol, syringic acid, guaiacol, eugenol and the like) generated after lignin degradation.

The invention has the beneficial effects that: according to the invention, the key enzymes of the bioconversion lignin, namely lignin multifunctional peroxidase VP and manganese peroxidase MNP, are expressed in the yeast to construct a bioconversion way of lignin degradation products, so that the yeast is endowed with the properties of growth and oil accumulation by using the lignin degradation products, the inhibition of the lignin degradation products (lignin-based phenolic compounds, furfural, hydroxymethyl furfural and the like) on the growth of the yeast and the oil synthesis is removed, and the yield of the yeast full-resource conversion lignocellulose synthetic oil is obviously improved.

The oil-producing yeast engineering bacteria provided by the invention can utilize lignin degradation products as a carbon source to grow and produce oil, has higher performance of growing and producing oil by utilizing the lignin degradation products, can realize high-efficiency conversion of lignin to oil, provides a core technology for full resource utilization and high-efficiency conversion of lignocellulose biomass raw materials, has wide industrial application prospect, and can be used as basic raw materials of biological energy and oil chemical industry, such as production of biodiesel, aviation fuel, surfactants, paint, lubricants, medicinal cosmetics and the like.

Drawings

FIG. 1 is a schematic structural diagram of a VP-HYG expression cassette in example 1 of the present invention.

FIG. 2 is a schematic structural diagram of the MNP-HYG expression cassette in example 1 of the present invention.

FIG. 3 shows the growth and sugar consumption of the strains VP9 and VP28 during fermentation in example 1 of the present invention.

FIG. 4 shows the growth and sugar consumption of the strains MNP28, MNP47 during fermentation in example 1 of the present invention.

FIG. 5 shows the biomass and oil content of the strains VP9, VP28, MNP28 and MNP47 at the end of fermentation in example 1, wherein the black packed column is biomass and the white packed column is oil content.

FIG. 6 shows TLC monitoring of the degradation of lignin degradation products by the strain during fermentation in example 1 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

In the following examples, Rhodosporidium toruloides (Rhodosporidium toruloides) CGMCC No.2.1389 and Trichosporon cutaneum (Trichosporon cutaneum) CGMCC No.2.571 were purchased from China general microbiological culture Collection center; lipomyces starkeyi NRRL Y-11557 was purchased from NITE Bioresource center, Rhodotorula glutinis (Rhodotorula glutinis) ATCC 204091 and Cryptococcus curvatus ATCC 20509 was purchased from American Standard Biolabs Collection (ATCC).

Rhodosporidium toruloides haploid NP11 strain was deposited at Guangdong province center for microbial cultures at 9/16 of 2020, deposit number GDMCC No.2.224, and classified under the name Rhodosporidium toruloides, which is disclosed in the documents Jiano Xiao, Zhang Qi, Zhang dough, Yang Xiao gang, Wang Qian, Zha Zongbao, instant co-expression of multiple enzymes, front simple promoter treated by viruses 2A sequence in the ocean Yeast Rhodosporidium toruloides, FEMS Yeast 2018,18(8), foy 086.

Agrobacterium tumefaciens AGL1 was purchased from American Standard Biolabs Collection (ATCC) under the accession number: ATCC BAA-100.

Example 1 construction, screening and application of Rhodosporidium toruloides haploid engineering strains expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP

1. Construction of Rhodosporidium toruloides haploid engineering strain for expressing lignin multifunctional peroxidase VP

(1) Synthesis of lignin multifunctional peroxidase gene VP

The VP gene is derived from Pleurotus eryngii (GenBank ID: AF175710, the amino acid sequence of the coded protein is shown in SEQ ID NO. 1), optimized according to the codon preference of rhodosporidium toruloides haploid (the optimized VP gene sequence is shown in SEQ ID NO. 3), and subjected to complete sequence synthesis, and the synthesized gene is connected to a pUC57 vector to obtain a plasmid pUC 57-VP.

(2) Construction of pZPK-VP vector

The target vector is constructed by using an RF cloning method. With plasmid pZPK-P_PGK-HYG-P2A-BLE-T_HSP(Jiano Xiao, Zhuang Qi, Zhuang Sufang, Yang Xiaobing, Wang Qian, Zhuao Zongbao. efficient co-expression of multiple enzymes from a single promoter mediated by viruses 2A sequence in the enzymology of Rhodosporidium toruloides, FEMS Yeast Research 2018,18(8), foy086) as templates, using primers P2A-HYG-F (5'-CTGGCGACGTCGAAGAGAACCCTGGCCCTATGCCGGAGCTCACGGCGAC-3') and HYG-T_HSP-R(5’-TGCATGCTGCAGGTCGA CTCTAGAGGATCCCGCGCACTTCTCTGCACTGC-3') to obtain a P2A-HYG-THSP fragment, and then integrating the fragment into the template again by a RF cloning method to obtain a vector pZPK-P_PGK-HYG-P2A-HYG-T_HSP. Plasmid pUC57-VP was used as a template, and primer P was used_PGKAmplifying VP-F (5'-CAACTCCCACCCTCCCCCGTGCA GCCCACCATGGCCTTCGCCAAGCTCTC-3') and VP-P2A-R (5'-GA GGAGCGAGAAGTTGGTAGCGCCCGAGCCCGAAGGCGGGACCGGCG-3') to obtain PPGK-VP-P2A fragment, and integrating the fragment into the vector pZPK-P by using an RF cloning method_PGK-HYG-P2A-HYG-T_HSPObtaining the target vector pZPK-P_PGK-VP-P2A-HYG-T_HSP。

The method for RF cloning was as follows: 10.0. mu.L of 5 XPCR buffer, 1.0. mu.L of dNTPs (10mM), 300ng of the above-mentioned amplified fragment as a large primer (mega-primer), 20ng of plasmid (pZPK-P)_PGK-HYG-P2A-HYG-T_HSP) As a template; PrimeSTAR DNA polymerase 0.5. mu.L, ddH₂Adding O to 50 mu L, keeping the temperature at 94 ℃ for 3min, then keeping the temperature at 98 ℃ for 10s, at 62 ℃ for 10s, at 72 ℃ for 10min, performing 15 cycles, at 72 ℃ for 15min, and finishing the reaction at 4 ℃.

DpnI digestion and shock transformation: adding 1 μ L of DpnI (purchased from TaKaRa) and 1 μ L of DpnI buffer solution into 8 μ L of RF reaction product, uniformly mixing, acting at 37 ℃ for 120min to remove the original template, taking 2 μ L of DH10B competent cells transformed by electric shock, preparing the competent cells according to a standard method (third edition of molecular cloning instruction, translation of SammBruker, Huangpetang, etc., published by scientific publishing Co., Ltd.), and transforming parameters by electric shock: 2200 ℃ and 2500V, 400 omega, 25 muF, 0 ℃ and 4-8 ms. And (3) selecting Kan resistant transformants for enrichment culture and plasmid extraction, and sending the recombinant vector to Shanghai bio-chemical company for sequencing to obtain a vector with a correct sequence. The recombinant vector is named as pZPK-VP and carries a VP gene expression cassette with a sequence shown in SEQ ID NO. 5. The composition of pZPK-VP vector is shown in FIG. 1.

(3) Construction of Rhodosporidium toruloides haploid engineering strain

Agrobacterium tumefaciens was transformed by electric shock transformation of the correctly sequenced pZPK-VP plasmid using Rhodosporidium toruloides haploid NP11(GDMCC No.2.224) as the starting strain, the resulting transformed strain was named AGL-PGK-VP, which was then transformed by Agrobacterium tumefaciens-mediated transformation (Lin Xinping, Wang Yanan, Zhang Sufang, Zhu Zhiwei, Zhou Yongjin, Yang Fan, Sun Wenyi, Wang Xueying, Zha Zongbao. functional integration of multiple genes inter the genome of the ocean engineering, Rhodosporidium japonica, Wems Yeast Research 2014,14(6), 547-42) or by electrotransformation (tissue culture, Japan Yeast strain, Yang, Wang Yeast strain, Zong Xueying, Zhang discovery, Zong Xueing, Zhang discovery, Zong discovery, Zhang discovery, strain, Zhang discovery, strain, Zhang discovery, III, Zhang discovery, strain, III, Zhang discovery, strain, III, Zhang discovery, strain, III, Zhang discovery, III, Zhang discovery, III, Zhang discovery, III, Zhang discovery, III, Zhang discovery, III, Zhang discovery, III, screening was performed on YEPD plates containing hygromycin. The resistance screening plate culture medium comprises the following components: 1% yeast extract powder, 2% glucose, 2% peptone, 1.5% agar, 50. mu.g/ml hygromycin.

2. Construction of Rhodosporidium toruloides haploid engineering strain for expressing manganese peroxidase MNP

(1) Synthesis of manganese peroxidase MNP Gene

The MNP gene is derived from Phanerochaete chrysosporium (GenBank ID: M60672.1, the amino acid sequence of the coded protein is shown as SEQ ID NO. 2), optimized according to the codon preference of rhodosporidium toruloides haploid (the sequence of the optimized MNP gene is shown as SEQ ID NO. 4), and subjected to complete sequence synthesis, and the synthesized gene is connected to a pUC57 vector, pUC 57-MNP.

(2) pZPK-MNP vector construction

The target vector is constructed by using an RF cloning method. With plasmid pZPK-P_PGK-HYG-P2A-BLE-T_HSPUsing primer P2A-HYG-F (5'-CTGGCGACGTCGAAGAGAACCCTGGCCCTATGCCGGAGCTCACGGCGAC-3') and HYG-T as template_HSPAmplification of-R (5'-TGCATGCTGCAGGTC GACTCTAGAGGATCCCGCGCACTTCTCTGCACTGC-3') to give P2A-HYG-T_HSPThen the fragment is integrated into the template again by the method of RF cloning to obtain the vector pZPK-P_PGK-HYG-P2A-HYG-T_HSP. Plasmid pUC57-MNP was used as a template, and primer P was used_PGKMNP-F (5'-CCTCCCCCGTGCAGCCCACCATGCACCACCATCACCATCACGCCTTCAAGTCGCTCAT-3') and MNP-P2A-R (5 ' -GAGGAGCGAGAAGTTGGTAGCGCCCGAGC CGG)CTGGGCCGTTGAACT-3') to obtain P_PGK-MNP-P2A fragment, which is integrated into the vector pZPK-P by means of RF cloning_PGK-HYG-P2A-HYG-T_HSPObtaining the target vector pZPK-P_PGK-MNP-P2A-HYG-T_HSP。

RF cloning: 10.0. mu.L of 5 XPCR buffer, 1.0. mu.L of dNTPs (10mM), 300ng of the above-mentioned amplified fragment as a large primer (mega-primer), 20ng of plasmid (pZPK-P)_PGK-HYG-P2A-HYG-T_HSP) As a template; PrimeSTAR DNA polymerase 0.5. mu.L, ddH₂Adding O to 50 mu L, keeping the temperature at 94 ℃ for 3min, then keeping the temperature at 98 ℃ for 10s, at 62 ℃ for 10s, at 72 ℃ for 10min, performing 15 cycles, at 72 ℃ for 15min, and finishing the reaction at 4 ℃.

DpnI digestion and shock transformation: mu.L of the RF reaction product was added to 1. mu.L of DpnI (from TaKaRa) and 1. mu.L of DpnI buffer, mixed and reacted at 37 ℃ for 120min to remove the original template, 2. mu.L of electric shock transformed DH10B competent cells were obtained, prepared according to standard methods (third edition of molecular cloning instructions, translation of Sammu Brooko, Huang Peigang, published by scientific Press), electric shock transformation parameters: 2200 ℃ and 2500V, 400 omega, 25 muF, 0 ℃ and 4-8 ms. And (3) selecting Kan resistant transformants for enrichment culture and plasmid extraction, and sending the recombinant vector to Shanghai bio-chemical company for sequencing to obtain a vector with a correct sequence. The recombinant vector is named as pZPK-MNP and carries an MNP gene expression cassette with the sequence shown in SEQ ID NO. 6. The composition of pZPK-MNP vector is shown in figure 2.

(3) Construction of Rhodosporidium toruloides haploid engineering strain for expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP

The method comprises the steps of taking rhodosporidium toruloides haploid NP11 as an original strain, transforming agrobacterium tumefaciens by adopting an electric shock transformation mode on a plasmid with correct sequencing, obtaining a transformed strain named AGL-pZPK-MNP (H), transforming a manganese peroxidase gene into the original strain by utilizing an agrobacterium tumefaciens mediated transformation or electric transformation mode, and screening on a YEPD (YEPD) plate containing hygromycin.

The method for preparing the soil agrobacterium tumefaciens competence and the method for mediating and transforming and electrically transforming are described in the specification 1. The primer used for colony PCR verification is P in (2)_PGK-MNP-F withP2A-R(5’-AAGTTGGTAGCGCCCGAGCC-3’)。

3. Screening of rhodosporidium toruloides haploid engineering strain expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP

And (3) carrying out high-throughput primary screening and secondary screening on transformants with correct genotypes obtained in the steps 1 and 2. The process of primary screening of transformants comprises the following steps: selecting a single colony of a yeast strain, inoculating the single colony of the yeast strain into 5mL of YEPD culture medium, and performing shaking culture at 30 ℃ and 200rpm for 24 h; transferring 40 mul of seed liquid to a 96-deep-hole plate, adding 400 mul of screening culture medium, and arranging three parallel plates; carrying out vibration culture at 30 ℃ and 800rpm for 48 h; detection of initial and terminal OD₆₀₀The value is obtained.

The YEPD culture medium consists of 1% of yeast extract powder, 2% of glucose and 2% of peptone; the screening culture medium is as follows: 1 XYNB medium (commercial mixed nitrogen source medium); respectively adding 2.5g/L of one of p-hydroxybenzaldehyde, alcohol, acid, coumaric aldehyde, alcohol, acid, coniferyl aldehyde, alcohol, ferulic acid, vanillin, alcohol, acid, syringaldehyde, alcohol, acid, sinapine aldehyde, alcohol, acid, hydroxymethyl furfural and furfural as a unique carbon source; the buffer system is K₂HPO₄With KH₂PO₄(ii) a The initial pH was adjusted to 5.6 by NaOH.

Starting strain NP11 as control, OD comparison under different inhibitor conditions₆₀₀And (4) selecting and storing the obviously increased transformant as a dominant strain, and re-screening. The inoculation mode is that dominant strains are selected to be monoclonal, liquid culture is carried out on YEPD culture medium for 24-48h, and seed liquid is prepared; then the seed liquid is mixed according to the initial OD6₀₀Transferring to gradient screening medium at 0.2, shaking culturing at 30 deg.C for 24-96 hr, and determining the concentration and OD of inhibitor₆₀₀And (4) carrying out variation screening on the engineering strains with enhanced tolerance or degradation capability.

The gradient medium used for rescreening was: 1 XYNB medium; adding p-hydroxybenzaldehyde, coumaric aldehyde, coniferyl aldehyde, vanillin, syringaldehyde, sinapildehyde, hydroxymethyl furfural and furfural respectively, and setting four concentration gradients (0.6, 1.3, 2.5 and 5.0 g/L); the buffer system is K₂HPO₄With KH₂PO₄(ii) a The initial pH was adjusted to 5.6 by NaOH.

Inoculation, sampling and OD of the above preliminary screening and secondary screening experiments₆₀₀The detection link is completed by the operation of a liquid workstation Biomek i7, and an automatic process SAMI EX and control software Beckman software are integrated; shaking culture was carried out in a full-temperature shaking incubator (ZQZY-98CV, Shanghai Zhichu), and shaking culture was carried out in a high-speed shaking incubator (ZQZY-88BH, Shanghai Zhichu). The starting strain NP11 is used as a control, and OD is compared and analyzed under different inhibitors and different concentrations₆₀₀Obviously increased dominant strains, engineering strains for expressing lignin multifunctional peroxidase obtained by screening are named as VP9 and VP28, and engineering strains for expressing manganese peroxidase are named as MNP28 and MNP 47.

4. Rhodosporidium toruloides haploid engineering strains VP9, VP28, MNP28 and MNP47 are fermented by simulated concentrated corn straw hydrolysate

The Rhodosporidium toruloides haploid engineering strains VP9, VP28, MNP28 and MNP47 are subjected to fermentation test in artificially synthesized concentrated corn straw hydrolysate, and the starting strain NP11 is used as a control strain.

The specific experimental method is as follows:

seed culture medium: YEPD medium.

The simulated hydrolysate comprises the following components: glucose 70 g/L; 4.8g/L of vanillin, 0.3g/L of p-hydroxybenzaldehyde, 4.5g/L of syringaldehyde, 1.5g/L of vanillic acid, 1.3g/L of p-hydroxybenzoic acid, 1.2g/L of vanillyl alcohol, 1.5g/L of syringanic acid, 0.3g/L of guaiacol, 0.8g/L of eugenol, 5g/L of hydroxymethyl furfural and 5g/L of furfural; the buffer system is K₂HPO₄With KH₂PO₄(ii) a The initial pH was adjusted to 5.6 by NaOH.

Inoculating the strain into 5mL of seed culture medium, activating for 24h to prepare seed liquid, transferring the seed liquid to fresh 50mL of simulated hydrolysate, and setting three biological parallels; control of initial OD₆₀₀Culturing at 30 deg.C and 200rpm/min for 156h, detecting the content change of lignin degradation product in the supernatant of fermentation liquid during fermentation culture, and collecting thallus cells to extract oil.

The oil extraction method comprises the following steps: taking 30mL of fermentation liquor, and calculating biomass after centrifugal drying. Then extracting oil by an acid heating method. 5mL of 4M hydrochloric acid was added to the dried cells, and the mixture was treated in a 78 ℃ water bath for 1.5 hours. After cooling, adding chloroform/methanol solvent with the same volume, carrying out vortex oscillation for 5min, centrifuging for 5min at 8000g, and transferring the lower organic phase to a new centrifuge tube. The upper layer was extracted with an equal volume of chloroform and repeated twice. Adding equal volume of 0.1% sodium chloride solution into the collected organic phase, vortex and shake for 1min, centrifuging for 5min at 8000g, transferring the lower organic phase into a glass funnel containing anhydrous sodium sulfate, drying, and continuously adding chloroform until the red color on the anhydrous sodium sulfate disappears. And removing the solvent from the oil bottle for collecting the oil through reduced pressure distillation, and weighing to calculate the yield of the oil.

The content of the lignin degradation products is detected by adopting a thin-layer chromatography dot plate: at different stages of fermentation, 1ml of fermentation liquid is respectively taken, centrifuged at 8000g/min, and directly dipped into supernatant by a capillary tube and dropped onto a silica gel thin layer chromatography plate (20 × 20cm, Merck, USA); the developing solvent is chloroform: ethyl acetate (3: 1); color development was performed with iodine vapor.

The results of the growth experiments are shown in fig. 3 and 4, and show that the control strain NP11 is inhibited by lignin degradation products with higher concentration during the fermentation period, and hardly grows by using glucose, while the constructed engineering strains VP9, VP28, MNP28 and MNP47 rapidly recover the growth after 72h of lag time.

The results of the oil and fat content measurements are shown in FIG. 5, which shows that at the end of fermentation, the biomass of NP11 was only 0.2g/L, and the oil and fat content was undetectable with too little biomass and was recorded as 0, while the biomass and oil content of strains VP9, VP28, MNP28, MNP47 were 1.9g/L and 13%, 5.1g/L and 37%, 2.9g/L and 12%, and 5.0g/L and 18%, respectively.

The detection results of the lignin degradation products are shown in fig. 6, after 48h of fermentation of the engineering strains VP9, VP28, MNP28 and MNP47, the lignin degradation products are almost completely consumed, and when 96h of fermentation of the strain NP11, much phenol alcohol and phenol acid are still accumulated. The result shows that the lignin multifunctional peroxidase and the manganese peroxidase are expressed in rhodosporidium toruloides haploid NP11, so that the metabolism of the strain on lignin degradation products in lignocellulose hydrolysate is promoted, and the growth of the strain by using the lignin degradation products and the fermentation performance of synthesized grease are improved.

The VP28 and MNP47 strains are sequenced, and sequencing results show that 3 copies of the lignin multifunctional peroxidase expression cassette are inserted into the chromosome of the VP28 strain, and the insertion sites are respectively: the non-coding region at 366954bp of the Scaffold 14 of the rhodosporidium toruloides is inserted into 2 expression cassettes, the 2 expression cassettes are sequentially connected, and the non-coding region at 317063bp of the Scaffold 22 is inserted into 1 expression cassette. The MNP47 strain has 2 copies of manganese peroxidase expression cassette inserted into its chromosome at the following sites: 40034bp of Scaffold 46 and the intron region of autophagy protein gene (ATG4) are inserted into 1 expression cassette, and 58108bp of Scaffold1 and the exon region of hypothetical protein RHTO _00026 are inserted into 1 expression cassette.

Example 2 construction, screening and application of Endomyces dermatioides engineering strains expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP

1. Construction and screening of engineering strain of Trichosporon dermatomyces for expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP

The method for constructing the filamentous fungus engineering strain respectively over-expressing MNP and VP genes by using filamentous fungus (Trichosporon cutaneum) CGMCC No.2.571 as an initial strain comprises the following steps: the recombinant plasmids pZPK-MNP and pZPK-VP are transferred into an engineering strain obtained by an original strain, wherein the construction process and the ATMT transformation process of expression vectors of the pZPK-MNP and the pZPK-VP are as described in example 1, and the electric shock competence preparation and transformation steps are as follows:

(1) selecting a single colony of a yeast strain, inoculating the single colony of the yeast strain into 5mL of YEPD culture medium, and culturing the single colony at 30 ℃ and 200rpm for 16h overnight;

(2) 2mL of the overnight culture was transferred to 50mL of YEPD medium and incubated overnight for 16h at 30 ℃ and 200rpm until the cell density reached 1X 10⁸cell/mL (OD)₆₀₀＝0.8-1.0)；

The following 3-7 steps are all carried out at 0-4 deg.C:

(3) centrifuging at 2000g for 10min, and collecting thallus cells;

(4)20mL of ice-precooled sterile water is used for washing the bacterial cells for 2 times, and the bacterial cells are collected by centrifugation at 2000g for 10 min;

(5)20mL of ice-precooled 1moL/L sorbitol is used for washing the bacterial cells for 2 times, and the bacterial cells are collected after centrifugation for 10min at 2000 g;

(6)0.4mL of ice-precooled 1moL/L sorbitol resuspended bacterial cells, and 50 mu L of each tube is subpackaged;

(7) transferring the cells and DNA to be transformed (200- & lt600 ng, total volume less than or equal to 5 mu L) into an ice-precooled electric transfer cup with a gap of 0.2cm, and performing pulse transformation by using 1.5KV, 25 mu F and 200 omega;

(8) 1mL of ice-precooled 1moL/L sorbitol is rapidly added into an electric rotating cup, the electric rotating cup is lightly blown and sucked by a gun head and uniformly mixed, and the mixture is cultured for 1 to 3 hours at the temperature of 30 ℃;

(9) yeast was spread on sorbitol selective medium plates (sorbitol 180g/L, YNB 1.7g/L, NH)₄SO₄5g/L, 20g/L glucose, 20mg/L histidine and 13g/L agar powder) and culturing at 30 ℃ for 3-6 days until transformants appear.

Engineering strain screening procedure as described in example 1; the obtained engineering strains are named as TC-MNP and TC-VP.

2. Trichosporon cutaneum engineering strains TC-MNP and TC-VP are fermented by utilizing simulated concentrated corn straw hydrolysate

The fermentation test of the above-mentioned engineering strains TC-MNP and TC-VP of Trichosporon dermatomyces in artificially synthesized concentrated corn stalk hydrolysate was carried out in the same manner as in 4 of example 1.

The results show that during the fermentation of the oil of the Trichosporon pisiferum, the control strain Trichosporon cutaneum CGMCC No.2.571 does not use glucose to grow, the constructed engineering strains TC-MNP and TC-VP resume growth after 72h of lag time, and at the end point of the fermentation (156h), the biomass and the oil content of the TC-MNP and the TC-VP are respectively 1.3g/L, 13 percent, 1.8g/L and 15 percent. The results show that the lignin multifunctional peroxidase and the manganese peroxidase are expressed in the filamentous fungi, which is beneficial to improving the metabolism of the strain on lignin degradation products in lignocellulose hydrolysate and improving the growth of the strain on the lignin degradation products and the fermentation performance of synthetic grease.

Example 3 construction, screening and application of Lipomyces starkeyi engineering strains expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP

1. Construction and screening of MNP and MNP engineering strains of grease yeast of Sdalbergia for expressing lignin

The recombinant plasmids pZPK-MNP and pZPK-VP are transferred into L.starkeyi by taking Lipomyces starkeyi NRRL Y-11557 as an original strain to respectively obtain the Lipomyces starkeyi engineering strain for over-expressing MNP and VP. The construction of pZPK-MNP and pZPK-VP expression vectors, the ATMT transformation procedure, the shock transformation procedure, the engineering strain screening procedure, and the identification of the engineering strains LS-MNP and LS-VP were as described in example 1, example 2, and example 1.

2. S-MNP and LS-VP of engineering strains of grease yeast of Sdaffia are fermented by utilizing simulated concentrated corn straw hydrolysate

The fermentation test of the above-mentioned engineering strains LS-MNP and LS-VP of Lipomyces starkeyi was carried out in artificially synthesized concentrated corn stalk hydrolysate by the same method as that described in 4 of example 1.

The results show that the control strain, Lipomyces starkkeyi NRRL Y-11557, does not use glucose to grow during the Lipomyces starkkeyi oil fermentation, while the constructed engineering strains LS-MNP and LS-VP resume growth after 72h of lag time, and the biomass and the oil content of the LS-MNP and the LS-VP are respectively 1.1g/L, 10 percent and 1.6g/L, 14 percent at the end point of the fermentation (156 h). The results show that the expression of the lignin multifunctional peroxidase and the manganese peroxidase in the grease yeast of Sdaffia is beneficial to improving the metabolism of the strain on lignin degradation products in lignocellulose hydrolysate and improving the growth of the strain on the lignin degradation products and the fermentation performance of synthesized grease.

Example 4 construction, screening and application of Rhodotorula glutinis engineering strains expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP

1. Construction and screening of engineering strain of rhodotorula glutinis expressing lignin multifunctional peroxidase VP and manganese peroxidase MNP

Rhodotorula glutinis (Rhodotorula glutinis) ATCC 204091 is used as an original strain, recombinant plasmids pZPK-MNP and pZPK-VP are transferred into the original strain, and engineering strains for respectively over-expressing MNP and VP genes are obtained. Wherein the construction process of pZPK-MNP and pZPK-VP expression vectors and the ATMT transformation process are described in example 1, the electric shock transformation process is described in example 2, the screening process of engineering strains is described in example 1, and the obtained engineering strains are named as RG-MNP and RG-VP.

2. Rhodotorula glutinis engineering strains RG-MNP and RG-VP are fermented by utilizing simulated concentrated corn straw hydrolysate

The fermentation test of the engineering strains RG-MNP and RG-VP of the rhodotorula glutinis in the artificially synthesized concentrated corn stalk hydrolysate is carried out by the same method as that in 4 of the embodiment 1.

The results show that the constructed engineering strains RG-MNP and RG-VP can resume growth after 72h of delay time during the fermentation period of the Rhodotorula glutinis oil and fat, and the biomass and the oil content of the RG-MNP and the RG-VP are respectively 1.3g/L, 10 percent, 1.5g/L and 12 percent at the end point of the fermentation (156 h). The results show that the expression of the lignin multifunctional peroxidase and the manganese peroxidase in the rhodotorula glutinis is beneficial to improving the metabolism of the strain on lignin degradation products in lignocellulose hydrolysate and improving the growth of the strain on the lignin degradation products and the fermentation performance of synthesized grease.

Example 5 construction, screening and application of Cryptococcus curvatus engineering strains expressing Lignin multifunctional peroxidase VP, manganese peroxidase MNP

Cryptococcus curvatus ATCC 20509 is used as an original strain, and recombinant plasmids pZPK-MNP and pZPK-VP are transferred into the original strain to respectively obtain engineering strains for over-expressing MNP and VP genes. Wherein the construction process of pZPK-MNP and pZPK-VP expression vectors and the ATMT transformation process are described in example 1, the electric shock transformation process is described in example 2, the screening process of engineering strains is described in example 1, and the obtained engineering strains are named as CC-MNP and CC-VP.

2. Cryptococcus sinuatus engineering strains CC-MNP and CC-VP are fermented by utilizing simulated concentrated corn straw hydrolysate

The fermentation test of the cryptococcus curvatus engineering strains CC-MNP and CC-VP in the artificially synthesized concentrated corn stalk hydrolysate is carried out, and the specific method is the same as that in 4 of the embodiment 1.

The results show that the control strain Cryptococcus curvatus ATCC 20509 does not grow by glucose during the fermentation of Cryptococcus curvatus oil, while the constructed engineering strains CC-MNP and CC-VP resume growth after 72h of delay, and the biomass and the oil content of the CC-MNP and CC-VP are respectively 1.0g/L, 10 percent and 1.1g/L, 18 percent at the end point of the fermentation (156 h). The results show that the expression of the lignin multifunctional peroxidase and the manganese peroxidase in the cryptococcus flexus is beneficial to improving the metabolism of the strain on lignin degradation products in lignocellulose hydrolysate and improving the growth of the strain on the lignin degradation products and the fermentation performance of synthetic grease.

Example 6 construction, screening and application of Rhodosporidium toruloides engineering strains expressing Lignin multifunctional peroxidase VP, manganese peroxidase MNP

Rhodosporidium toruloides (Rhodosporidium toruloides) CGMCC No.2.1389 is used as an original strain, and recombinant plasmids pZPK-MNP and pZPK-VP are transferred into the original strain to respectively obtain engineering strains for over-expressing MNP and VP genes. Wherein the construction process of pZPK-MNP and pZPK-VP expression vectors and the ATMT transformation process are described in example 1, the electric shock transformation process is described in example 2, the screening process of engineering strains is described in example 1, and the obtained engineering strains are named as RT-MNP and RT-VP.

2. Rhodosporidium toruloides engineering strains RT-MNP and RT-VP are fermented by utilizing simulated concentrated corn straw hydrolysate

The fermentation test of the rhodosporidium toruloides engineering strains RT-MNP and RT-VP in the artificially synthesized concentrated corn straw hydrolysate is carried out, and the specific method is the same as that in 4 of the embodiment 1.

The results show that during the lipid fermentation period of Rhodosporidium toruloides, the control strain Rhodosporidium toruloides CGMCC No.2.1389 does not use glucose for growth, while the constructed engineering strains RT-MNP and RT-VP resume growth after 72h of lag phase, and at the end of fermentation (156h), the biomass and lipid content of RT-MNP and RT-VP are respectively 1.1g/L, 13 percent, 1.2g/L and 15 percent. The results show that the lignin multifunctional peroxidase and the manganese peroxidase are expressed in rhodosporidium toruloides, which is beneficial to improving the metabolism of the strain on lignin degradation products in lignocellulose hydrolysate and improving the growth of the strain by using the lignin degradation products and the fermentation performance of synthetic grease.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga 900

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tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg 1080

ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc 1140

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gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc 1260

ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc 1320

gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg 1380

attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc 1440

caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg 1500

cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc 1560

attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc 1620

acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag 1680

tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt 1740

gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa 1800

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gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca 1920

cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga 1980

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ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga 2160

acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga 2220

acccccaagc ccgaggaatc ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg 2280

cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca 2340

acgcatcgag gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg 2400

caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg 2460

cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg 2520

cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt 2580

gatccgctac gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc 2640

cagtgtgtgg gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa 2700

ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga 2760

cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac 2820

ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg 2880

ccgcctggtg acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag 2940

cgaaaccggg cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat 3000

cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc 3060

cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag 3120

atggttgttc aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg 3180

tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga 3240

ggcggggcag gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc 3300

atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa 3360

aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat 3420

tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat 3480

gtaagtgact gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact 3540

tattaaaact cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga 3600

agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg 3660

tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc 3720

agggcgcgga caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct 3780

gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg 3840

tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 3900

gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 3960

ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 4020

aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 4080

cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 4140

ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 4200

ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 4260

cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 4320

actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 4380

cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 4440

tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 4500

gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 4560

caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 4620

agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 4680

tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 4740

tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 4800

gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 4860

gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gccatattca 4920

acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg 4980

gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg 5040

gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt 5100

tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa 5160

gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac 5220

agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc 5280

agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg 5340

cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga 5400

ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact 5460

tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat 5520

ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg 5580

ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa 5640

acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt 5700

gatgctcgat gagtttttct aatcacaggc agcaacgctc tgtcatcgtt acaatcaaca 5760

tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg 5820

aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg 5880

tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg 5940

agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa 6000

cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt gaattcgagc 6060

tcggtaccca gacggacctt gagaaccctc aatcgctcgc ggtactcgtc cgccctgcga 6120

tccagcatcg aaaccgagtg cagcgcgttc aacaaatccg agtcgtctcc tcctgctcct 6180

tcgcgctgtt tcggcgcggg tggcgcaggg acagccggag ggagaggggg aggaggagga 6240

ggggctgggg cgaccttttt cttcttcttc ctgttcttgc ccttcttctt cgccgcctct 6300

gcttctccat cacccaccgc ccccacattc gccgccgaag caccgaccac ggcccccctc 6360

atccgcctct gcaacctcct cgcctcgctc gcctccaaac tcaatcgcgc gacgcactgc 6420

tccaactcgg cgatggcgct catcaagctt gggagggagg cgggtgagag agccgagtcg 6480

gagaggatgc cgtctgcggg gatttgggag ggtgagaggt gggtttgagg cggttgagag 6540

gaggaggacg agagggggag ggcggaggag agggctgtca agtccgagag ggagaggggg 6600

atcgaggtcg tgaaggatgt cgaggacgag gaagaggcgt tccgctgttg ttgttgctgg 6660

acggcggaga ggacgcccag gaaggctgcg tcggccgctt gcgagggcga gtcgatcacc 6720

tcgtaggcgg cgtcgtcgtg ctcggacggc gacgacgcgt ggtcctccga ctcgacccag 6780

tctggcccgt tgaagaggca ttcggcggcg aagacgacgg ccgggtcgtc cgaggctgtc 6840

gaggcaagtg gctgtaagca agcgcggcag aagaggcaga acgctgcgac gcactcctag 6900

gatcccacgg ctgcgcatcc ggatcctccc cgcgctgctg gcgcctcgcc cgctcccgct 6960

cgtactccgc catgaccgtc aacccctggc agagcaggcg gtactgctcg accagggccc 7020

agtcctcgct cgtctgtggg aaaggttgga cgggacatcc tggcggcaat ggaagcgagt 7080

acagccagtc gagcgtgaga ggggagggcc gaaagtgctc cgtcgagatg ctggcgagcg 7140

agacgagggg ctgggtgagc gacggcgggc ggatccctgc gcctgtgttg tccatccctg 7200

cagtgcactc tgttgctcgt atcatgtccc actcccttgt atccctcgag tcggtcgact 7260

cttccctggc gagtccaagc ggaggaggtg gtcgtcgcct gacccgctcg gagtgcgccg 7320

ctcgacttgg ccctgggaga acaagcctgt gtgagtctgt ctagcctgtc agcgaatgcg 7380

ccagacgagt gcaagcgggt gagcgaggtc gaccctgctc gtcactcgct cgtcgggtgc 7440

ggccgcatcg ttgaacttgc acttctcact cgcactcgct ctggtacagc tacagtcact 7500

cgcttactac tctgcaggtt cacagcaact cacccgtcca actcccaccc tcccccgtgc 7560

agcccaccat ggccttcgcc aagctctcgg ccttcgtcct cgctctcggc gccaccgtcg 7620

ccctcggcga gtcgccgacc caccgctgcc tcaacaagcg cgtcacctgc gccaccggcc 7680

agaccaccgc taacgaggct tgctgcgccc tcttcccgat cctcgacgac atccagacca 7740

acctcttcga cggcgctcag tgcggagagg aggtccacga gtcgctccgc ctcaccttcc 7800

acgacgctat cgccttctcg ccggctctca ccaacgccgg acagttcgga ggcggcggcg 7860

ccgacggctc gatgatcatc ttctcggaca ccgagccgaa cttccacgcc aacctcggca 7920

tcgacgagat cgtcgaggcc cagaagccgt tcatcgcccg ccacaacatc tcggccgccg 7980

acttcatcca gttcgccggc gccatcggag tctcgaactg cgccggagct ccgcgcctca 8040

acttcttcct cggacgccca gacgctaccc agatccctcc agacggactc gtcccagagc 8100

cgttcgacga cgtcaccaag atcctctcgc gcatgggaga cgccggattc tcgaccgtcg 8160

aggtcgtctg gctcctcgct tcgcacacca tcgcggctgc tgaccacgtc gacccttcga 8220

tcccaggcac cccgttcgac tcgaccccgt cgaccttcga ctcgcagttc ttcctcgaga 8280

ccatgctcca gggcaccgct ttcccgggca ccccgggcaa ccagggcgag gtcgagtcgc 8340

cgctcgccgg cgagatgcgc ctccagtcgg acttcctcct cgcccgcgac tcgcgctcgg 8400

cttgcgagtg gcagtcgatg gtcaacaaca tgccgaagat ccagaaccgc ttcacccagg 8460

tcatgaagaa gctctcgctc ctcggccaca accaggccga cctcatcgac tgctcggacg 8520

tcatcccggt cccgaagacc ctcaccaagg ccgctacctt cccagccgga aagtcgcagg 8580

ccgacgtcga gatcgtctgc aacgccgccg ctaccccttt cccggccctc gcctcggacc 8640

cgggccctgt caccgccgtc ccgccggtcc cgccttcggg ctcgggcgct accaacttct 8700

cgctcctcaa gcaggctggc gacgtcgaag agaaccctgg ccctatgccg gagctcacgg 8760

cgacgtcggt cgagaagttc ctcatcgaaa agttcgactc ggtctcggac ctcatgcaac 8820

tctcggaggg agaggaatcg cgcgcgttct cgttcgacgt cggaggccgc ggatacgtcc 8880

tccgcgtcaa ctcgtgtgcg gacggattct acaaggatcg gtacgtctac cgccattttg 8940

cgtcggcggc gctcccgatc cccgaagtcc tcgacatcgg agagttctcg gaatccctca 9000

cgtactgtat ctcgcgccgg gcgcaaggag tcacgctcca agatctcccg gagacggaac 9060

tcccggcggt cctccaaccg gtcgcggaag cgatggacgc gatcgcggcc gcggacctct 9120

cgcaaacgtc gggattcgga ccgtttggac cgcaaggaat cggacaatac acgacgtggc 9180

gcgacttcat ctgtgcgatc gcggatcccc atgtctacca ctggcaaacg gtcatggatg 9240

acacggtctc ggcgtcggtc gcgcaagcgc tcgacgagct catgctctgg gcggaggact 9300

gtccggaggt ccgccacctc gtccacgcgg actttggatc gaacaacgtc ctcacggaca 9360

acggacgcat cacggcggtc atcgactggt cggaggcgat gtttggagac tcgcaatacg 9420

aggtcgcgaa catcttcttc tggcgcccgt ggctcgcgtg catggagcaa cagacgcgct 9480

acttcgagcg ccgccacccg gagctcgcgg gatcgccgcg cctccgcgcg tacatgctcc 9540

gcatcggcct cgaccaactc taccagtcgc tcgtcgacgg aaacttcgac gatgccgcgt 9600

gggcccaagg acgctgcgac gcgattgtcc gctcgggagc gggaaccgtg ggacgcacgc 9660

aaattgcgcg gcgctcggcg gccgtctgga cggatggatg tgtcgaagtc ctcgcggatt 9720

cgggaaaccg gcgcccgtcg acgcgcccgc gggcgaaaga acaccaccat caccatcact 9780

agacgattcc gccccgtctc acctcgcatc cgacctgtgt agccacgcct tttctctttc 9840

cgcccccgac actaaaagga gttcagtcgt ccgcttttcc tctcttccgt tctccacgcc 9900

tgcagtactg catgcctcag ctgtgtaatt tcgatagagt actcgcacgt atgctcgcgc 9960

ggacttgtcg agagagcggg cgagaggtcg agaggagctg acttgagtga gcagcgagag 10020

cggagcggct tgttgcggca cacaacggtc cggcttacgg acaatcagcg atgaagccgg 10080

aagagcggca ggagtggacg aagacgtgcg aagaggaaag ggggcgagga agagaagcgg 10140

aggaggaaac gcagcgaagc agcacaactt ccgcagagac gacggttcac atagtcaaag 10200

atgcagtgca gagaagtgcg cgggatcctc tagagtcgac ctgcagcatg caagcttgga 10260

gcttgagctt ggatcagatt gtcgtttccg ccttcagttt aaactatcag tgtttgacag 10320

gatatattgg cgggtaaacc taagagaaaa gagcgtttat tagaataatc ggatatttaa 10380

aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac cacagggttc 10440

ccctcgggat caa 10453

<210> 6

<211> 10479

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag 60

ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg 120

cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac 180

tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc 240

cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc 300

tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc 360

aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg 420

gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc 480

ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg 540

accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc 600

gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg 660

gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg 720

gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa 780

gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc 840

gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg 900

acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg 960

ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg 1020

gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc 1080

gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg 1140

cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc 1200

tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc 1260

cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg 1320

cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac 1380

gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc 1440

ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt 1500

gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg 1560

cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg 1620

cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga 1680

gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct 1740

tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa 1800

atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta 1860

agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac 1920

acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag 1980

atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag 2040

ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg 2100

cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg 2160

aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg 2220

aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg 2280

gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc 2340

aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc 2400

gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg 2460

gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc 2520

gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg 2580

tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg 2640

ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga 2700

accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg 2760

acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa 2820

cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg 2880

gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga 2940

gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga 3000

tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc 3060

ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca 3120

gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct 3180

gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg 3240

aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag 3300

catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa 3360

aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca 3420

ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca 3480

tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac 3540

ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg 3600

aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc 3660

gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac 3720

cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc 3780

tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 3840

gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 3900

ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 3960

actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 4020

aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 4080

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4140

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4200

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4260

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4320

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4380

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4440

atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4500

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4560

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4620

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 4680

ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 4740

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 4800

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4860

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agccatattc 4920

aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg 4980

ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg 5040

ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg 5100

ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca 5160

agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa 5220

cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg 5280

cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc 5340

gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg 5400

attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac 5460

ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta 5520

tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc 5580

gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga 5640

aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt 5700

tgatgctcga tgagtttttc taatcacagg cagcaacgct ctgtcatcgt tacaatcaac 5760

atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg ccgttcttcc 5820

gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc cgctgacgcc 5880

gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct gccggtcggg 5940

gagctgttgg ctggctggtg gcaggatata ttgtggtgta aacaaattga cgcttagaca 6000

acttaataac acattgcgga cgtttttaat gtactgaatt aacgccgaat tgaattcgag 6060

ctcggtaccc cagacggacc ttgagaaccc tcaatcgctc gcggtactcg tccgccctgc 6120

gatccagcat cgaaaccgag tgcagcgcgt tcaacaaatc cgagtcgtct cctcctgctc 6180

cttcgcgctg tttcggcgcg ggtggcgcag ggacagccgg agggagaggg ggaggaggag 6240

gaggggctgg ggcgaccttt ttcttcttct tcctgttctt gcccttcttc ttcgccgcct 6300

ctgcttctcc atcacccacc gcccccacat tcgccgccga agcaccgacc acggcccccc 6360

tcatccgcct ctgcaacctc ctcgcctcgc tcgcctccaa actcaatcgc gcgacgcact 6420

gctccaactc ggcgatggcg ctcatcaagc ttgggaggga ggcgggtgag agagccgagt 6480

cggagaggat gccgtctgcg gggatttggg agggtgagag gtgggtttga ggcggttgag 6540

aggaggagga cgagaggggg agggcggagg agagggctgt caagtccgag agggagaggg 6600

ggatcgaggt cgtgaaggat gtcgaggacg aggaagaggc gttccgctgt tgttgttgct 6660

ggacggcgga gaggacgccc aggaaggctg cgtcggccgc ttgcgagggc gagtcgatca 6720

cctcgtaggc ggcgtcgtcg tgctcggacg gcgacgacgc gtggtcctcc gactcgaccc 6780

agtctggccc gttgaagagg cattcggcgg cgaagacgac ggccgggtcg tccgaggctg 6840

tcgaggcaag tggctgtaag caagcgcggc agaagaggca gaacgctgcg acgcactcct 6900

aggatcccac ggctgcgcat ccggatcctc cccgcgctgc tggcgcctcg cccgctcccg 6960

ctcgtactcc gccatgaccg tcaacccctg gcagagcagg cggtactgct cgaccagggc 7020

ccagtcctcg ctcgtctgtg ggaaaggttg gacgggacat cctggcggca atggaagcga 7080

gtacagccag tcgagcgtga gaggggaggg ccgaaagtgc tccgtcgaga tgctggcgag 7140

cgagacgagg ggctgggtga gcgacggcgg gcggatccct gcgcctgtgt tgtccatccc 7200

tgcagtgcac tctgttgctc gtatcatgtc ccactccctt gtatccctcg agtcggtcga 7260

ctcttccctg gcgagtccaa gcggaggagg tggtcgtcgc ctgacccgct cggagtgcgc 7320

cgctcgactt ggccctggga gaacaagcct gtgtgagtct gtctagcctg tcagcgaatg 7380

cgccagacga gtgcaagcgg gtgagcgagg tcgaccctgc tcgtcactcg ctcgtcgggt 7440

gcggccgcat cgttgaactt gcacttctca ctcgcactcg ctctggtaca gctacagtca 7500

ctcgcttact actctgcagg ttcacagcaa ctcacccgtc caactcccac cctcccccgt 7560

gcagcccacc atggccttca agtcgctcat cgccttcgtc gctctcgccg ccgccgtccg 7620

cgccgctcct accgccgtct gccccgacgg cacccgcgtc tcgcacgccg cttgctgcgc 7680

cttcatccct ctcgcccagg acctccagga gaccatcttc cagaacgagt gcggcgagga 7740

cgctcacgag gtcatccgcc tcaccttcca cgacgccatc gctatctcgc gctcgcaggg 7800

acctaaggcc ggcggcggcg ccgacggctc gatgctcctc ttcccgaccg tcgagccgaa 7860

cttctcggcc aacaacggca tcgacgactc ggtcaacaac ctcatcccgt tcatgcagaa 7920

gcacaacacc atctcggccg ccgacctcgt ccagttcgcc ggcgccgtcg ccctctcgaa 7980

ctgcccgggc gccccgcgcc tcgagttcct cgccggacgc ccgaacaaga ccatcgccgc 8040

cgtcgacgga ctcatcccag agccgcagga ctcggtcacc aagatcctcc agcgcttcga 8100

ggacgccgga ggattcaccc cgttcgaggt cgtctcgctc ctcgcttcgc actcggtcgc 8160

ccgcgccgac aaggtcgacc agaccatcga cgccgctcct ttcgactcga ccccgttcac 8220

cttcgacacc caggtcttcc tcgaggtcct cctcaagggc gtcggcttcc caggatcggc 8280

caacaacacc ggcgaggtcg cttcgcccct cccgctcggc tcgggctcgg acaccggcga 8340

gatgcgcctc cagtcggact tcgctctcgc tcacgaccct cgcaccgctt gcatctggca 8400

gggcttcgtc aacgagcagg ccttcatggc cgcttcgttc cgcgccgcta tgtcgaagct 8460

cgccgtcctc ggacacaacc gcaactcgct catcgactgc tcggacgtcg tcccggtccc 8520

taagccagct accggacagc cagccatgtt cccggcttcg accggacctc aggacctcga 8580

gctctcgtgc ccttcggagc gcttccctac cctcaccacc cagccaggag cttcgcagtc 8640

gctcatcgcc cactgcccag acggatcgat gtcgtgccct ggagtccagt tcaacggccc 8700

agccggctcg ggcgctacca acttctcgct cctcaagcag gctggcgacg tcgaagagaa 8760

ccctggccct atgccggagc tcacggcgac gtcggtcgag aagttcctca tcgaaaagtt 8820

cgactcggtc tcggacctca tgcaactctc ggagggagag gaatcgcgcg cgttctcgtt 8880

cgacgtcgga ggccgcggat acgtcctccg cgtcaactcg tgtgcggacg gattctacaa 8940

ggatcggtac gtctaccgcc attttgcgtc ggcggcgctc ccgatccccg aagtcctcga 9000

catcggagag ttctcggaat ccctcacgta ctgtatctcg cgccgggcgc aaggagtcac 9060

gctccaagat ctcccggaga cggaactccc ggcggtcctc caaccggtcg cggaagcgat 9120

ggacgcgatc gcggccgcgg acctctcgca aacgtcggga ttcggaccgt ttggaccgca 9180

aggaatcgga caatacacga cgtggcgcga cttcatctgt gcgatcgcgg atccccatgt 9240

ctaccactgg caaacggtca tggatgacac ggtctcggcg tcggtcgcgc aagcgctcga 9300

cgagctcatg ctctgggcgg aggactgtcc ggaggtccgc cacctcgtcc acgcggactt 9360

tggatcgaac aacgtcctca cggacaacgg acgcatcacg gcggtcatcg actggtcgga 9420

ggcgatgttt ggagactcgc aatacgaggt cgcgaacatc ttcttctggc gcccgtggct 9480

cgcgtgcatg gagcaacaga cgcgctactt cgagcgccgc cacccggagc tcgcgggatc 9540

gccgcgcctc cgcgcgtaca tgctccgcat cggcctcgac caactctacc agtcgctcgt 9600

cgacggaaac ttcgacgatg ccgcgtgggc ccaaggacgc tgcgacgcga ttgtccgctc 9660

gggagcggga accgtgggac gcacgcaaat tgcgcggcgc tcggcggccg tctggacgga 9720

tggatgtgtc gaagtcctcg cggattcggg aaaccggcgc ccgtcgacgc gcccgcgggc 9780

gaaagaacac caccatcacc atcactagac gattccgccc cgtctcacct cgcatccgac 9840

ctgtgtagcc acgccttttc tctttccgcc cccgacacta aaaggagttc agtcgtccgc 9900

ttttcctctc ttccgttctc cacgcctgca gtactgcatg cctcagctgt gtaatttcga 9960

tagagtactc gcacgtatgc tcgcgcggac ttgtcgagag agcgggcgag aggtcgagag 10020

gagctgactt gagtgagcag cgagagcgga gcggcttgtt gcggcacaca acggtccggc 10080

ttacggacaa tcagcgatga agccggaaga gcggcaggag tggacgaaga cgtgcgaaga 10140

ggaaaggggg cgaggaagag aagcggagga ggaaacgcag cgaagcagca caacttccgc 10200

agagacgacg gttcacatag tcaaagatgc agtgcagaga agtgcgcggg atcctctaga 10260

gtcgacctgc agcatgcaag cttggagctt gagcttggat cagattgtcg tttccgcctt 10320

cagtttaaac tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc 10380

gtttattaga ataatcggat atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt 10440

gtatgtgcat gccaaccaca gggttcccct cgggatcaa 10479

<210> 7

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctggcgacgt cgaagagaac cctggcccta tgccggagct cacggcgac 49

<210> 8

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgcatgctgc aggtcgactc tagaggatcc cgcgcacttc tctgcactgc 50

<210> 9

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caactcccac cctcccccgt gcagcccacc atggccttcg ccaagctctc 50

<210> 10

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gaggagcgag aagttggtag cgcccgagcc cgaaggcggg accggcg 47

<210> 11

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctggcgacgt cgaagagaac cctggcccta tgccggagct cacggcgac 49

<210> 12

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgcatgctgc aggtcgactc tagaggatcc cgcgcacttc tctgcactgc 50

<210> 13

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cctcccccgt gcagcccacc atgcaccacc atcaccatca cgccttcaag tcgctcat 58

<210> 14

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gaggagcgag aagttggtag cgcccgagcc ggctgggccg ttgaact 47

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aagttggtag cgcccgagcc 20

Claims (10)

1. A yeast engineering bacterium, which is characterized in that the yeast engineering bacterium expresses lignin multifunctional peroxidase and/or manganese peroxidase;

the yeast engineering bacteria belong to the genera Lipomyces (Lipomyces), Trichosporon (Trichosporon), Cryptococcus (Cryptococcus), Saccharomyces (Rhodosporidium) or Rhodotorula (Rhodotorula).

2. The engineered yeast strain of claim 1, wherein the lignin multifunctional peroxidase is derived from Pleurotus eryngii (Pleurotus eryngii),

and/or the presence of a gas in the gas,

the manganese peroxidase is derived from Phanerochaete chrysosporium (Phanerochaete chrysosporium).

3. The yeast engineering bacteria of claim 2, wherein the amino acid sequence of the lignin multifunctional peroxidase is shown as SEQ ID No.1, and/or the amino acid sequence of the manganese peroxidase is shown as SEQ ID No. 2;

preferably, the sequence of the encoding gene of the lignin multifunctional peroxidase is shown as SEQ ID No.3, and/or the sequence of the encoding gene of the manganese peroxidase is shown as SEQ ID No. 4.

4. The yeast engineering bacteria according to any one of claims 1 to 3, wherein the chromosome of the yeast engineering bacteria contains one or two of lignin multifunctional peroxidase expression cassettes and manganese peroxidase expression cassettes;

preferably, the lignin multifunctional peroxidase expression cassette contains a Ppgk promoter, a lignin multifunctional peroxidase-encoding gene VP and a transcription terminator;

the manganese peroxidase expression cassette contains a Ppgk promoter, a manganese peroxidase coding gene MNP and a transcription terminator;

more preferably, the transcription terminator is T_HSP。

5. The engineered yeast strain according to any one of claims 1 to 4, wherein the starting strain of the engineered yeast strain is any one selected from Rhodosporidium toruloides (Rhodosporidium toruloides) with a collection number of CGMCC No.2.1389, Rhodosporidium toruloides (Rhodosporidium toruloides) haploid NP11 with a collection number of GDMCC No.2.224, Trichosporon cutaneum (Trichosporon cutaneum) with a collection number of CGMCC No.2.571, Lipomyces stakkenyi (Lipomyces stakkenyi) with a collection number of NRRL Y-11557, Rhodotorula glutinis (Rhodotorula glutinis) with a collection number of ATCC 204091, and Cryptococcus curvatus (Cryptococcus curvatus) with a collection number of ATCC 20509.

6. The engineered yeast strain of claim 4 or 5, wherein the engineered yeast strain comprises 3 copies of the lignin multifunctional peroxidase expression cassette or 2 copies of the manganese peroxidase expression cassette on a chromosome.

7. The method for constructing engineered yeast strain according to any one of claims 1 to 6, comprising: introducing the lignin multifunctional peroxidase expression cassette and/or the manganese peroxidase expression cassette into an original strain, and screening to obtain a strain expressing the lignin multifunctional peroxidase and/or the manganese peroxidase;

preferably, the expression cassette is linked to a yeast episomal or integrative vector, which is introduced into the starting strain.

8. The use of the engineered yeast strain of any one of claims 1 to 6 in any one of the following applications:

(1) the application in the production of microbial oil;

(2) use in increasing the tolerance of yeast to lignin degradation products;

(3) the application of the yeast in improving the growth performance of the yeast in raw materials containing lignin degradation products and the yield of microbial oil;

(4) application in breeding of yeast production strains;

(5) the application in the degradation of lignin biomass.

9. A method for producing microbial oil, comprising: culturing the engineered yeast strain of any one of claims 1 to 6, and separating to obtain microbial oil.

10. The production method according to claim 9, wherein the cultured medium contains lignin degradation products.

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